Patent Application
20240175092
The present invention relates to the field of molecular biology detection technology, especially to a primer-probe composition, a kit, and a detection method.
Aldehyde-ketone reductase family 1 member C3 (AKR1C3) is an enzyme encoded by the AKR1C3 gene in humans. In various types of cancer, AKR1C3 is overexpressed at the protein level. Due to the high expression of AKR1C3 in tumors, compounds OBI-3424 (also referred as AST-3424 and TH-3424), OBI-3423 and OBI-2870 are designed to be specifically activated in tumors, but cannot be activated in normal cells expressing low level of AKR1C3 in order to achieve tumor-specific targeting. Compound OBI-3424 is currently being studied in multiple phase I clinical trials in the United States (NCT04315324 and NCT03592264) and China (CXHL1900137 and CXHL2000263) for treating more than 14 types of human cancers, including solid and hematological tumors.
Compound OBI-3424 is a novel prodrug dialkylating agent activated by AKR1C3. Studies have confirmed that compound OBI-3424 activation is AKR1C3-dependent, and its cytotoxicity and antitumor efficacy are highly correlated with the expression level of AKR1C3 enzyme. Compound OBI-3424 shows AKR1C3-dependent cytotoxicity ex vivo and antitumor activity in vivo in a variety of human cancer types, which supports the further development of compound OBI-3424 as an anticancer agent that can be used to treat different types of cancers. AKR1C3 is used as a biomarker to analyze the condition of cancer patients and further guides patients to choose compound OBI-3424 for treatment. Therefore, in practical application, the AKR1C3 enzyme content in an ex vivo sample of a patient can be detected, and then it is determined whether to administrate the compound OBI-3424 to patient for treatment according to the detection results.
In the prior art, Western blotting or immunochemical staining (IHC) is usually used to detect AKR1C3 enzyme content in an ex vivo sample of a patient. In the reference (Harvey D J, Singleton R S, Dachs G U, et al., The Bioreductive Prodrug PR-104A Is Activated under Aerobic Conditions by Human Aldo-Keto Reductase 1C3[J]. Cancer Research, 2010, 70(4):1573), 2700 tumor tissue samples were counted and analyzed by IHC method, wherein AKR1C3 enzyme was highly expressed in liver cancer, gastric cancer, esophageal cancer and bladder cancer, while it was low expressed in small cell lung cancer, breast cancer, leukemia and prostate cancer. Therefore, it is theoretically feasible to use IHC method to detect the expression level of AKR1C3 enzyme in solid tumor tissue samples.
However, there are many types of cancer or tumor, patient with hematological cancer (leukemia) other than solid tumor cannot provide tissue samples, so it is not possible to directly use western blotting or immunochemical staining to detect the expression level of AKR1C3 enzyme in hematological cancer (leukemia) sample.
The applicant's R&D team has found that the expression level of AKR1C3 enzyme and AKR1C3 RNA content of different solid tumor cancer cells are significantly correlated with the IC50 value of AST-3424 inhibition on cancer cell proliferation, and the expression level of AKR1C3 enzyme and the AKR1C3 RNA content of different hematological cancer cell lines are significantly correlated with the IC50 value of AST-3424 inhibition on cancer cell proliferation. Therefore, the enzyme content can also be predicted or characterized by detecting the ARKR1C3 RNA content, thereby guiding the drug administration.
However, there is no corresponding detection method for detecting AKR1C3 RNA content in the prior art. Assuming that a corresponding detection method can be used to assess AKR1C3 RNA content, patient with higher AKR1C3 RNA content and most likely to respond to the prodrug thus can be selected to administrate compound OBI-3424 for achieving better cancer treatment effect.
In view of this, the purpose of the present invention is to provide a primer-probe composition, a kit, and a detection method, so that the detection of AKRIC3 RNA content in an ex vivo sample of a patient can be achieved with good accuracy, analytical specificity, precision and low detection limit by using the primer-probe composition, the kit, and the detection method.
For the above purpose, the first aspect of the present invention provides a primer-probe composition for the detection of AKR1C3 RNA content in an ex vivo sample of a patient, selected from any one of the following groups:
In a preferred embodiment of the present invention, wherein the primer-probe composition is selected from any one of groups (iii), (iv) and (v);
more preferably, the primer-probe composition is selected from group (iv).
In a preferred embodiment of the present invention, wherein the 5′-end reporters of the probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 are FAM, and the 3′-end quenchers of the probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 are MGB.
Based on the same inventive concept, the second aspect of the present invention provides a kit comprising the primer-probe composition as described above.
In a preferred embodiment of the present invention, wherein the kit further comprises a primer-probe composition of reference gene;
preferably, the reference gene is ACTB.
In a preferred embodiment of the present invention, wherein the primer-probe composition of reference gene comprises upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1, and the nucleotide sequences of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 are shown as SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.
In a preferred embodiment of the present invention, wherein the 5′-end reporter of the probe ACTB-P1 is VIC, and the 3′-end quencher of the probe ACTB-P1 is BHQ1.
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a polymerase mixture, and the polymerase mixture mainly comprises: DNA polymerase, MgCl2, buffer and dNTPs;
preferably, the polymerase mixture is KAPA PROBE FAST RT-PCR Master Mix(2x).
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a reverse transcriptase mixture;
preferably, the reverse transcriptase mixture is Superscript VILO MARSTER MIX.
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a negative control and a positive control;
preferably, the negative control is nuclease-free water; and/or
preferably, the positive control is a reference with a known copy number.
Based on the same inventive concept, the third aspect of the present invention provides use of the primer-probe composition or the kit as described above in the preparation of a drug for treating cancers.
In a preferred embodiment of the present invention, wherein the AKR1C3 RNA content in an ex vivo sample of a patient is obtained by using the primer-probe composition or the kit as described above;
patient with AKR1C3 RNA content greater than or equal to the predetermined content is administrated with AKR1C3 activated anticancer drugs.
In a preferred embodiment of the present invention, wherein the AKR1C3 RNA content is obtained according to the ratio of AKR1C3 copy number/reference gene copy number;
preferably, the predetermined content is 0.0001˜1;
more preferably, the predetermined content is 0.00011˜0.5;
even more preferably, the predetermined content is 0.00013˜0.05.
In a preferred embodiment of the present invention, wherein the ex vivo sample comprises blood sample, bone marrow sample, or tissue sample.
In a preferred embodiment of the present invention, wherein the AKR1C3 activated anticancer drug of course includes the AKR1C3 activated anticancer prodrug in the present invention, i.e., the compounds in the form of prodrugs are reduced under the catalysis of AKR1C3 in the biochemical environment in the cells to obtain cytotoxic toxins, thereby exerting toxic effect on cancer cells., see the following patent applications:
The compounds of the general formula and specific compounds disclosed in the above patent applications all belong to AKR1C3 activated anticancer prodrugs (compounds), and the above applications are hereby incorporated into the present patent application text in their entirety.
Broadly speaking, AKR1C3 activated anticancer drug should meet the following conditions:
In the presence of an AKR1C3 inhibitor (such as TH-3021 disclosed in the three patents above, or compound 36, i.e.,
in Flanagan et al., Bioorganic and Medicinal Chemistry (2014), pp. 962-977), the inhibition effect detected of a compound on the proliferation of cancer cells is less than that of cancer cells in the absence of an AKR1C3 inhibitor (such as TH-3021 disclosed in the above three patents); and when the inhibition effect on cancer cell proliferation is quantified using the IC50, then if the IC50 detected of a compound on a certain cancer cell line in the presence of an AKR1C3 inhibitor is greater than that in the absence of an AKR1C3 inhibitor, then the compound can be determined to be an AKR1C3-activated anticancer drug.
preferably, the AKR1C3 activated anticancer drug is a compound selected from the compounds with the following structures:
In a preferred embodiment of the present invention, wherein the cancers comprise:
lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, gastric cancer, bone cancer, esophageal cancer, mastocarcinoma, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular carcinoma, melanoma, 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, osteocyte carcinoma, epithelial carcinoma, carcinoma of bile duct, choriocarcinoma, embryonal carcinoma, seminoma, Wilm's tumor, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemocytoblastoma, vocal cords neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromyxoma, fibroosteoma, fibromyxosarcoma, fibropapilloma, myxosarcoma, myxocystoma, myxochondroma, myxochondrosarcoma, myxochondrofibrosarcoma, myxadenoma, myxoblastoma, liposarcoma, lipoma, lipoadenoma, lipoblastoma, lipochondroma, lipofibroma, lipoangioma, myxolipoma, chondrosarcoma, chondroma, chondromyoma, chordoma, choriocarcinoma, chorioepithelioma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, osteofibroma, fibrosarcoma of bone, angiosarcoma, hemangioma, angiolipoma, angiochondroma, hemangioblastoma, angiokeratoma, angioglioma, angioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipoleiomyoma, angiomyolipoma, angiomyoneuroma, angiomyxoma, angioreticuloma, lymphangiosarcoma, lymphogranuloma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, peripheral T-cell lymphoma, nodular NK/T-cell lymphoma, endothelioma, endoblastoma, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoblastoma, leiomyofibroma, rhabdomyoma, rhabdomyosarcoma, rhabdomyomyxoma, acute lymphatic leukemia, acute myelogenous leukemia, chronic disease cells, polycythemia, lymphoma, endometrial cancer, glioma, colorectal cancer, thyroid cancer, urothelial cancer or multiple myeloma;
preferably, the cancers comprise: ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, stomach cancer, liver cancer, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma, nodular NK/T-cell lymphoma, acute lymphoblastic leukemia or acute myelogenous leukemia.
