WO2020215313A1 - Procédé et kit de détection du niveau d'expression de pd-l1 - Google Patents

Procédé et kit de détection du niveau d'expression de pd-l1 Download PDF

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WO2020215313A1
WO2020215313A1 PCT/CN2019/084550 CN2019084550W WO2020215313A1 WO 2020215313 A1 WO2020215313 A1 WO 2020215313A1 CN 2019084550 W CN2019084550 W CN 2019084550W WO 2020215313 A1 WO2020215313 A1 WO 2020215313A1
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cdna
internal reference
rna
target
seq
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PCT/CN2019/084550
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Chinese (zh)
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张道允
巩子英
孙永华
叶建伟
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嘉兴允英医学检验有限公司
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Priority to PCT/CN2019/084550 priority Critical patent/WO2020215313A1/fr
Priority to CN201980001127.6A priority patent/CN110268071A/zh
Priority to CN202110517370.2A priority patent/CN113249447A/zh
Publication of WO2020215313A1 publication Critical patent/WO2020215313A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • This application relates to the field of biotechnology, in particular to a method and kit for detecting the expression level of PD-L1.
  • drugs based on PD-L1 Programing Death-Ligand 1
  • NCCN Advanced non-small cell lung cancer
  • the efficacy of PD-1/PD-L1 inhibitors is closely related to the expression level of PD-L1.
  • monitoring the expression level of PD-L1 mainly uses immunohistochemical methods. Specifically, specific antibodies (such as 28-8, 22C3, SP263, SP142) are used to detect the expression level of PD-L1 in non-small cell lung cancer tissue specimens by hybridization. However, it is very difficult to evaluate PD-L1 for some patients who are unable or difficult to obtain tumor tissue. In addition, the current immunohistochemistry method also faces some problems.
  • test results may not truly reflect the expression level of PD-L1; in terms of tissue sources, for cytology specimens, archive specimens, and fresh specimens, the expression levels of PD-L1 in the primary site and metastases will be different.
  • Liquid biopsy technology using blood (or other body fluids) as a sample can detect circulating tumor DNA fragments released by tumor cells or metastatic cells into the blood. It is a breakthrough technology for tumor detection and adjuvant therapy, and samples are obtained in a non-invasive manner. , Can avoid the heterogeneity of tumor cells, and can also dynamically detect the changes of tumor cells periodically. Platelets and exosomes in the blood carry proteins and nucleic acids expressed by tumor cells, which can be used for cancer diagnosis, recurrence detection and drug resistance research. At the same time, the number of platelets and exosomes is large, easy to enrich, and the nucleic acids carried are secreted The protection of vesicles from nuclease degradation has a wide range of clinical applications.
  • this application provides a method and kit for detecting the expression level of plasma PD-L1.
  • One of the embodiments of the present application provides a method for detecting the expression level of PD-L1.
  • the method includes: obtaining a body fluid sample of a mammal; wherein the body fluid sample contains target RNA and an internal reference RNA, and the target RNA is derived from the gene of PD-L1; and extracting the target RNA from the body fluid sample And internal reference RNA; reverse transcribing the target RNA and internal reference RNA into target cDNA and internal reference cDNA; subject the target cDNA and internal reference cDNA to PCR amplification reaction; according to the number of target cDNA and internal reference cDNA after amplification To determine the expression level of PD-L1 in the mammal.
  • the body fluid includes peripheral blood, tissue fluid, lymph fluid, or cerebrospinal fluid.
  • the internal reference RNA includes GAPDH, ACTB, or 18S RNA.
  • the step of extracting target RNA and internal reference RNA includes at least high temperature denaturation, extraction, precipitation, washing and dissolution.
  • the specific reverse transcription primer used for reverse transcription of the target RNA includes: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.:1.
  • the specific reverse transcription primer used for reverse transcription of the internal reference RNA includes: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 2.
  • the specific reverse transcription primer used for reverse transcription of the internal reference RNA includes: a nucleotide sequence of SEQ ID NO.:1 and a nucleotide sequence of SEQ ID NO.:2.
