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WO2024001668A1 - Marqueur moléculaire de méthylation pour la détection de nodules pulmonaires bénins et malins et son utilisation - Google Patents

Marqueur moléculaire de méthylation pour la détection de nodules pulmonaires bénins et malins et son utilisation Download PDF

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WO2024001668A1
WO2024001668A1 PCT/CN2023/098075 CN2023098075W WO2024001668A1 WO 2024001668 A1 WO2024001668 A1 WO 2024001668A1 CN 2023098075 W CN2023098075 W CN 2023098075W WO 2024001668 A1 WO2024001668 A1 WO 2024001668A1
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seq
primers
dna methylation
molecular marker
sequences
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叶竹佳
杨昊
刘艳英
陶锦胜
罗茜
许洁涵
陈志伟
范建兵
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AnchorDx Medical Co Ltd
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AnchorDx Medical Co Ltd
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention is required to be submitted to the China Patent Office on June 28, 2022, with the application number 202210753404.2, and the application name is "Methylation molecular markers for detecting benign and malignant pulmonary nodules and their applications.”
  • the present invention also requires the priority of the Chinese patent application submitted to the China Patent Office on May 19, 2023, with the application number 202310565785.6 and the application title "Methylation Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Their Applications” , the entire contents of which are incorporated herein by reference.
  • the invention belongs to the field of biotechnology, and specifically relates to a methylation molecular marker for detecting benign and malignant pulmonary nodules and its application.
  • the application includes a detection kit and a detection method.
  • Pulmonary nodules also known as solitary pulmonary nodules, refer to high- and low-density images that are round shadows, single, well-defined, less than or equal to 3cm in diameter, and surrounded by air-containing lung tissue. Solid or subsolid lesions without atelectasis, hilar enlargement or pleural effusion. It often invades the lungs, bilateral hilar lymph nodes, eyes, skin and other organs, and its chest invasion rate is as high as 80% to 90%. A considerable number of pulmonary nodules cannot rule out the possibility of early malignancy. Pulmonary nodules are an important indicator of primary lung cancer
  • Pulmonary nodules are usually divided into two categories: benign and malignant, but both benign and malignant often have no obvious symptoms. Benign nodules require treatment based on the cause, while malignant nodules require early surgery. The causes of benign pulmonary nodules are often related to autoimmune diseases or various infections, while the causes of malignant pulmonary nodules are often related to lung cancer.
  • lung cancer The clinical manifestations of lung cancer are relatively complex. The presence, severity, and timing of symptoms and signs depend on the location of the tumor, pathological type, metastasis and complications, as well as differences in patient response and tolerance. Early symptoms of lung cancer are often mild and may even cause no discomfort. Symptoms of central lung cancer appear early and are severe, while symptoms of peripheral lung cancer appear late and are mild, or even asymptomatic. Therefore, once clinical symptoms appear or are detected during routine testing, lung cancer is in the late stage. Therefore, early screening is crucial. Lung cancer generally manifests itself as malignant pulmonary nodules in the early stages. Therefore, early screening generally starts with the detection of pulmonary nodules.
  • liquid biopsy In terms of sample collection, compared with tissue biopsy, liquid biopsy has the advantages of simple operation, non-invasiveness, strong reproducibility, and is conducive to dynamic monitoring of diseases.
  • Lung cancer liquid biopsy uses the patient's blood, sputum and alveolar lavage fluid as samples to detect and analyze the tumor cell DNA and its modification levels, such as DNA methylation.
  • sputum and bronchoalveolar lavage fluid sputum can be collected clinically through atomization to induce sputum, and bronchoalveolar lavage fluid can be obtained through fiberoptic bronchoscopy.
  • sputum collection is a non-invasive operation and is safer; fiberoptic bronchoscopy collection alveolar lavage (bronohoalveolarlavage, BAL) uses a bronchoscope to inject normal saline into the bronchoalveoli and then aspirates it out to collect the alveolar surface.
  • Effective liquid a method to examine its cellular components and soluble substances. Compared with percutaneous lung puncture and surgical biopsy, it is a safer minimally invasive biopsy method.
  • DNA methylation molecular markers that can well detect benign and malignant pulmonary nodules.
  • DNA methylation molecular markers or combinations thereof that are suitable for detecting benign and malignant pulmonary nodules, in order to achieve highly sensitive and specific detection of benign and malignant pulmonary nodules, especially for respiratory fluid samples. Sensitive detection, thereby providing technical support for non-invasive diagnosis of lung cancer, especially early-stage lung cancer.
  • one of the purposes of the present invention is to provide a DNA methylation molecular marker for detecting benign and malignant pulmonary nodules.
  • the DNA methylation molecular marker combination has very good performance in detecting benign and malignant pulmonary nodules. Good sensitivity and specificity can effectively improve the detection rate of malignant pulmonary nodules.
