WO2021243904A1 - 一种基因标志物组合及其应用 - Google Patents

一种基因标志物组合及其应用 Download PDF

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WO2021243904A1
WO2021243904A1 PCT/CN2020/118998 CN2020118998W WO2021243904A1 WO 2021243904 A1 WO2021243904 A1 WO 2021243904A1 CN 2020118998 W CN2020118998 W CN 2020118998W WO 2021243904 A1 WO2021243904 A1 WO 2021243904A1
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seq
gene
methylation
lung cancer
detection
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English (en)
French (fr)
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吴孝林
李仕良
张志伟
陈新周
吴幽治
古云娟
邹鸿志
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Creative Biosciences Guangzhou Co Ltd
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Creative Biosciences Guangzhou Co Ltd
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Priority to EP20939371.9A priority Critical patent/EP4163386A4/en
Priority to KR1020227046371A priority patent/KR20230017885A/ko
Priority to US18/008,100 priority patent/US20230279500A1/en
Priority to CA3185836A priority patent/CA3185836A1/en
Priority to JP2022573626A priority patent/JP7612715B2/ja
Priority to BR112022023887A priority patent/BR112022023887A2/pt
Priority to AU2020451827A priority patent/AU2020451827B2/en
Publication of WO2021243904A1 publication Critical patent/WO2021243904A1/zh
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present disclosure belongs to the field of biomedicine, and particularly relates to a gene marker combination and its application.
  • 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. It is a necessary routine examination item 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.
  • One of the objectives of the present disclosure is to provide a combination of gene markers, a detection/diagnostic reagent for detecting the combination of gene markers, and applications thereof.
  • the present disclosure provides a combination of genetic markers, the genetic markers including HOXB4 and SCRIN1.
  • the gene marker combination of the present disclosure also includes fragments of any length in each gene marker, that is to say, any combination of fragments from each of HOXB4 and SCRIN1 (the fragments can be of any length) fall into the present disclosure. In the scope of protection.
  • the HOXB4 gene is a member of the Antp homeobox gene family and belongs to the homeobox B cluster gene on chromosome 17. It encodes a nuclear protein with a homeobox DNA binding domain, and the encoded protein acts as a specific sequence transcription factor involved in development.
  • the intracellular or ectopic expression of the protein can expand hematopoietic stem and progenitor cells in vivo and in vitro, making it a potential candidate for therapeutic stem cell expansion.
  • SRCIN1 (SRC kinase signaling inhibitor 1) is called SRC kinase signaling inhibitor 1.
  • SRCIN1 gene and protein as negative regulators of SRC, inhibit SRC activity and downstream signal transduction by activating CSK, leading to impaired cell proliferation and migration. Regulate the morphology of dendritic spines. Participate in calcium-dependent exocytosis.
  • the present disclosure also provides the application of the multi-gene methylation combined detection reagent in the preparation of lung cancer detection reagents or kits, and the genes include HOXB4 and SCRIN1.
  • 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 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 present disclosure also provides a multi-gene methylation combined detection reagent or kit, including a reagent for the methylation detection of HOXB4 and SRCIN1 genes.
  • Methods include the following: reagents for detecting any smaller/shorter sequence of the gene or gene content. That is to say, any detection and detection reagent for any site (for example, a smaller fragment) in the gene falls into the protection scope of the present disclosure.
  • the genetic markers HOXB4 and SCRIN1 in the present disclosure are jointly detected, that is, several genetic markers in the present disclosure are detected at the same time.
  • 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.
  • lung cancer markers HOXB4 and SCRIN1 makes the early diagnosis of lung cancer possible.
  • a gene 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 the combination of lung cancer-specific HOXB4 and SCRIN1 genes in normal appearance cells.
  • early diagnosis includes the possibility of detecting cancer before metastasis, preferably before the morphological changes of tissues or cells can be observed.
  • 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 HOXB4 and SCRIN1 gene combination obtained from the sample by the progression of lung cancer in different stages or periods.
  • the degree of methylation of the HOXB4 and SCRIN1 gene combination of nucleic acids isolated from samples of each stage of lung cancer with the HOXB4 and SCRIN1 of one or more nucleic acids isolated from samples in tissues without abnormal cell proliferation Due to the combined degree of methylation, the specific stage of lung cancer in the sample can be detected.
  • CpG islands refer to regions rich in CpG dinucleotides, which are usually located in the promoter and its vicinity.
  • the CpG islands in this disclosure not only refer to the promoter and its surrounding regions being rich in CpG dinuclei.
  • Glycolic acid also includes heteromethylated CpG sites or isolated CpG sites.
  • the methylation combined detection reagent of the HOXB4 and SCRIN1 gene combination 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 enzymes 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 the HOXB4 and SCRIN1 gene combination.
  • the present disclosure also provides a methylation combined detection reagent for the HOXB4 and SCRIN1 gene combination, including primers and/or probes for each gene in the HOXB4 and SCRIN1 gene combination.
  • primers and/or probes obtained for the CpG islands of each gene in the HOXB4 and SCRIN1 gene combination are included.
  • the primers and/or probes detect the methylation of each gene in the HOXB4 and SCRIN1 gene combination by quantitative Methylation-Specific PCR (qMSP).
  • qMSP quantitative Methylation-Specific PCR
  • the methylation detection reagents provided in the present disclosure detect changes in the gene body, intergenic region or promoter region of each gene in the HOXB4 and SCRIN1 gene combination and the region near the promoter region. Methylation level.
  • the methylation detection reagent provided by the present disclosure includes primers and/or probes obtained from the promoter region of each gene in the HOXB4 and SCRIN1 gene combination or the CpG islands in the vicinity of the promoter region .
  • the upstream primer in the methylation detection primer of the HOXB4 gene in the methylation detection reagent provided in the present disclosure has any one of the following nucleotide sequences:
  • the downstream primer in the primer for methylation detection of the HOXB4 gene has any one of the nucleotide sequences shown below:
  • the upstream primer in the primer for methylation detection of the SRCIN1 gene has any one of the following nucleotide sequences:
  • V It 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% of the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 22 Or a nucleotide sequence that is at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical; and
  • the downstream primer in the primer for methylation detection of the SRCIN1 gene has any one of the following nucleotide sequences:
  • the primer pair for methylation detection of the HOXB4 gene is shown in SEQ ID NO.: 1 and SEQ ID NO.: 2.
  • the primer pair for methylation detection of the HOXB4 gene is shown in SEQ ID NO.: 16 and SEQ ID NO.: 17.
  • the primer pair for the methylation detection of the HOXB4 gene is shown in SEQ ID NO.: 19 and SEQ ID NO.: 20.
  • the primer pair for methylation detection of the SRCIN1 gene is shown in SEQ ID NO.: 4 and SEQ ID NO.: 5.
  • the primer pair for methylation detection of the SRCIN1 gene is shown in SEQ ID NO.: 22 and SEQ ID NO.: 5.
  • the HOXB4 gene methylation detection probe has any one of the following nucleotide sequences:
  • SEQ ID NO.: 3 SEQ ID NO.: 18 and SEQ ID NO.: 21 have 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 sequences;
  • the SRCIN1 gene methylation detection probe has any one of the following nucleotide sequences:
  • XI 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 the nucleotide sequence shown in SEQ ID NO.: 6
  • 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 kit for detecting lung cancer, including the methylation combined detection reagent.
  • the kit provided by the present disclosure also includes common reagents in the kit, such as a common conversion agent in qMSP, which is used to convert all unmethylated cytosine bases into uracil, while methylation The cytosine bases remain unchanged.
  • the conversion agent is not particularly limited.
  • the reagents reported in the prior art that can convert cytosine to uracil can be used, such as hydrazine salt, bisulfite and bisulfite (for example, sodium metabisulfite, One or more of potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.).
  • Another example is DNA polymerase, dNTPs, Mg 2+ ions and buffers commonly used in gene amplification.
  • the reagents or kits further include detection reagents for internal reference genes.
  • 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: 13 and SEQ ID NO: 14 and a probe shown in SEQ ID NO: 15.
  • the present disclosure provides the application of the methylation combined detection reagent of HOXB4 and SCRIN1 genes in the preparation of lung cancer detection reagents or kits.
  • the present disclosure provides a primer selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 22 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 A nucleotide sequence that is at least 96% or at least 97% or at least 98% or at least 99%, or 100% identical, or at least any one of their complementary sequences.
  • the present disclosure provides a primer which is selected from the group consisting of at least 85% or at least 90% of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5 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 sequences, or they are complementary At least any one in the sequence.
  • the primer is selected from at least one primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5
  • the primer is selected from the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5.
  • 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 present disclosure also provides a nucleic acid probe selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO.: 18 and SEQ ID NO.: 21. Show 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 at least 98% or at least 99%, or 100%.
  • SEQ ID NO: 3 SEQ ID NO: 6
  • SEQ ID NO.: 18 and SEQ ID NO.: 21 Show 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 at least 98% or at least 99%, or 100%
  • the nucleic acid probe is selected from the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 6.
  • the kit provided by the present disclosure includes: a first container, which contains a primer pair for amplification; and a second container, which 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 applications of the above-mentioned methylation detection reagents, kits, primers, and probes in preparing a reagent or kit for methylation detection, or preparing a reagent or kit for detecting lung cancer.
  • the present disclosure also provides applications of the aforementioned methylation detection reagents, kits, primers, and probes in methylation detection, or applications in the detection of lung cancer.
  • the present disclosure also provides a lung cancer detection system.
  • the system includes the following components:
  • the methylation detection component contains a methylation detection instrument.
  • the methylation detection component further contains the methylation combined detection reagents, kits, primers, and probes.
  • the methylation detection instrument includes one or more of a fluorescent quantitative PCR machine, a PCR machine, and a sequencer.
  • the data processing component includes a data processing machine.
  • the data processing machine includes any equipment or instrument or device that can perform data processing that can be used by those skilled in the art.
  • the data processing machine includes one or more of a calculator and a computer.
  • the computer is attached with any software or program that can be used by those skilled in the art that can perform data processing or statistical analysis.
  • the computer includes a computer loaded with one or more software of SPSS, SAS, and Excel.
  • the result output means includes a result output device.
  • the output device includes any equipment or instrument or device that can display the data processing result as readable content.
  • the methylation detection component further contains the multi-gene methylation combined detection reagent.
  • the result output device includes one or more of a screen and a paper report.