  • the PCR amplification reaction of the target cDNA is real-time fluorescent quantitative PCR (Q-PCR);
  • Q-PCR specific primers used for the amplification of the target cDNA include: and SEQ ID NO. : Nucleotides with a similarity of ⁇ 95% in the sequence shown in 3; nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 4.
  • the PCR amplification reaction of the internal reference cDNA is real-time fluorescent quantitative PCR (Q-PCR);
  • Q-PCR specific primers used for the amplification of the internal reference cDNA are: and SEQ ID NO. : Nucleotides with a similarity of ⁇ 95% in the sequence shown in 5; nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 6.
  • the probe used for the amplification of the target cDNA is: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.:7.
  • the probe used for the amplification of the internal reference cDNA is: nucleotides with a similarity of ⁇ 92% to the sequence shown in SEQ ID NO.:8.
  • the number of the target cDNA and the number of the internal reference cDNA are characterized as the CT value of the target cDNA and the CT value of the internal reference cDNA, and the number of the target cDNA after amplification and the number of the internal reference cDNA are determined.
  • the expression level of PD-L1 in a mammal includes determining the expression level of PD-L1 in the mammal according to the ratio of the CT value of the amplified target cDNA and the CT value of the internal reference cDNA.
  • the method further includes performing clinical prediction based on the ratio of the CT value of the amplified target cDNA and the CT value of the internal reference cDNA.
  • the clinical prediction based on the ratio of the CT value of the amplified target cDNA and the CT value of the internal reference cDNA includes: the ratio is greater than or equal to a first threshold, predicting that the mammal will undergo PD-L1 After immunotherapy, the benefit is low; the ratio is less than the first threshold, which predicts that the mammal will benefit from PD-L1 immunotherapy; wherein, the first threshold is obtained by processing the clinical results through a mathematical model.
  • the mammal is a human.
  • kits for detecting the expression level of PD-L1 includes a reverse transcription system, and the reverse transcription system is configured to reverse transcribe an RNA sample including target RNA and internal reference RNA extracted from a mammalian body fluid sample into target cDNA and internal reference cDNA, respectively .
  • the kit further includes a PCR amplification system configured to amplify the target cDNA and the internal reference cDNA in the PCR amplification reaction to determine the PD in the mammal. -The expression level of L1.
  • the reverse transcription system includes:
  • the PCR amplification system includes:
  • the total volume is 20uL.
  • the specific reverse transcription primer used for reverse transcription of the target RNA includes: a nucleotide sequence of SEQ ID NO.:1 and a nucleotide sequence of SEQ ID NO.:2.
  • the PCR amplification reaction performed on the target cDNA is real-time fluorescent quantitative PCR (Q-PCR);
  • Q-PCR specific primers used for the amplification of the target cDNA include: the sequence is SEQ ID NO
  • the nucleotide and sequence of .:3 are the nucleotide of SEQ ID NO.: 4; or the nucleotide of the sequence of SEQ ID NO.: 5 and the nucleotide of SEQ ID NO.: 6.
  • the probe used for the amplification of the target cDNA is: the nucleotide sequence of SEQ ID NO.: 7; or the nucleotide sequence of SEQ ID NO.: 8.
  • Fig. 1 is a distribution diagram of CT ratios of 142 blood samples according to some embodiments of the present application
  • Fig. 2 is a ROC curve diagram according to some embodiments of the present application.
  • Figure 3 is a diagram showing the relationship between progression-free survival rate and time after treatment with PD-L1 inhibitors according to some embodiments of the present application;
  • Fig. 4 is a graph showing the fluorescence signal and cycle threshold of PCR amplification according to some embodiments of the present application.
  • Fig. 5 is a graph of the fluorescence signal and cycle threshold of PCR amplification according to another embodiment of the present application.
  • PD-1 Programmed Death 1
  • PD-L1 is the ligand of PD-1 and a protein expressed on the surface of tumor cells. It is related to the suppression of the immune system and can transmit inhibitory signals.
  • PD-1 and PD-L1 Once PD-1 and PD-L1 are combined, they will send a negative regulatory signal to T cells, induce T cells to enter a resting state, reduce the proliferation of CD8+ T cells in lymph nodes, make it unable to recognize cancer cells, and make T cells The reduction in self-proliferation or apoptosis effectively relieves the body's immune response, so that cancer cells can grow unscrupulously.
