WO2021243905A1 - Réactif et kit de détection pour le cancer du poumon - Google Patents

Réactif et kit de détection pour le cancer du poumon Download PDF

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WO2021243905A1
WO2021243905A1 PCT/CN2020/119000 CN2020119000W WO2021243905A1 WO 2021243905 A1 WO2021243905 A1 WO 2021243905A1 CN 2020119000 W CN2020119000 W CN 2020119000W WO 2021243905 A1 WO2021243905 A1 WO 2021243905A1
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seq
lung cancer
sequence
methylation
detection
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PCT/CN2020/119000
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Chinese (zh)
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李仕良
吴孝林
张志伟
吴幽治
陈新周
肖丽雯
邹鸿志
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广州康立明生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure belongs to the field of genetic testing. More specifically, the present disclosure relates to a lung cancer detection reagent and kit.
  • Lung cancer is a malignant tumor of the lung that originates from the bronchial mucosa, glands, or alveolar epithelium.
  • SCLC Small cell lung cancer
  • NSCLC Non-small cell lung cancer
  • central lung cancer lung cancer that grows at or above the bronchial opening of the lung
  • peripheral lung cancer lung cancer that grows beyond the bronchial opening of the lung. Lung cancer.
  • the relative risk of lung cancer for people aged 45-64 who smoked 1-19 cigarettes a day and 20 or more cigarettes a day was 4.27 and 8.61, respectively.
  • long-term daily smoking was 1-19
  • the relative risk of dying from lung cancer for those with more than 20 branches and those with more than 20 branches was 6.14 and 10.73, respectively.
  • the 5-year survival rate has only increased from 4% to about 12%.
  • Existing anti-tumor drugs can only relieve the disease, and the progression-free survival of patients is only extended by an average of 3 months to 5 months. Months, but for patients with stage I lung cancer, the 5-year survival rate after surgery is as high as about 60% to 70%. Therefore, early diagnosis and early surgery of lung cancer is one of the most effective methods to improve the 5-year survival rate of lung cancer and reduce the mortality rate.
  • the current clinical auxiliary diagnosis of lung cancer mainly includes the following types, but none of them can fully achieve early detection and early diagnosis:
  • Blood biochemical test For primary lung cancer, there is currently no specific blood biochemical test. For lung cancer patients, elevated blood alkaline phosphatase or blood calcium may consider the possibility of bone metastasis, and elevated blood alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase or bilirubin may consider the possibility of liver metastasis.
  • CEA 30% to 70% of lung cancer patients have abnormally high levels of CEA in the serum, but they are mainly seen in patients with advanced lung cancer. At present, the examination of CEA in serum is mainly used to estimate the prognosis of lung cancer and monitor the treatment process.
  • NSE It is the first choice marker for small cell lung cancer. It is used for the diagnosis and monitoring of treatment response of small cell lung cancer. The reference value is different according to the different detection methods and reagents used.
  • CYFRA21-1 It is the first choice marker for non-small cell lung cancer, with a sensitivity of up to 60% in the diagnosis of lung squamous cell carcinoma. The reference value is different depending on the detection method and reagents used.
  • Chest X-ray examination It should include frontal and lateral chest radiographs. In primary hospitals, chest radiographs are still the most basic and preferred imaging diagnosis method for lung cancer at the initial diagnosis. Once lung cancer is diagnosed or suspected, a CT scan of the chest is performed. 2) CT examination: Chest CT is the most commonly used and most important examination method for lung cancer. It is used for the diagnosis and differential diagnosis, staging and follow-up after treatment of lung cancer. CT-guided lung biopsy is an important diagnostic technique for lung cancer. Hospitals with conditions can use it for the diagnosis of lung lesions that are difficult to characterize. The clinical diagnosis of lung cancer needs to be confirmed by cytology and histology, and other methods are difficult to obtain. Case.
  • LDCT low-dose CT
  • NLST National Lung Cancer Screening Study
  • Low-dose spiral CT is recommended as an important method for early lung cancer screening, but there are many man-made factors and the false positive rate is very high.