  • the data processor is configured to a. receive the test data of the test sample and the normal control sample; b. store the test data of the test sample and the normal control sample; c. compare the same type The test data of the test sample and the normal control sample; d. According to the comparison result, respond to the probability or possibility of the tester suffering from lung cancer.
  • the result output component is used to output the probability or likelihood that the tester will develop lung cancer.
  • the judgment standard of the data processing component is: judging lung cancer specimens and normal specimens according to the cut-off value.
  • the combined detection of HOXB4 and SRCIN1 of the present disclosure can also be achieved by multiplex PCR.
  • the ⁇ Cp value of one of the genes in the tissue specimen is less than the threshold value of the ⁇ Cp value, it is determined to be a lung cancer specimen, and only two genes in the tissue specimen have a ⁇ Cp value greater than The boundary value equal to the ⁇ Cp value is judged to be a normal specimen.
  • the critical value of the Cp value in the specimen ranges from 35 to 39, and the critical value of the ⁇ Cp value ranges from 4 to 12.
  • HOXB4 is combined with SRCIN1.
  • the ⁇ Cp value of HOXB4 has a cutoff value of 5.4
  • the ⁇ Cp value of SRCIN1 has a cutoff value of 6.5.
  • HOXB4 and SRCIN1 are jointly detected.
  • the threshold line Cp values of HOXB4 and SRCIN1 are: 36.9 and 37.0, respectively.
  • the cut-off value of the Cp value in the sputum specimen is 36.7
  • the cut-off value of the Cp value in the lavage fluid specimen is 37.2
  • the cut-off value of the ⁇ Cp value is 9 .
  • HOXB4 and SRCIN1 multiplex PCR detection, when the Cp value of the sputum specimen is less than the threshold value of the Cp value, it is determined to be a lung cancer specimen, and the Cp value of the sputum specimen is greater than or equal to the Cp value The boundary value of is judged to be a normal specimen.
  • HOXB4 and SRCIN1 multiplex PCR detection, if any one of the Cp value and ⁇ Cp value of the lavage fluid sample is less than the boundary value of the Cp value and ⁇ Cp, it is judged to be a lung cancer sample, and the lavage fluid sample If the Cp value and the ⁇ Cp value of the lotion sample are both greater than or equal to the boundary value of the Cp value and the ⁇ Cp value, it is judged as a normal sample.
  • the tumor is lung cancer.
  • the tumor is 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 target sample or sample type is selected from at least one of alveolar lavage fluid, tissue, pleural fluid, sputum, blood, serum, plasma, urine, prostate fluid, or feces.
  • the sample described in the present disclosure is selected from at least one of alveolar lavage fluid, tissue, and sputum.
  • the sample described in the present disclosure is selected from at least one of alveolar lavage fluid or sputum.
  • the present disclosure also provides a method for diagnosing lung cancer, which includes the following steps:
  • the present disclosure provides a method for diagnosing lung cancer, the method comprising the following steps: (1) detecting the methylation level of HOXB4 and SRCIN1 genes in a test sample derived from a subject; the detection This includes contacting the test sample of the subject with a detection reagent for detecting the methylation level of the HOXB4 and SRCIN1 genes; (2) comparing the methylation level of the HOXB4 and SRCIN1 genes of the test sample with the normal control sample; and (3) Based on the deviation of the methylation level of the test sample and the normal control sample, lung cancer is diagnosed.
  • the present disclosure provides a method for diagnosing lung cancer.
  • the method includes the following steps: adding a gene methylation detection reagent to a test sample from a subject, and detecting HOXB4 in the test sample And SRCIN1 gene methylation level; compare the HOXB4 and SRCIN1 gene methylation levels of the test sample and the normal control sample; and diagnose lung cancer based on the deviation of the methylation level of the test sample and the normal control sample.
  • the deviation in step (3) refers to the deviation of the methylation level of any one of the two genes, HOXB4 and SRCIN1.
  • the detection includes contacting the test sample of the subject with a detection reagent for the methylation level of the HOXB4 and SRCIN1 genes.
  • methylation-specific quantitative PCR (qMSP) is used to detect the methylation level of HOXB4 and SRCIN1 genes.
  • the methylation results of the test sample and the normal sample are compared by the results.
  • the result determines that the test sample has a high risk of disease .
  • 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.
  • a method for treating lung cancer comprising the following steps:
  • 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.
  • lung cancer samples can be well distinguished from the samples.
  • the detection sensitivity and specificity of lung cancer are extremely high.
  • Figure 1 shows the ROC curve of different marker combinations detected in tissue samples
  • Figure 2 shows the ROC curve of different marker combinations detected in sputum samples
  • Figure 3 shows the amplification curve of the combined detection of HOXB4 and SRCIN1 in lavage fluid samples.
  • 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 genes and screened them in tissue samples. Using ⁇ -actin gene as an internal reference gene, the pairwise combined detection results of HOXB4, SRCIN1, PCDHGA12, and HOXD8 genes were compared.
  • the detection primer probes for each gene are as follows:
  • the detection primers and probes of HOXB4 are:
  • SEQ ID NO: 2 HOXB4-R1 primer R TACTAACCGCCTCGCTAC
  • the detection primers and probes of SRCIN1 are:
  • SEQ ID NO: 4 SRCIN1 primer F TCGTGTGTCGTCGTTCAGAC
  • the detection primers and probes of PCDHGA12 are:
  • the detection primers and probes of HOXD8 are:
  • the detection primers and probes for ⁇ -actin are:
  • SEQ ID NO: 13 ⁇ -actin primer F: GGAGGTTTAGTAAGTTTTTTGGATT
  • SEQ ID NO: 14 ⁇ -actin primer R CAATAAAACCTACTCCTCCCTTA
  • Amplification system See Table 2 and Table 3 for the amplification system of each detected gene.
  • 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. 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-cancerous control samples were included.
  • ACTB is used as the internal reference gene
  • Figure 1 shows the ROC curves of HOXB4 combined with SRCIN1, HOXB4 combined with PCDHGA12, and HOXB4 combined with HOXD8 in all tissue samples.
  • the statistical results of the detection of each gene in the tissue are shown in Table 3.
  • the combined detection of HOXB4 and SRCIN1 in the tissue samples has a specificity of 97.8% and a sensitivity of 89.7% compared with the normal group and the entire cancer group.
  • the combined detection of HOXB4 and SRCIN1 is more effective.
  • the combined detection of HOXB4 and SRCIN1 significantly improves the sensitivity compared to the detection of HOXB4 and SRCIN1 alone.
  • HOXB4 and SRCIN1 still have high sensitivity in tissue samples with high specificity. Especially through the combined detection, the sensitivity is greatly improved without affecting the specificity.
  • sputum is of greater significance in the diagnosis of lung cancer. For this reason, the inventors tested the two markers of HOXB4 and SRCIN1 in sputum.
  • Sample information A total of 107 sputum samples were tested, including 51 samples from the normal control group, 56 samples from the cancer group, and 56 samples from the cancer group including 20 cases of squamous cell carcinoma, 8 cases of small cell carcinoma, 20 cases of adenocarcinoma, and large cell There was 1 case of cancer, 1 case of giant cell carcinoma, and 6 cases of lung cancer not clearly classified.
  • the ROC curve of HOXB4 and SRCIN1 detected in sputum samples is shown in Figure 2, and the statistical results are shown in Table 4. From the above results, it can be seen that in the sputum samples, when HOXB4 and SRCIN1 are jointly detected, the normal group and all cancer groups are compared, The sensitivity to lung cancer is increased to 76.8%; the sensitivity of the normal group and all small cell carcinoma groups can reach 100%. Compared with a single gene marker, the detection rate of HOXB4 is 64.3%, and the detection rate of SRCIN1 is 48.2%. When the two are jointly detected, the sensitivity to lung cancer is increased to 76.8%, and the two are synergistic.
  • the detection primers and probe sequences of HOXB4, SRCIN1 and ⁇ -actin are the same as in Example 1.
  • liquid dosing system is as follows:
  • Reaction component Addition amount ( ⁇ l) HOXB4-F1(100 ⁇ M) 0.125 HOXB4-R1(100 ⁇ M) 0.125 HOXB4-P1(100 ⁇ M) 0.05 SRCIN1-F1(100 ⁇ M) 0.125 SRCIN1-R1(100 ⁇ M) 0.125 SRCIN1-P1(100 ⁇ M) 0.05 ⁇ -actin-F1(100 ⁇ M) 0.125 ⁇ -actin-R1(100 ⁇ M) 0.125 ⁇ -actin-P2(100 ⁇ M) 0.05 Magnesium ion (25mM) 6 dNTPs (10mM) 1 Taq polymerase (5unit/ ⁇ l) 0.5 5X buffer 6 Sterilized water 10.6 Template DNA 5 total capacity 30
  • the amplification system is the same as the amplification system in Table 2 of Example 1;
  • the results show that the detection results of the HOXB4 and SRCIN1 multiplex PCR system are basically consistent with those in Example 2. It shows that the detection result of the multiplex PCR system can be used as the result of the combined detection of lung cancer with HOXB4 and SRCIN1 genes.
  • Sample information A total of 387 samples of alveolar lavage fluid were tested, including 303 samples from the normal control group, 84 samples from the cancer group, and 21 samples from the cancer group including 21 cases of squamous cell carcinoma, 40 cases of adenocarcinoma, and 10 cases of small cell carcinoma. , 13 cases of lung cancer type were not clear.
  • the detection primers and probe sequences of HOXB4, SRCIN1 and ⁇ -actin are the same as in Example 1.
  • the test results of 387 lavage fluid samples are as follows:
  • the amplification curve of the combined detection of HOXB4 and SRCIN1 in all lavage fluid samples is shown in Figure 3, and the statistical results are shown in Table 7. From the above results, it can be seen that the combined detection of HOXB4 and SRCIN1 has a high specificity of 96.0%, and the sensitivity reaches 77.4%. According to the comparison and analysis of the subtypes of lung cancer, the detection rate of the combined detection of HOXB4 and SRCIN1 in the squamous cell carcinoma group is 71.4%. . Especially for the detection effect of adenocarcinoma, the sensitivity of the combined detection of HOXB4 and SRCIN1 is as high as 75.0%.
  • 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.