Based on the same inventive concept, the fourth aspect of the present invention provides a method for detecting AKR1C3 RNA content, including the following steps:
In a preferred embodiment of the present invention, wherein in step (1), the concentration of the extracted RNA is detected;
preferably, Qubit RNA HS Assay Kits are used to detect the concentration of the extracted RNA; and/or
the reverse transcription system comprises reverse transcriptase;
preferably, the mass-to-volume ratio of the extracted RNA and reverse transcriptase is (0.5˜2):4, with the unit of μg/μL;
more preferably, the mass-to-volume ratio of the extracted RNA and reverse transcriptase is (1˜1.8):4, with the unit of μg/μL;
even more preferably, the mass-to-volume ratio of the extracted RNA and reverse transcriptase is 2:4, with the unit of μg/μL.
In a preferred embodiment of the present invention, wherein in step (2), in qPCR reaction system, the primer-probe compositions selected from any one of groups (i) to (ix) is mixed with the primer-probe composition of reference gene;
preferably, in qPCR reaction system, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is (2˜10):(2˜10):3;
more preferably, in qPCR reaction system, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is (3˜7):(3˜7):3;
even more preferably, in qPCR reaction system, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is 5:5:3; and/or, in qPCR reaction system,
preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is (2˜10):(2˜10):3;
more preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is (3˜7):(3˜7):3;
even more preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is 5:5:3; and/or, in qPCR reaction system,
preferably, the amount of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is the same as the amount of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1, respectively.
In a preferred embodiment of the present invention, wherein in step (2), in qPCR reaction system, dUTP, UNG enzyme, cDNA template and polymerase mixture are also added;
preferably, the volume of the polymerase mixture is (0.3˜0.8) of the total volume of qPCR reaction system;
more preferably, the volume of the polymerase mixture is 0.5 of the total volume of qPCR reaction system.
In a preferred embodiment of the present invention, wherein in step (2), AKR1C3 and reference gene are amplified by AKR1C3 digital PCR detection system and reference gene digital PCR detection system, respectively;
preferably, in AKR1C3 digital PCR detection system, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is (5˜15):(5˜15):3;
more preferably, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is (8˜14):(8˜14):3;
even more preferably, the molar ratio of AKR1C3 upstream primer, AKR1C3 downstream primer and AKR1C3 probe is 12:12:3; and/or, in the reference gene digital PCR detection system,
preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is (5˜15):(5˜15):3;
more preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is (8˜14):(8˜14):3;
even more preferably, the molar ratio of upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1 is 12:12:3.
In a preferred embodiment of the present invention, wherein in step (3), the step for obtaining AKR1C3 RNA content of ex vivo sample to be detected according to qPCR or digital PCR amplification results includes:
In a preferred embodiment of the present invention, wherein when the qPCR method is used, step (A) further includes:
In a preferred embodiment of the present invention, wherein the above method is used to detect AKR1C3 RNA content in blood sample, bone marrow sample or tissue sample.
Based on the same inventive concept, the fifth aspect of the present invention provides a method for detecting the expression level of AKR1C3 enzyme, wherein the method is used to detect the AKR1C3 RNA content of ex vivo sample to be detected, and then the expression level of AKR1C3 enzyme of ex vivo sample to be detected is obtained according to the AKR1C3 RNA content of ex vivo sample to be detected.
In a preferred embodiment of the present invention, wherein there is a linear correlation between AKR1C3 RNA content ex vivo sample to be detected and the expression level of AKR1C3 enzyme ex vivo sample to be detected.
It is necessary to indicate that, unless otherwise defined, technical terms or scientific terms used in one or more examples of the present specification shall have the ordinary meaning as understood by people having ordinary skill in the field to which the present disclosure belongs.
The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials of the medicaments, the reagents and the like used in the following examples are all commercially available products unless otherwise specified.
“Administering” drug to patient refers to direct administration (which may be administered to patient by a medical professional or self-administered) and/or indirect administration, which may be an operation of making drug prescriptions. For example, physicians who instruct patient to self-administer drugs and/or provide patient with drug prescriptions will administer drugs to patient.
“Cancer” refers to leukemia, lymphoma, cancer and other malignant tumors (including solid tumors) that can spread locally by invasion and spread systemically by metastasis with potential unrestricted growth. Examples of cancers include (but are not limited to) cancers of adrenal gland, bone, brain, breast, bronchus, colon and/or rectum, gallbladder, head and neck, kidney, throat, liver, lung, nervous tissue, pancreas, prostate, accessory thyroid gland, skin, stomach and thyroid gland. Certain other examples of cancers include acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, poorly differentiated cervical epithelium and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforme, hairy cell tumor, intestinal ganglion cell tumor, proliferative corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, myeloid epithelial carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, granuloma fungoide, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian tumors, pheochromocytoma, true erythrocytosis, primary brain tumor, small cell lung cancer, squamous cell carcinoma of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, local skin lesion, reticulum cell sarcoma, and Wilm's tumor.
The terms “patient” and “individual” can be used interchangeably herein and refer to mammals in need of treatment for cancer. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain examples, “patient” or “individual” may refer to a non-human mammal used in screening, characterizing, and evaluating drugs and therapies, such as, a non-human primate, a dog, cat, rabbit, pig, mouse or a rat.
“Prodrug” refers to a compound that, after administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug, relative to the drug, is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered. A prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference). A prodrug may be synthesized using reactants other than the corresponding drug.
“Solid tumor” refers to solid tumors including (but not limited to) metastatic tumor in bone, brain, liver, lung, lymph node, pancreas, prostate, skin and soft tissue (sarcoma).
“Treatment of” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or improvement of one or more symptoms of cancer; diminishment of extent of disease; delay or slowing of disease progression; alleviation, palliation, or stabilization of the disease state; or other beneficial results. Treatment of cancer may, in some cases, result in partial response or stable disease.
“Tumor cells” refers to tumor cells of any appropriate species, e.g., mammalian such as murine, canine, feline, equine or human.
“Therapeutically effective amount” of a drug refers to an amount of a drug that, when administered to a patient with cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer in the patient. A therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.
In describing optically active compounds, the prefixes R and S are used to represent the absolute configuration of the molecule around its chiral center. (+) and (−) are used to represent the optical activity of the compound, i.e., the direction in which the plane of polarization rotates through the optically active compound. The prefix (−) represents that the compound is levorotatory, i.e., the compound rotates the direction of the plane of polarization to left or anticlockwise. The prefix (+) represents that the compound is dextrorotary, i.e. the compound rotates the direction of the plane of polarization to right or clockwise. However, the optical signs (+) and (−) are independent of the absolute configurations of R and S of the molecule.
The structure of compounds OBI-3424 (also referred to as AST-3424 and TH-3424), OBI-3423 and OBI-2870 are shown below:
wherein OBI-3424 is an S-enantiomer, OBI-3423 is an R-enantiomer, and OBI-2870 is a racemic mixture of OBI-3424 and OBI-3423 at a ratio of 1:1.
Previous studies have shown that compounds OBI-3424, OBI-3423 and OBI-2870 have good therapeutic effects on a variety of human cancers, especially compound OBI-3424 has good therapeutic effects on solid tumors and hematological malignancies; wherein the therapeutic effects of compound OBI-3424 on solid tumors (including liver cancer, hepatocellular carcinoma (HCC), non-small cell lung cancer, melanoma, prostate cancer, mastocarcinoma, esophageal cancer, renal cancer, gastric cancer, colon cancer, cerebral cancer, bladder cancer, cervical cancer, ovarian cancer, head and neck cancer, endometrial cancer, pancreas cancer, sarcomatoid carcinoma and rectal cancer and the like.) are recited in patent CN108290911A; and the therapeutic effects of compound OBI-3424 on hematological malignancies (including B lineage acute lymphoblastic leukemia (B-ALL) and T lineage acute lymphoblastic leukemia (T-ALL)) are recited in patent WO2019/062919A1.
AKR1C3 is overexpressed at the protein level in many types of cancer, especially in liver cancer, gastric cancer, renal cancer, CRPC and non-small cell lung cancer. Due to the high expression of AKR1C3 in tumors, compound OBI-3424 is designed to be specifically activated in tumors, but compound OBI-3424 cannot be activated in normal cells expressing low level of AKR1C3 to achieve tumor-specific targeting. OBI-3424 is developed as a highly potent DNA alkylation prodrug selectively activated by AKR1C3. In the presence of NADPH, OBI-3424 is reduced from AKR1C3 to an intermediate which is spontaneously hydrolyzed to OBI-2660, and OBI-2660 is a DNA dialkylating agent similar to thioTEPA (N,N′,N″-triethylenethiophosphoramide) which leads to DNA cross-linking at N7 (or O6) position of guanine, and subsequently leads to cell death, as shown below.