  • a first aspect of the present invention provides a DNA methylation molecular marker that can be used to detect benign and malignant pulmonary nodules.
  • a DNA methylation molecular marker that can be used to detect benign and malignant pulmonary nodules includes SEQ ID NO.6 or its complete complement, or SEQ ID NO.6 or its complete complement At least 55% of the entire length of the sequence is contiguous.
  • a second aspect of the present invention provides the use of the DNA methylation molecular marker and/or a reagent for detecting its methylation level in preparing a kit for detecting benign and malignant pulmonary nodules and/or lung cancer.
  • a third aspect of the present invention provides a kit for detecting benign and malignant pulmonary nodules, which kit includes a reagent for detecting the methylation level of the above-mentioned DNA methylation molecular marker.
  • the fourth aspect of the present invention provides a methylation level detection method for the above-mentioned DNA methylation molecular marker combination, including the following steps:
  • step (3) The transformation product obtained in step (2) is detected by multiplex fluorescence quantitative PCR with a probe targeting the above-mentioned DNA methylation molecular marker.
  • a fifth aspect of the present invention also provides a method for detecting benign and malignant pulmonary nodules, including the following steps:
  • the present invention has found a combination of DNA methylation-specific specific molecular markers that are highly correlated with benign and malignant pulmonary nodules.
  • benign and malignant pulmonary nodules can be better detected, and it has Higher sensitivity and specificity can improve the sensitivity and specificity of detecting benign and malignant pulmonary nodules, which can effectively increase the detection rate of early malignant pulmonary nodules, enable early treatment and intervention, and improve patient survival rate; at the same time, it can reduce the detection rate of malignant pulmonary nodules. It can reduce the false positive rate and avoid over-diagnosis and treatment of benign pulmonary nodules.
  • DNA methylation molecular markers provided by the present invention is highly correlated with benign and malignant pulmonary nodules, and is particularly suitable for respiratory tract samples, including respiratory fluid samples obtained through minimally invasive or non-invasive means, to achieve non-invasive detection of pulmonary nodules.
  • the fluorescent quantitative PCR detection primers and probes of the DNA methylation molecular markers designed by the present invention use the primers to pair the DNA treated with bisulfite, and then perform multiplex fluorescent quantitative PCR detection on the transformed products.
  • Figure 1 is a ROC curve chart of the methylation molecular marker combination of the present invention for identifying benign and malignant nodules in 134 lung tissue samples in Example 4.
  • Figure 2 is an ROC curve chart of the methylation molecular marker combination of the present invention for identifying benign and malignant nodules in 173 respiratory fluid samples in Example 5.
  • Figure 3 is a ROC curve chart of the methylation molecular marker combination of the present invention for identifying benign and malignant nodules in 61 respiratory fluid samples in Example 6.
  • the "plurality” mentioned in the present invention means two or more.
  • “And/or” describes the relationship between related objects, indicating that there can be three relationships.
  • a and/or B can mean: A exists alone, A and B exist simultaneously, and B exists alone.
  • the character “/” generally indicates that the related objects are in an "or” relationship.
  • the DNA methylation molecular markers provided by the present invention for detecting benign and malignant pulmonary nodules are directed at six methylated regions in the four detection genes HOXB4, PTGER4, LHX9 and ZSCAN31 or a combination thereof.
  • the DNA methylation molecular marker includes SEQ ID NO. 6 or its complete complementary sequence, or at least 55% of the continuous fragment of the full length of SEQ ID NO. 6 or its complete complementary sequence.
  • a combination of molecular markers obtained from at least one of the sequences shown in SEQ ID NO.1 to SEQ ID NO.5 or their completely complementary sequences is also included; or selected from SEQ ID A combination of molecular markers obtained from at least one of at least 55% of the continuous fragments of the full length of the sequence shown in NO.1 to SEQ ID NO.5 or its completely complementary sequence.
  • the DNA methylation molecular marker includes SEQ ID NO.6 and SEQ ID NO.4, or the complete complementary sequence of SEQ ID NO.6 and SEQ ID NO.4.
  • the DNA methylation molecular marker also includes SEQ ID NO. 2 or its complete complementary sequence
  • the DNA methylation molecular marker contains SEQ ID NO.6, or SEQ ID NO.6 and SEQ ID NO.4, and also includes SEQ ID NO.5 or its exact complementary sequence.