  • Example 5 Test results of different marker combinations in sputum samples
  • the inventors also compared the detection conditions of different combinations of markers in sputum samples, and the comparison groups are as follows:
  • Combination 1 Combination 2
  • Combination 3 Combination 4
  • Combination 5 Combination 6
  • Combination 7 Sensitivity 76.8% 64.3% 46.4% 66.1% 53.6% 53.6% 76.8% Specificity 92.2% 92.2% 92.2% 94.1% 94.1% 90.2% 92.2%
  • 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.
  • liquid preparation systems are all the same, the liquid preparation systems and the amplification procedures are the same, and the amplification procedures are the same as in Example 2.
  • the results show that different primer pairs for the same region will have an impact on the detection results.
  • the primer and probe combinations of HOXB-F1, HOXB-R1, HOXB-P1 and SRCIN1-F1, SRCIN1-R1, SRCIN1-P1 have higher sensitivity.

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Abstract

本申请公开一种肿瘤基因标志物组合、甲基化检测试剂、试剂盒及其应用。通过检测HOXB4、SRCIN1基因组合的甲基化水平，可以区分肺癌标本。本申请的试剂经试验证实，可以检测和诊断肺癌，有临床应用价值。

Description

一种基因标志物组合及其应用 技术领域
本公开属于生物医药领域，特别涉及一种基因标志物组合及其应用。
发明背景
肺癌是起源于支气管粘膜、腺体或肺泡上皮的肺部恶性肿瘤。按照病理类型可以分为：1)小细胞肺癌(small cell lung cancer，SCLC)：一种特殊病理学类型的肺癌，有明显的远处转移倾向，预后较差，但多数病人对放化疗敏感；2)非小细胞肺癌(non-small cell lung cancer，NSCLC)：除小细胞肺癌以外其他病理学类型的肺癌，包括鳞状细胞癌、腺癌、大细胞癌等。在生物学行为和临床病程方面具有一定差异。按照发生位置又可以分为：1)中心型肺癌(central lung cancer)：生长在肺段支气管开口及以上的肺癌；2)周围型肺癌(peripheral lung cancer)：生长在肺段支气管开口以远的肺癌。
近年来，人口老龄化、大气污染及吸烟等因素的影响，致使中国肺癌的发病率和死亡率逐年递增，根据国家癌症中心发布的《2017中国肿瘤登记年报》显示，全国每分钟约7人确诊患癌，其中肺癌发病率与死亡率均为第一位。中国已成为世界上肺癌人数最多的国家，专家预测到2025年中国肺癌的人数将达到100万。而根据流行病学研究显示：吸烟是引起肺癌的重要因素。世界上约80％-90％的肺癌可归因于吸烟。与非吸烟者相比，45-64岁每日吸香烟1-19支和20支以上者患肺癌的相对危险度分别为4.27和8.61，与从不吸烟者比较，长期每日吸烟1-19支和20支以上者死于肺癌的相对危险度分别为6.14和10.73。虽然肺癌的治疗技术日新月异，但5年生存率从4％仅上升至12％左右，现有的抗肿瘤药物仍只能起到缓解病情作用，患者无进展生存期平均仅延长3个月～5个月，但对于Ⅰ期肺癌患者，手术后5年生存率高达约60％～70％。因此肺癌的早期诊断与早期手术是提高肺癌5年生存率、降低死亡率最有效的方法之一。
肺癌目前的临床辅助诊断主要有以下几种，但是他们都不能完全做到早发现，早诊断：
(1)血液生化检查：对于原发性肺癌，目前无特异性血液生化检查。肺癌病人血液碱性磷酸酶或血钙升高考虑骨转移的可能，血液碱性磷酸酶、谷草转氨酶、乳酸脱氢酶或胆红素升高考虑肝转移的可能。
(2)肿瘤标志物检查：1)CEA：30％～70％肺癌患者血清中有异常高水平的CEA，但主要见于较晚期肺癌患者。目前血清中CEA的检查主要用于估计肺癌预后以及对治疗过程的监控。2)NSE：是小细胞肺癌首选标志物，用于小细胞肺癌的诊断和监测治疗反应，根据检测方法和使用试剂的不同，参考值不同。3)CYFRA21-1：是非小细胞肺癌的首选标记物，对肺鳞癌诊断的敏感性可达60％，根据检测方法和使用试剂的不同，参考值不同。
(3)影像学检查：1)胸部X线检查：应包括胸部正位和侧位片。在基层医院，胸部正侧位片仍是肺癌初诊时最基本和首选的影像诊断方法。一旦诊断或疑诊肺癌，即行胸部CT检查。2)CT检查：胸部CT是肺癌的最常用和最重要的检查方法，用于肺癌的诊断与鉴别诊断、分期及治疗后随诊。CT引导下肺穿刺活检是肺癌的重要诊断技术，有条件的医院可将其用于难以定性的肺内病变的诊断，以及临床诊断肺癌需经细胞学、组织学证实而其它方法又难以取材的病例。近年来，多层螺旋CT和低剂量CT(LDCT)是发现早期肺癌和降低死亡率的有效筛查工具，全美国家肺癌筛查研究(NLST)已经表明LDCT相比胸部X线筛查可降低20％肺癌的死亡率。低剂量螺旋CT被推荐为早期肺癌筛查的重要手段，但是人为影响因素较多，假阳性率非常高。3)超声检查：主要用于发现腹部重要器官及腹腔、腹膜后淋巴结有无转移，也用于颈部淋巴结的检查。对于贴邻胸壁的肺内病变或胸壁病变，可鉴别其囊实性及进行超声引导下穿刺活检；超声还常用于胸水抽取定位。4)骨扫描：对肺癌骨转移检出的敏感性较高，但有一定的假阳性率。可用于以下情况：肺癌的术前检查；伴有局部症状的病人。
(4)其它检查：1)痰细胞学检查：目前肺癌简单方便的无创诊断方法，连续涂片检查可提高阳性率约达60％，是可疑肺癌病例的常规诊断方法。2)纤维支气管镜检查：肺癌诊断中最重要的手段之一，对于 肺癌的定性定位诊断和手术方案的选择有重要的作用。对拟行手术治疗的患者为必需的常规检查项目。而经支气管镜穿刺活检检查(TBNA)，虽利于治疗前分期，但因技术难度和风险较大，有需要者应转上级医院进一步检查。3)其他：如经皮肺穿刺活检、胸腔镜活检、纵隔镜活检、胸水细胞学检查等，在有适应证的情况下，可根据现有条件分别采用以协助诊断。
影像学检查中的多层螺旋CT和低剂量CT(LDCT)是发现早期肺癌和降低死亡率的有效筛查工具，全美国家肺癌筛查研究(NLST)已经表明LDCT相比胸部X线筛查可降低20％肺癌的死亡率。在临床实践工作中证明，任何肺癌筛查项目的成败取决于高危人群的识别，融合多重高危因素的风险预测模型已被世界公认是识别肺癌高危人群的方法之一。风险模型通过协助临床医生改进干预措施或治疗手段，从而进一步改善肺癌患者的疗效。虽然世界已经认同针对高危人群的筛查能够降低肺癌目前较高的死亡率，但高危人群界定仍然是难以解决的问题。为了使肺癌筛查的效益-伤害比达到最大化，关键的问题第一是如何界定高危患病风险的人群；第二是用什么方法对该人群进行筛查，包括高危因素的界定，总体风险的量化汇总以及筛查效益界值的选择。
随着技术的飞速发展，肿瘤标志物检测成为继影像学诊断，病理诊断之后的肿瘤诊断治疗新领域，能够对肿瘤的诊断，检测，治疗产生重大的影响。肿瘤标志物可以在体液或者是组织中检测到，能够反映肿瘤的存在，分化程度，预后估计和个性化用药以及治疗效果等。早期肺癌患者没有明显的症状，难以被医生和患者察觉，再加之其在血液或生化项目上没有明显的特异性的标识物，因此难以通过常规的诊断方法进行早期发现和早期诊断，因此对肺癌早期诊断，尤其是大规模应用人群筛查上较为困难。
越来越多的研究表明，在肿瘤形成过程中包含两大类机制。一个是通过DNA核苷酸序列改变而形成突变，即遗传学机制。肿瘤作为一种遗传学疾病在分子生物学领域已经得到证实。另外一个就是表观遗传学(epigenetics)机制，即不依赖DNA序列改变导致基因表达水平的变化，它在肿瘤形成过程中的作用越来越受到重视。遗传学与表观遗传学两种机制相互交叉存在，共同促进了肿瘤的形成。基因的异常甲基化在肿瘤发生的早期就可出现，并且在肿瘤逐步发展的过程中，基因异常甲基化的程度增加。对常见的98种人类原发肿瘤的基因组进行分析，发现每种肿瘤至少有600个异常甲基化的CpG岛。
许多研究显示启动子异常甲基化在许多肿瘤的发生过程中是一个频发的早期事件，因此肿瘤相关基因的甲基化状态是肿瘤发生的一个早期敏感指标，被认为是一种有前景的肿瘤分子生物标志物(biomarker)。更为重要的是，癌变细胞可以释放DNA到外周血中。正常人外周血中都存在纳克级的游离DNA。研究发现外周血血浆/血清、肿瘤累及器官相关的体液(如唾液、痰等)中同样可以检测到肿瘤组织中存在的肿瘤相关基因的启动子异常甲基化。这些生物样品比较容易获得，并且通过PCR技术将其中的DNA进行大量的扩增后能够很灵敏的检测到，因此检测其中某些肿瘤相关基因的启动子区甲基化状态，可以为肿瘤的早期诊断提供非常有价值的信息。与其它类型的肿瘤分子标记物相比，检测启动子异常甲基化有更多的优点。某一基因在不同类型肿瘤中，其启动子异常甲基化的区域是相同的，检测比较方便；另外与等位基因缺失这样的标志物相比，异常甲基化是一个阳性信号，很容易与正常组织中的阴性背景区分。Esteller等检测了22例非小细胞肺癌(NSCLC)的肿瘤组织和血清中p16、DAPK、GSTP1及MGM T等基因的启动子区异常甲基化状态，发现68％(15/22)的肿瘤组织中存在至少一种基因的启动子甲基化；而在15例组织阳性病例中，有11例同时在血清中也检测到了启动子异常甲基化的存在。另有许多研究者也分别从肝癌、头颈部癌、食管癌及结肠癌患者的肿瘤组织和血清中同时检测出了某些肿瘤相关基因的启动子甲基化。
