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WO2020063898A1 - Utilisation de réactif de détection de méthylation hoxa7 dans la préparation d'un réactif de diagnostic pour le cancer du poumon - Google Patents

Utilisation de réactif de détection de méthylation hoxa7 dans la préparation d'un réactif de diagnostic pour le cancer du poumon Download PDF

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WO2020063898A1
WO2020063898A1 PCT/CN2019/108645 CN2019108645W WO2020063898A1 WO 2020063898 A1 WO2020063898 A1 WO 2020063898A1 CN 2019108645 W CN2019108645 W CN 2019108645W WO 2020063898 A1 WO2020063898 A1 WO 2020063898A1
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seq
lung cancer
methylation
detection
hoxa7
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李仕良
赵霞
牛智通
黄龙武
赵荣淞
吴幽治
邹鸿志
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Creative Biosciences Guangzhou 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present disclosure belongs to the field of genetic diagnosis, and more particularly, the present disclosure relates to an application of a human HOXA7 gene methylation detection reagent in preparing a lung cancer detection reagent, and a method for detecting human HOXA7 gene methylation detection.
  • Lung cancer is a malignant tumor of the lungs that originates from the bronchial mucosa, glands, or alveolar epithelium. According to the pathological type, it can be divided into: 1. Small cell lung cancer (SCLC): A special pathological type of lung cancer with a significant distant metastasis tendency and a poor prognosis, but most patients are sensitive to chemoradiotherapy. 2. Non-small cell lung cancer (NSCLC): In addition to small cell lung cancer, other pathological types of lung cancer, including squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. There are certain differences in biological behavior and clinical course. According to the occurrence location, it can be divided into: 1. Central lung cancer: Lung cancer that grows in the bronchus opening of the lung segment and above. 2. Peripheral lung cancer: Lung cancer that grows beyond the bronchial openings in the lung segment.
  • SCLC Small cell lung cancer
  • NSCLC Non-small cell lung cancer
  • the current clinical auxiliary diagnosis of lung cancer mainly includes the following types, but they can not completely detect and diagnose early:
  • Blood biochemical examination For primary lung cancer, there is currently no specific blood biochemical examination. Elevated blood alkaline phosphatase or calcium in patients with lung cancer considers the possibility of bone metastasis, and elevated blood alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, or bilirubin consider the possibility of liver metastasis.
  • CEA 30% to 70% of lung cancer patients have abnormally high levels of CEA in serum, but mainly found in patients with advanced lung cancer.
  • the current examination of CEA in serum is mainly used to estimate the prognosis of lung cancer and to monitor the treatment process.
  • NSE It is the first choice marker of small cell lung cancer. It is used for the diagnosis and monitoring of treatment response of small cell lung cancer. Depending on the detection method and the reagents used, the reference value is different.
  • CYFRA21-1 It is the first choice marker for non-small cell lung cancer, and the sensitivity to lung squamous cell carcinoma can reach 60%. The reference value varies according to the detection method and the reagents used.
  • Imaging examination (1) X-ray examination of the chest: it should include chest upright and lateral radiographs. In primary hospitals, chest orthotopic radiography is still the most basic and preferred imaging diagnostic method for the first diagnosis of lung cancer. Once lung cancer is diagnosed or suspected, a chest CT scan is performed.
  • CT examination Chest CT is the most common and important examination method for lung cancer. It is used for the diagnosis and differential diagnosis of lung cancer, staging and follow-up after treatment. Conditional hospitals should include adrenal glands when performing chest CT scans of lung cancer patients. Enhanced scanning should be used as much as possible, especially in patients with lung-centric lesions.
  • CT is the basic examination method for showing brain metastases. Patients with clinical symptoms or advanced patients should undergo brain CT scans, and enhanced scans should be used whenever possible.
  • CT-guided lung biopsy is an important diagnostic technique for lung cancer.
  • Conditional hospitals can use it for the diagnosis of difficult-to-characterize lung lesions, and clinical diagnosis of lung cancer requires cytological and histological confirmation, but other methods are difficult to obtain.
  • Ultrasound examination It is mainly used to detect the presence of metastases in the abdominal organs and lymph nodes in the abdominal cavity and retroperitoneum. It is also used in the examination of cervical lymph nodes. For lung lesions or chest wall lesions adjacent to the chest wall, cystic solidity can be identified and ultrasound-guided puncture biopsy can be performed; ultrasound is also commonly used for pleural fluid extraction and localization.
  • Bone scan The sensitivity to lung cancer bone metastasis is high, but there is a certain false positive rate. It can be used in the following cases: preoperative examination of lung cancer; patients with local symptoms.