  • the above-mentioned combinations including SEQ ID NO.5 or its complete complementary sequence also include SEQ ID NO.2 and/or SEQ ID NO.3, or its complete complementary sequence;
  • the above-mentioned combinations including SEQ ID NO.5 or its complete complementary sequence also include SEQ ID NO.1 and/or SEQ ID NO.3, or its complete complementary sequence;
  • the DNA methylation molecular markers include SEQ ID NO.6 and SEQ ID NO.1, the sequence shown or its exact complement; or
  • DNA methylation molecular markers include SEQ ID NO.6 and SEQ ID NO.2, the sequences shown or their complete complements; or
  • DNA methylation molecular markers include SEQ ID NO.6 and SEQ ID NO.3, the sequences shown or their complete complements; or
  • DNA methylation molecular markers include SEQ ID NO.6 and SEQ ID NO.4, the sequences shown or their complete complements; or
  • DNA methylation molecular markers include the sequences shown in SEQ ID NO.6, SEQ ID NO.1 and SEQ ID NO.2 or their complete complementary sequences; or
  • the DNA methylation molecular markers include SEQ ID NO.6, SEQ ID NO.2, and SEQ ID NO.4, or include SEQ ID NO.6, SEQ ID NO.2, and the completely complementary sequence of SEQ ID NO.4;
  • the DNA methylation molecular markers include SEQ ID NO.6, SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, or include SEQ ID NO.
  • SEQ ID NO.6 SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4;
  • the DNA methylation molecular markers include SEQ ID NO.6, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, or include SEQ ID NO. The completely complementary sequences of ID NO.6, SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5;
  • the DNA methylation molecular markers include SEQ ID NO.1 to SEQ ID NO.6, or the complete complementary sequences of SEQ ID NO.1 to SEQ ID NO.6;
  • the DNA methylation molecular marker is a continuous fragment of at least 55% of the full length of the sequence shown in SEQ ID NO.1 to SEQ ID NO.6, respectively:
  • the fragment amplified by using primers as any one of SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14 is in SEQ The sequence corresponding to ID NO.1;
  • the DNA methylation molecular marker is a continuous fragment of at least 55% of the full length of the sequence shown in SEQ ID NO.1 to SEQ ID NO.6, respectively:
  • the primers are the sequences corresponding to the fragments amplified by SEQ ID NO.7 and SEQ ID NO.8 in SEQ ID NO.1;
  • primers as the sequences corresponding to the fragments amplified by SEQ ID NO.16 and SEQ ID NO.17 in SEQ ID NO.2;
  • primers as the sequences corresponding to the fragments amplified by SEQ ID NO.28 and SEQ ID NO.29 in SEQ ID NO.3;
  • primers as the sequences corresponding to the fragments amplified by SEQ ID NO.37 and SEQ ID NO.38 in SEQ ID NO.4;
  • primers as the sequences corresponding to the fragments amplified by SEQ ID NO.43 and SEQ ID NO.44 in SEQ ID NO.5;
  • primers as the sequences corresponding to the fragments amplified by SEQ ID NO.52 and SEQ ID NO.53 in SEQ ID NO.6.
  • the DNA methylation molecular marker combination is a molecular marker for respiratory tract samples, preferably lung tissue samples, or respiratory tract fluid samples.
  • the DNA methylation molecular marker is a combination of any two or more of the sequences shown in SEQ ID NO.1 ⁇ SEQ ID NO.6 or their completely complementary sequences; or is selected from SEQ ID NO.1 ⁇ A combination of any two or more of the full length of the sequence shown in SEQ ID NO.6 or at least 55% of the contiguous fragments of its completely complementary sequence.
  • a DNA methylation molecular marker that can be used to detect benign and malignant pulmonary nodules is also provided, and the DNA methylation molecular marker includes SEQ ID NO.2 and SEQ ID NO.4 the sequence shown or its exact complement; or
  • DNA methylation molecular markers include the sequences shown in SEQ ID NO.1 to SEQ ID NO.4 or their complete complementary sequences; or
  • DNA methylation molecular markers include the sequences shown in SEQ ID NO.1 to SEQ ID NO.5 or their complete complementary sequences; or
  • DNA methylation molecular markers include the sequences shown in SEQ ID NO.3 and SEQ ID NO.6 or their completely complementary sequences;
  • a combination of any two or more methylation detection regions of six DNA methylation molecular markers suitable for detecting benign and malignant pulmonary nodules in respiratory samples is included.
  • One embodiment of the present invention relates to the use of a detection reagent for the above DNA methylation molecular marker combination in preparing a kit for detecting benign and malignant pulmonary nodules.
  • the present invention involves the application of the above-mentioned DNA methylation molecular markers in detecting benign pulmonary nodules. malignant, and/or lung cancer.
  • a kit for detecting benign and malignant pulmonary nodules includes a reagent for detecting the methylation level of the above-mentioned DNA methylation molecular marker.
  • the kit includes PCR amplification method, fluorescence quantitative PCR method, digital PCR method, liquid phase chip method, next-generation sequencing method, third-generation sequencing method, second-generation sequencing method, pyrosequencing method, repeat sequencing method, etc.