现有的肺癌检测技术中主要存在灵敏度低、假阳性高，有创，并且，目前常规检测技术难以检出早期肺癌。
而肺癌的无创检测，例如，痰液检测，难度则更大。尽管也有研究者研究肺癌患者痰液中的肿瘤标志物，然而，对比起其他肿瘤患者血液样本的肿瘤标志物检测及评估，痰液样本的成功率却很低。这主要由于以下原因：①痰液的成分比较复杂，不同的人群在不同的疾病或者环境下痰液的成分和粘度等差异比较大；②痰液中含有较多的气管上皮细胞和细菌，口腔黏膜细胞等非肺癌细胞的成分，一般的样本处理方法无法有 效的富集到数目充足的肺癌来源的DNA；③有很多的吸烟患者并不表现出咳痰。A J Hubers等人在《Molecular sputum analysis for the diagnosis of lung cancer》中对过去10篇文献研究显示，肺癌组织中标志物的中位数的甲基化程度为48％，而痰液的中位数的甲基化程度为38％，结果显示甲基化标志物在组织中的检出率明显高于痰液。同时，Rosalia Cirincione(Methylation
Figure PCTCN2020118998-appb-000001
in tumor and sputum samples of lung cancer patientsdetected by spiral computed tomography:A nested case–contro)报道了RARbeta2、P16、RASSF1A在肺癌组织中检出率分别达到65.5％、41.4％、51.7％，而在痰液中分别只有44.4％、5％、5％。
发明内容
本公开的目的之一在于提供一种基因标志物组合，以及检测该基因标志物组合的检测/诊断试剂及其应用。
一方面，本公开提供了一种基因标志物组合，所述基因标志物包括HOXB4和SCRIN1。
本公开的基因标志物组合，还包括了各基因标志物中的任意长度的片段，也就是说，来自HOXB4、SCRIN1各自任意一个片段(该片段可以为任意长度)组合，均落入本公开的保护范围中。
HOXB4基因是Antp同源盒基因家族的一员，属于染色体17上的同源盒B簇基因。编码带有同源盒DNA结合结构域的核蛋白，编码的蛋白作为参与发育的特异性序列转录因子。该蛋白的细胞内或异位表达可在体内和体外扩增造血干细胞和祖细胞，使其成为治疗性干细胞扩增的潜在候选者。
SRCIN1(SRC kinase signaling inhibitor 1)全称为SRC激酶信号传导抑制基因1。SRCIN1基因和蛋白质作为SRC的负调控因子，通过激活CSK抑制SRC活性和下游信号传导，导致细胞扩散和迁移受损。调节树突棘的形态。参与钙依赖性的胞吐作用。
本公开还提供了多基因的甲基化联合检测试剂在制备肺癌检测试剂或者试剂盒中的应用，所述基因包括HOXB4和SCRIN1。
目前肺癌的漏检率较高。特别是，对于腺癌这种类型，痰液无创检测更加是难上加难，检出率极其低。这是因为，多数腺癌起源于较小的支气管，为周围型肺癌，肺深部的脱落细胞更加难以通过痰液咳出。因此，目前腺癌的痰液检测手段几乎为零。
降低漏检率在肿瘤早期筛查中是尤其重要的。如果一个肿瘤早期筛查产品无法将所有或绝大部分的病患筛查出来的话，那么漏检的那些将无法得到足够的风险提示，从而延误的治疗时机，这对患者来说是一个巨大的损失。
尽管现有技术中已经发现了一些肺癌相关的肿瘤标志物，但是，受限于针对这些肿瘤标志物的检测试剂或者检测手段，导致这些肿瘤标志物的灵敏度和特异性不能满足需求，因此，目前本领域中仍然需要进一步研究能够切实地应用于肺癌的筛查手段。然而，虽然无创式的筛查具有取样方面独到的优势，然而，其也具有其他方面的一些局限，例如，肺癌中的腺癌这种类型，由于其肺深部的脱落细胞难以通过痰液咳出，通常说来，本领域技术人员会认为该种类型的肺癌不适宜采用无创筛查。另一方面，即使是其他类型的肺癌，目前已报道的无创筛查方法也很难达到临床使用的要求。尽管相关研究已进展多年，但至今仍未有可以推向临床的肺癌无创筛查方法。
本公开还提供了一种多基因甲基化联合检测试剂或试剂盒，包括HOXB4和SRCIN1基因的甲基化检测的试剂。
“甲基化检测试剂”，包括以下内容：针对所述基因或基因内容的更小/短的任意序列进行检测的试剂。也就是说，任意针对所述基因中的任意位点(例如，一段更小的片段)进行的检测及检测试剂，均落入本公开的保护范围中。
本公开中的基因标志物HOXB4和SCRIN1是联合检测的，也就是说，本公开中的几个基因标志物是同时被检测的。
本公开中的“检测”同诊断，除了肺癌的早期诊断，还包括肺癌中期和晚期的诊断，且也包括肺癌筛选、风险评估、预后、疾病识别、病症阶段的诊断和治疗性靶标的选择。
肺癌标志物组合HOXB4和SCRIN1的应用使得肺癌的早期诊断成为可能。当确定在癌症细胞中甲基化的基因在临床上或形态学上正常表象的细胞中甲基化时，这就表明该正常表象的细胞向癌症发展。这样，肺癌可在早期通过在正常表象的细胞中的肺癌特异性HOXB4和SCRIN1基因组合的甲基化而诊断。
其中，早期诊断包括在转移之前发现癌症的可能性，优选在可观察到组织或者细胞的形态学变化之前。
除了肺癌的早期诊断，本公开的试剂/试剂盒还有希望用于肺癌筛选、风险评估、预后诊断、疾病识别、病症阶段的诊断和治疗性靶标的选择。
作为病症阶段可选的实施方式，可通过在肺癌在不同阶段或时期的进展可通过从样品中获取的HOXB4和SCRIN1基因组合的甲基化程度的测量进行诊断。通过比较从肺癌的每个阶段的样品中分离出的核酸的HOXB4和SCRIN1基因组合甲基化程度与从没有细胞增殖性异常的组织中的样品中分离出的一个或多个核酸的HOXB4和SCRIN1因组合甲基化程度，可检测样品中肺癌的具体阶段。
通常来说，CpG岛是指富含CpG二核苷酸的一些区域，通常位于启动子及其附近的区域，本公开中的CpG岛，不仅指启动子及其附近的区域富含CpG二核苷酸，也包括杂合甲基化的CpG位点，或者是孤立的CpG位点。
所述的HOXB4和SCRIN1基因组合的甲基化联合检测试剂可以是现有技术中的甲基化检测试剂。现有技术中，已经有多种方法可以检测目的基因的甲基化，如甲基化特异性PCR(MSP)、甲基化特异性定量PCR(qMSP)、甲基化DNA特异性结合蛋白的PCR，定量PCR以及DNA芯片、甲基化敏感的限制性内切酶、重亚硫酸盐测序法或者焦磷酸测序等等。除此之外，其他的甲基化检测方法可以通过专利US62007687引入。每种检测方法均有其相对应的试剂，这些试剂均可以用于本公开检测HOXB4和SCRIN1基因组合的甲基化。
本公开还提供了HOXB4和SCRIN1基因组合的甲基化联合检测试剂，包括针对HOXB4和SCRIN1基因组合中每个基因的引物和/或探针。
在本公开的一些具体的实施方案中，包括针对HOXB4和SCRIN1基因组合中每个基因的CpG岛获得的引物和/或探针。
在本公开的一些具体的实施方案中，引物和/或探针通过quantitative Methylation-Specific PCR(qMSP)检测HOXB4和SCRIN1基因组合中的每个基因的甲基化。
在本公开的一些具体的实施方案中，本公开提供的甲基化检测试剂通过检测HOXB4和SCRIN1基因组合中的每个基因的基因体、基因间区或启动子区及启动子区附近区域的甲基化水平。
在一些实施方案中，本公开提供的甲基化检测试剂包括针对HOXB4和SCRIN1基因组合中的每个基因的启动子区或所述启动子区附近区域的CpG岛获得的引物和/或探针。
在一些实施方案中，本公开提供的甲基化检测试剂中所述HOXB4基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
I、与如SEQ ID NO:1、SEQ ID NO：16和SEQ ID NO：19所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
II、如I所示序列的互补序列；和/或
所述HOXB4基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
III、与如SEQ ID NO:2、SEQ ID NO：17和SEQ ID NO：20所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；
IV、如III所示序列的互补序列；和/或
所述SRCIN1基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
V、与如SEQ ID NO:4和SEQ ID NO:22所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核 苷酸序列；及
VI、如V所示序列的互补序列；和/或
所述SRCIN1基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
VII、与如SEQ ID NO:5所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
VIII、如VII所示序列的互补序列；
在一些实施方案中，所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:1和SEQ ID NO.:2所示。
在一些实施方案中，所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:16和SEQ ID NO.:17所示.