  • the principle of PD-L1 immunotherapy is: use PD-1 or PD-L1 as the target to design and synthesize antibody preparations (human-derived monoclonal antibody preparations) to bind to PD-1 or PD-L1 protein, respectively. It can block the connection between the original two proteins, so that T cells can regain immunity to tumor cells.
  • the effect of PD-L1 immunotherapy is related to the expression of PD-L1 in patients.
  • the PD-L1 expression is strong, the better the effect of PD-L1 immunotherapy.
  • the less PD-L1 expression the worse the effect of PD-L1 immunotherapy.
  • the number of cDNA of PD-L1 can be normalized according to the number of internal reference cDNA reverse-transcribed from the internal reference RNA. Measure the expression level of PD-L1 to predict the effect of patients after PD-L1 immunotherapy.
  • the cancer may include breast cancer, triple negative breast cancer, metaplastic breast cancer (MpBC), head and neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV) positive HNSCC, HPV negative/TP53 Mutant HNSCC, metastatic HNSCC, oropharyngeal HNSCC, non-oropharyngeal HNSCC, melanoma, luminal A breast cancer, luminal B breast cancer, HER2+ breast cancer, high microsatellite instability (MSI-H) colorectal cancer, Microsatellite stable colorectal cancer (MSS), non-small cell lung cancer (NSCLC), chordoma or adrenocortical carcinoma.
  • MpBC metaplastic breast cancer
  • HNSCC head and neck squamous cell carcinoma
  • HPV human papillomavirus
  • HPV human papillomavirus
  • HPV HPV negative/TP53 Mutant HNSCC
  • metastatic HNSCC oropharyngeal
  • the cancer can be breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate, Merkel cells, ovary, liver, endometrial, bladder, kidney, or cancer unknown primary (CUP).
  • the sarcoma can be liposarcoma, chondrosarcoma, extraskeletal myxoid chondrosarcoma, or uterine sarcoma.
  • the sarcoma comprises alpha alveolar soft tissue sarcoma (ASPS), angiosarcoma, breast angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma, proliferative small round cell tumor (DSRCT), epithelioid cell hemangioendothelioma ( EHE), epithelioid sarcoma, endometrial stromal sarcoma (ESS), Ewing sarcoma, fibromatosis, fibrosarcoma, giant cell tumor, leiomyosarcoma (LMS), uterine LMS, liposarcoma, malignant fibrous histiocytoma (MFH) /UPS), malignant peripheral nerve sheath tumor (MPNST), osteosarcoma, perivascular epithelioid cell tumor (PEComa), rhabdomyosarcoma, solitary fibroma (SFT), synovialpha alveolar
  • the cancer may include one or more of the following: melanoma, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, stomach cancer, kidney cancer, colon cancer, esophageal cancer, hepatocellular carcinoma, breast cancer, lymphoma Or leukemia.
  • cancer may include one or more of the following: kidney cancer, lung cancer, especially non-small cell lung cancer, melanoma, lymphoma, mesothelioma, colon cancer, pancreatic cancer, breast cancer, melanoma, and glial Blastoma.
  • the present invention provides a method and a kit for detecting the expression level of PD-L1, in particular, a method and a kit for detecting the expression level of PD-L1 using mammalian body fluid samples. Further, the present invention provides a method for predicting the effect of cancer patients receiving PD-1/PD-L1 targeted therapy based on the expression level of PD-L1.
  • the first aspect of the present invention provides a method for detecting the expression level of PD-L1, which uses mammalian body fluid samples to detect the expression level of PD-L1.
  • the detection method uses a kit for detecting the expression level of PD-L1 for detection. The method includes at least the following steps:
  • Step 1 Obtain samples.
  • PD-L1 may include the amino acid sequence of the PD-L1 protein and/or the nucleotide sequence of the PD-L1 gene.
  • the sample may include a tissue sample or a body fluid sample.
  • the body fluid sample may include one or a combination of peripheral blood, tissue fluid, lymph fluid, or cerebrospinal fluid sample.
  • the body fluid sample may include a mammalian blood sample, tissue fluid sample, or lymph fluid sample.