  • Ultrasound examination It is mainly used to find out whether there are metastases in vital organs of the abdomen, abdominal cavity and retroperitoneal lymph nodes. It is also used to check the lymph nodes in the neck.
  • Bone scan It is highly sensitive to detecting bone metastases from lung cancer, but has a certain false positive rate. It can be used in the following situations: preoperative examination of lung cancer; patients with local symptoms.
  • Sputum cytology examination The current simple and convenient non-invasive diagnosis method for lung cancer, continuous smear examination can increase the positive rate by about 60%, and it is a routine diagnosis method for suspected lung cancer cases.
  • Fiberoptic bronchoscopy one of the most important methods in the diagnosis of lung cancer, which plays an important role in the qualitative diagnosis of lung cancer and the selection of surgical options. Routine examination items are necessary for patients who are to be treated by surgery.
  • the bronchoscopy needle biopsy (TBNA) is good for pre-treatment staging, but due to technical difficulties and risks, those in need should be transferred to a higher-level hospital for further examination.
  • Others such as percutaneous lung biopsy, thoracoscopic biopsy, mediastinoscopy biopsy, pleural effusion cytology, etc., if there are indications, they can be used according to existing conditions to assist in diagnosis.
  • Multi-slice spiral CT and low-dose CT (LDCT) in imaging examinations are effective screening tools for detecting early lung cancer and reducing mortality.
  • the National Lung Cancer Screening Study (NLST) has shown that LDCT is more effective than chest X-ray screening. Reduce the death rate of lung cancer by 20%.
  • NLST National Lung Cancer Screening Study
  • the risk prediction model integrating multiple high-risk factors has been recognized worldwide as one of the methods for identifying high-risk groups of lung cancer.
  • the risk model can further improve the efficacy of lung cancer patients by assisting clinicians to improve interventions or treatment methods.
  • Tumor markers can be detected in body fluids or tissues, and can reflect the existence of tumors, the degree of differentiation, prognostic estimation, personalized medication and treatment effects, etc.
  • Early-stage lung cancer patients have no obvious symptoms and are difficult to be detected by doctors and patients. In addition, they have no obvious specific markers in blood or biochemical items. Therefore, it is difficult to carry out early detection and early diagnosis through conventional diagnostic methods. Therefore, lung cancer Early diagnosis, especially in large-scale population screening, is more difficult.
  • tumor-related genes are an early sensitive indicator of tumorigenesis and is considered to be a promising Tumor molecular biomarker (biomarker). More importantly, cancerous cells can release DNA into peripheral blood. There are nanogram-level free DNA in the peripheral blood of normal people. Studies have found that peripheral blood plasma/serum, tumor-related organ-related body fluids (such as saliva, sputum, etc.) can also detect abnormal methylation of the promoters of tumor-related genes in tumor tissues. These biological samples are relatively easy to obtain, and the DNA in them can be detected sensitively after a large amount of DNA amplification by PCR technology.
  • the detection of the methylation status of the promoter regions of some tumor-related genes can be used for the early stage of tumors. Diagnosis provides very valuable information. Compared with other types of tumor molecular markers, detecting abnormal promoter methylation has more advantages. In different types of tumors, the abnormally methylated region of the promoter of a certain gene is the same, which is more convenient to detect. In addition, compared with markers such as allelic deletion, abnormal methylation is a positive signal, which is easy Distinguish from the negative background in normal tissues. Esteller et al.
  • Existing lung cancer detection techniques mainly have low sensitivity, high false positives, and are invasive, and it is difficult to detect early lung cancer with conventional detection techniques.
  • Rosalia Cirincione Method of treatment in tumor and sputum samples of lung cancer patients detected by spiral computed tomography: A nested case–contro
  • the detection rates of RARbeta2, P16, and RASSF1A in lung cancer tissues reached 65.5%, 41.4%, 51.7%, and in sputum They are only 44.4%, 5%, and 5% respectively.
  • the present disclosure provides the application of the nucleic acid fragment shown in SEQ ID NO: 4 (hereinafter referred to as "nucleic acid fragment”) in the preparation of lung cancer detection reagents or kits.