  • the reagents include primers and probes for fluorescence quantitative PCR detection of DNA methylation molecular markers, and the primers and probes are:
  • the primers and probes for SEQ ID NO.1 are selected from at least one of the following groups: the primers shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe shown in SEQ ID NO.9; SEQ ID NO.10 and The primers shown in SEQ ID NO.11, and the probe shown in SEQ ID NO.12; the primers shown in SEQ ID NO.13 and SEQ ID NO.14, and the probe shown in SEQ ID NO.15;
  • the primers and probes for SEQ ID NO.2 are selected from at least one of the following groups: the primers shown in SEQ ID NO.16 and SEQ ID NO.17, and the probe shown in SEQ ID NO.18; SEQ ID The primers shown in NO.19 and SEQ ID NO.20, and the probe shown in SEQ ID NO.21; the primers shown in SEQ ID NO.22 and SEQ ID NO.23, and the probe shown in SEQ ID NO.24;
  • the primers and probes for SEQ ID NO.3 are selected from at least one of the following groups: the primers shown in SEQ ID NO.25 and SEQ ID NO.26, and the probe shown in SEQ ID NO.27; SEQ ID The primers shown in NO.28 and SEQ ID NO.29, and the probe shown in SEQ ID NO.30; the primers shown in SEQ ID NO.31 and SEQ ID NO.32, and the probe shown in SEQ ID NO.33;
  • the primers and probes for SEQ ID NO.4 are selected from at least one of the following groups: the primers shown in SEQ ID NO.34 and SEQ ID NO.35, and the probe shown in SEQ ID NO.36; SEQ ID The primers shown in NO.37 and SEQ ID NO.38, and the probe shown in SEQ ID NO.39; the primers shown in SEQ ID NO.40 and SEQ ID NO.41, and the probe shown in SEQ ID NO.42;
  • the primers and probes for SEQ ID NO.5 are selected from at least one of the following groups: the primers shown in SEQ ID NO.43 and SEQ ID NO.44, and the probe shown in SEQ ID NO.45; SEQ ID The primers shown in NO.46 and SEQ ID NO.47, and the probe shown in SEQ ID NO.48; the primers shown in SEQ ID NO.49 and SEQ ID NO.50, and the probe shown in SEQ ID NO.51;
  • the primers and probes for SEQ ID NO.6 are selected from at least one of the following groups: the primers shown in SEQ ID NO.52 and SEQ ID NO.53, and the probe shown in SEQ ID NO.54; SEQ ID The primers shown in NO.55 and SEQ ID NO.56, and the probe shown in SEQ ID NO.57; the primers shown in SEQ ID NO.58 and SEQ ID NO.59, and the probe shown in SEQ ID NO.60;
  • primers and probes having at least 70%, 80%, 90%, 95% or 99% sequence identity to the above-described sequence over multiple contiguous nucleotides.
  • the primers and probes are:
  • the primers and probes for SEQ ID NO.1 are: the primers shown in EQ ID NO.7 and SEQ ID NO.8, and the probe shown in SEQ ID NO.9;
  • the primers and probes for SEQ ID NO.2 are: the primers shown in SEQ ID NO.16 and SEQ ID NO.17, and the probe shown in SEQ ID NO.18;
  • the primers and probes for SEQ ID NO.3 are: the primers shown in SEQ ID NO.28 and SEQ ID NO.29, and the probe shown in SEQ ID NO.30;
  • the primers and probes for SEQ ID NO.4 are: the primers shown in SEQ ID NO.37 and SEQ ID NO.38, and the probe shown in SEQ ID NO.39;
  • the primers and probes for SEQ ID NO.5 are: the primers shown in SEQ ID NO.43 and SEQ ID NO.44, and the probe shown in SEQ ID NO.45;
  • the primers and probes for SEQ ID NO.6 are: the primers shown in SEQ ID NO.52 and SEQ ID NO.53, and the probe shown in SEQ ID NO.54.
  • the kit further includes primers and probes for fluorescence quantitative PCR detection of the internal reference gene ACTB.
  • Further described primers and probes for the internal reference gene ACTB are: the primers shown in SEQ ID NO.61 and SEQ ID NO.62, and the probe shown in SEQ ID NO.63.
  • the present invention designs primers and probes for each marker of the DNA methylation molecular marker combination of the above-mentioned specific methylation region, and uses the amplification primers of the DNA methylation molecular marker combination to analyze the DNA methylation molecular marker combination.
  • Genomic DNA gDNA
  • Genomic DNA extracted from respiratory samples and treated with bisulfite; using a specific probe for the DNA methylation molecular marker to perform multiplex fluorescence quantitative PCR detection of the methylation signal in this detection area.