  • the mammal may be a human.
  • the body fluid sample may contain target RNA and internal reference RNA, and the target RNA may be derived from the gene of PD-L1.
  • the target RNA may be transcribed from the gene of PD-L1.
  • the gene of PD-L1 may be the mRNA of PD-L1.
  • the internal reference RNA can be a gene with a constant expression level during cell growth or under a specific environment, and it can be used as a reference when detecting changes in protein expression levels.
  • the internal reference RNA may be derived from genes of GAPDH, ACTB, or 18S.
  • the internal reference RNA can be transcribed from the GAPDH gene.
  • Step 2 Extract target RNA and internal reference RNA from body fluid samples.
  • Methods for extracting RNA can include guanidine isothiocyanate cesium chloride ultracentrifugation method, guanidine hydrochloride-organic solvent method, lithium chloride-urea method, hot phenol method, rapid extraction method, cytoplasmic RNA extraction method, phenol-lithium chloride Method for simultaneous extraction of cellular RNA and DNA, one-step rapid hot phenol extraction method.
  • RNA can be extracted from blood by methods of extracting RNA.
  • the step of extracting target RNA and internal reference RNA may at least include high-temperature denaturation, extraction, precipitation, washing, and dissolution.
  • the target RNA and the internal reference RNA can be directly extracted from the body fluid sample.
  • the kit for extracting target RNA and internal reference RNA may be the kit shown in Table 1.
  • Step 3 Reverse transcription of target RNA and internal reference RNA into target cDNA and internal reference cDNA, respectively.
  • Reverse transcription can use RNA transcribed separately from target gene and internal reference gene as template, and synthesize complementary single-stranded DNA (cDNA) by reverse transcriptase.
  • cDNA complementary single-stranded DNA
  • the specific reverse transcription primer used for reverse transcription of the target RNA may include nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.:1.
  • the specific reverse transcription primer used for reverse transcription of the target RNA may include: a nucleotide sequence of SEQ ID NO.:1 and a nucleotide sequence of SEQ ID NO.:2.
  • the specific reverse transcription primer used for reverse transcription of the internal reference RNA may include: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 2.
  • the specific reverse transcription primer used for reverse transcription of the internal reference RNA may include: a nucleotide sequence of SEQ ID NO.: 1 and a nucleotide sequence of SEQ ID NO.: 2.
  • Step 4 Perform PCR amplification reaction of target cDNA and internal reference cDNA.
  • the PCR amplification reaction of the target cDNA is Quantitative Real-time PCR (Q-PCR).
  • the basic principle of PCR amplification is: use single-stranded DNA (cDNA) as a template, 4 kinds of dNTPs as substrates, in the presence of primers at the 3'end of the template, use enzymes to extend the complementary strands, repeated cycles It can greatly amplify a small amount of template DNA.
  • cDNA single-stranded DNA
  • dNTPs as substrates
  • enzymes to extend the complementary strands, repeated cycles It can greatly amplify a small amount of template DNA.
  • In the microcentrifuge tube add two primers that are complementary to the known sequences at both ends of the DNA fragment to be amplified, an appropriate amount of buffer, a small amount of DNA membrane, four dNTP solutions, heat-resistant Taq DNA polymerase, and Mg 2+ etc.
  • the above-mentioned solution is heated to denature the template DNA at high temperature, and the double strands are unwound into a single-stranded state; then the temperature of the solution is lowered so that the synthetic primers are paired with their target sequences at low temperatures to form partial double strands, which is called annealing; Then raise the temperature to an appropriate temperature.
  • the primer Under the catalysis of Taq DNA polymerase, using dNTP as the raw material, the primer extends in the 5' ⁇ 3' direction to form a new DNA fragment, which can be used as a template for the next round of reaction.
  • Such repeated changes in temperature consist of high temperature denaturation, low temperature renaturation and temperature suitable extension to form a cycle, repeated cycles, so that the target gene can be rapidly amplified.
  • the Q-PCR specific primers used for the amplification of the target cDNA may include: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 3; and SEQ ID NO.: 4 The similarity of the sequence shown is ⁇ 95% nucleotides.