Further studies made by the applicant have confirmed that compound OBI-3424 activation is AKR1C3-dependent, and its cytotoxicity and antitumor efficacy are highly correlated with the expression level of AKR1C3 protein. Compound OBI-3424 shows AKR1C3-dependent cytotoxicity ex vivo and antitumor activity in vivo in a variety types of human cancer, which supports the further development of compound OBI-3424 as an anticancer agent that can be used to treat different types of cancer. AKR1C3 is used as a biomarker to analyze the condition of cancer patients and further guides patients to choose compound OBI-3424 for treatment.
Example 1 of the present application has confirmed that AKR1C3 enzyme and AKR1C3 RNA content in solid tumors are linearly correlated with the amount after mathematical transformation for IC50 value of compound OBI-3424 on cancer cells and the linear correlation coefficient is high enough; Examples 2 and 3 have confirmed that AKR1C3 enzyme and AKR1C3 RNA content in hematological tumors are linearly correlated with the amount after mathematical transformation for IC50 value of compound OBI-3424 on cancer cells, and the linear correlation coefficient is high enough. It is well known to those skilled in the art that the therapeutic effect of a drug on a patient can be directly confirmed only by association with IC50 value; in theory and in practice, the lower the IC50 value, the better the efficacy of a drug on a patient. All of the experimental data of examples 1-3 detected a relevant biometric value that can be objectively measured to correlate the IC50 value, and then predicted the efficacy of a drug on a specific cancer patient and achieved targeted drug delivery (in fact, it is best to directly obtain the tumor tissue of a specific patient and then detect the IC50 value under the condition of cancer cell survival. This is the best, most intuitive and accurate, but it is not feasible in practice, so only relevant biometric values that can be objectively measured can be detected to correlate the IC50 value.). After extensive detections, the present application has confirmed that AKR1C3 enzyme and AKR1C3 RNA content in solid tumors and hematological tumors are not only linearly correlated with the IC50 value of compound OBI-3424 on cancer cells, but also the linear correlation coefficient is high enough, which is enough to illustrate that AKR1C3 enzyme and AKR1C3 RNA content in solid and hematological tumors are highly correlated with the IC50 value of compound OBI-3424 on cancer cells. Therefore, in practical application, AKR1C3 enzyme content and AKR1C3 RNA content of ex vivo sample of a patient can be detected, and then compound OBI-3424 can be administrated to a patient for treatment according to the detection results.
In the prior art, Western blotting or immunochemical staining is usually used to detect AKR1C3 enzyme content, however, there is no corresponding detection method for detecting AKR1C3 RNA content in the prior art. Assuming that a corresponding assay can be used to assess AKR1C3 RNA content, patient with higher AKR1C3 RNA content and most likely to respond to the prodrug thus can be selected to administrate compound OBI-3424 for achieving better cancer treatment effect.
Based on the above purpose, based on real-time fluorescence quantitative PCR technology (qPCR), the present invention adopts RNA reverse transcription reaction, polymerase chain reaction and Taqman probe technology to detect the expression of AKR1C3 gene, and establishes a method and the corresponding kit of human AKR1C3 gene expression which can be applied to LDT (Laboratory developed test) system, finally they can be used for the drug detection of clinical trial samples companion diagnostics.
Basic principles for designing primer and probe: 1) length of primer-probe: the length of each primer is 1530 bases. 2) GC % in the primer-probe is required to range from 30 to 80%, and Tm value of primer ranges from between 55 to 60° C. 3) base pairing of the primer-probe itself and the 3′ end between primers should be avoided as much as possible. 4) more than 6 consecutive base pairs of the primer-probe itself and between primer should be avoided as much as possible. 5) the length of the target fragment amplified by upstream and downstream primers ranges from 50 to 180 bp, and the probe is located between the upstream and downstream primers, as close as possible to the upstream primer, and the use of guanine should be avoided at the 5′ end of the probe. When the annealing temperature of the probe is too low, LNA probe or MGB probe is considered for design. 6) The selected primers and probes should be designed in the conserved region of AKR1C3 gene which can specifically detect the expression of AKR1C3 gene.
Technical principles: RNA reverse transcription reaction, polymerase chain reaction and Taqman probe technology are used to design specific primers and probes directing AKR1C3 gene expression. The target sequence is amplified by PCR using specific primers, and the Taqman probe bound to the template is cut by Taq enzyme (5′→3′ exonuclease activity), the reporter is separated from the quencher, generates and accumulates fluorescent signals. The real-time amplification curve can be obtained according to the relationship between the fluorescent signals and the number of amplification cycles, and then the detection for the expression level of AKR1C3 RNA can be achieved.
The present invention designs 9 pairs of primers and probes for 3 target regions of AKR1C3 (exons 2-3, 4-5 and 5-6, respectively). The position information of the primer-probes can be seen in
The first aspect of the present invention provides a primer-probe composition, selected from any one of the following groups:
The present invention performed AKR1C3 amplification by using the above 9 groups of primer-probe compositions, and selected groups (iii), (iv) and (v) from the 9 groups of primer-probe compositions as candidate primer-probe compositions according to the amplification efficiency and specificity. In order to determine the best primer-probe composition, further AKR1C3 amplification was performed in different samples by using primer-probe composition from groups (iii), (iv) and (v), respectively. According to the amplification efficiency, the primer-probe composition of group (iv) (upstream primer AKR1C3-F6, downstream primer AKR1C3-R2 and probe AKR1C3-P1) was finally determined as the best primer-probe composition of the present invention.
It should be noted that the 9 groups of primer-probe compositions of the present invention can be used not only for the detection of AKR1C3 RNA content by qPCR method, but also for the detection of AKR1C3 RNA content by digital PCR method. The steps of the detection of AKR1C3 RNA content by qPCR method and digital PCR method are described in detail below.
In a preferred embodiment of the present invention, wherein the 5′-end reporters of probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 are FAM, and the 3′-end quenchers of probes AKR1C3-P1, AKR1C3-P2 and AKR1C3-P3 are MGB.
The preferred probe for the implementation of the present invention is the probe labeled according to the TaqMan system. The TaqMan system is available from the manufacturer, Life Technologies Inc. According to the TaqMan system, oligonucleotide probes that are specifically designed to hybridize to the amplified target DNA are covalently linked to a reporter at 5′ end and to a quencher at 3′ end. Examples of appropriate reporters for use in TaqMan system include 6-carboxyfluorescein (FAM) or tetrachlorofluorescein (TET). The typical quencher is minor groove binder (MGB). The principle of this TaqMan system is that the quencher inhibits the fluorescence of the reporter as long as the position of the quencher and the reporter are very close to each other on the probe. Once the oligonucleotide probe hybridizes to the target DNA during qPCR amplification, it will be degraded by Taq polymerase because the enzyme makes that the oligonucleotide primer extends along the DNA corresponding to the target DNA. This degradation releases the reporter and the quencher, and the quencher becomes no longer very close to the reporter, thus enabling the reporter to emit its fluorescence, which can then be detected and measured by appropriate tools typically integrated in qPCR equipment (i.e. thermal cyclers).
Based on the same inventive concept, the second aspect of the present invention provides a kit comprising the primer-probe composition as described above.
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a primer-probe composition of reference gene; and the primer-probe composition of the reference gene comprises upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1, and the nucleotide sequences of the upstream primer ACTB-F1, the downstream primer ACTB-R1 and the probe ACTB-P1 are shown as SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, respectively.
When using qPCR technology to measure the expression level of gene, the absolute expression level of the target gene is not often directly measured, but the expression levels of the target gene and the reference gene are measured, respectively. Specifically, the expression level of the reference gene as a standard is taken to measure the relative expression level of the target gene, and then the relative expression levels between samples are compared. When the relative quantitative method is used to measure the expression level of gene, the Ct value obtained by qPCR is an exponential relationship, not a linear relationship. Therefore, the Ct value cannot be directly used for statistical analysis methods that require normal distribution of data, such as t test and analysis of variance (ANOVA) method. Therefore, the original Ct value should be converted to 2−Ct first to makes the data reach a linear relationship before statistical analysis. After the data are obtained by the relative quantitative method, the comparative CT value method (2ΔΔCt method) is often used for data analysis. This method is based on two assumptions: {circle around (1)} the amplification efficiency is 100%, i.e., the amount of products in each PCR cycle is doubled, which can be solved by the verification of amplification efficiency; {circle around (2)} there are appropriate reference genes to correct the error of the loading quantity of samples. According to the formula, calculating the fold change =2−ΔΔCt; ΔΔCt=(Ct of target gene−Ct of reference gene) of treatment group−(Ct of target gene−Ct of reference gene) of control group. According to the calculation formula, in relative quantitative method, the relative expression of the target gene cannot be calculated without the Ct value of the reference gene, suggesting that the reference gene plays a very important role.
The reference gene refers to a known reference gene whose expression level is not affected by the study conditions and can be expressed consistently among multiple samples, and the expression level of this gene can be used to accurately quantify the loading of the initial material. Since the expression level of housekeeping gene is less affected by environmental factors and can be continuously expressed in almost all tissues and various growth stages of an organism, housekeeping gene is usually used as a reference gene in experiments.