  • This "application” includes the application of any part of the nucleic acid fragment shown in SEQ ID NO: 4, that is, any part of the nucleic acid fragment shown in SEQ ID NO: 4 (for example, a smaller fragment
  • the application of) in the preparation of lung cancer detection reagents or kits all fall within the protection scope of the present disclosure.
  • the present disclosure provides that the nucleic acid fragment as shown in SEQ ID NO: 20 has at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%. Or at least 96%, or at least 97%, or 98%, or at least 99%, or 100% identical sequence, or the application of its complementary sequence in the preparation of lung cancer detection reagents or kits.
  • the present disclosure also provides a primer, the primer is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 9 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identity, or at least any one of its complementary sequences.
  • the primer is selected from SEQ ID NO.: 1 and SEQ ID NO: 2; SEQ ID NO: 5 and SEQ ID NO: 6; and SEQ ID NO: 8 and SEQ ID NO: 9
  • the sequence has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identity At least one primer pair in the sequence.
  • the primer is selected from the primer pair shown in SEQ ID NO:1 and SEQ ID NO:2.
  • the present disclosure also provides a probe which is selected from the group consisting of at least 85% or at least 90% of the sequence shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 10 Or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence, or its complementary sequence At least any one.
  • the probe is selected from the sequence shown in SEQ ID NO: 3.
  • the present disclosure also provides the application of the above-mentioned primers and/or probes in the preparation of lung cancer detection reagents or kits.
  • Detection in the present disclosure is the same as diagnosis, in addition to the early diagnosis of lung cancer, it also includes the diagnosis of middle and late stages of lung cancer, and also includes lung cancer screening, risk assessment, prognosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets.
  • nucleic acid fragments of the present disclosure makes the early diagnosis of lung cancer possible.
  • a nucleic acid fragment methylated in a cancer cell is methylated in a clinically or morphologically normal cell, this indicates that the normal cell is developing into cancer.
  • lung cancer can be diagnosed at an early stage by methylation of lung cancer-specific nucleic acid fragments in normal-appearing cells.
  • early diagnosis refers to the possibility of detecting cancer before metastasis, optionally before observing the morphological changes of tissues or cells.
  • the reagents/kits of the present disclosure are also promising for lung cancer screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets.
  • the diagnosis can be made by measuring the degree of methylation of the nucleic acid fragment obtained from the sample through the progress of lung cancer in different stages or periods.
  • the degree of methylation of the nucleic acid fragments isolated from samples of each stage of lung cancer By comparing the degree of methylation of the nucleic acid fragments isolated from samples of each stage of lung cancer with the methylation of the nucleic acid fragments in one or more nucleic acids isolated from samples with no abnormal cell proliferation The degree can detect the specific stage of lung cancer in the sample.
  • the present disclosure provides a lung cancer detection reagent, which contains at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or the nucleic acid fragment shown in SEQ ID NO: 4
  • Methods for nucleic acid fragments include the following: reagents for detecting the sequence of the nucleic acid fragment or any smaller/shorter sequence in the nucleic acid fragment. That is to say, any detection and detection reagents performed on any site in the nucleic acid fragment (for example, a smaller fragment) fall within the protection scope of the present disclosure.
  • the methylation detection reagent can be a methylation detection reagent in the prior art.
  • MSP methylation-specific PCR
  • qMSP methylation-specific quantitative PCR
  • DNA binding protein PCR quantitative PCR and DNA chips
  • methylation-sensitive restriction endonucleases bisulfite sequencing or pyrosequencing, etc.
  • other methylation detection methods can be introduced through patent US62007687. Each detection method has its corresponding reagents, and these reagents can be used in the present disclosure to detect the methylation of nucleic acid fragments.
  • the primers and/or probes detect the methylation of nucleic acid fragments by quantitative Methylation-Specific PCR (qMSP).
  • qMSP quantitative Methylation-Specific PCR
  • the sequence of the nucleic acid fragment is at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96 % Or at least 97% or 98% or at least 99%, or 100% identical to the sequence, or its complementary sequence.