  • the Q-PCR specific primers used for the amplification of the target cDNA may include: the nucleotide sequence of SEQ ID NO.: 3 and the nucleotide sequence of SEQ ID NO.: 4; or The nucleotide sequence of SEQ ID NO.: 5 and the nucleotide sequence of SEQ ID NO.: 6.
  • the PCR amplification reaction of the internal reference cDNA is real-time fluorescent quantitative PCR (Q-PCR).
  • Q-PCR specific primers used for the amplification of the internal reference cDNA may include: nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 5; and SEQ ID NO.: 6 The similarity of the sequence shown is ⁇ 95% nucleotides.
  • the probe used for the amplification of the target cDNA may include nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.:7.
  • the probe used for the amplification of the target cDNA may include: the nucleotide sequence of SEQ ID NO.: 7; or the nucleotide sequence of SEQ ID NO.: 8.
  • the probe used for amplification of the internal reference cDNA may include nucleotides with a similarity of ⁇ 92% to the sequence shown in SEQ ID NO.:8.
  • Step 5 Determine the expression level of PD-L1 in mammals according to the number of target cDNAs after amplification and the number of internal reference cDNAs.
  • the number of target cDNA and the number of internal reference cDNA can be characterized as the CT value (Cycle Threshold, cycle threshold) of the target cDNA and the CT value of the internal reference cDNA.
  • the CT value refers to the cycle threshold experienced when the fluorescent signal in each reaction tube reaches the set threshold.
  • determining the expression level of PD-L1 in mammals based on the number of amplified target cDNAs and the number of internal reference cDNAs may include the ratio of the CT value of the amplified target cDNA to the CT value of the internal reference cDNA ( Hereinafter, referred to as the ratio of the CT value or the CT ratio) determines the expression level of PD-L1 in the mammal.
  • the ratio of the CT value or the CT ratio determines the expression level of PD-L1 in the mammal.
  • the mammal may be a human.
  • the method for detecting the expression level of PD-L1 may further include performing clinical prediction based on the ratio of the CT value of the amplified target cDNA and the CT value of the internal reference cDNA.
  • the ratio is greater than or equal to the first threshold, which predicts that the mammal will benefit less after PD-L1 immunotherapy; the ratio is less than the first threshold, and it is predicted that the mammal will undergo PD-L1 The benefit is higher after immunotherapy; wherein, the first threshold is obtained after the clinical results are processed by a mathematical model.
  • the mathematical model processing method may be to draw the ROC curve through the ROC package of the R language, so as to obtain the first threshold.
  • the first threshold may be 3.0.
  • whether the selection of the first threshold is reasonable can be confirmed by an existing clinical case (eg, the actual effect of the patient after receiving PD-L1 immunotherapy). For example, a certain number of body fluid samples of patients who have received PD-L1 immunotherapy can be collected. By measuring the ratio of the CT value of the target cDNA and the CT value of the internal reference cDNA in the body fluid samples of these patients after RNA extraction, reverse transcription, and amplification, the first threshold is used to predict the patient's PD-L1 immunotherapy The benefit, combined with the actual effect of the patient after receiving PD-L1 immunotherapy (ie, the benefit of the patient after PD-L1 immunotherapy), confirm whether the selected first threshold is reasonable.
  • an existing clinical case eg, the actual effect of the patient after receiving PD-L1 immunotherapy.
  • test materials used in the following examples are all purchased from conventional biochemical reagent companies.
  • the quantitative tests in the following examples are all set to three repeated experiments, and the results are averaged.
  • Example 1 Extraction of the target RNA and internal reference RNA.
  • RNA-containing centrifuge tube On ice, mash it, add 600ul Buffer A, vortex and mix well, add 200ul Buffer B, vortex and mix well, centrifuge at 15000rpm 4°C for 20 minutes.
  • RNA-containing centrifuge tube On ice, extract the supernatant and place it in a T2 centrifuge tube (divide into two tubes, each tube is about 500ul), add 50ul Buffer C and 1mL absolute ethanol, and invert it until it is mixed. Place at -80°C and settle for 2 hours.
  • Example 2 The target RNA and internal reference RNA were reverse transcribed into target cDNA and internal reference cDNA, respectively.