According to the reference (DONG Enni, LIANG Qing, LI Li, et al., The Selection Of Reference Gene in Real-time Quantitative PCR [J]. China Animal Industry, 49(11):92-96. DOI:10.3969/j.issn.0258-7033.2013.11.025) and the reference (ZHAO Wen-Jing, XU Jie, BAO Qiuhua, et al., Selection of Reference Genes for Real-time Quantitative PCR [J]. Microbiology China, 2010(12):1825-1829. DOI:CNKI:SUN:WSWT.0.2010-12-019), the reference genes used in q-PCR are shown in the following table:
As for reference genes, 4 types of reference genes: HPRT1 (hypoxanthine phosphoribosyl transferase), GAP (glyceraldehyde-3-phosphate dehydrogenase), ACTB (also known as β-actin) and GOLGA1 (Golgi protein, specifically Golgi autoantigen A1) were screened out in present invention. The primer-probe compositions corresponding to each of 4 types of reference genes (as shown in Table 1) were used and detected with the cell line CCRF-CEM to evaluate the fluorescence intensity and amplification. Different samples (including healthy blood samples and bone marrow samples of leukemia patients) and cell lines with different expression levels (including cell lines with high AKR1C3 expression and cell lines with low AKR1C3 expression) were further detected to verify the expression stability and whether the samples and cell lines with high and low expression levels can be distinguished. The detected results showed that cell lines RPMI8226 and CCRF-CEM with high expression levels can be better distinguished from cell line Jurkat with low expression level by using ACTB as reference as compared to using other three reference genes as references. In healthy people, the values remained relatively low level, while high expression level of AKR1C3 was detected in bone marrow RNA samples of leukemia patients. ACTB was selected as the reference gene based on the result of amplification curve. Accordingly, the primer-probe composition of the reference gene in the above kit comprises upstream primer ACTB-F1, downstream primer ACTB-R1 and probe ACTB-P1.
In a preferred embodiment of the present invention, wherein the 5′-end reporter of the probe ACTB-P1 is VIC (green fluorescent protein), and the 3′-end quencher of the probe ACTB-P1 is BHQ1 (Black Hole Quencher 1).
The best AKR1C3 primer-probe composition and reference ACTB primer-probe composition finally determined after screening are shown in Table 1.
All the sequences without source indicated were new sequences or newly developed or selected sequences for the kit.
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a polymerase mixture, and the polymerase mixture mainly comprises: DNA polymerase, MgCl2, buffer and dNTPs; preferably, the polymerase mixture is selected as KAPA PROBE FAST RT-PCR Master Mix(2x), which has been used in the laboratory for a long time and has been repeatedly verified to have good amplification efficiency.
In a preferred embodiment of the present invention, wherein the kit as described above further comprises a reverse transcriptase mixture;
preferably, the reverse transcriptase mixture is Superscript VILO MARSTER MIX.
In each PCR reaction, it must be detected and analyzed together with positive control and negative control (NC, nuclease-free water). In a preferred embodiment of the present invention, wherein the kit as described above further comprises a negative control and a positive control;
preferably, the negative control (NC) is nuclease-free water; and/or
preferably, the positive control is a reference with a known copy number.
The kit as described above may further comprises the elements necessary to perform qPCR, for example, reagents, as known to those skilled in the art. In addition to primer pairs and probes, the kit may further comprises one or more enzymes (Taq polymerase) or reagents used in qPCR reactions. The enzyme can be present in a lyophilized form or in a suitable buffer. In addition, the kit may comprises all the additional elements necessary to perform qPCR, such as buffer, extraction reagent, enzyme, pipette, plate, nucleic acid, filter paper, gel material, transfer material, autoradiography equipment, instructions (recording relevant operation methods), etc.
Based on the same inventive concept, the third aspect of the present invention provides use of the primer-probe composition or the kit as described above in the preparation of a drug for treating cancers.
In a preferred embodiment of the present invention, wherein the AKR1C3 RNA content in an ex vivo sample of a patient is obtained by using the primer-probe composition or the kit as described above;
patient with AKR1C3 RNA content greater than or equal to the predetermined content are administrated AKR1C3 activated anticancer drugs.
Accordingly, the present invention provides a method for treating a patient suffering from cancer, wherein the method includes:
obtaining the AKR1C3 RNA content in ex vivo sample of a patient by using the primer-probe composition or the kit as described above; administering AKR1C3 activated anticancer drugs to a patient with AKR1C3 RNA content greater than or equal to the predetermined content ; preferably, a therapeutically effective amount of compounds OBI-3424, OBI-3423 and OBI-2870 is administrated to the patient.
In a preferred embodiment of the present invention, wherein the AKR1C3 RNA content is obtained according to the ratio of AKR1C3 copy number/reference gene copy number, for example, the ratio of AKR1C3 copy number/ACTB copy number is calculated, if the ratio of AKR1C3 copy number/ACTB copy number>X, then it is determined that the expression of AKR1C3 is high, and the patient will be given a therapeutically effective amount of compounds OBI-3424, OBI-3423 and OBI-2870; if the ratio of AKR1C3 copy number/ACTB copy number≤X, then it is determined that the expression of AKR1C3 is low, and the patient will not be given a therapeutically effective amount of compounds OBI-3424, OBI-3423 and OBI-2870, but other cancer drugs can be given to the patient.
The value of predetermined content X needs to be determined by detecting a large number of samples. According to a large number of clinical trial samples and through statistical methods, the inventors finally determined that the predetermined content X is 0.0001˜1; more preferably, the predetermined content X is 0.00011˜0.5; even more preferably, the predetermined content X is 0.00013˜0.05; in particular preferably, the predetermined content X is 0.00014˜0.015. For example, the predetermined content X is 0.0001˜0.0005, 0.0001˜0.001, 0.0001˜0.005, 0.0001˜0.01, 0.0001˜0.05, 0.0001˜0.1, 0.0001˜0.5, 0.0001˜0.9, 0.00011˜0.0005, 0.00011˜0.001, 0.00011˜0.005, 0.00011˜0.01, 0.00011˜0.05, 0.00011˜0.1, 0.00011˜0.5, 0.00011˜1, 0.00013˜0.0005, 0.00013˜0.001, 0.0001˜30.01, 0.00013˜0.05, 0.00013˜0.1, 0.00013˜0.5, 0.00013˜1, 0.0005˜0.001, 0.0005˜0.005, 0.0005˜0.01, 0.0005˜0.05, 0.0005˜0.1, 0.0005˜0.5, 0.0005˜1, 0.00091˜0.001, 0.00091˜0.05, 0.00091˜0.1, 0.00091˜0.5, 0.00091˜1, 0.001˜0.005, 0.001˜0.01, 0.001˜0.05, 0.001˜0.1, 0.001˜0.5, 0.001˜1, 0.005˜0.01, 0.005˜0.05, 0.005˜0.1, 0.005˜0.5, 0.005˜1, 0.01˜0.05, 0.01˜0.1, 0.01˜0.5, 0.01˜1, 0.05˜0.1, 0.05˜0.5 or 0.05˜1; more preferably, the predetermined content X is 0.0001, 0.00011, 0.00012, 0.00013, 0.00014, 0.00015, 0.000156, 0.00016, 0.000163, 0.000165, 0.00017, 0.000177, 0.00018, 0.000181, 0.00019, 0.000195, 0.0002, 0.000201, 0.000216, 0.000221, 0.00023, 0.000239, 0.00025, 0.00029, 0.0003, 0.00035, 0.0004, 0.00045, 0.0005, 0.00054, 0.0006, 0.00065, 0.0007, 0.00075, 0.0008, 0.00085, 0.0009, 0.00095, 0.001, 0.0015, 0.00177, 0.002, 0.00238, 0.0025, 0.00266, 0.00296, 0.003, 0.00315, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.00679, 0.007, 0.00710, 0.0075, 0.00778, 0.00791, 0.008, 0.0085, 0.009, 0.00939, 0.0095, 0.01, 0.011, 0.012, 0.01207, 0.013, 0.01365, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.
Based on the same inventive concept, the fourth aspect of the present invention provides a method for detecting AKR1C3 RNA content, including the following steps:
Methodological principle of the method for detecting AKR1C3 RNA content of the present invention: firstly, the sample RNA is reverse transcribed into cDNA, and then the cDNA is amplified by specific primers and probes, wherein the AKR1C3 gene and reference gene are indicated by FAM and VIC signals, respectively; and the quantitative expression levels of AKR1C3 gene in peripheral blood RNA sample is calculated by samples and quality control products. At the same time, VIC signal in AKR1C3 reaction solution is used to monitor the sample quality.
In the present invention, two methods of qPCR and digital PCR are used to conduct a comparative experiment at the same time under the condition of sufficient samples. The same set of primer-probe is used for digital PCR and qPCR. Firstly, the temperature gradient of qPCR is used to optimize the primer-probe and the most appropriate temperature and primer-probe composition is selected for the digital PCR platform for quantitative reference materials and as a comparative method.
The detection process of digital PCR mainly includes two parts, i.e., PCR amplification and fluorescence signal analysis. Before PCR reaction, samples are divided into tens of thousands of units (reaction chambers) so that only a single DNA molecule is present in each unit. The amplification procedure and system of amplification stage of digital PCR are basically the same as those of ordinary PCR, but different from traditional qPCR method, digital PCR adopts direct counting method for quantitative analysis, that is, after the end of PCR amplification, if fluorescence signal (product) is present, marked as 1, and if no fluorescence signal (product) is present, marked as 0. A reaction unit with a fluorescent signal contains at least one copy of the target molecule. Theoretically, in the case of the concentration of target DNA in the sample is very low, the number of reaction units with fluorescence signal is equal to the copy number of target DNA molecule. However, under normal circumstances, the reaction units of digital PCR may contain two or more target molecules, which can be calculated by Poisson distribution.