所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:19和SEQ ID NO.:20所示。
在一些实施方案中，所述SRCIN1基因的甲基化检测的引物对如SEQ ID NO.:4和SEQ ID NO.:5所示。
在一些实施方案中，所述SRCIN1基因的甲基化检测的引物对如SEQ ID NO.:22和SEQ ID NO.:5所示。
在一些实施方案中，所述HOXB4基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
IX、与如SEQ ID NO.:3、SEQ ID NO.:18和SEQ ID NO.:21所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
X、如IX所示序列的互补序列；和/或
所述SRCIN1基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
XI、与如SEQ ID NO.:6所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
XII、如XI所示序列的互补序列。
在一些实施方案中，所述肺癌选自小细胞肺癌和非小细胞肺癌。
在一些实施方案中，所述的非小细胞肺癌选自鳞状细胞癌、腺癌。
本公开还提供了一种检测肺癌的试剂盒，包括所述的甲基化联合检测试剂。
在一些实施方案中，本公开提供的试剂盒还包括试剂盒中的常用试剂，如qMSP中常用转化剂，用于将非甲基化的胞嘧啶碱基都转化为尿嘧啶，而甲基化的胞嘧啶碱基保持不变。所述的转化剂无特别限制，现有技术中报道的可实现胞嘧啶到尿嘧啶转化的试剂均可以，如肼盐、重亚硫酸氢盐和亚硫酸氢盐(例如偏亚硫酸氢钠、亚硫酸氢钾、亚硫酸氢铯、亚硫酸氢铵等)中的一种或几种。又如扩增基因中常用的DNA聚合酶、dNTPs、Mg 2+离子和缓冲液等等。
在一些实施方案中，所述的试剂或试剂盒还包括内参基因的检测试剂。
在一些实施方案中，所述的内参基因为β-actin。
在一些实施方案中，所述的内参基因的检测试剂为针对内参基因的引物和探针。
在一些实施方案中，所述的内参基因的检测试剂为SEQ ID NO：13和SEQ ID NO：14所示的引物对和SEQ ID NO：15所示的探针。
本公开提供了HOXB4和SCRIN1基因的甲基化联合检测试剂在制备肺癌检测试剂或者试剂盒中的应用。
本公开提供了一种引物，所述引物选自与如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：22所示序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列，或它们互补序列中的至少任意一条。
本公开提供了一种引物，所述引物选自与如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：5所示序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列，或它们互补序列中的至少任意一条。
在一些实施方案中，所述引物选自SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：5 所示的至少一对引物对
在一些实施方案，所述引物选自SEQ ID NO：1和SEQ ID NO：2、SEQ ID NO：4和SEQ ID NO：5所示的引物对。
所述的引物用于扩增所述核酸片段。本领域公知，引物的成功设计对于PCR是至关重要。相对于一般的PCR，在甲基化检测中，引物的设计影响更为关键，这是由于甲硫化反应促使DNA链中的“C”转化为“U”，导致GC含量降低，使PCR反应后在序列中出现长的连续“T”，容易引起DNA链的断裂，导致很难选择具有合适的Tm值及稳定的引物；另一方面，为了区别硫化处理和没有硫化处理以及未完全处理的DNA，需要引物有足够数量的“C”，这些都增加了选择稳定引物的困难。因此，DNA甲基化检测中，引物所针对的扩增片段的选择，如扩增片段长短和位置，以及引物的选择等等都对检测的灵敏度和特异性产生影响。发明人经实验也发现，不同的扩增目的片段和引物对检测效果有所区别。很多时候，发现了某些基因或核酸片段在肿瘤和非肿瘤中具有表达差异，然而其距离转化为肿瘤的标志物，应用到临床中，仍存在很长的距离。其中最主要的原因是因为检测试剂的限制，导致该潜在肿瘤标志物的检测灵敏度和特异性难以满足检测需求，或者检测方法操作复杂、成本高，难以在临床中大规模应用。
另一方面，本公开还提供了一种核酸探针，所述核酸探针选自与如SEQ ID NO：3、SEQ ID NO：6、SEQ ID NO.:18和SEQ ID NO.:21所示序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列，或它们互补序列中的至少任意一条。
作为本公开优选的实施方式，所述核酸探针选自SEQ ID NO：3和SEQ ID NO：6所示的序列。
在一些实施方案中，本公开提供的试剂盒包括：第一容器，其包含用于扩增的引物对；第二容器，其包含探针。
在一些实施方案中，所述试剂盒还包含说明书。
在一些实施方案中，所述试剂盒还包含核酸提取试剂。
在一些实施方案中，所述试剂盒还包含取样装置。
本公开还提供了上述的甲基化检测试剂、试剂盒、引物、探针在制备甲基化检测的试剂或试剂盒，或者制备检测肺癌的试剂或试剂盒中的应用。
本公开还提供了上述的甲基化检测试剂、试剂盒、引物、探针在甲基化检测中的应用，或者在检测肺癌中的应用。
本公开还提供了一种肺癌的检测系统，所述的系统包含有以下构件：
(1)HOXB4和SRCIN1基因的甲基化联合检测构件：
(2)数据处理构件；
(3)结果输出构件。
在一些实施方案中，所述的甲基化检测构件含有甲基化检测仪器。
在一些实施方案中，所述的甲基化检测构件还含有所述的甲基化联合检测试剂、试剂盒、引物、探针。
在一些实施方案中，所述的甲基化检测仪器包含荧光定量PCR仪、PCR仪、测序仪中的一种或多种。
在一些实施方案中，所述的数据处理构件含有数据处理机器。
所述的数据处理机器包括本领域技术人员可使用的任何可以进行数据处理的设备或仪器或装置。
在一些实施方案中，所述的数据处理机器包含计算器、计算机中的一种或多种。
所述的计算机中附载有本领域技术人员可使用的任何可以进行数据处理或统计分析的软件或程序。
在一些实施方案中，所述的计算机包含附载有SPSS、SAS、Excel中一种或多种软件的计算机。
在一些实施方案中，所述的结果输出构件含有结果输出器。
所述的输出器包含任何可以将数据处理结果显示为可阅读的内容的设备或仪器或装置。
在一些实施方案中，所述的甲基化检测构件还含有所述的多基因的甲基化联合检测试剂。
在一些实施方案中，所述的结果输出器包含屏幕、纸质报告中的一种或多种。
在一些实施方案中，所述的数据处理器被配置于a.接收待测样本以及正常对照样本的测试数据；b.储存待测样本以及正常对照样本的测试数据；c.比对同种类型的待测样本以及正常对照样本的测试数据；d.根据比对结果，响应于测试者罹患肺癌的概率或者可能性。
在一些实施方案中，所述的结果输出构件用于输出测试者罹患肺癌的概率或者可能性。
在一些实施方案中，数据处理构件的判断标准为：根据界值判断肺癌标本和正常标本。
在一些实施方案中，本公开的HOXB4与SRCIN1联合检测，还可以通过多重PCR的方式实现。
在一些实施方案中，根据目标基因即HOXB4、SRCIN1的Cp值和/或△Cp值(△Cp值＝Cp 靶向基因-Cp 内参 基因)来判断标本的甲基化水平。
在一些实施方案中，只要其中一个基因在所述组织标本中的△Cp值小于所述△Cp值的界值则判断为肺癌标本，只有两个基因在所述组织标本的△Cp值同时大于等于所述△Cp值的界值则判断为正常标本。
在一些实施方案中，标本中的Cp值的界值取值范围为35～39，△Cp值的界值取值范围为4～12。
在一些实施方案中，HOXB4与SRCIN1联合检测，在组织标本中，HOXB4的△Cp值的界值为5.4，SRCIN1的△Cp值的界值6.5。
在一些实施方案中，HOXB4与SRCIN1联合检测，在痰液标本中，HOXB4与SRCIN1的阈值线Cp值分别为：36.9和37.0。当单个基因检测结果中的一项小于前述阈值，则可判定为阳性(即肺癌标本)，若检测结果中2项结果均大于或等于相对应的阈值，则可判定为阴性(即正常标本)。
在一些实施方案中，HOXB4与SRCIN1多重PCR检测，在痰液标本中的Cp值的界值为36.7，在灌洗液标本中的Cp值的界值为37.2和△Cp值的界值为9。
在一些实施方案中，HOXB4与SRCIN1多重PCR检测，在所述痰液标本的Cp值小于所述Cp值的界值则判断为肺癌标本，所述痰液标本的Cp值大于等于所述Cp值的界值则判断为正常标本。
在一些实施方案中，HOXB4与SRCIN1多重PCR检测，在所述灌洗液标本的Cp值和△Cp值任何一个数值小于所述Cp值和△Cp的界值则判断为肺癌标本，所述灌洗液标本的Cp值和△Cp值均大于等于所述Cp值和△Cp值的界值则判断为正常标本。
在一些实施方案中，所述肿瘤为肺癌。
在一些实施方案中，所述肿瘤为小细胞肺癌和非小细胞肺癌。
在一些实施方案中，所述非小细胞肺癌选自鳞状细胞癌、腺癌。
在一些实施方案中，所述针对的待测样本或者样本类型选自肺泡灌洗液、组织、胸水、痰液、血液、血清、血浆、尿液、前列腺液或粪便中的至少一种。
在一些实施方案中，本公开所述的样本选自肺泡灌洗液、组织、痰液中的至少一种。
在一些实施方案中，本公开所述的样本选自肺泡灌洗液或痰液中的至少一种。
本公开还提供了一种肺癌的诊断方法，所述方法包括以下步骤：
(1)检测来源于受试者的待测样本HOXB4和SRCIN1基因的甲基化水平；
(2)将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及
(3)基于待测样本与正常对照样本的甲基化水平的偏离，诊断肺癌。
在一些实施方案中，本公开提供了一种肺癌的诊断方法，所述方法包括以下步骤：(1)检测来源于受试者的待测样本HOXB4和SRCIN1基因的甲基化水平；所述检测包括将受试者待测样本与检测HOXB4和SRCIN1基因的甲基化水平的检测试剂接触；(2)将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及(3)基于待测样本与正常对照样本的甲基化水平的偏离，诊断肺癌。
在一些实施方案中，本公开提供了一种肺癌的诊断方法，所述方法包括以下步骤：将基因的甲基化检测试剂加入来自受试者的待测样本中，检测该待测样本中HOXB4和SRCIN1基因的甲基化水平；将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及基于待测样本与正常对照样本的甲基化水平 的偏离，诊断肺癌。
在一些实施方案中，所述步骤(3)中所述的偏离，是指HOXB4和SRCIN1这2个基因中任意一个的甲基化水平的偏离。
在一些实施方案中，所述步骤(1)中，所述检测包括将受试者的待测样本与HOXB4和SRCIN1基因的甲基化水平的检测试剂接触。
在一些实施方案中，采用甲基化特异性定量PCR(qMSP)检测HOXB4和SRCIN1基因的甲基化水平。
在一些实施方案中，通过结果比较待测样本与正常样本的甲基化结果，当待测样本与正常样本的甲基化具有显著差异或极显著差异时，结果判断待测样本患病风险高。
本公开的诊断方法可以在肺癌治疗前后使用或者与肺癌治疗联合使用，治疗后使用如评价治疗的成功或者监测治疗后肺癌的缓解、复发和/或进展(包括转移)。
一方面，还提供了一种肺癌的治疗方法，所述方法包括以下步骤：
(1)检测来源于受试者的待测样本HOXB4和SRCIN1基因的甲基化水平；
(2)将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及
(3)基于待测样本与正常对照样本的甲基化水平的偏离，诊断肺癌；
(4)向被诊断为肺癌的受试者施用抗肺癌的药物。