  • Methylation occurs when a methyl group is added to cytosine. After being treated with bisulfite or bisulfite or hydrazine salt, cytosine will become uracil, because uracil is used for PCR amplification. Similar to thymine, it will be recognized as thymine. It is reflected in the PCR amplified sequence that cytosine that has not been methylated becomes thymine (C becomes T), and methylated cytosine (C) is Will not change.
  • the technology for detecting methylated genes by PCR is usually MSP. Primers are designed for the treated methylated fragments (that is, the unchanged C in the fragments), and PCR amplification is carried out. If there is amplification, it means that methylation has occurred. There is no methylation for amplification.
  • the methylation detection reagent detects the sequence of the nucleic acid fragment modified by bisulfite, bisulfite, or hydrazine.
  • the sequence detected is the sequence of the nucleic acid fragment modified with bisulfite.
  • the methylation detection reagent includes primers and/or probes for methylation detection of the nucleic acid fragment shown in SEQ ID NO: 4.
  • the upstream primer in the primer has any one of the nucleotide sequences shown below:
  • the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 8 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94 % Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or a sequence of 100% identity;
  • the downstream primer in the primer has any one of the nucleotide sequences shown below:
  • the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 9 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94 % Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or a sequence of 100% identity;
  • the missed detection rate of lung cancer is relatively high.
  • non-invasive detection of sputum is even more difficult, and the detection rate is extremely low.
  • most adenocarcinomas originate from smaller bronchial tubes, which are peripheral lung cancers.
  • the exfoliated cells in the deep lungs are more difficult to expectorate through sputum. Therefore, the current sputum detection methods for adenocarcinoma are almost zero.
  • Reducing the missed detection rate is especially important in the early screening of tumors. If an early tumor screening product fails to screen all or most of the patients, those who missed the test will not be able to get enough risk prompts, which will delay the timing of treatment, which is a huge for patients. loss.
  • the primers are used to amplify the nucleic acid fragments. It is well known in the art that the successful design of primers is essential for PCR. Compared with general PCR, in methylation detection, the influence of primer design is more critical. This is because the methylsulfurization reaction promotes the conversion of "C” in the DNA chain to "U”, resulting in a decrease in GC content, which makes the PCR reaction Long continuous "T” in the sequence can easily cause DNA strand breaks, making it difficult to select suitable Tm and stable primers; on the other hand, in order to distinguish between sulfurized and non-sulfurized and incompletely processed DNA , It is necessary to have a sufficient number of "C” in the primers, all of which increase the difficulty of selecting stable primers.
  • the selection of the amplified fragments targeted by the primers such as the length and position of the amplified fragments, and the selection of primers, all have an impact on the sensitivity and specificity of the detection.
  • the inventor also found through experiments that different amplification target fragments and primers have different detection effects. In many cases, some genes or nucleic acid fragments are found to have differences in expression between tumors and non-tumor. However, their distances are transformed into tumor markers, and there is still a long way to be applied to the clinic. The main reason is that the detection sensitivity and specificity of the potential tumor markers are difficult to meet the detection requirements due to the limitation of detection reagents, or the detection methods are complicated and costly, and it is difficult to apply them in clinics on a large scale.
  • the probe has any one of the nucleotide sequences shown below:
  • SEQ ID NO: 3 The nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 10 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94 % Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or a sequence of 100% identity;
  • the reagent includes a detection reagent including an internal reference gene.
  • the internal reference gene is ⁇ -actin.
  • the detection reagents for the internal reference gene are primers and probes for the internal reference gene.
  • the detection reagent for the internal reference gene is a primer pair shown in SEQ ID NO: 11 and SEQ ID NO: 12 and a probe shown in SEQ ID NO: 13.
  • the reagent further includes at least one of bisulfite, bisulfite, or hydrazine salt to modify the nucleic acid fragment, which of course may not be included.
  • the PCR reagent containing DNA polymerase including, dNTPs, Mg 2+ ions, buffer one or more, preferably comprising a DNA polymerase, dNTPs, Mg 2+ ions and buffer
  • the reaction system is used for the amplification of modified nucleic acid fragments.