In the above formula, where λ is the average copy number (concentration) of target DNA molecules contained in each reaction unit and p is the probability of k copies of target DNA molecules contained in each reaction unit under certain λ conditions. λ is determined by the dilution coefficient m of sample solution, with λ=cm, where c is the original copy number (concentration) of the sample. When k=0 (without the target DNA molecule), the above formula can be simplified as p=e−λ=e−cm, p can be regarded as the ratio of the number of reaction units without fluorescence signal to the total number of reaction units, i.e,
where n is the total number of reaction units and ƒ is the number of reaction units with fluorescence signal. Take the logarithm (ln) of both sides of the above formula to obtain
The initial copy number (concentration) of sample can be obtained from the total number of reaction units in the digital PCR and the number of units with fluorescent signals, as well as the dilution coefficient of sample. The quantitative method of digital PCR does not depend on the cyclic threshold of the amplification curve, so it will not affected by the amplification efficiency and does not need to use the standard curve. It has good accuracy and reproducibility, and absolute quantitative analysis can be achieved.
In the present invention, the specific reaction system of digital PCR differs from the specific reaction system of qPCR in that: in qPCR reaction system, a group of primer-probe composition selected from groups (i) to (ix) is mixed with the primer-probe composition of reference gene ACTB; whereas in digital PCR reaction system, due to the large difference in the expression levels of AKR1C3 and ACTB in samples and cell lines, the maximum difference in Ct may reach 10 Ct values. Therefore, the copy number of VIC may be much larger than that of FAM during the digital PCR detection. In view of this, a group of primer-probe composition selected from groups (i) to (ix) is not mixed with the primer-probe composition of reference gene ACTB, but AKR1C3 digital PCR detection system and reference gene digital PCR detection system are used for the amplification of AKR1C3 and reference genes, respectively.
PCR amplification results are affected by the changes in various factors of reaction system (including primer concentration, probe concentration, etc.) and amplification procedures. In order to obtain the best amplification efficiency and the least non-specific products, the present application determines the reaction system and amplification procedure of digital PCR and qPCR based on years of research and development experience. In combination with the selection of probe-primers, the correlation coefficient of standard curve is ensured to be greater than or equal to 0.98 and the E value (amplification efficiency) is between 85% and 110%.
In a preferred embodiment of the present invention, wherein in step (3), the step for obtaining AKR1C3 RNA content of ex vivo sample to be detected according to qPCR or digital PCR amplification results includes:
Wherein when the qPCR method is used, step (A) further includes:
Another difference between digital PCR and qPCR is that the copy numbers of AKR1C3 and reference gene of the ex vivo sample to be detected are obtained by qPCR according to the standard curve, while the copy numbers of AKR1C3 and reference gene of the ex vivo sample to be detected can be directly obtained by digital PCR according to the amplification result, without drawing the standard curve.
The Ct value of target gene AKR1C3 and the Ct value of reference gene should be interpreted. The Ct value of target gene AKR1C3 is the Ct value corresponding to the amplification signal (FAM signal) of target gene AKR1C3. The Ct value of reference gene is the Ct value corresponding to the amplification signal (VIC signal) of reference gene. The threshold values of FAM and VIC are set during the exponential amplification stage.
In a preferred embodiment of the present invention, wherein the method is used to detect AKR1C3 RNA content in blood samples, bone marrow samples, or tissue samples. Digital PCR and qPCR of the present invention can not only detect blood samples or bone marrow samples, but also detect tissue samples of solid tumors, except that the pre-processing process (such as RNA extraction process) is different. For example, as for blood samples, RNA extraction can be performed directly using the Qiagen made-up extraction kit; as for bone marrow samples or tissue samples, the mortar can be pre-cooled with liquid nitrogen, the bone marrow samples or tissue samples are ground to powder in the mortar, the dried tissue powder is placed in a 1.5 ml EP tube, and then the RNA extraction kit is used for extraction.
The present application has conducted a performance test on the above kit and detection method, and the test results are as follows:
Through the performance verification of minimum detection limit, precision, negative coincidence rate, positive coincidence rate, template amount, analytical specificity and actual sample detection, all of the 7 performances met the verification standards. The performance of the detection method met the requirements of clinical application and can be used for clinical sample detection.
Based on the same inventive concept, the fifth aspect of the present invention provides a method for detecting the expression level of AKR1C3 enzyme, wherein the method is used to detect the AKR1C3 RNA content of ex vivo sample to be detected, and then the expression level of the AKR1C3 enzyme of ex vivo sample to be detected is obtained according to the AKR1C3 RNA content of ex vivo sample to be detected.
In a preferred embodiment of the present invention, wherein there is a linear correlation between AKR1C3 RNA content ex vivo sample to be detected and the expression level of AKR1C3 enzyme ex vivo sample to be detected.
In the prior art, administration of compounds OBI-3424, OBI-3423 and OBI-2870 are usually judged based on whether the AKR1C3 reductase content is equal to or greater than the predetermined content, and example 4 of the present invention proves that the expression level of AKR1C3 enzyme is highly correlated with the expression level of AKR1C3 RNA. Therefore, in practical application, the AKR1C3 enzyme content can be calculated according to AKR1C3 RNA content measured by the above method, and then administration of compounds OBI-3424, OBI-3423 and OBI-2870 are judged based on whether the AKR1C3 enzyme content reaches the predetermined content.
The technical embodiments provided by the present invention are further described in the following examples. The following examples are only used to illustrate the invention and do not limit the protection scope of the present invention.
The synthetic method for the AKR1C3 activated anticancer prodrug OBI-3424 (also referred to as AST-3424 or TH-3424) used in the following examples can be seen in WO2017087428A1 or CN108290911A.
All human cancer cell lines were from American Type Culture Collection (ATCC, Manassas, VA) or Japanese Collection of Research Bioresources (JCRB, Osaka, Japan) or Cobioer Biosciences (NanJing, China).
Exponentially growing cells were inoculated. After 24 h, the test compound OBI-3424 was added. After addition of the test compound OBI-3424, plates were incubated in a standard tissue incubator at 37° C. for the indicated hours. At the end of the experiment, viable cells were detected using the CellTiter Glo (CTG) assay kit or AlamarBlue. Drug concentrations (IC50) resulting in 50% growth inhibition relative to untreated control were calculated using XLfit (IDBS, Boston, MA) or Prism 6 (GraphPad, San Diego, CA).
Human cell extract was prepared and protein concentration was determined. Proteins were detected using antibodies that recognize human AKR1C3 and tubulin or β-actin. Odyssey Laser Imaging System and software (LI-COR Biosciences, Lincoln, NE) or UVP ChemStudio imaging system and VisionWorks software (Analytik Jena AG) were used to scan and quantify the band densities of AKR1C3 and tubulin or β-actin, and the ratio of AKR1C3 to tubulin or β-actin was calculated.
After exposing the hepatocellular carcinoma cell lines to compound OBI-3424 for 96 h and exposing the NSCLC cell lines to compound OBI-3424 for 72 h, the IC50 value was determined by ex vivo proliferation assay, and the expression of AKR1C3 protein in hepatocellular carcinoma cell lines was determined by Western blotting (tubulin was used as loading control). The expression data of AKR1C3 RNA in the hepatocellular carcinoma cell lines and in the NSCLC cell lines were from CrownBio database (https://db.crownbio.com/Crownbio/OncoExpress_Login.aspx). The results were showed in Tables 2 and 3.
As shown in Tables 2 and 3, the hepatocellular carcinoma cell lines with high AKR1C3 expression at both protein and RNA levels were more sensitive to OBI-3424, where IC50 values were in the low nanomolar range. On the other hand, cells expressing low AKR1C3 were less sensitive to OBI-3424, where IC50 values were higher than 1000 nM. Similarly, NSCLC cells also showed AKR1C3-dependent cytotoxic properties after exposing NSCLC cell to compound OBI-3424 for 72 h (Table 3). 3424 IC50 in hepatocytes was highly correlated with the level of AKR1C3 protein (R2=0.71,
1.1 Cell lines and PDX Ex Vivo Studies
All cell lines were purchased from HD Biosciences. All experimental work was conducted under the approval of the respective institutional review board and the animal ethics committee of each institution, and the use of human relevant tissue samples was in accordance with the ethical and relevant legal regulations of the location of the experiments. Serial PDX previously established in 20-25 g female non-obese/SCID (NOD.CB17-Prkdcscid/SzJ, NOD/SCID) or NOD/SCID/IL2 receptor γ-negative (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJAusb, NSG) was used in the experiments, as described elsewhere (Lock RB, Liem N, Farnsworth ML, Milross CG, Xue C, Tajbakhsh M, et al. The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse. Blood 2002; 99:4100-8.). The development of lentivirus-transduced ALL-11 PDX [empty vector (EV) and AKR1C3 overexpression] has been described previously (Jamieson SM, Gu Y, Manesh DM, El-Hoss J, Jing D, Mackenzie KL, et al. A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells. Biochem Pharmacol 2014; 88:36-45.).