  • the naive Bayes algorithm is used to establish a benign and malignant prediction model, and finally the benign and malignant pulmonary nodules are diagnosed through the established model.
  • a method for detecting the methylation level of the above DNA methylation molecular marker combination including the following steps:
  • step (3) The transformation product obtained in step (2) is detected by multiplex fluorescence quantitative PCR with a probe targeting the above-mentioned DNA methylation molecular marker.
  • the multiplex fluorescence quantitative PCR reaction conditions are as follows:
  • Amplification I 95°C15s; 10 ⁇ 20 cycles, 60 ⁇ 66°C30S;
  • Amplification II 95°C for 15s; 40 ⁇ 60 cycles, 60°C ⁇ 64°C for 30s.
  • a logistic regression (Logistic Regression) algorithm is used to establish a benign and malignant pulmonary nodule prediction model.
  • a logistic regression algorithm uses the cross-validation method (Cross-validation) to randomly divide the data set into 3 equal parts, and combine any 2 parts as the training set, and the remaining 1 part as the test set, then for any 3 equal parts
  • Cross-validation Cross-validation
  • three different training-test set combinations can be obtained, and then a logistic regression algorithm is used to establish a benign and malignant prediction model for the combinations containing different DNA methylation molecular markers in the training set, and the benign and malignant prediction models are established based on the combinations containing specific DNA methylation molecular markers.
  • a test set of DNA methylation molecular marker panels evaluates the model's classification ability.
  • the classification ability of the model of chemical molecular markers is determined by the average classification ability of 100 models.
  • the DNA methylation molecular marker provided by the present invention for detecting benign and malignant pulmonary nodules is the DNA methylation molecular marker Marker1 targeting 6 methylation regions in the 4 detection genes HOXB4, PTGER4, LHX9 and ZSCAN31.
  • the detection primers and probes to Marker6, the detection region sequences and sequence numbers of each DNA methylation molecular marker are specifically shown in Table 1 (the underlined part of each region is amplified by the primers preferably used in the following examples of the present invention) Marker of the corresponding sequence of the fragment):
  • the kit has designed three pairs of primers and three probes for each of the six specific methylation sites in the molecular markers Marker1 to Marker5 used to detect benign and malignant pulmonary nodules in respiratory samples (the probes can be fluorescently labeled. Labeled with fluorescent groups such as FAM, VIC, and NED), and labeled as combinations 1, 2, and 3 respectively.
  • the selected primers for each molecular marker The combination of primers and probes can be arbitrarily selected to be combined with combinations 1, 2, and 3 of primers and probes from other molecular markers and detected on the same platform.
  • the specific primer and probe sequences corresponding to each molecular marker are shown in Table 2:
  • primer and probe combinations used are as follows: primer and probe combination 1 for Marker1, primer and probe combination 1 for Marker2, primer and probe combination 2 for Marker3, Primer and probe combination 2 for Marker4, primer and probe combination 1 for Marker5, and primer and probe combination 1 for Marker6.
  • the kit also contains primers and probes for the internal reference gene ACTB, whose sequences are specifically shown in Table 3:
  • the kit described in Example 1 is used to detect the methylation levels of Marker1 to Marker6 in respiratory samples.
  • a method for detecting methylation levels of DNA methylation molecular markers including the following steps:
  • gDNA is extracted from paraffin section samples of lung tissue
  • the following method can be followed: The specific steps for gDNA extraction from paraffin tissue should be carried out according to Qiagen's ALLPrep DNA/RNA FFPE Kit instructions.
  • the components of the fluorescent quantitative PCR reaction are: primer-probe mixture, in which the concentration of each primer is 100-500 nM, preferably 200 nM in this embodiment; the probe concentration is 50-150 nM, preferably 100 nM in this embodiment.
  • Methy Tect Taq HS PCR kit (Ekore Bio, Cat# AG11209) was used, and one reaction was a 25ul system.
  • the specific reaction conditions are: predenaturation, 95°C, 5min; amplification I: 95°C for 15s; 10 to 20 cycles, annealing at 60 to 66°C for 30S. In this embodiment, 15 cycles, 65°C are preferred; amplification II: 95°C for 15s; 40 to 60 cycles, annealing at 60°C to 64°C for 30s. In this embodiment, 50 cycles at 62°C are preferred.
  • the preparation of qPCR fluorescence quantitative reaction system is shown in Table 5:
  • This embodiment further provides a method for detecting benign and malignant pulmonary nodules, which further includes the following steps:
  • test sample is a valid sample based on the C T value of the internal reference gene in the sample measured by the fluorescence quantitative PCR reaction. If the C T value of the internal reference gene of the test sample is between 10-25, the sample is judged to be a valid sample. ; If the initial investment sample is determined to be invalid, it will not be included in the testing and analysis.