本公开另一方面提供了一种肺癌的治疗方法，所述方法包括对经上述诊断方法诊断为肺癌的患者，施用手术、化疗、放疗、放化疗、免疫疗法、溶瘤病毒疗法、或其他本领域所用的任何其他类型肺癌治疗方法以及这些治疗方法的组合。
本公开通过研究发现：在一些具体的实施方案中，通过检测HOXB4和SRCIN1基因组合，可以很好的从样本中区分出肺癌样本。对肺癌的检测敏感性和特异性极高。
附图说明
图1为不同标志物组合在组织样本中检测的ROC曲线；
图2为不同标志物组合在痰液样本中检测的ROC曲线；
图3为HOXB4与SRCIN1联合检测灌洗液标本中检测的扩增曲线。
具体实施方式
以下通过具体的实施例进一步说明本公开的技术方案，具体实施例不代表对本公开保护范围的限制。其他人根据本公开理念所做出的一些非本质的修改和调整仍属于本公开的保护范围。
本公开中的“引物”或“探针”是指一种寡核苷酸，其包含与靶分子(例如靶核酸片段)的至少6个连续核苷酸的序列互补的区域。在一些实施方案中，所述引物或探针至少一部分序列与扩增的序列不互补。在一些实施方案中，引物或探针包含与靶分子的至少9、至少10、至少11、至少12、至少13、至少14、至少15、至少16、至少17、至少18、至少19或至少20连续核苷酸的序列互补的区域。当引物或探针包含“与靶分子的至少x个连续核苷酸互补”的区域时，所述引物或探针与靶分子的至少x个连续或不连续的分块核苷酸至少95％互补。在一些实施方案中，引物或探针与靶分子至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、96％、至少97％、至少98％、至少99％或100％互补。
本公开中，“正常”样本指分离自已知无所述癌症或肿瘤的个体的相同类型的样本。
本公开甲基化检测的样本包括但不限于DNA，或RNA，或含mRNA的DNA和RNA样品、或DNA-RNA杂交体。其中DNA或者RNA可为单链或双链。
本公开中，所述“受试者”是哺乳动物，例如是人。
本公开中，“甲基化水平”同“甲基化程度”，通常可以表示为甲基化胞嘧啶的百分比，其为甲基化的胞嘧啶数量除以甲基化胞嘧啶的数量与未甲基化胞嘧啶数量的总和；以及目前普遍采用甲基化靶向基因数量除 以内参基因数量的方法来表示甲基化水平；以及其他现有技术中甲基化水平表示方法。
本公开中“样本”同“标本”。
如本公开所使用的术语“和/或”是指并且涵盖一个或多个相关联的所列项目的任何和所有可能的组合。当在两个或多个项目的列表中使用时，术语“和/或”表示所列出的项目中的任何一个可以单独使用，或者可以使用两个或多个所列出的项目的任何组合。例如，如果组合物，组合，构造等被描述为包括(或包含)组分A，B，C和/或D，则该组合物可以单独包含A；单独包含B；单独包含C；单独包含D；包含A和B的组合；包含A和C的组合；包含A和D的组合；包含B和C的组合；包含B和D的组合；包含C和D的组合；包含A，B和C的组合；包含A，B和D组合；包含A，C和D的组合；包含B，C和D组合；或A，B，C和D组合使用。
实施例1
发明人筛选了数百个基因，在组织样本中进行筛选，以β-actin基因作为内参基因，比较HOXB4、SRCIN1、PCDHGA12、HOXD8基因的两两联合检测结果，各基因检测引物探针如下：
HOXB4的检测引物和探针为：
SEQ ID NO：1 HOXB4-F1引物F：TTCGTCGTTTTCGTTATCATTC
SEQ ID NO：2 HOXB4-R1引物R：TACTAACCGCCTCGCTAC
SEQ ID NO：3 HOXB4-P1探针P：FAM-CGGGTTTTTGCGTCGTTATTCGTC-BQ1
SRCIN1的检测引物和探针为：
SEQ ID NO：4 SRCIN1引物F：TCGTGTGTCGTCGTTCAGAC
SEQ ID NO：5 SRCIN1引物R：GAAATACCCGCGAAAATACTG
SEQ ID NO：6 SRCIN1探针P：
FAM-AGTTTTACGTTGGAGAAGCGTCGG-BQ1
PCDHGA12的检测引物和探针为：
SEQ ID NO：7 PCDHGA12引物F：TTGGTTTTTACGGTTTTCGAC
SEQ ID NO：8 PCDHGA12引物R：AAATTCTCCGAAACGCTCG
SEQ ID NO：9 PCDHGA12探针P：
FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1
HOXD8的检测引物和探针为：
SEQ ID NO：10 HOXD8引物F：TTAGTTTCGGCGCGTAGC
SEQ ID NO：11 HOXD8引物R：CCTAAAACCGACGCGATCTA
SEQ ID NO：12 HOXD8探针P：FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
β-actin的检测引物和探针为：
SEQ ID NO：13 β-actin引物F：GGAGGTTTAGTAAGTTTTTTGGATT
SEQ ID NO：14 β-actin引物R：CAATAAAACCTACTCCTCCCTTA
SEQ ID NO：15 β-actin探针P：FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
实验过程：
1、提取DNA
收集确诊肺癌患者的标本和非肺癌患者的标本，分别包括石蜡组织标本、痰液标本、灌洗液标本。样品经过预处理及分离细胞后，按美基生物公司试剂盒HiPure FFPE DNA Kit(D3126-03)说明书进行DNA提取。
2、DNA修饰
以ZYMO RESEARCH生物公司试剂盒EZ DNA Methylation TM KIT(D5002)说明进行重亚硫酸氢盐修饰。
3、扩增与检测
表1 配液体系
  HOXB4 SRCIN1 PCDHGA12 HOXD8 β-actin
反应组份 加入量(μl) 加入量(μl) 加入量(μl) 加入量(μl) 加入量(μl)
上游引物(100μM) 0.125 0.125 0.125 0.05 0.125
下游引物(100μM) 0.125 0.125 0.125 0.125 0.125
探针(100μM) 0.05 0.05 0.05 0.05 0.05
镁离子(25mM) 6 6 6 6 6
dNTPs(10mM) 1 1 1 1 1
Taq聚合酶(5unit/μl) 0.5 0.5 0.5 0.5 0.5
5X缓冲液 6 6 6 6 6
灭菌水 11.2 11.2 11.2 11.275 11.2
模板DNA 5 5 5 5 5
总体积 30 30 30 30 30
扩增体系：各检测基因的扩增体系见表2、表3。
表2 HOXB4、SRCIN1和β-actin扩增体系
Figure PCTCN2020118998-appb-000002
表3 PCDHGA12和HOXD8的扩增体系
Figure PCTCN2020118998-appb-000003
4、检测结果
样本信息：肺组织样本共计169例，其中正常组织样本91例，癌组织样本78例，78例癌症组样本中有鳞癌27例，腺癌38例，小细胞癌3例，大细胞癌4例，复合型癌1例，未明确分类的肺癌5例，其中癌和癌旁对照样本77对。
以ACTB作为内参基因，根据靶向基因即HOXB4、SRCIN1的△Cp值(△Cp值＝Cp 靶向基因-Cp ACTB)来判断标本的甲基化水平，HOXB4与SRCIN1的阈值线分别为：△Cp值＝5.4，△Cp值＝6.5。当检测结果中的一项小于以上阈值，则可判定为阳性，若检测结果中2项结果均大于或等于相对应的阈值，则可判定为阴性。
PCDHGA12的阈值线为Cp值＝25.9，HOXD8的阈值线为Cp值＝27.4，当各标志物检测结果大于或等于对应的阈值线时则可判定为阴性；若标志物检测结果小于对应的阈值线则可判定为阳性。
HOXB4与SRCIN1联合、HOXB4与PCDHGA12联合、HOXB4与HOXD8联合在所有组织标本中检测的ROC曲线图1所示。各基因在组织中检测的统计结果表3所示。
表3 组织中的检测结果
Figure PCTCN2020118998-appb-000004
表3(续)
Figure PCTCN2020118998-appb-000005
从以上结果可以看出，在组织样本中HOXB4与SRCIN1联合检测，正常组和全部癌症组比较，特异性为97.8％，灵敏度89.7％，与另外两组联合检测相比，HOXB4与SRCIN1联合检测在特异性一致的情况下有更高的灵敏度。此外，HOXB4与SRCIN1联合检测相较于HOXB4、SRCIN1单独检测，非显而易见地，显著提升了灵敏度。
根据以上结果，HOXB4和SRCIN1在组织样本中，在高特异性下，仍然具有较高的灵敏度。特别是通过联合检测，在基本不影响特异性的情况下，灵敏度有非常大的提升。痰液作为无创性的检测样本，在肺癌诊断上更具重要意义，为此，发明人对HOXB4和SRCIN1这2个标志物在痰液中进行检测。
实施例2：HOXB4和SRCIN1基因在痰液中的检测
样本信息：测试痰液样本共计107例，其中正常对照组样本51例，癌症组样本56例，56例癌症组样本中有鳞癌20例，小细胞癌8例，腺癌20例，大细胞癌1例，巨细胞癌1例，未明确分类的肺癌6例。
试验过程：
a.收集确诊为肺癌患者和非肺癌患者的痰液标本，使用NaOH解稠后，离心取沉淀分离细胞，使用PBS洗涤2遍，然后使用美基生物(Magen)公司的DNA提取试剂盒(HiPure FFPE DNA Kit，D3126-03)提取DNA。
b.使用ZYMO RESEARCH生物公司的DNA转化试剂盒(EZ DNA Methylation Kit，D5002)进行DNA的重亚硫酸氢盐修饰。
c.各基因标志物的引物探针序列、配液体系及扩增体系与实施例1相同。
d.根据靶向基因即HOXB4、SRCIN1的Cp值来判断标本的甲基化水平，HOXB4与SRCIN1的阈值线Cp值分别为：36.9和37.0。当单个基因检测结果中的一项小于前述阈值，则可判定为阳性，若检测结果中2项结果均大于或等于相对应的阈值，则可判定为阴性。
PCDHGA12、HOXD8：PCDHGA12的阈值线为Cp值＝23.48，HOXD8的阈值线为Cp值＝26.4，当各标志物检测结果大于或等于对应的阈值线时则可判定为阴性；若标志物检测结果小于对应的阈值线则可判定为阳性。
e.检测结果如下：
表4 痰液中的检测结果
Figure PCTCN2020118998-appb-000006
表4(续)
Figure PCTCN2020118998-appb-000007
HOXB4和SRCIN1在痰液标本中检测的ROC曲线见图2，统计结果见表4，从以上结果可以看出，在痰液样本中，HOXB4和SRCIN1联合检测时，正常组和全部癌症组比较，对肺癌的灵敏度提达到76.8％；正常组和全部小细胞癌组比较，灵敏度能达到100％。相对于单个基因标志物，HOXB4的检出率为64.3％，SRCIN1的检出率为48.2％，二者联合检测时，对肺癌的灵敏度提升到76.8％，二者具有协同性。
实施例3：HOXB4和SRCIN1基因多重PCR体系优化
基于以上结果显示HOXB4和SRCIN1基因的联合检测可明显提高肺癌的检出率，本发明人通过多重PCR方式对PCR体系进行优化，从而简化检测流程，并在实施例2的样本基础上进行验证。各基因检测引物探针如下：
HOXB4、SRCIN1和β-actin的检测引物和探针序列与实施例1相同。
a.痰液样本处理同实施例2；
b.配液体系如下：
表5 配液体系
反应组份 加入量(μl)
HOXB4-F1(100μM) 0.125
HOXB4-R1(100μM) 0.125
HOXB4-P1(100μM) 0.05
SRCIN1-F1(100μM) 0.125
SRCIN1-R1(100μM) 0.125
SRCIN1-P1(100μM) 0.05
β-actin-F1(100μM) 0.125
β-actin-R1(100μM) 0.125
β-actin-P2(100μM) 0.05
镁离子(25mM) 6
dNTPs(10mM) 1
Taq聚合酶(5unit/μl) 0.5
5X缓冲液 6