  • sputum cytology examination currently a simple and convenient non-invasive diagnosis method for lung cancer. Continuous smear examination can increase the positive rate by about 60%, which is a routine diagnosis method for suspicious lung cancer cases.
  • Fiber bronchoscopy One of the most important methods in the diagnosis of lung cancer, it plays an important role in the qualitative localization diagnosis of lung cancer and the selection of surgical schemes. It is a necessary routine examination item for patients who are going to undergo surgery. Transbronchoscopic biopsy (TBNA) is helpful for staging before treatment, but because of technical difficulties and risks, those in need should be transferred to a higher level hospital for further examination.
  • Others such as percutaneous lung biopsy, thoracoscopy biopsy, mediastinoscopy biopsy, pleural fluid cytology, etc., if there are indications, they can be used separately to assist diagnosis according to the existing conditions.
  • Multi-slice spiral CT and low-dose CT (LDCT) in imaging studies are effective screening tools to detect early lung cancer and reduce mortality.
  • the National Lung Cancer Screening Study (NLST) has shown that LDCT is more effective than chest X-ray screening. Reduce lung cancer mortality by 20%. It has been proved in clinical practice that the success of any lung cancer screening program depends on the identification of high-risk populations.
  • a risk prediction model incorporating multiple high-risk factors has been recognized by the world as one of the methods to identify high-risk populations of lung cancer. Risk models further improve the efficacy of lung cancer patients by assisting clinicians to improve interventions or treatments. Although the world has agreed that screening for high-risk groups can reduce the current high mortality rate of lung cancer, the definition of high-risk groups is still a difficult problem.
  • the key question is first how to define the population at high risk of the disease; the second is how to screen the population, including the definition of high-risk factors, and the overall risk. Quantitative summary and selection of screening benefit cutoffs.
  • Existing lung cancer detection technologies mainly include low sensitivity, high false positives, and invasiveness. Moreover, it is difficult to detect early lung cancer with current conventional detection technologies.
  • Noninvasive tests for lung cancer are more difficult.
  • some researchers have also studied tumor markers in the sputum of patients with lung cancer, compared with the detection and evaluation of tumor markers in blood samples of other tumor patients, the success rate of sputum samples is very low.
  • the current rate of missed detection of lung cancer is high.
  • noninvasive detection of sputum is more difficult and the detection rate is extremely low.
  • most adenocarcinomas originate from smaller bronchial tubes, which are peripheral lung cancers. It is more difficult to cough out sputum cells in the deep lungs. Therefore, the current sputum detection method for adenocarcinoma is almost zero.
  • Reducing the rate of missed tests is particularly important in early tumor screening. If an early cancer screening product cannot detect all or most of the patients, those who miss the test will not be able to get enough risk prompts, which will delay the timing of treatment, which is huge for patients. loss.
  • non-invasive screening has unique advantages in sampling, it also has some other limitations.
  • the type of adenocarcinoma in lung cancer because the exfoliated cells in the deep lungs are difficult to cough through sputum, usually In other words, those skilled in the art would think that this type of lung cancer is not suitable for non-invasive screening.
  • the currently reported non-invasive screening methods are difficult to meet the requirements for clinical use.
  • An object of the present disclosure is to provide an application of a methylation detection reagent for a lung cancer tumor marker in preparing a lung cancer diagnostic reagent.
  • Another object of the present disclosure is to provide an application of a methylation detection reagent for detecting lung cancer tumor markers in sputum and lavage fluid with a sensitivity that is not less than that in tissues, in preparing a diagnostic reagent for lung cancer.
  • Another object of the present disclosure is to provide an application of a detection reagent for methylation of tumor markers with high sensitivity and specificity for adenocarcinoma in sputum and lavage fluid for preparing a diagnostic reagent for lung cancer.
  • Another object of the present disclosure is to provide a method for detecting methylation of HOXA7 gene.
  • the gene is the human HOXA7 gene.
  • the inventors not only verified that the detection of HOXA7 methylation has high specificity and sensitivity in the detection of lung cancer in tissue samples, but also verified that they have the same high specificity and sensitivity in sputum samples and lavage fluid samples. Sex.
  • a first aspect of the present disclosure provides an application of a detection reagent for methylation of the HOXA7 gene in the preparation of a diagnostic reagent for lung cancer.