  • the sample detected by the detection/diagnostic reagent of the present disclosure may be selected from at least one of alveolar lavage fluid, tissue, pleural fluid, sputum, blood, serum, plasma, urine, prostate fluid, or feces.
  • the sample is selected from at least one of alveolar lavage fluid, tissue, and sputum.
  • the sample is selected from at least one of alveolar lavage fluid or sputum.
  • the present disclosure also provides a kit containing the aforementioned primers, or probes, or lung cancer detection reagents.
  • the kit includes: a first container that contains a primer pair for amplification; and a second container that contains a probe.
  • the kit further includes instructions.
  • the kit further includes nucleic acid extraction reagents.
  • the kit further includes a sampling device.
  • the present disclosure also provides the primers, or the probes, or the application of the reagents in the preparation of methylation detection reagents or kits, or the preparation of the detection of lung cancer Application in reagents or kits.
  • the present disclosure also provides the primer, or the probe, or the reagent, or the kit, for use in methylation detection, or in the detection of lung cancer Applications.
  • the tissues targeted by the detection reagents of the present disclosure are selected from lung cancer tissues and adjacent normal tissues (or benign lung disease tissues).
  • the lung cancer is selected from small cell lung cancer and non-small cell lung cancer.
  • the non-small cell lung cancer is selected from squamous cell carcinoma and adenocarcinoma.
  • the present disclosure also provides a method for detecting the methylation of the nucleic acid fragment, which is characterized in that it comprises the following steps:
  • step (2) real-time fluorescence quantitative methylation-specific polymerase chain reaction is used for detection.
  • the present disclosure also provides a lung cancer detection system.
  • the described system contains:
  • the nucleic acid fragment as shown in SEQ ID NO: 4 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence, or its complementary sequence methylation detection component, and,
  • the system contains:
  • the nucleic acid fragment as shown in SEQ ID NO: 20 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93%
  • the methylation detection component contains the aforementioned detection reagent or kit.
  • the result judgment component is used to have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% of the nucleic acid fragment as shown in SEQ ID NO: 4 detected by the detection system. % Or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence, or the methylation result of its complementary sequence, output the risk of lung cancer and / Or type of lung cancer.
  • the result judging component is used to have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% of the nucleic acid fragment as shown in SEQ ID NO: 20 detected by the detection system. % Or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence, or the methylation result of its complementary sequence, output the risk of lung cancer and / Or type of lung cancer.
  • the disease risk is based on the result of comparing the methylation results of the test sample and the normal sample.
  • the methylation of the test sample and the normal sample is significantly different or extremely significant, As a result, it is judged that the sample to be tested has a high risk of disease.
  • the nucleic acid fragment if the nucleic acid fragment is positive for methylation, it indicates that the provider of the sample to be tested is a high-risk lung cancer patient or a lung cancer patient.
  • the “positive” refers to comparing the obtained detection result with the detection result of a normal sample. When the amplification result of the sample to be tested and the normal sample has a significant or extremely significant difference, the waiting The donor of the test sample was positive.
  • the judgment standard of the judgment system includes: judging lung cancer specimens and normal specimens according to the cut-off value.
  • the critical value of the Cp value in the specimen ranges from about 35 to 39, and the critical value of the ⁇ Cp value ranges from about 4 to 12.
  • the cutoff value of the ⁇ Cp value in the tissue specimen is about 5.4
  • the cutoff value of the Cp value in the sputum specimen is about 36.9
  • the cutoff value of the ⁇ Cp value in the lavage fluid specimen is about 11.2.
  • the ⁇ Cp value of the tissue and lavage fluid specimen is less than the threshold value of the ⁇ Cp value, and it is determined as a lung cancer specimen, and the ⁇ Cp value of the tissue and lavage fluid specimen is greater than or equal to the ⁇ Cp value.