  • the reverse transcription system is as follows:
  • the reverse transcription product can be directly used in the subsequent PCR amplification reaction.
  • Embodiment 3 The target cDNA and internal reference cDNA are subjected to PCR amplification reaction.
  • the PCR amplification system is as follows:
  • the total volume is 20uL.
  • the conditions of the PCR amplification reaction are as follows:
  • Example 4 Determine the expression level of PD-L1.
  • the threshold line of the PCR amplification reaction is set at 10000, and the CT value of the target cDNA and the CT value of the internal reference cDNA are calculated respectively, and the ratio of the two is calculated.
  • 142 clinical blood samples were selected, ranging in age from 15 to 89 years old, with a median age of 62 years.
  • the characteristics of the treatment effect of the corresponding patients after PD-L1 immunotherapy eg, CT ratio, the patient's benefit after PD-L1 immunotherapy
  • the benefit of patients after PD-L1 immunotherapy can be divided and labeled in advance according to the patient's survival time after PD-L1 immunotherapy.
  • the patient's progression-free survival time after PD-L1 immunotherapy is 10.3 months, it is marked as "higher benefit”; the patient's progression-free survival time after PD-L1 immunotherapy is 6 months, then Mark it as "low return” and use the marked sample as a feature for drawing the ROC curve.
  • Fig. 1 is a distribution diagram of CT ratios of 142 blood samples according to some embodiments of the present application.
  • Figure 2 is an ROC curve diagram according to some embodiments of the present application.
  • the abscissa represents the blood sample number
  • the ordinate represents the CT ratio.
  • the characteristics corresponding to the 142 blood samples in Figure 1 such as CT ratio, the patient's benefit after PD-L1 immunotherapy
  • enter the ROC package of the R voice enter the ROC (Receiver Operating Operating) as shown in Figure 2.
  • Characteristic Characteristic
  • Characteristic to get the corresponding AUC (Area Under Curve) value.
  • the AUC value represents the area under the ROC curve.
  • the ordinate of the ROC curve is sensitivity, and the abscissa is 1-specificity.
  • the sensitivity range of the ROC curve is (6.5, 1)
  • the 1-specificity range is (0, 0.7)
  • the AUC value is 0.942
  • the corresponding first threshold is 3.0.
  • the CT ratio is greater than or equal to 3.0, which predicts that the patient will benefit less after PD-L1 immunotherapy; the CT ratio is less than 3.0, and it is predicted that the patient will benefit more after PD-L1 immunotherapy.
  • the CT ratio detected by 71 blood samples was less than 3.0, and the CT ratio detected by 71 blood samples was greater than or equal to 3.0.
  • Fig. 3 is a diagram showing the relationship between progression-free survival (PFS) and time after treatment with PD-L1 inhibitors according to some embodiments of the present application.
  • the 71 blood samples with a CT ratio less than 3.0 in Figure 1 are divided into 3-1 groups, and 71 blood samples with a CT ratio greater than or equal to 3.0 are divided into 3-2 groups, based on the CT ratio of 142 blood samples and the corresponding patients
  • the therapeutic effect (eg, progression-free survival time) of PD-L1 immunotherapy is shown in Figure 3.
  • the abscissa represents the time after PD-L1 immunotherapy
  • the ordinate represents the progression-free survival rate of patients corresponding to 142 blood samples after PD-L1 immunotherapy.
  • the median survival time of patients in group 3-1 after being treated with PD-L1 inhibitors was greater than 15 months (the progression-free survival rate at 15 months was about 75%) , Indicating that this part of the patients benefited more from PD-L1 immunotherapy.
  • the CT ratio of the 71 blood samples of this part of the patients after PCR amplification reaction was less than 3.0; the 3-2 group was the patients after PD-L1 inhibitor treatment
  • the median survival time is 6 months, and the progression-free survival rate at 15 natural months is as low as 25%, indicating that this part of patients benefited from PD-L1 immunotherapy.
  • the 71 blood samples corresponding to this part of patients The CT ratio of the PCR amplification reaction is greater than or equal to 3.0.
  • the CT ratio determined based on 142 clinical blood samples and the corresponding patients after PD-L1 immunotherapy is more accurate; further, clinical predictions can be made based on the CT ratio.