灭菌水 10.6
模板DNA 5
总体积 30
c.扩增体系与实施例1表2的扩增体系相同；
d.根据靶向基因即HOXB4、SRCIN1多重PCR的Cp值来判断标本的甲基化水平，HOXB4与SRCIN1多重PCR检测的阈值线Cp值为：36.7。当单个基因检测结果中的一项小于前述阈值，则可判定为阳性，若检测结果大于或等于阈值，则可判定为阴性。
e.检测结果如下表6：
表6 检测结果
Figure PCTCN2020118998-appb-000008
结果显示HOXB4与SRCIN1多重PCR体系的检测结果与实施例2中的检测结果基本保持一致。表明该多重PCR体系检测结果可作为HOXB4与SRCIN1基因联合检测肺癌的结果判定。
实施例4：HOXB4和SRCIN1基因在灌洗液液中的检测
样本信息：测试肺泡灌洗液样本共计387例，其中正常对照组样本303例，癌症组对照样本84例，84例癌症组样本中有鳞癌21例，腺癌40例，小细胞癌10例，未明确肺癌类型13例。
HOXB4、SRCIN1和β-actin的检测引物和探针序列与实施例1相同。
试验过程：
a.收集确诊为肺癌患者和非肺癌患者的肺泡灌洗液标本，离心分离细胞，然后使用美基生物公司的DNA提取试剂盒(HiPure FFPE DNA Kit，D3126-03)提取DNA。
b.使用ZYMO RESEARCH生物公司的DNA转化试剂盒(EZ DNA Methylation Kit，D5002)进行DNA的重亚硫酸氢盐修饰。
c.配液体系和扩增体系与实施例3相同。
d.检测结果如下：
以ACTB作为内参基因，根据靶向基因即HOXB4、SRCIN1的Cp值和△Cp值(△Cp值＝Cp 靶向基因-Cp ACTB)来判断标本的甲基化水平，HOXB4与SRCIN1的阈值线为：Cp值＝37.2，△Cp值＝9。当检测结果中的一项小于以上阈值，则可判定为阳性，若检测结果中2项结果均大于或等于阈值，则可判定为阴性。387例灌洗液标本的检测结果如下：
表7 检测结果
Figure PCTCN2020118998-appb-000009
HOXB4与SRCIN1联合检测在所有灌洗液标本中检测的扩增曲线见图3，统计结果见表7。从以上结果可以看出，HOXB4与SRCIN1联合检测在96.0％的高特异性下，灵敏度达到77.4％；按照肺癌的亚型进行比较分析，鳞癌组HOXB4与SRCIN1联合检测的检出率为71.4％。特别是对腺癌的检测效果，HOXB4与SRCIN1联合检测的灵敏性高达到75.0％，这一突破对腺癌的检测具有重大的意义。因为腺癌一般为周围型，由于支气管的树状生理结构，肺泡灌洗液不容易接触到肺深部的肺泡或者癌组织。
实施例5 不同标志物组合在痰液样本的检测结果
发明人同时比较了不同标志物组合在痰液样本中的检测情况，对比的组别如下：
组合1：HOXB4+SRCIN1
组合2：HOXB4+PCDHGA12
组合3：SRCIN1+PCDHGA12
组合4：HOXB4+HOXD8
组合5：SRCIN1+HOXD8
组合6：SRCIN1+PCDHGA12+HOXD8
组合7：SRCIN1+HOXB4+HOXD8
具体实验条件及操作同实施例2。
在107例痰液样本对不同标志物组合进行检测，其中正常对照组样本51例，癌症组样本56例，各组合的检测结果如表8：
表8(正常组vs.全部癌症组)
  组合1 组合2 组合3 组合4 组合5 组合6 组合7
灵敏度 76.8％ 64.3％ 46.4％ 66.1％ 53.6％ 53.6％ 76.8％
特异性 92.2％ 92.2％ 92.2％ 94.1％ 94.1％ 90.2％ 92.2％
AUC 0.870 0.810 0.848 0.833 0.883 0.832 0.892
表8的结果表明不同标志物组合对痰液样本肺癌检出率有较大的影响。分析组合1、组合2、组合3、组合4和组合5的结果显示，不同标志物两两组合的检测结果差异较大，而组合1中通过HOXB4与SRCIN1联合检测其特异性为92.2％，灵敏度为76.8％，综合检测性能远远高于其他4个组合。分析组合3、组合5、组合6或组合1、组合7，结果显示额外增加标志物并不一定能提高检出率，反而有可能降低其特异性。结果表明，并非任意的基因标志物组合能够如本公开的组合1一样，在灵敏度、特异性及AUC这3个指标上有综合最佳的效果。非显而易见地，增加目标基因标志物的数量进行联合检测(如组合6，7)，也并未相应地提升特异性和灵敏度。
实施例6 引物探针组合的选择
引物和探针也对肿瘤标志物的检测效果有极大的影响，发明人在研究过程中，设计了多对引物及其对应的探针，以寻找到尽可能提高检测灵敏度和特异性的探针和引物，以使本发明的检测试剂能够实际应用到临床检测中。
在40例痰液样本对不同引物探针组合进行检测，其中正常对照组样本15例，癌症组对照样本25例。
表9 引物和探针
名称 序列编号 序列 作用
HOXB4-F1 SEQ ID NO：1 TTCGTCGTTTTCGTTATCATTC HOXB4上游引物
HOXB4-R1 SEQ ID NO：2 TACTAACCGCCTCGCTAC HOXB4下游引物
HOXB4-P1 SEQ ID NO：3 FAM-CGGGTTTTTGCGTCGTTATTCGTC-BQ1 HOXB4检测探针
HOXB4-F2 SEQ ID NO：16 ATTCGTTCGGGTATTACGTC HOXB4上游引物
HOXB4-R2 SEQ ID NO：17 CCAAAATCCCGACAAACCG HOXB4下游引物
HOXB4-P2 SEQ ID NO：18 FAM-CGGTTAGAGGCGAGAGAGTAGTTT-BQ1 HOXB4检测探针
HOXB4-F3 SEQ ID NO：19 CGGGTTTCGGGCGGCGCGC HOXB4上游引物
HOXB4-R3 SEQ ID NO：20 CGAACGATAACGAAAACGACG HOXB4下游引物
HOXB4-P3 SEQ ID NO：21 FAM-CGTGTATCGTGTAGCGTTACGCGG-BQ1 HOXB4检测探针
SRCIN1-F1 SEQ ID NO：4 TCGTGTGTCGTCGTTCAGAC SRCIN1上游引物
SRCIN1-R1 SEQ ID NO：5 GAAATACCCGCGAAAATACTG SRCIN1下游引物
SRCIN1-P1 SEQ ID NO：6 AGTTTTACGTTGGAGAAGCGTCGG SRCIN1检测探针
SRCIN1-F2 SEQ ID NO：22 TATCGTGTATCGTCGTTCGGAC SRCIN1上游引物
SRCIN1-R1 SEQ ID NO：5 GAAATACCCGCGAAAATACTG SRCIN1下游引物
SRCIN1-P1 SEQ ID NO：6 AGTTTTACGTTGGAGAAGCGTCGG SRCIN1检测探针
A3-TqMF SEQ ID NO：13 GGAGGTTTAGTAAGTTTTTTGGATT β-actin基因上游引物
A3-TqMR SEQ ID NO：14 CAATAAAACCTACTCCTCCCTTA β-actin基因下游引物
A3-TqP SEQ ID NO：15 FAM-TTGTGTGTTGGGTGGTGGTT-BQ1 β-actin基因检测探针
各配液体系均一致，配液体系、各扩增程序均一致，扩增程序同实施例2。
检测结果如表10、11所示。
表10 HOXB4在痰液样本中的检测结果(正常组vs.全部癌症组)
组别 特异性 灵敏性
F1，R1，P1 93.3％ 72.0％
F2，R2，P2 93.3％ 44.0％
F3，R3，P3 93.3％ 68.0％
表11 SRCIN1在痰液样本中的检测结果(正常组vs.全部癌症组)
组别 特异性 灵敏性
F1，R1，P1 93.3％ 56.0％
F2，R1，P1 93.3％ 52.0％
结果表明针对同一区域的不同引物对，对检测结果会产生影响。在特异性一致的情况下，HOXB-F1，HOXB-R1，HOXB-P1和SRCIN1-F1，SRCIN1-R1，SRCIN1-P1的引物和探针组合有更高的灵敏性。

Claims (12)

  1. 一种基因标志物组合，其特征在于，所述基因标志物包括HOXB4和SRCIN1。
  2. 多基因的甲基化联合检测试剂在制备肺癌检测试剂或者试剂盒中的应用，其特征在于，所述基因包括HOXB4和SRCIN1。
  3. 一种多基因甲基化联合检测试剂/试剂盒，包括HOXB4和SRCIN1基因的甲基化检测的试剂。
  4. 如权利要求2所述应用或权利要求3所述的试剂，其特征在于，所述多基因甲基化联合检测试剂，包括每个基因的甲基化检测的引物和/或探针；
    可选地，包括针对每个基因的CpG岛获得的引物和/或探针；
    可选地，包括针对每个基因的基因体、基因间区、启动子区或所述启动子区附近区域的CpG岛获得的引物和/或探针。
  5. 如权利要求2所述应用或权利要求3所述的试剂，其特征在于，所述HOXB4基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    I、与如SEQ ID NO:1、SEQ ID NO：16和SEQ ID NO：19所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    II、如I所示序列的互补序列；和/或
    所述HOXB4基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    III、与如SEQ ID NO:2、SEQ ID NO：17和SEQ ID NO：20所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    IV、如III所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    V、与如SEQ ID NO:4和SEQ ID NO:22所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    VI、如V所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    VII、与如SEQ ID NO:5所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    VIII、如VII所示序列的互补序列；
    可选的，所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:1和SEQ ID NO.:2所示；
    可选地，所述SRCIN1基因的甲基化检测的引物对如SEQ ID NO.:4和SEQ ID NO.:5所示；
    可选地，所述HOXB4基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    IX、与如SEQ ID NO.:3、SEQ ID NO.:18和SEQ ID NO.:21所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    X、如IX所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    XI、与如SEQ ID NO.:6所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    XII、如XI所示序列的互补序列。
  6. 根据权利要求1所述的基因标志物组合、或权利要求2所述应用或权利要求3所述的试剂，其特征在于：所述的肺癌选自小细胞肺癌和非小细胞肺癌；更优选地，所述的非小细胞肺癌选自鳞状细胞癌、腺癌。
  7. 根据权利要求1所述的基因标志物组合、或权利要求2所述应用或权利要求3所述的试剂，其特征在于：所述检测试剂所针对的待测样本选自肺泡灌洗液、组织、胸水、痰液、血液、血清、血浆、尿液、前列腺液或粪便中的至少一种；
    优选地，所述样品选自肺泡灌洗液、组织、痰液中的至少一种；