  • the boundary value of Cp value is judged to be a normal specimen. If the Cp value of the sputum specimen is less than the threshold value of the Cp value, it is judged as a lung cancer specimen, and the sputum specimen is judged to be a normal specimen if the Cp value of the sputum specimen is greater than or equal to the threshold value of the Cp value.
  • the present disclosure also provides a method for diagnosing lung cancer, which includes the following steps:
  • test sample derived from the subject has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94 % Or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or a sequence of 100% identity, or its complementary sequence, or a nucleic acid fragment shown in SEQ ID NO: 20 that has at least 85 % Or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99% or a sequence of 100% identity, or The methylation level of its complementary sequence;
  • a method for diagnosing lung cancer includes the following steps:
  • test sample derived from the subject with the primer, or the probe, or the reagent, or the kit to detect the methylation of the nucleic acid fragment of the test sample level;
  • the present disclosure also provides a method for treating lung cancer, which includes the following steps:
  • test sample derived from the subject with the primer, or the probe, or the reagent, or the kit to detect the methylation of the nucleic acid fragment of the test sample level;
  • methylation-specific quantitative PCR is used to detect the methylation level of genes.
  • the present disclosure provides a method for treating lung cancer, the method includes the following steps: detecting a nucleic acid fragment shown in SEQ ID NO: 4 in a test sample derived from a subject that has at least 85 % Or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or 100% identical nucleosides
  • the detection includes combining the test sample of the subject and the detection with the nucleic acid fragment shown in SEQ ID NO: 4 that has at least 85% or at least 90% or at least 91 % Or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical nucleotide sequence, or its complement
  • the detection reagent for methylation level of the test sample the nucleic acid fragment shown in
  • the present disclosure provides a method for the treatment of lung cancer, the method comprising the following steps: detecting that a nucleic acid fragment shown in SEQ ID NO: 20 in a test sample derived from a subject has at least 85 % Or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or 100% identical nucleosides The methylation level of the acid sequence or its complementary sequence; the detection includes combining the test sample of the subject and the detection with the nucleic acid fragment shown in SEQ ID NO: 20 that has at least 85% or at least 90% or at least 91 % Or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical nucleotide sequence, or its complement Contact with the detection reagent of methylation level of the test sample and the normal control sample;
  • the diagnostic method of the present disclosure can be used before and after treatment of lung cancer or in combination with treatment of lung cancer, after treatment, such as evaluating the success of the treatment or monitoring the remission, recurrence and/or progress (including metastasis) of lung cancer after treatment.
  • Another aspect of the present disclosure provides a method for the treatment of lung cancer, the method comprising administering surgery, chemotherapy, radiotherapy, radiotherapy and chemotherapy, immunotherapy, oncolytic virus therapy, or other medicines to a patient diagnosed with lung cancer by the above-mentioned diagnostic method. Any other types of lung cancer treatment methods used in the field and combinations of these treatment methods.
  • the detection reagent for specific nucleic acid fragments of the present disclosure has high sensitivity and specificity for lung cancer, and is very promising as a tumor marker for clinical diagnosis of lung cancer.
  • lung cancer can be detected in tissue samples with a specificity of 100% and a sensitivity of 73.1%.
  • tissue samples with a specificity of 100% and a sensitivity of 73.1%.
  • squamous cell carcinoma can all be detected.
  • adenocarcinoma its specificity reached 100.0% and its sensitivity reached 78.9%.
  • the nucleic acid fragments of the present disclosure have high specificity and sensitivity for different types of lung cancer, including squamous cell carcinoma and adenocarcinoma in small cell lung cancer and non-small cell lung cancer. They have a wide range of applications and can basically be used for all lung cancers.
  • Tumor markers The existing lung cancer markers for clinical use are generally only applicable to the detection of one type of lung cancer. For example, NSE is used for the diagnosis of small cell lung cancer and monitoring treatment response, while CYFRA21-1 is the first choice for non-small cell lung cancer. .
  • the detection reagents and methods for specific nucleic acid fragments provided in the present disclosure can easily and accurately determine patients with lung cancer and benign lung diseases.
  • the gene detection method is expected to be transformed into a gene detection kit and serve for the screening of lung cancer. Clinical testing and prognostic monitoring.