  • This embodiment provides a method for detecting molecular markers in standards.
  • the detection steps are as follows:
  • test sample is a valid sample based on the C T value of the internal reference gene ACTB in the sample measured by the fluorescence quantitative PCR reaction. If the C T value of the internal reference gene of the test sample is between 10-25, the sample is judged to be valid. sample;
  • the target DNA methylation molecular marker C T value is ⁇ 50, it is judged that the DNA methylation molecular marker has been detected. If the target DNA methylation molecular marker C T If the value is "Undetermined", it is judged that the DNA methylation molecular marker has not been detected.
  • the primer-probe combinations of each molecular marker are the preferred combinations in Example 1.
  • a negative control will be set for each experiment, and the negative control uses water as a template to perform fluorescence quantitative PCR measurement of each specific molecular marker. If there is no detection signal in the negative control, it is judged that there is no external contamination in the entire experimental operation.
  • Example 4 Detection performance of molecular markers on benign and malignant pulmonary nodule tissue samples
  • Example 6 molecular markers in 134 lung tissue samples were detected respectively.
  • 57 samples were identified as benign by surgical biopsy and 77 were malignant.
  • the malignant samples included 66 stage I samples, 4 stage II samples, and 5 stage III malignant samples.
  • the specific detection kit, test method and data judgment processing are as described in Example 2.
  • the primer and probe combinations are as preferred in Example 1.
  • the combination of molecular marker combination 1 was selected for modeling analysis of pulmonary nodule tissue samples. Its average AUC was 0.91 (specificity: 91%; sensitivity: 82%), and its sensitivity for stage I malignant samples was 82%. , the phase II sensitivity is 100%, the phase III sensitivity is 100%, and the ROC is shown in Figure 1.
  • the combination of molecular marker combination 2 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 96%; sensitivity: 75%), and its sensitivity for stage I malignant samples was 74%.
  • the sensitivity of phase II is 100%
  • the sensitivity of phase III is 100%
  • the sensitivity of phase IV samples is 100%
  • the ROC is shown in Figure 1.
  • a combination of molecular marker combination 3 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.87 (specificity: 91%; sensitivity: 75%), and its sensitivity for stage I malignant samples was 74%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • a combination of molecular marker combination 4 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 96%; sensitivity: 79%), and its sensitivity for stage I malignant samples was 79%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • a combination of 5 molecular markers was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 91%; sensitivity: 82%), and its sensitivity to stage I malignant samples was 82%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • the performance of this combination is similar to that of combination 1, but there is less Marker1 than combination 1, indicating that these five molecular markers can achieve the performance of a combination of six molecular markers.
  • a combination of 6 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 96%; sensitivity: 75%), and its sensitivity to stage I malignant samples was 74%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • the only difference between this combination and combination 1 is marker 2.
  • the performance of combination 1 containing marker 2 is better than that of the combination without marker 1.
  • a combination of molecular marker combinations 7 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.90 (specificity: 96%; sensitivity: 78%), and its sensitivity to stage I malignant samples was 77%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • the only difference between this combination and combination 1 is marker 6.
  • the performance of combination 1 containing marker 6 is better than that of the combination without marker 6.
  • a combination of 8 molecular markers was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 95%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%
  • the ROC is shown in Figure 1.
  • the only difference between this combination and combination 1 is marker 5.
  • Combination 1 containing marker 5 is slightly more sensitive to stage I malignancy than the combination without marker 1, but the overall performance of the two is similar.
  • a combination of molecular marker combinations 9 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.92 (specificity: 96%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%. , the phase II sensitivity is 100%, and the phase III sensitivity is 100%.
  • a combination of 10 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.92 (specificity: 96%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%.
  • Phase II sensitivity is 100%
  • III Period sensitivity is 100%.
  • a combination of molecular marker combinations 11 was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.88 (specificity: 81%; sensitivity: 86%), and its sensitivity to stage I malignant samples was 86%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%.
  • a combination of 12 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.81 (specificity: 82%; sensitivity: 75%), and its sensitivity to stage I malignant samples was 74%.
  • the phase II sensitivity is 100%, and the phase III sensitivity is 100%.
  • a combination of 13 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.79 (specificity: 82%; sensitivity: 75%), and its sensitivity to stage I malignant samples was 75%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%.
  • a combination of 14 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.82 (specificity: 79%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%.
  • a combination of 15 molecular markers was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.91 (specificity: 96%; sensitivity: 79%), and its sensitivity to stage I malignant samples was 79%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%.
  • a combination of 16 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.81 (specificity: 75%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%.
  • the phase II sensitivity is 100%
  • the phase III sensitivity is 100%.