  • the HOXA7 gene is a member of the HOX (homebox homology box) gene family. It belongs to the HOXA cluster gene on chromosome 7p15-p14. Like other HOX genes, it contains a 180-bp DNA fragment and transcribes the same 60-amino acid homolog. Source domain. HOXA7 plays a regulatory role in the proliferation and differentiation of normal hematopoietic cells. At present, more studies have focused on that the abnormal expression of HOXA7 plays an important role in the occurrence and development of leukemia. There are also reports that methylation of HOXA7 gene is related to lung cancer.
  • the detection reagent for methylation of the HOXA7 gene detects a sequence of the HOXA7 gene modified by a transformation reagent.
  • the transformation reagent refers to a reagent that deaminates cytosine in DNA into uracil, while leaving 5-MeC substantially unaffected.
  • Exemplary conversion reagents include hydrazine, bisulfite (e.g., sodium bisulfite, etc.), bisulfite (e.g., sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite) Etc.), or one or more compounds which can generate hydrazine, bisulfite, bisulfite under appropriate reaction conditions.
  • the detection reagent for methylation of the HOXA7 gene detects a sequence modified by bisulfite.
  • Methylation occurs when there is an additional methyl group on cytosine. After treatment with bisulfite or bisulfite or hydrazine, cytosine will become uracil, because uracil and Thymine is similar and will be recognized as thymine. It is reflected in the PCR amplified sequence that thymine that has not been methylated has become thymine (C becomes T), and methylated cytosine (C) is not Will change.
  • the technique for detecting methylated genes by PCR is usually methylation-specific PCR (MSP). Primers are designed for the methylated fragments after treatment (that is, the unchanged C in the fragments), and PCR amplification is performed. The presence of amplification indicates methylation, and the absence of amplification indicates no methylation.
  • MSP methylation-specific PCR
  • the detection region of the HOX7 gene targeted by the detection reagent for methylation of the HOXA7 gene includes a CG-enriched region or a non-CG-enriched region or a CTCF (CTCF-binding sites) region of the HOXA7 gene.
  • the detection region of the reagent includes a CG-enriched region or a CTCF (CTCF-binding sites) region of the HOXA7 gene.
  • the detection region of the HOXA7 gene targeted by the detection reagent for methylation of the HOXA7 gene includes the HOXA7 gene body or a promoter region thereof.
  • the detection region targeted by the detection reagent for methylation of the HOXA7 gene comprises the sequence shown in SEQ ID NO: 22 (region 1) or SEQ ID NO: 24 (region 2).
  • the inventors have found through experiments that the selection of the HOXA7 gene detection region will affect the detection efficiency of the tumor. Primers designed based on the CG-rich region of the HOXA7 gene, and the primers designed in different regions have significant differences in detection results. The detection rate of tumors is 50% to 60% higher than that of poor areas. The inventors found through experiments and comparisons that the detection results of specific CG-enriched regions or CTCF (CTCF-binding sites) regions were significantly better than other regions.
  • CTCF CTCF-binding sites
  • the reagent for detecting methylation of the HOXA7 gene of the present disclosure includes an amplification primer.
  • the primer includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO : 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46 At least one of SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53.
  • the primers include primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the reagent for detecting methylation of the HOXA7 gene of the present disclosure further includes a probe.
  • the probe includes SEQ ID NO: 3, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54.
  • the probe is selected from SEQ ID NO: 3.
  • the label of the detection probe includes a radioisotope, a fluorescent group, a bioluminescent compound, a chemiluminescent compound, a metal chelate, or an enzyme.
  • the label of the probe is a fluorescent group, which includes but is not limited to VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, Texas Red, etc .; the quenching group includes but is not limited to TAMRA, BHQ, MGB, Dabcyl, etc., are suitable for multi-channel PCR detection systems commonly used in clinical detection at present, and realize multi-color fluorescence detection in one reaction tube.
  • the detection reagent for methylation of the HOXA7 gene of the present disclosure includes a primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 and a probe shown in SEQ ID NO: 3.
  • the reagent for detecting methylation of the HOXA7 gene in the present disclosure further includes bisulfite, bisulfite, or hydrazine for modifying methylated cytosine to thymine.
  • bisulfite bisulfite
  • hydrazine for modifying methylated cytosine to thymine.
  • it may not be included in the reagent of the present disclosure, and may be purchased separately when used.
  • the detection reagent for the methylation of the HOXA7 gene of the present disclosure further comprises one or more of a DNA polymerase, dNTPs, Mg 2+ ions, and a buffer solution; , Mg 2+ ions and buffers for amplification of HOXA7 gene.
  • the detection reagent for methylation of the HOXA7 gene of the present disclosure further includes a detection reagent for an internal reference gene.
  • the internal reference gene comprises ⁇ -actin or COL2A1.