  • Figure 1 is the ROC curve of nucleic acid fragments detected in tissue samples in Example 1;
  • Figure 2 shows the ROC curve of nucleic acid fragments detected in sputum samples in Example 2;
  • Figure 3 is the ROC curve detected in the nucleic acid fragment lavage fluid sample in Example 3.
  • FIG. 4 is the amplification curve of the nucleic acid fragment in Example 3.
  • the "primer” or “probe” in the present disclosure refers to an oligonucleotide that includes a region complementary to a sequence of at least 6 consecutive nucleotides of a target molecule (for example, a target nucleic acid fragment). In some embodiments, at least a portion of the sequence of the primer or probe is not complementary to the amplified sequence. In some embodiments, the primer or probe contains at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 relative to the target molecule. A region where the sequence of consecutive nucleotides is complementary.
  • the primer or probe When a primer or probe contains a region "complementary to at least x consecutive nucleotides of the target molecule", the primer or probe is at least 95% of at least x consecutive or discontinuous block nucleotides of the target molecule Complementary.
  • the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.
  • normal samples refer to samples of the same type isolated from individuals who are known to be free of the cancer or tumor.
  • the samples for methylation detection in the present disclosure include but are not limited to DNA, or RNA, or DNA and RNA samples containing mRNA, or DNA-RNA hybrids.
  • the DNA or RNA can be single-stranded or double-stranded.
  • the "subject” is a mammal, such as a human.
  • methylation level and “methylation degree” can usually be expressed as the percentage of methylated cytosine, which is the number of methylated cytosine divided by the number of methylated cytosine and the amount of unmethylated cytosine.
  • the sum of the number of methylated cytosines; and the method of dividing the number of methylation target genes by the number of internal reference genes is generally used to express the methylation level; and other methods of expressing the methylation level in the prior art.
  • sample is the same as “specimen”.
  • compositions may include A alone; B alone; C alone; D alone Contains the combination of A and B; Contains the combination of A and C; Contains the combination of A and D; Contains the combination of B and C; Contains the combination of B and D; Contains the combination of C and D; Contains the combination of A, B and C Combination; including A, B and D combination; including A, C and D combination; including B, C and D combination; or A, B, C and D in combination.
  • the inventor screened hundreds of gene markers and nucleic acid fragments, studied the distribution of methylation sites of each gene, and designed and detected primers and probes for real-time fluorescence quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) detection. Screening in tissue samples, using the ⁇ -actin gene as the internal reference gene, and finally screening to obtain the nucleic acid fragment shown in SEQ ID NO: 4 has a better detection result for lung cancer. Comparing the detection effect of the SEQ ID NO: 4 fragment with the commonly used lung cancer detection gene markers (PCDHGA12, HOXD8), the primer probes for each gene detection are as follows:
  • the detection primers and probes for nucleic acid fragments are:
  • the detection primers and probes of PCDHGA12 are:
  • the detection primers and probes of HOXD8 are:
  • SEQ ID NO: 18 HOXD8 primer R CCTAAAACCGACGCGATCTA
  • the detection primers and probes for ⁇ -actin are:
  • SEQ ID NO: 11 ⁇ -actin primer F GGAGGTTTAGTAAGTTTTTTGGATT
  • SEQ ID NO: 12 ⁇ -actin primer R CAATAAAACCTACTCCTCCCTTA
  • Amplification system see Table 2 for the amplification system.
  • Sample information There are a total of 169 lung tissue samples, including 91 normal tissue samples, 78 cancer tissue samples, and 78 cancer group samples including 27 cases of squamous cell carcinoma, 38 cases of adenocarcinoma, 3 cases of small cell carcinoma, and 4 cases of large cell carcinoma. Among them, there were 1 case of compound cancer and 5 cases of lung cancer that were not clearly classified. Among them, 77 pairs of cancer and para-cancer control samples were included.