    更优选地，所述样品选自肺泡灌洗液或痰液中的至少一种。
  8. 一种肺癌的检测系统，其特征在于，所述的系统包含有以下构件；
    (1)HOXB4和SRCIN1基因的甲基化联合检测构件：
    (2)数据处理构件；
    (3)结果输出构件；
    优选地，所述的甲基化检测构件含有甲基化检测仪器；
    优选地，所述的甲基化检测仪器包含荧光定量PCR仪、PCR仪、测序仪中的一种或多种；
    优选地，所述的数据处理构件含有数据处理机器；
    优选地，所述的数据处理机器包含计算器、计算机中的一种或多种；
    优选地，所述的计算机包含附载有SPSS、SAS、Excel中一种或多种软件的计算机；
    优选地，所述的结果输出构件含有结果输出器；
    优选地，所述的结果输出器包含屏幕、纸质报告中的一种或多种；
    优选地，所述的甲基化检测构件还含有如权利要求3-5任一所述的多基因的甲基化联合检测试剂；
    优选地，所述的数据处理构件被配置于a.接收待测样本以及正常对照样本的测试数据；b.储存待测样本以及正常对照样本的测试数据；c.比对同种同种类型的待测样本以及正常对照样本的测试数据；d.根据比对结果，响应于测试者罹患肺癌的概率或者可能性；
    优选地，所述的结果输出构件用于输出测试者罹患肺癌的概率或者可能性；
    优选地，所述数据处理构件的判断标准为：通过结果比较待测样本与正常样本的甲基化结果，当待测样本与正常样本的甲基化具有显著差异或极显著差异时，结果判断待测样本患病风险高。
  9. 如权利要求7所述的肺癌的检测系统，其特征在于，所述的肺癌选自小细胞肺癌和非小细胞肺癌；更优选地，所述的非小细胞肺癌选自鳞状细胞癌、腺癌。
  10. 如权利要求7所述的肺癌的检测系统，其特征在于，所述检测系统所针对的待测样本选自肺泡灌洗液、组织、胸水、痰液、血液、血清、血浆、尿液、前列腺液或粪便中的至少一种；
    优选地，所述样本选自肺泡灌洗液、组织、痰液中的至少一种；
    更优选地，所述样本选自肺泡灌洗液或痰液中的至少一种。
  11. 一种肺癌的诊断方法，所述方法包括以下步骤：
    (1)检测来源于受试者的待测样本HOXB4和SRCIN1基因的甲基化水平；
    (2)将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及
    (3)基于待测样本与正常对照样本的甲基化水平的偏离，诊断肺癌；
    可选地，采用甲基化特异性定量PCR(qMSP)检测HOXB4和SRCIN1基因的甲基化水平；
    可选地，所述步骤(1)中，所述检测包括将受试者的待测样本与HOXB4和SRCIN1基因的甲基化水平的检测试剂接触；
    可选地，通过结果比较待测样本与正常样本的甲基化结果，当待测样本与正常样本的甲基化具有显著差异或极显著差异时，结果判断待测样本患病风险高；
    可选地，所述的待测样品选自肺泡灌洗液、组织、胸水、痰液、血液、血清、血浆、尿液、前列腺液或粪便中的至少一种；
    可选地，所述样品选自肺泡灌洗液、痰液、组织中的至少一种；
    可选地，所述样品选自肺泡灌洗液或痰液中的至少一种；
    可选地，所述的肺癌选自小细胞肺癌和非小细胞肺癌；
    可选地，所述的非小细胞肺癌选自鳞状细胞癌、腺癌；
    可选地，所述步骤(1)中用多基因甲基化联合检测试剂检测HOXB4和SRCIN1基因的甲基化水平，所述联合检测试剂包括每个基因的甲基化检测的引物和/或探针；
    可选地，包括针对每个基因的CpG岛获得的引物和/或探针；
    可选地，包括针对每个基因的基因体、基因间区、启动子区或所述启动子区附近区域的CpG岛获得的引物和/或探针；
    可选地，所述HOXB4基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    I、与如SEQ ID NO:1、SEQ ID NO：16和SEQ ID NO：19所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    II、如I所示序列的互补序列；和/或
    所述HOXB4基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    III、与如SEQ ID NO:2、SEQ ID NO：17和SEQ ID NO：20所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    IV、如III所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    V、与如SEQ ID NO:4和SEQ ID NO:22所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    VI、如V所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    VII、与如SEQ ID NO:5所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同 一性的核苷酸序列；及
    VIII、如VII所示序列的互补序列；
    可选的，所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:1和SEQ ID NO.:2所示；
    可选地，所述SRCIN1基因的甲基化检测的引物对如SEQ ID NO.:4和SEQ ID NO.:5所示；
    可选地，所述HOXB4基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    IX、与如SEQ ID NO.:3、SEQ ID NO.:18和SEQ ID NO.:21所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    X、如IX所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    XI、与如SEQ ID NO.:6所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    XII、如XI所示序列的互补序列。
  12. 一种肺癌的治疗方法，所述方法包括以下步骤：
    (1)检测来源于受试者的待测样本HOXB4和SRCIN1基因的甲基化水平；
    (2)将待测样本与正常对照样本的HOXB4和SRCIN1基因甲基化水平比较；及
    (3)基于待测样本与正常对照样本的甲基化水平的偏离，诊断肺癌；
    (4)向被诊断为肺癌的受试者施用抗肺癌的药物；
    可选地，采用甲基化特异性定量PCR(qMSP)检测HOXB4和SRCIN1基因的甲基化水平；
    可选地，通过结果比较待测样本与正常样本的甲基化结果，当待测样本与正常样本的甲基化具有显著差异或极显著差异时，结果判断待测样本患病风险高；
    可选地，采用甲基化特异性定量PCR检测基因的甲基化水平；
    可选地，所述的待测样品选自肺泡灌洗液、组织、胸水、痰液、血液、血清、血浆、尿液、前列腺液或粪便中的至少一种；
    可选地，所述样品选自肺泡灌洗液、痰液、组织中的至少一种；
    可选地，所述样品选自肺泡灌洗液或痰液中的至少一种；
    可选地，所述的肺癌选自小细胞肺癌和非小细胞肺癌；
    可选地，所述的非小细胞肺癌选自鳞状细胞癌、腺癌；
    可选地，所述步骤(1)中用多基因甲基化联合检测试剂检测HOXB4和SRCIN1基因的甲基化水平，所述联合检测试剂包括每个基因的甲基化检测的引物和/或探针；
    可选地，包括针对每个基因的CpG岛获得的引物和/或探针；
    可选地，包括针对每个基因的基因体、基因间区、启动子区或所述启动子区附近区域的CpG岛获得的引物和/或探针；
    可选地，所述HOXB4基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    I、与如SEQ ID NO:1、SEQ ID NO：16和SEQ ID NO：19所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    II、如I所示序列的互补序列；和/或
    所述HOXB4基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意 一项：
    III、与如SEQ ID NO:2、SEQ ID NO：17和SEQ ID NO：20所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    IV、如III所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    V、与如SEQ ID NO:4和SEQ ID NO:22所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    VI、如V所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    VII、与如SEQ ID NO:5所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    VIII、如VII所示序列的互补序列；
    可选的，所述HOXB4基因的甲基化检测的引物对如SEQ ID NO.:1和SEQ ID NO.:2所示；
    可选地，所述SRCIN1基因的甲基化检测的引物对如SEQ ID NO.:4和SEQ ID NO.:5所示；
    可选地，所述HOXB4基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    IX、与如SEQ ID NO.:3、SEQ ID NO.:18和SEQ ID NO.:21所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    X、如IX所示序列的互补序列；和/或
    所述SRCIN1基因的甲基化检测的探针具有如下所示的核苷酸序列中的任意一项：
    XI、与如SEQ ID NO.:6所示的核苷酸序列具有至少85％或至少90％或至少91％或至少92％或至少93％或至少94％或至少95％或至少96％或至少97％或至少98％或至少99％，或100％同一性的核苷酸序列；及
    XII、如XI所示序列的互补序列。
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CN115807095A (zh) * 2022-12-07 2023-03-17 中国人民解放军总医院第八医学中心 一种肺腺癌甲基化位点检测的引物组合物及其应用
CN115807095B (zh) * 2022-12-07 2023-10-13 中国人民解放军总医院第八医学中心 一种肺腺癌甲基化位点检测的引物组合物及其应用

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AU2020451827B2 (en) 2025-01-09
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JP7612715B2 (ja) 2025-01-14
CN111676287B (zh) 2022-04-29
JP2023527868A (ja) 2023-06-30
TWI775168B (zh) 2022-08-21
EP4163386A1 (en) 2023-04-12
CN111676287A (zh) 2020-09-18
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