  • a combination of 17 molecular marker combinations was selected for modeling analysis of pulmonary nodule tissue samples.
  • the average AUC was 0.92 (specificity: 96%; sensitivity: 81%), and its sensitivity to stage I malignant samples was 80%. , the phase II sensitivity is 100%, and the phase III sensitivity is 100%.
  • the ROC of the combination of the above markers is shown in Figure 1. It can be seen from the above experimental results that the combined use of specific molecular markers will affect the performance of individual molecular markers.
  • the overall AUC, SP or SN of the molecular marker combinations 1-2, 4-11, 14, 15 and 17 described in this example are higher than those when using individual markers.
  • the molecular markers screened by the present invention have better sensitivity and specificity when combined with appropriate markers, and have potential clinical application value.
  • Example 5 Performance of molecular markers and combinations in detecting benign and malignant pulmonary nodules in respiratory fluid samples
  • Example 2 a combination of 6 molecular markers in 173 respiratory fluid samples was detected. Among them, 65 samples were identified as benign by surgical biopsy and 108 were malignant. Among them, the malignant samples included 33 stage I samples, 4 stage II samples, 4 stage III samples, 10 IV samples and 57 other malignant cases. sample.
  • the specific detection kit, test method and data judgment processing are as described in Example 2.
  • the primer and probe combinations are as preferred in Example 1.
  • This embodiment also detected a combination of six molecular markers.
  • the samples, detection methods, and data analysis methods were set up as described in Example 2.
  • the combination of molecular marker group 1 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.84 (specificity: 80%; sensitivity: 77%), and its sensitivity for stage I malignant samples was 68%, and its sensitivity for stage II malignant samples was 68%.
  • the sensitivity was 75% for phase III, 100% for phase III, and 88% for phase IV samples.
  • the ROC is shown in Figure 2.
  • the combination of molecular marker combination 2 was selected for modeling analysis of respiratory fluid samples. Its average AUC was 0.80 (specificity: 83%; sensitivity: 60%), and its sensitivity for stage I malignant samples was 42%, and its sensitivity for stage II malignant samples was 42%. The sensitivity was 50% for phase III, 100% for phase III, and 81% for phase IV samples.
  • the ROC is shown in Figure 2.
  • the combination of molecular marker combination 3 was selected for modeling analysis of respiratory fluid samples. Its average AUC was 0.77 (specificity: 71%; sensitivity: 75%), and its sensitivity for stage I malignant samples was 58%, and its sensitivity for stage II malignant samples was 58%. The sensitivity was 100% for phase III, 88% for phase III, and 88% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of molecular marker combination 4 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.81 (specificity: 69%; sensitivity: 81%), and its sensitivity for stage I malignant samples was 74%, and its sensitivity for stage II malignant samples was 74%. The sensitivity was 75% for phase III, 88% for phase III, and 88% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of molecular marker combinations 5 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.83 (specificity: 82%; sensitivity: 71%), and its sensitivity to stage I malignant samples was 47%, and its sensitivity to stage II malignant samples was 47%.
  • the sensitivity was 100% for phase III, 100% for phase III, and 88% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of molecular marker combinations 6 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.80 (specificity: 88%; sensitivity: 61%), and its sensitivity for stage I malignant samples was 45%, and its sensitivity for stage II malignant samples was 45%.
  • the sensitivity was 50% for phase III, 100% for phase III, and 79% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of molecular marker combinations 7 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.82 (specificity: 82%; sensitivity: 71%), and its sensitivity for stage I malignant samples was 58%, and its sensitivity for stage II malignant samples was 58%.
  • the sensitivity for phase III samples was 75%, the sensitivity for phase III samples was 88%, and the sensitivity for phase IV samples was 86%.
  • the ROC is shown in Figure 2
  • a combination of 8 molecular markers was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.84 (specificity: 86%; sensitivity: 68%). Its sensitivity to stage I malignant samples was 53%, and its sensitivity to stage II malignant samples was 53%. The sensitivity was 50% for phase III, 88% for phase III, and 83% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of molecular marker combinations 9 was selected for modeling analysis of respiratory fluid samples, and its average AUC was 0.80 (specificity: 82%; sensitivity: 70%), and its sensitivity to stage I malignant samples was 50%, and its sensitivity to stage II malignant samples was 50%.
  • the sensitivity was 75% for phase III, 100% for phase III, and 86% for phase IV samples.
  • the ROC is shown in Figure 2.
  • a combination of 10 molecular marker combinations was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.81 (specificity: 71%; sensitivity: 80%). Its sensitivity to stage I malignant samples was 66%, and its sensitivity to stage II malignant samples was 66%. Sensitivity was 50% for Phase III samples, 100% for Phase III samples, and 93% for Phase IV samples.