  • the CT ratio is greater than or equal to 3.0, which predicts that the patient will benefit less after PD-L1 immunotherapy; the CT ratio is less than 3.0, which predicts that the patient will benefit more after PD-L1 immunotherapy.
  • the present invention is used to test clinical samples, and the PD-L1 expression level detection method and kit of the present invention are used to detect the PD-L1 gene expression of the two clinical samples.
  • the detection method for detecting the expression level of PD-L1 is shown in steps 1 to 5, which will not be repeated here. Two groups of clinical samples with PCR amplified CT ratios greater than 3.0 and less than 3.0 were selected for testing.
  • Fig. 4 is a graph of the fluorescence signal value and cycle threshold value of PCR amplification according to some embodiments of the present application.
  • FIG. 5 is a graph of the fluorescence signal value and cycle threshold value of PCR amplification according to another embodiment of the present application.
  • the abscissa represents the cycle threshold experienced by the reaction tube
  • the ordinate represents the fluorescence signal value.
  • curve 4-1 represents the curve of the fluorescence signal value of the internal reference cDNA amplification and the cycle threshold
  • curve 4-2 represents the curve of the fluorescence signal value of the target cDNA amplification and the cycle threshold.
  • the fluorescence signal threshold line When the fluorescence signal threshold line is set Set at 10000, the ratio of the CT value of the amplified target cDNA to the CT value of the internal reference cDNA is greater than 3.0.
  • curve 5-1 represents the curve of the fluorescence signal value of the internal reference cDNA amplification and the cycle threshold
  • curve 5-2 represents the curve of the fluorescence signal value of the target cDNA amplification and the cycle threshold.
  • the fluorescence signal threshold line When the fluorescence signal threshold line is set When set at 10000, the ratio of the CT value of the amplified target cDNA to the CT value of the internal reference cDNA is less than 3.0.
  • Curve 4-1 and curve 5-1 are graphs of PCR amplification of internal reference cDNA.
  • the CT ratio is greater than 3.0, according to the present invention, it is predicted that the mammal will benefit less after PD-L1 immunotherapy; as shown in Figure 5, the CT ratio is less than 3.0, and the mammal is predicted according to the present invention Benefits are higher after PD-L1 immunotherapy.
  • the progression-free survival period of the patient corresponding to the clinical sample shown in Figure 4 is 6 months, and the progression-free survival period of the patient corresponding to the clinical sample shown in Figure 5 is 10.3 months. It can be seen that the prediction results of the above two clinical samples are consistent with the clinical results, which further illustrates that the present invention can more accurately predict the benefit of mammals after PD-L1 immunotherapy.
  • the possible beneficial effects of the PD-L1 expression level detection method and kit disclosed in this application include but are not limited to: (1) The patient's body fluid (for example, blood) is used as a sample for detection, the sampling process is more convenient, and the detection result More accurate; (2) Use the CT ratio after PCR amplification to predict the effect of PD-L1 immunotherapy, which can guide PD-L1 immunotherapy. It should be noted that different embodiments may have different beneficial effects. In different embodiments, the possible beneficial effects may be any one or a combination of the above, or any other beneficial effects that may be obtained.
  • the patient's body fluid for example, blood
  • the possible beneficial effects may be any one or a combination of the above, or any other beneficial effects that may be obtained.

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Abstract

L'invention concerne un procédé et un kit pour détecter le niveau d'expression de PD-L1. Le procédé comprend les étapes consistant à obtenir un échantillon de fluide corporel provenant d'un mammifère, l'échantillon de fluide corporel contenant un ARN cible et un ARN de référence, et l'ARN cible étant dérivé du gène PD-L1 ; à extraire l'ARN cible et l'ARN de référence de l'échantillon de fluide corporel ; à effectuer respectivement une transcription inverse sur l'ARN cible et l'ARN de référence pour obtenir un ADNc cible et un ADNc de référence ; à effectuer une réaction d'amplification par PCR sur l'ADNc cible et l'ADNc de référence ; et à déterminer le niveau d'expression de PD-L1 chez le mammifère en fonction du nombre de l'ADNc cible et du nombre de l'ADNc de référence après amplification. Le procédé de détection peut détecter le niveau d'expression de PD-L1 par collecte d'échantillons de fluide corporel prélevé chez un mammifère, et effectuer une prédiction d'immunothérapie sur PD-L1, de manière à guider l'immunothérapie sur PD-L1.