  • the specificity of the nucleic acid fragments for each analysis group is as high as 100%, and when the specificity is 100%, the sensitivity of the normal group and all cancer groups is 73.1. %; Comparing the normal group and the adenocarcinoma group, the sensitivity reached 78.9%. It shows that the nucleic acid fragment still has high sensitivity under the condition of zero false positive. However, other genetic markers still have the problem of false positives in the detection of tissue samples.
  • sputum is more important in the diagnosis of lung cancer. For this reason, the inventors detected nucleic acid fragments in sputum.
  • Sample information A total of 107 sputum samples were tested, including 51 normal control samples, 56 cancer control samples, and 56 cancer group samples including 20 squamous cell carcinomas, 8 small cell carcinomas, and 20 adenocarcinomas. 1 case was cell carcinoma, 1 case was giant cell carcinoma, and 6 cases were unclearly classified lung cancer.
  • the primer probe sequence of the nucleic acid fragment the primer probe sequence of ⁇ -actin, and the DNA modification are the same as those in Example 1.
  • liquid dosing system is as follows:
  • the amplification system is as follows:
  • Nucleic acid fragment ACTB is used as the internal reference gene, and the methylation level of the specimen is judged according to the Cp value of the target gene, that is, the nucleic acid fragment.
  • the ROC curve of nucleic acid fragments detected in sputum samples is shown in Figure 2, and the statistical results are shown in Table 7. From the above results, it can be seen that in sputum samples, the specificity of nucleic acid fragments is as high as 96.1%.
  • the detection rate of lung cancer is 64.3%, and the detection rate of all squamous cell carcinomas can reach 80.0%.
  • Sample information A total of 176 samples of alveolar lavage fluid were tested, including 94 samples from the normal control group, 82 samples from the cancer group, and 20 samples from the cancer group, including 20 cases of squamous cell carcinoma, 40 cases of adenocarcinoma, and 9 cases of small cell carcinoma. , 13 cases of lung cancer type were not clear.
  • the primer probes for each gene detection are as follows:
  • the detection primers and probes for nucleic acid fragments are:
  • the detection primers and probes for ⁇ -actin are:
  • SEQ ID NO: 11 ⁇ -actin primer F GGAGGTTTAGTAAGTTTTTTGGATT
  • SEQ ID NO: 12 ⁇ -actin primer R CAATAAAACCTACTCCTCCCTTA
  • the amplification detection system is as follows:
  • the detection system is as follows:
  • the ROC curve of nucleic acid fragments detected in the lavage fluid sample is shown in Figure 3, the amplification curve is shown in Figure 4, and the statistical results are shown in Table 10. It can be seen from the above results that the sensitivity of nucleic acid fragments in the detection of 97.9% high specificity for all lung cancer groups reaches 69.5%; according to the comparison and analysis of the subtypes of lung cancer, the detection rate of nucleic acid fragments in the squamous cell carcinoma group is 65.0 %. Especially for the detection effect of adenocarcinoma, the detection sensitivity of nucleic acid fragments is as high as 80.0%. This breakthrough is of great significance for the detection of adenocarcinoma. Because adenocarcinoma is generally peripheral, due to the tree-like physiological structure of the bronchi, the alveolar lavage fluid is not easy to contact the deep lung alveoli or cancer tissue.
  • Primers and probes also have a great impact on the detection effect of tumor markers.
  • the inventor designed many pairs of primers and their corresponding probes to find a probe that can improve the detection sensitivity and specificity as much as possible. Needle and primers, so that the detection reagent of the present invention can be practically applied to clinical detection.
  • Some primers and probes are shown in Table 11 below, and the detection results are shown in Table 11.
  • primer probe detection results of each group are as follows:

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Abstract

La présente invention concerne le domaine des tests génétiques. La présente invention concerne un réactif et un kit de détection pour le cancer du poumon Le réactif ou le kit comprend un réactif de détection pour la méthylation d'un fragment d'acide nucléique spécifique et est utilisé pour détecter une séquence modifiée du fragment d'acide nucléique spécifique.
PCT/CN2020/119000 2020-06-03 2020-09-29 Réactif et kit de détection pour le cancer du poumon WO2021243905A1 (fr)

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