Leukemia cell lines were suspended in RPMI medium supplemented with FBS (Biosera), while ALL PDX cells were cultured in QBSF medium (Quality Biological Inc.,) supplemented with Flt-3 ligand (20 ng/mL, BioNovus Life Sciences) or IL7 (10-20 ng/mL, Jomar Life Research). Cells were inoculated according to the optimal cell density and incubated for 3 h or overnight (37° C., 5% CO2). PDX cells and leukemia cell lines were treated with OBI-3424 (10 mmol/L−1 pmol/L) or medium control for 48 or 72 h, respectively. Viability was determined using Alamar Blue reduction assay or Cell Titer-Glo Luminescent Cell Viability Assay (Promega). The half-maximal inhibitory concentration (IC50) was used to calculate the interpolation of nonlinear regression curves by GraphPad Prism 7 software.
Cryopreserved leukemia cells were thawed and lysed in RIPA lysis buffer, and protein concentrations were quantified by BCA assay. Each sample was loaded with 20 μg of protein lysate in NuPAGE 4-12% Bis-Tris protein gel, then electrophoresed at 120V and transferred to polyvinylenedifluoride membrane at 30V for 1 h. Mouse anti-AKR1C3 (#A6229, Sigma-Aldrich, St. Louis, MO) or rabbit anti-actin primary antibody (#A2066, Sigma-Aldrich) was used, followed by horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG secondary antibody (GE Healthcare, Buckingham, UK) respectively to probe the membrane. Immobilon Western chemiluminescent HRP substrate (Merck Millipore, Billerica, MA) was used to detect conjugated secondary antibodies by quantifying the signals in the BioRad Chemidoc touch imaging system.
The ex vivo cytotoxicity of AKR1C3-related OBI-3424 was observed in 11 types of T-ALL cell lines, one B-ALL cell line transfected with granulocyte-colony stimulating factor, and one BCP-ALL cell line. The expression levels of AKR1C3 protein was determined by Western blot analysis. The ex vivo cytotoxicity of OBI-3424 was determined using CellTiter-Glo assay and calculated as 50% maximum inhibitory concentration (IC50).
Ex vivo cytotoxicity demonstrated by OBI-3424, IC50 ranged from 3.0 to 30.0 nM in 6 types of cell lines expressing high (strong) levels of AKR1C3. As for cell lines with medium AKR1C3 expression levels, IC50 ranged from 3.0 to 84.0 nM (Table 4).
In order to evaluate the potential anti-leukemia activity of OBI-3424, ex vivo cytotoxicity assays were performed on a variety of leukemia cell lines. It proved that OBI-3424 can treat T-ALL, B-ALL, acute myeloid leukemia, acute promyelocytic leukemia (APL) and erythroleukemia. OBI-3424 showed potent cytotoxicity, in particular against cell lines derived from T-ALL (T-lineage acute lymphoblastic leukemia) with high AKR1C3 expression, where IC50 values were in the low nmol/L range (see Table 5). The difference in IC50 values between cell lines with high/medium AKR1C3 expression and low expression was statistically significant (P=0.0016).
Similar to the results obtained from leukemia cell lines, OBI-3424 exerted potent cell-killing effects on all leukemia cell lines.
After exposing the hematological cancer cell lines to compound OBI-3424 for 72 h, the IC50 value was determined by ex vivo proliferation assay. The expression data of AKR1C3 RNA in hematological cancer cell lines was from CrownBio database (https://db.crownbio.com/Crownbio/OncoExpress_Login.aspx). The results were showed in Table 6.
The expression levels of AKR1C3 RNA (Log2 FPKM) and IC50 (nM) in Table 6 above were plotted as a curve graph, as shown in
All cell lines were purchased from HD Biosciences. All experimental work was conducted under the approval of the respective institutional review board and the animal ethics committee of each institution. Serial PDX previously established in 20-25 g female non-obese/SCID (NOD.CB17-Prkdcscid/SzJ, NOD/SCID) or NOD/SCID/IL2 receptor γ-negative (NOD.Cg-Prkdcscid Il2rgtm1wil/SzJAusb, NSG) was used in the experiments, as described elsewhere (Lock RB, Liem N, Farnsworth ML, Milross CG, Xue C, Tajbakhsh M, et al. The nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of childhood acute lymphoblastic leukemia reveals intrinsic differences in biologic characteristics at diagnosis and relapse. Blood 2002; 99:4100-8.). The development of lentivirus-transduced ALL-11 PDX [empty vector (EV) and AKR1C3 overexpression] has been described previously (Jamieson SM, Gu Y, Manesh DM, El-Hoss J, Jing D, Mackenzie KL, et al. A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells. Biochem Pharmacol 2014; 88:36-45.).
Cryopreserved leukemia cells were thawed and lysed in RIPA lysis buffer, and protein concentrations were quantified by BCA assay. Each sample was loaded with 20 μg of protein lysate in NuPAGE 4-12% Bis-Tris protein gel, then electrophoresed at 120V and transferred to polyvinylenedifluoride membrane at 30V for 1 h. Mouse anti-AKR1C3 (#A6229, Sigma-Aldrich, St. Louis, MO) or rabbit anti-actin primary antibody (#A2066, Sigma-Aldrich) was used, followed by horseradish peroxidase conjugated anti-mouse or anti-rabbit IgG secondary antibody (GE Healthcare, Buckingham, UK) respectively to probe the membrane. Immobilon Western chemiluminescent HRP substrate (Merck Millipore, Billerica, MA) was used to detect conjugated secondary antibodies by quantifying the signals in the BioRad Chemidoc touch imaging system.
In order to analyze AKR1C3 expression in aspirates from primary patients, the patients were divided into B-ALL and T-ALL and their related subgroups. Paired end readings were mapped by STAR (Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15-21.) to GRCh37 human genome reference via a recommended round-trip mapping pipeline with default parameters, and Picard MarkDuplicates module was used to mark the duplication rate. Gene annotation files were downloaded from Ensembl (http://www.ensembl.org/) for STAR localization and subsequent evaluation of gene expression levels. In order to evaluate gene expression profiles, the count of readings of annotated genes was invoked by HTSeq (Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with highthroughput sequencing data. Bioinformatics 2015;31:166-9.) and processed by the DESeq2 R program package (Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol 2010;11:R106.) to normalize gene expression to a regularized log2 value.
In order to analysis PDX samples, Illumina paired end RNA-seq data were aligned to human genome assembly (construct hg38) using STAR with the quantMode parameters set to TranscriptomeSAM, and the alignment was converted to transcript coordinates. The alignment was run with rsem-calculate-expression commanded by RSEM (version 1.2.31) to calculate raw gene counts, TPM, FPKM, and isoform expression. All RNA-seq values were expressed as number of fragments per kilobase million (FPKM). The FPKM data were performed with loge conversion.
The expression levels of AKR1C3 mRNA and protein in various types of ALL were detected by RNA-Seq analysis (
The cell lines used in this example were purchased from Nanjing Cobioer Biosciences Co., Ltd and National Infrastructure of Cell Line Resource; Leukemia samples were obtained from acute lymphoblastic leukemia patient volunteers (clinical) and normal samples were obtained from healthy volunteers (donors), and the volunteers had signed informed consent. Fresh blood samples were preserved in PAXGENE tubes and stored in a refrigerator at −20° C.
The collection, use and subsequent processing of all human samples were conducted in accordance with Chinese relevant regulatory regulations of the location of the experiments and in accordance with the relevant requirements of international ethics.
The RNA extraction kit was Qiagen made-up extraction kit: PAXGENE BloodRNA Kit (catalog number: 762174, used for blood preserved in PAXGENE tubes) and Qiagen made-up extraction kit: QlAamp RNA Blood Mini Kit (catalog number: 52304, used for blood preserved in EDTA tubes).
According to the instructions, RNA extraction was performed on blood preserved in PAXgene tubes using an extraction and purification Kit (PAXgene Blood RNA Kit-IVD), and RNA extraction was performed on blood preserved in EDTA tubes using QIAamp RNA Blood Mini Kit. Concentration detection was performed on the extracted RNA using the recommended RNA nucleic acid quantification kit (Qubit RNA HS Assay Kits, Thermo Fisher Scientific, Q32852).
4 μL of AKR1C3 reverse transcriptase mixture (Superscript VILO MARSTER MIX from Thermo Fisher (catalog number: 11755050)) was absorbed and added to 16 μL of RNA sample (replenish water to 16 μL if the volume was insufficient), slightly vortexed and mixed, and then centrifuged transiently. PCR tubes containing the reaction solution were placed in PCR instrument, the PCR program was set for the synthesis of cDNA.
9 pairs of primers and probes were designed for a total of 3 target regions of AKR1C3. The position information of primer-probes can be seen in
Polymerase mixture is a key component of PCR success or failure and affects the efficiency of amplification. The main components of polymerase mixture comprise: polymerase, MgCl2, buffer and dNTPs. The above components in commercially available polymerase mixture have been optimized to the best state in a certain proportion to make concentrated mother liquor. When using, the corresponding detection can be carried out as long as the polymerase buffer was added in proportion according to the multiplier. The polymerase KAPA PROBE FAST RT-PCR Master Mix (2x) (catalog number: KK4702) which has been repeatedly verified to have good amplification efficiency, was used in this example.