  • other internal reference genes for methylation detection in the prior art can also be used.
  • the detection reagent for the internal reference gene includes a primer and a probe for the internal reference gene.
  • the detection reagent for the internal reference gene ⁇ -actin includes a primer pair represented by SEQ ID NO: 16, SEQ ID NO: 17, and a probe of SEQ ID NO: 18.
  • the detection reagent for the internal reference gene COL2A1 includes a primer pair represented by SEQ ID NO: 55 (TTTTGGATTTAAGGGGAAGATAAA), a primer pair represented by SEQ ID NO: 56 (TTTTTCCTTCTCTCTACATCTTTCTACCT), and a probe represented by SEQ ID NO: 57 (AAGGGAAATTGAGAAATGAGAGAGAAGGGA).
  • Another aspect of the present disclosure provides a method for detecting DNA methylation of the HOXA7 gene, which includes the following steps:
  • the above-mentioned detection reagent for methylation of HOXA7 gene is used to detect the methylation of HOXA7 gene on the modified detection sample in step (1).
  • a methylation-specific polymerase chain reaction (MSP) or real-time fluorescent methylation-specific PCR (qMSP) is used for detection.
  • MSP methylation-specific polymerase chain reaction
  • qMSP real-time fluorescent methylation-specific PCR
  • the result judgment component is configured to output a diagnosis result such as a risk of lung cancer and / or a type of lung cancer according to a DNA methylation level of the HOXA7 gene detected by the detection component.
  • the result determination component includes a data processing machine.
  • the data processing machine includes any device or instrument or device that can be used by those skilled in the art to perform data processing.
  • the data processing machine includes one or more of a calculator and a computer.
  • the computer is loaded with any software or program that can be used by those skilled in the art to perform data processing or statistical analysis.
  • the computer includes a computer with one or more of SPSS, SAS, Excel software attached.
  • the result judgment component further includes a result outputter.
  • the output device includes any device or instrument or device capable of displaying data processing results as readable content.
  • the result outputter includes one or more of a screen and a paper report.
  • Another aspect of the present disclosure provides a method for diagnosing lung cancer.
  • the method includes the following steps:
  • the sample to be tested when the methylation level of the sputum sample to be tested is greater than 0.77%, the sample to be tested is a lung cancer sample, and when the methylation level is 0.77% or less, the sample to be tested is a non-lung cancer sample. .
  • the sample to be tested when the methylation level of the test lavage sample is greater than 0.7%, the sample to be tested is a lung cancer sample, and when the methylation level is 0.7% or less, the sample to be tested is determined to be non- Lung cancer sample.
  • the diagnostic method of the present disclosure can be used before and after lung cancer treatment or in combination with lung cancer treatment.
  • Post-treatment use can be used to evaluate the success of treatment or to monitor the remission, recurrence, and / or progression (including metastasis) of lung cancer after treatment.
  • Another aspect of the present disclosure provides a method for treating lung cancer.
  • the method includes administering surgery, chemotherapy, radiotherapy, radiochemotherapy, immunotherapy, oncolytic virus therapy, or other methods for patients diagnosed with lung cancer by the above-mentioned diagnostic method. Any other type of lung cancer treatment used in the art and a combination of these treatments.
  • the detection sample of the detection reagent for methylation of HOXA7 gene includes sputum, lung lavage fluid, lung tissue, pleural fluid, blood, serum, plasma, urine, prostate fluid, tear fluid, or feces. and many more.
  • the detection sample of the detection reagent for methylation of HOXA7 gene includes sputum, tissue, or lung lavage fluid.
  • the detection sample of the detection reagent for methylation of HOXA7 gene includes sputum or lung lavage fluid.
  • the present disclosure found that the methylation level of the HOXA7 gene in tissues is highly correlated with the incidence of lung cancer.
  • the normal HOXA7 gene group compared with all lung cancer groups has a specificity of 95% and a sensitivity of 63.3%.
  • the sensitivity is lower than the SHOX2 gene of another tumor marker in the experiment disclosed in this disclosure, it was surprisingly found.
  • the methylation level of HOXA7 gene detected in sputum and lung lavage fluid has also maintained a high degree of correlation with the incidence of lung cancer.
  • the sensitivity is 88.6% and the specificity is 95%.
  • the sensitivity is 76.2% and specificity is 95%.
  • the sensitivity is even higher than in tissues, which is rare in molecular markers. , Even unique.
  • SHOX2, PCDHGA12, HOXD8, and GATA3 have been reported to be related to lung cancer.