  • a combination of molecular marker combination 11 was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.79 (specificity: 55%; sensitivity: 85%). Its sensitivity to stage I malignant samples was 71%, and its sensitivity to stage II malignant samples was 71%. Phase sensitivity is 100%, III The sensitivity was 88% for stage IV samples and 95% for stage IV samples.
  • a combination of 12 molecular marker combinations was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.78 (specificity: 54%; sensitivity: 91%). Its sensitivity to stage I malignant samples was 87%, and its sensitivity to stage II malignant samples was 87%. Sensitivity was 100% for phase III, 88% for phase III, and 95% for phase IV samples.
  • a combination of molecular marker combination 13 was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.67 (specificity: 86%; sensitivity: 57%). Its sensitivity to stage I malignant samples was 63%, and its sensitivity to stage II malignant samples was 63%. The sensitivity was 75% for Phase III samples and 79% for Phase IV samples.
  • a combination of 14 molecular marker combinations was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.73 (specificity: 86%; sensitivity: 60%). Its sensitivity to stage I malignant samples was 39%, and its sensitivity to stage II malignant samples was 39%. Sensitivity was 75% for Phase III, 75% for Phase III, and 81% for Phase IV samples.
  • a combination of 15 molecular markers was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.82 (specificity: 68%; sensitivity: 81%). Its sensitivity to stage I malignant samples was 74%, and its sensitivity to stage II malignant samples was 74%. The sensitivity was 75% for Phase III samples, 88% for Phase III samples, and 88% for Phase IV samples.
  • a combination of 16 molecular marker combinations was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.79 (specificity: 52%; sensitivity: 93%). Its sensitivity to stage I malignant samples was 87%, and its sensitivity to stage II malignant samples was 87%. Sensitivity was 100% for phase III, 88% for phase III, and 93% for phase IV samples.
  • a combination of 17 molecular markers was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.84 (specificity: 60%; sensitivity: 90%). Its sensitivity to stage I malignant samples was 79%, and its sensitivity to stage II malignant samples was 79%. Sensitivity is 100% for phase III, 100% for phase III, and 95% for phase IV samples.
  • the ROC of the above marker combinations is shown in Figure 2. It can be seen from the above experimental results that the combined use of specific molecular markers will affect the performance of individual molecular markers. For the molecular marker combinations 1-11 and 15-17 described in this example, the overall performance (such as AUC, SP or SN) of the combination of multiple markers is higher than that of the individual markers.
  • the molecular markers screened by the present invention have better sensitivity and specificity when combined with appropriate markers, and have potential clinical application value.
  • Example 6 Performance of molecular markers and combinations in detecting benign and malignant pulmonary nodules in independent validation set of respiratory fluid samples
  • Example 2 a combination of 6 molecular markers in 61 naturally admitted respiratory tract fluid samples was detected.
  • 19 benign samples identified by surgical biopsy and 42 were malignant samples (the stage of the malignant samples is unknown).
  • the specific detection kit, test method and data judgment processing are as described in Example 2.
  • the primer and probe combinations are as preferred in Example 1.
  • This embodiment also detected a combination of six molecular markers.
  • the samples, detection methods, and data analysis methods were set up as described in Example 2.
  • the combination of molecular marker group 1 was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.919 (specificity: 94.4%; sensitivity: 79.5%), and the ROC is shown in Figure 3.
  • a combination of molecular marker combinations 7 was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.915 (specificity: 94.4%; sensitivity: 72.7%), and the ROC is shown in Figure 3.
  • a combination of 10 molecular marker combinations was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.878 (specificity: 94.4%; sensitivity: 72.7%), and the ROC is shown in Figure 3.
  • the combination of molecular marker combination 11 was selected for modeling analysis of respiratory fluid samples.
  • the average AUC was 0.876 (specificity: 91.4%; sensitivity: 70.5%), and the ROC is shown in Figure 3.
  • the molecular markers screened by the present invention have better sensitivity and specificity when combined with appropriate markers, and have potential clinical application value.

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Abstract

La présente invention concerne un marqueur moléculaire de méthylation d'ADN pour détecter des nodules pulmonaires bénins et malins. Le marqueur moléculaire de méthylation d'ADN comprend SEQ ID NO. 6 ou une séquence complémentaire complète de celui-ci, ou un fragment continu ayant au moins 55 % de la longueur totale de SEQ ID NO. 6 ou la séquence complémentaire complète de celui-ci. La présente invention concerne en outre l'utilisation du marqueur moléculaire de méthylation d'ADN décrit, un kit de détection correspondant et un procédé de détection correspondant.
PCT/CN2023/098075 2022-06-28 2023-06-02 Marqueur moléculaire de méthylation pour la détection de nodules pulmonaires bénins et malins et son utilisation Ceased WO2024001668A1 (fr)

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