In each PCR reaction, it must be detected and analyzed together with positive reference and negative control (NC, nuclease-free water). qPCR detection process was as follows:
The above steps 5.2-5.5 were adopted, 9 pairs of primer-probes were amplified first, the amplification results were shown in
Total RNA was extracted from the peripheral blood of healthy volunteers, and cDNA was synthesized via reverse transcription. It can be seen from the amplification results that F1R1P1, F2R2P1, F2R6P1, F6R2P1, F6R6P1 and F3R3P1 were higher than other F5R5P2, F4R3P2 and F7R7P3 in terms of fluorescence intensity values.
Considering the specificity of the primer-probes, BLAST was performed on 9 pairs of primer, and F2R6P1, F6R2P1 and F6R6P1 displayed better specificity, and no non-specific products appeared.
cDNA was obtained by reverse transcription with total RNA extracted from peripheral blood of healthy volunteers using 3 sets of primer-probes (F2R6P1, F6R2P1 and F6R6P1), and 6 samples were selected for testing. The results were shown in the following table.
As can be seen from Table 10 that Ct value of the target gene AKR1C3 expression is relatively stable in the range of 27 to 30 in healthy population.
cDNA was obtained by reverse transcription with RNA extracted from peripheral blood of one healthy volunteer using 3 sets of primer-probes (F2R6P1, F6R2P1 and F6R6P1) for amplification efficiency test. The concentration of cDNA was 100 ng/μL, after 5-fold dilution, it was further diluted 25-fold, 125-fold and 625-fold, and there were a total of four concentration gradients for 5 to 625-fold, with two replicates for each concentration for qPCR testing. The standard curve and amplification efficiency were determined based on the linear relationship between Ct value and log value of initial concentration. The results were shown in
It can be seen from the results that the amplification efficiency of F6R2P1 and F6R6P1 was higher than that of F2R6P1, with greater than 95%. Therefore, F6R2P1 and F6R6P1 primer-probes were selected for subsequent tests.
RNA samples extracted from the blood of 4 healthy volunteers were selected for amplification efficiency test of F6R2P1 and F6R6P1, respectively. The results were shown in the following table:
According to the results, it can be seen that there was little difference in the amplification efficiency of the 2 pairs of primer-probes, and there were slight fluctuations in different samples. However, R2 of F6R2P1 was better, and the R2 was equal to 1 in sample 3. Therefore, F6R2P1 was selected as the primer-probe of AKR1C3 in this kit.
After dissolving in 40 μL nuclease-free water, the AKR1C3 plasmid dry powder was diluted 10-fold to a concentration of 5.78 ng/μL measured by Qubit. The plasmid stock solution was diluted by gradient, which was 105, 106, 107 and 108-fold, respectively, to obtain a total of 4 gradient concentrations of samples for qPCR test. Each gradient was repeated twice, and the standard curve of AKR1C3 was measured as shown in
As can be seen from the standard curve in
As for the selection of reference genes, the references on leukemia gene expression ([1] Yanlan Wang, Yue Liu, Changhua Zhou et al. An AKR1C3-specific prodrug with potent antitumor activities against T-ALL. [J] .Leukemia&Lymphoma, 2020, Feb. DOI: 10.1080/10428194.2020.1728746; [2] Donya Moradi Manesh, Jad El-Hoss, Kathryn Evans et al. Growth factor AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemiaas treatment targets in clinical oncology.[J]. BLOOD, 2015, 125: 1193-1201; [3] Yuantong Tian, Lijing Zhao, Haitao Zhang et al. AKR1C3 overexpression may serve as a promising biomarker for prostate cancer progression. [J]. Diagnostic Pathology, 2014, 9: 42.) were referred to, and in combination with the primer-probes previously used, 4 reference genes (HPRT1, GAP, ACTB and GOLGA1) were selected for testing with the cell line CCRF-CEM to evaluate their fluorescence intensity and amplification. The results were shown in
In terms of fluorescence intensity: ACTB was the highest, followed by HPRT1.
In terms of amplification curves: the amplification curve of GOLGA1 are not typical S-shaped, and the other three are typical S-shaped amplification curves.
In terms of Ct: the Ct values of GAP and ACTB were low and their expression levels were relatively high, while the Ct values of HPRT1 and GOLGA1 were high and their expression levels were relatively low.
Samples and cell lines were tested to verify their expression stability and whether high and low expression samples could be distinguished.
RNA numbers 5, 6 and 7 of blood samples from healthy volunteers, bone marrow RNA numbers BZQ and QQY of leukemia patients, and cell lines expressing AKR1C3 (RPMI8226, CCRF-CEM and Jurkat) were used in this experiment. According to the data and previous results, the cell lines RPMI8226 and CCRF-CEM were high-expression cell lines, and Jurkat was low-expression cell line (as shown in
ΔΔCt calculating method was adopted for the experimental results, i.e., ΔΔCt=unknown sample FAM Ct−unknown sample VIC Ct−(quality control FAM Ct−quality control VIC Ct), and CCRF-CEM was used as quality control in each group. The results were shown in
It can be seen from the results that ACTB can better distinguish high-expression cell lines RPMI8226 and CCRF-CEM from low-expression cell line Jurkat when ACTB was used as the reference gene compared with the other three reference genes. In healthy people, the values remained relatively low level, while high expression of AKR1C3 was detected in bone marrow RNA samples of leukemia patients. ACTB was selected as the reference gene based on the results of the amplification curve.
After ACTB was determined as the reference gene, 5 types of cell lines were tested, respectively, and the test results were shown in
It can be seen from the results that the test results of the cell lines were consistent with the validation results of Western blot (
The quality of qPCR reaction system was a necessary condition for successful development and detection, and its main components must be optimized.
The reverse transcription system was shown below:
The reverse transcription reaction conditions were shown below:
Polymerase mixture was the key component that affects PCR amplification efficiency. Contained components of the polymerase mixture KAPA PROBE FAST RT-PCR Master Mix (2x) used in this example have been optimized to the best state at a certain ratio to make concentrated mother liquor. When in use, the corresponding detection can be carried out as long as the polymerase buffer was added in proportion according to the multiplier. According to the instructions, the recommended amount was half of the reaction system, i.e., in the reaction system of 2 μL, the amount should be 10 μL. The specific system was shown in the following table:
The reaction conditions used in this example were shown in the following table 15. The reaction conditions were tested as the preferred reaction conditions for RNA reaction after long-term verification in the early stage.
Cycle number was one of the direct factors affecting the results of PCR, and the cycle number determined the degree of PCR amplification. The cycle number of qPCR used was usually between 30 and 45 cycles. The more cycles there were, the more significant the non-specific amplification was, while the less cycles there were, the more significant was the effect on detection sensitivity.
In optimized reaction system, the primer-probes of AKR1C3 and ACTB were mixed, and the test of standard curve was conducted to investigate the amplification efficiency of the primer-probes. CCRF-CEM was used as the cell line, and reverse transcription was carried out according to the reverse transcription system of section 7.1. After cDNA was obtained, four gradients of stock solution, 5, 25 and 125-fold dilution were obtained according to the 5-fold gradient dilution with two replicates for each concentration and the detection was performed by qPCR reaction system. The standard curve was obtained based on the linear relationship between the Ct value and initial copy number lg value, as shown in
It can be seen from
The AKR1C3 and ACTB plasmid dry powders used were dissolved in 40 μL nuclease-free water. After diluting 10-fold, the concentrations were measured by Qubit to be 1.67 ng/μL, 5.78 ng/μL, respectively. The plasmid of AKR1C3 was diluted to 10−5, and the plasmid of ACTB was diluted to 10−3. 100 μL of each plasmid was mixed, and then the gradient of 10, 100 and 1000-fold was diluted. Each gradient was repeated twice for qPCR test. The standard curve was made according to the linear relationship between Ct value and log value of initial concentration. The results were shown in
It can be seen from the result in the figures that the amplification efficiency of AKR1C3 reached 101.9% and that of ACTB reached 94.1%, with R2 greater than 0.99, meeting the requirements.
A digital PCR test using AKR1C3-ACTB primer-probes was tried in this example. Since the expression levels of AKR1C3 and ACTB in samples and cell lines were quite different, the maximum Ct difference may reach 10 Ct values.
Therefore, in digital PCR detection, the copy number of VIC will be much larger than that of FAM, as shown in
In view of this, AKR1C3 and ACTB were detected separately. Two cell lines, CCRF-CEM and MOLT-4, were taken as examples. After 2 ug RNA was reverse transcribed, the transcribed product was diluted 5-fold, and the copy number of AKR1C3 was detected by digital PCR using the following system:
The reaction procedure of the digital PCR was shown in the following table:
The detection results were shown in
As for the detection of ACTB, 5-fold dilution of cDNA was required, followed by 200-fold dilution, and then the copy number of ACTB was detected using the following system:
The detection results were shown in
Sample No. 9 of healthy volunteer, 8 cases of leukemia patients (numbered as PSS, GZJ, HXB, HCL, ZYQ, LBJ, JRZ, LSX), and 5 types of cell lines (TF-1, CCRF-CEM, RPMI8226, MOLT-4, Jurkat) were used. The plasmid and concentration used in the standard curve were as described in step 7.3.2 of Example 7. The preparation method was as follows: the plasmid of AKR1C3 was diluted to 10−4 and the plasmid of ACTB was diluted to 10−2; 100 μL of each plasmid was mixed, and then the gradient of 10, 100 and 1000-fold was diluted. The copy numbers were quantified by digital PCR. qPCR tests were performed jointly, with two replicates for each sample. The copy numbers of AKR1C3 and ACTB of each unknown sample can be obtained by standard curve, and the ratio of AKR1C3 copy number/ACTB copy number was used as the way to measure the expression of AKR1C3. The measured standard curve was shown in
It can be seen from
The sample test results were shown in the following table:
The results of the FAM copy number NIC copy number were shown in
As can be seen from
Based on the studies of the above AKR1C3 primer-probe design, reaction system and reaction conditions, the detection method of AKR1C3 expression at RNA level was established, and certain sensitivity and accuracy were achieved.