PCT/CN2019/084550 2019-04-26 2019-04-26 Procédé et kit de détection du niveau d'expression de pd-l1 WO2020215313A1 (fr)

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CN201980001127.6A CN110268071A (zh) 2019-04-26 2019-04-26 一种pd-l1表达水平检测的方法和试剂盒
CN202110517370.2A CN113249447A (zh) 2019-04-26 2019-04-26 一种pd-l1表达水平检测的方法和试剂盒

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023069604A1 (fr) * 2021-10-20 2023-04-27 Life Technologies Corporation Compositions, kits et procédés pour la quantification de séquences d'acides nucléiques à l'aide d'une norme quantitative interne

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110699435A (zh) * 2019-11-08 2020-01-17 宁波胤瑞生物医学仪器有限责任公司 一种用于pd-l1表达水平检测的试剂盒
CN112725418A (zh) * 2021-01-25 2021-04-30 深圳乐土生物科技有限公司 基于游离rna检测pd-l1表达量的方法及其试剂盒

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106950371A (zh) * 2017-02-17 2017-07-14 张灏 Pd‑l1、cdk5和ctla4中的至少一种在制备肿瘤诊断试剂盒中的用途
CN107338305A (zh) * 2017-07-23 2017-11-10 嘉兴允英医学检验有限公司 一种用于pd‑1表达水平检测的试剂盒
CN107460239A (zh) * 2017-07-23 2017-12-12 嘉兴允英医学检验有限公司 一种用于pd‑l1表达水平检测的试剂盒
US20180010191A1 (en) * 2016-07-06 2018-01-11 Genetics Development Corporation Cutoff point delta ct method for genetic pcr testing in human cancer
CN109628596A (zh) * 2019-01-18 2019-04-16 臻悦生物科技江苏有限公司 Rna水平检测pd-1和pd-l1表达量的试剂盒及方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2768524T3 (da) * 2011-10-17 2022-07-04 Io Biotech Aps PD-L1-baseret immunterapi
CN106987631A (zh) * 2017-04-01 2017-07-28 武汉赛云博生物科技有限公司 一种用于pd‑1/pd‑l1阻断治疗伴随诊断的免疫组测序技术

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180010191A1 (en) * 2016-07-06 2018-01-11 Genetics Development Corporation Cutoff point delta ct method for genetic pcr testing in human cancer
CN106950371A (zh) * 2017-02-17 2017-07-14 张灏 Pd‑l1、cdk5和ctla4中的至少一种在制备肿瘤诊断试剂盒中的用途
CN107338305A (zh) * 2017-07-23 2017-11-10 嘉兴允英医学检验有限公司 一种用于pd‑1表达水平检测的试剂盒
CN107460239A (zh) * 2017-07-23 2017-12-12 嘉兴允英医学检验有限公司 一种用于pd‑l1表达水平检测的试剂盒
CN109628596A (zh) * 2019-01-18 2019-04-16 臻悦生物科技江苏有限公司 Rna水平检测pd-1和pd-l1表达量的试剂盒及方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
VASSILAKOPOULOU, M. ET AL.: "Evaluation of PD-L1 Expression and Associated Tumor-Infiltrating Lymphocytes in Laryngeal Squamous Cell Carcinoma", CLIN. CANCER RES., 25 September 2015 (2015-09-25), XP055746493, DOI: 20200108183840A *
WU, JIANG ET AL.: "Establishment of real-time quantitative RT-PCR assays for detecting PD-L1 of liver transplantation in rat", CHINA JOURNAL OF MODERN MEDICINE, vol. 2, no. 22, 31 January 2012 (2012-01-31), DOI: 20200108182451A *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023069604A1 (fr) * 2021-10-20 2023-04-27 Life Technologies Corporation Compositions, kits et procédés pour la quantification de séquences d'acides nucléiques à l'aide d'une norme quantitative interne

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