According to examples 5-8, the best primer-probe composition, reference gene and corresponding primer-probe composition, reaction system and reaction conditions in qPCR method and digital PCR method were determined. Based on the above conditions, the performance test was performed in this example by combining qPCR method and digital PCR method, including sensitivity, specificity, repeatability, accuracy and the like.
The qPCR reaction process included the following steps:
The concentration of RNA should be more than 5 ng/μL, 20˜200 ng RNA was taken and replenished to a certain volume with nuclease-free water;
As shown in Table 12, the reverse transcription mixture was successively added to the above PCR tubes, and the mixture was mixed by vortex or by blowing with a pipettor. After short centrifugation, the mixture was placed on an ordinary PCR instrument. Reverse transcription was performed as shown in Table 13, and the reverse transcription product cDNA was used as the template of qPCR reaction system and could be stored at −20° C.
Dry powders of primers and probes were dissolved in TE buffer for 2 h at room temperature (or dissolved overnight at 4° C.). The concentration of primer working solution was 10 μM. After dissolving, the primers and probes were mixed according to a certain proportion and combination. After mixing thoroughly, it was stored in refrigerator at −20° C. The primer working solution can be stably stored at −20° C. for 1 year.
Before each use, the centrifuge tube containing DNA primer amplification mixture was shaken and mixed, then centrifuged transiently. According to the indicated quantity (20 μL/reaction), the required amount for single-use was dispensed and reserved.
Positive control product (STD), negative control product (Neg) and blank control product (NTC) should be used for quality control each time. The qPCR reaction system was prepared according to Table 14, mixed and centrifuged transiently. Then qPCR amplification was performed under the amplification conditions as shown in Table 15.
The corresponding meanings of the sample numbers involved in the following tests are: W1—standard with no AKR1C3 expression, P1—strong positive cell lines with AKR1C3 expression, P2—gradient of standards for AKR1C3-reference with different copy numbers, which were used to detect standard curves, P3—weak positive cell lines with AKR1C3 expression, S1—AKR1C3 standard with the lowest detection limit, R1—strong positive cell lines with AKR1C3 expression, R2—standard with no AKR1C3 expression.
RNA extracted from cell lines with low expression was selected for reverse transcription, and the amount of RNA was 2 μg. cDNA after reverse transcription was diluted 25-fold as the lowest detection limit reference, the copy numbers of reference were determined by digital PCR, and each reference was repeated for 10 times.
copy number calculation: target copy number=copies(target)×14.5/loading amount.
9.1.3 qPCR Verification Results
As can be seen from Table 20, the lowest detection limit reference was detected by qPCR with 10 times repeat. The positive detection rate was 100%, and the lowest detection limit met the verification standard.
Precision referred to the degree of closeness between multiple parallel test results under the same conditions. The closer the test values were to each other, the higher the precision. Precision reflected the repeatability of the kit operation. In this example, negative references, precision references with weak expression, and a precision references with high expression were selected to determine the intra-batch precision. The precision performance of the kit was evaluated by calculating the negative coincidence rate of negative precision references, the coefficient of variation (CV) of the relative expression level of AKR1C3 for precision references with weak expression and high expression.
In this experiment, 2 precision references were used as detection samples, 2 instruments, 2 operators and 3 verification dates were used, and each reference was detected 24 times. All negative references were negative, and the positive CV value was <15%.
CV value=(standard deviation SD/Mean)×100%
The CV value of AKR1C3 Ct for 24 times of positive precision reference was 0.78%, and the CV value of ACTB CT was 1.73%. The 24 times of negative precision reference were all negative.
As can be seen from Table 21, the samples of precision and repeatability detection were detected by qPCR for 24 times, and the CV values were all <5%, suggesting that precision and repeatability met the verification standards.
Negative references (AKR1C3 homologous plasmid AKR1C4, 9.06×103 copies) were selected and detected by the kit for 3 times repeat to evaluate for false positives.
As can be seen from Table 22, the repeated detection results were all negative, and the negative coincidence rate was 100%, which met the verification standards.
Two positive references (AKR1C3 cell lines verified by Western blot) were selected and detected by kit to evaluate the accuracy of the detection. AKR1C3 and ACTB plasmids (quantified by digital PCR) were used for gradient dilution to make standard curves, which were detected by kit to evaluate amplification efficiency and linear relationship.
As can be seen from Table 23, the selected positive references (cell lines verified by Western blot) were detected using the above detection method. P1 was a strong positive standard and the ratio of FAM copy number/VIC copy number was high, P3 was a weak positive standard and the ratio of FAM copy number/VIC copy number was low, and CV met the requirements. The detection accuracy of the method met the requirements.
2 concentrations of negative references (AKR1C3 homologous plasmid AKR1C2 with copy numbers of 1.14×104 and 1.14×103, respectively) were selected. Each concentration level was repeated for 3 times to evaluate the specificity.
2 gradients of the negative references were repeated for 3 times, and the results were all negative. The specificity of kit test met the requirements.
8 normal blood samples were selected for detection, and the loading amount was 40 ng cDNA of RNA reverse transcription. Each sample was detected twice, and the accuracy of FAM copy number/VIC copy number ratio was calculated.
The detection results were shown in the following table:
8 normal blood samples were detected twice, the ratio of FAM copy number/VIC copy number was <0.001, and the detection CV was <5%. The accuracy of the kit met the requirements.
200 ng and 10 ng RNA were used as the initial amount of reverse transcription, and the range of loading amount of bone marrow samples from 3 clinical leukemia patients was verified.
Conclusions: the detection results of 10 ng and 200 ng loading samples were consistent.
Conclusions: through the performance verification of the lowest detection limit, precision, negative coincidence rate, positive coincidence rate, template amount, analytical specificity and actual sample detection, the seven performances all met the verification standards. The performance of this detection method met the requirements of clinical application, and can be used for clinical sample detection.
In this example, the kit for detecting AKR1C3 RNA content comprises:
As described in example 8, separate detections were needed for AKR1C3 and ACTB in digital PCR method. Therefore, the kit in this example differs from the kit in example 10 in that the AKR1C3 reaction mixture only comprises primer-probe composition for detecting AKR1C3 gene, and does not comprise primer-probe composition for detecting reference gene ACTB. The kit in this example comprises both AKR1C3 reaction mixture (comprising a primer-probe composition for detecting AKR1C3 gene) and reference gene ACTB reaction mixture (comprising a primer-probe composition for detecting reference gene ACTB), the remaining components being the same.
As described in example 10 or 11, the kit for detecting AKR1C3 RNA content was used together with AKR1C3 activated anticancer drugs, usually screening patients. The use of the kit makes it easier for medical personnel to detect in different laboratories using a uniform detection kit standard operation procedure (SOP) before deciding to administer drugs to patients. In this way, the AKR1C3 detection results obtained by the same reagent and same procedure can match the recommended detection results for a specific cancer in the drug instructions of the AKR1C3 activated cancer drug.
The specific operation method of the kit is recorded in the instructions, i.e., the specific operating conditions in the above instructions. Optionally or as a preferred embodiment, the instructions also provide a predetermined amount X value for the AKR1C3 activated anticancer drug for different cancer (tumor) types. For example, as for B lineage acute lymphoblastic leukemia (B-ALL) and T lineage acute lymphoblastic leukemia (T-ALL), the predetermined X value was 0.00014 to 0.015, and the ratio of AKR1C3 copy number/ACTB copy number of an ex vivo peripheral blood sample from a patient detected by the above kit was 0.0092. According to statistics, the ratio of AKR1C3 copy number/ACTB copy number of B-ALL patients using AKR1C3 activated anticancer drugs should not be less than 0.00014. Therefore, doctors can prescribe AKR1C3 activated anticancer drugs for this patient. Another example, as for T lineage acute lymphoblastic leukemia (T-ALL), the ratio of AKR1C3 copy number/ACTB copy number of an ex vivo peripheral blood sample from a patient detected by the above kit was 0.00012. According to statistics, the ratio of AKR1C3 copy number/ACTB copy number of T-ALL patients using AKR1C3 activated anticancer drugs should not be less than 0.00014. Therefore, doctors should not prescribe AKR1C3 activated anticancer drugs for this patient.
The ratio of AKR1C3 copy number/ACTB copy number of an ex vivo peripheral blood sample from a B-ALL patient detected by the detection method for AKR1C3 RNA content established in examples 5-9 or the kit for detecting AKR1C3 RNA content established in example 10 or 11 was 0.0092, greater than the predetermined content of 0.00014.
The B-ALL patient was administered with AKR1C3 activated anticancer drug.
Through the verification of existing experiments, AKR1C3 activated anticancer drugs selected from the following structure have better therapeutic effect.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/079299 | 3/5/2021 | WO |