  • the detection sensitivity of SHOX2 in tissues is 80.6%, which is higher than the sensitivity of the HOXA7 gene, and the sensitivity is reduced in sputum. To 62.9%, it is significantly lower than 88.6% of the HOXA7 gene (Note, comparison between normal group and all cancer groups).
  • the sensitivity of the SHOX2 gene decreased to 52.4%, which seriously affected the diagnosis of lung cancer, while the sensitivity of HOXA7 was 76.2%, which was higher than that in tissues.
  • the sensitivity of the HOXA7 gene in sputum and lung lavage samples does not decrease, but maintains a specificity as high as 95%, which makes this gene extremely useful as a sample of sputum and lung lavage fluid. Reliable lung cancer marker.
  • the lung cancer is selected from the group consisting of small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC); further, the non-small cell lung cancer is selected from squamous cell carcinoma Squamous cell carcinoma, adenocarcinoma or large cell carcinoma. As a preferred embodiment, the lung cancer is selected from adenocarcinoma.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • HOXA7 gene has high specificity and high sensitivity in many different types of lung cancer. Even in lung adenocarcinoma, it has higher sensitivity than other tumor markers. At present, the rate of missed detection of lung adenocarcinoma is high. On the one hand, because lung adenocarcinoma is more likely to occur in women and non-smokers, the incidence is lower than that of squamous cell carcinoma and undifferentiated cancer. Bronchus is a peripheral lung cancer. It is more difficult to cough out sputum from deep lungs. On the other hand, lung adenocarcinoma generally does not have obvious clinical symptoms in the early stage. Therefore, the detection of lung adenocarcinoma is more difficult and valuable.
  • HOXA7 for adenocarcinoma detection in sputum has reached 88.9%, which is a breakthrough improvement over SHOX2 (adenocarcinoma sensitivity of 33.3%).
  • SHOX2 adenocarcinoma sensitivity of 33.3%
  • HOXA7 is effective for adenocarcinoma.
  • the detection sensitivity has reached 72.7%, which has also been greatly improved compared to SHOX2 (adenocarcinoma sensitivity of 36.4%), and it is not obvious that it is higher than the sensitivity in tissue. Therefore, for adenocarcinoma, the preferred test sample is sputum.
  • HOXA7 has great application significance for the detection of adenocarcinoma.
  • tissue can be used as a detection sample, but also prominent, it has higher sensitivity in sputum and lung lavage fluid, and can be easily used in sputum and lung Department of lavage fluid as a test sample for reliable diagnosis of lung cancer.
  • Sputum samples are easy to obtain and do not cause any pain or inconvenience to the patient.
  • the sample size is very small, the sampling process is very convenient and has no impact on the patient.
  • samples are easy to mail or take to the hospital for examination.
  • a technical solution of the above technical solution has the beneficial effects that it can detect multiple types of lung cancer, and it also has higher sensitivity than other markers for difficult-to-detect adenocarcinoma.
  • a beneficial effect of one of the above technical solutions is that the diagnostic reagent for lung cancer does not need to consider the detection target and age, and has a wide application range.
  • the beneficial effect of one of the above technical solutions is that cancer is detected and diagnosed through methylation levels. More and more studies have confirmed that methylation changes are an early event in tumorigenesis, and it is easier to detect abnormal methylation. Find early lesions.
  • FIG. 1 ROC curves of HOXA7, SHOX2, PCDHGA12, HOXD8, and GATA3 detected in tissue specimens;
  • Figure 4 Amplification curve of HOXA7 and SHOX2_n3 in sputum samples (A is the amplification map of HOXA7, B is the amplification map of SHOX2_n3;
  • FIG. 6 Amplification curves of HOXA7 and SHOX2_n3 in lavage fluid samples (A is the amplification map of HOXA7, and B is the amplification map of SHOX2_n3).
  • a “primer” or “probe” in this disclosure refers to an oligonucleotide comprising a region that is complementary to the sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (eg, a target gene). 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 comprises 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 with the target molecule. A region of complementary sequence of consecutive or discontinuous block nucleotides.
  • the primer or probe When a primer or probe comprises a region "complementary to at least x consecutive nucleotides of a target molecule", the primer or probe is at least 95% complementary to at least x consecutive nucleotides of the target molecule.
  • 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% are complementary.
  • lung cancer marker HOXA7 makes early diagnosis of lung cancer possible.
  • a methylated gene is determined to be methylated in a clinically or morphologically normal cell in a cancer cell, this indicates that the normally expressed cell is developing towards cancer.
  • lung cancer can be diagnosed at an early stage by methylation of the lung cancer-specific gene HOXA7 in normal-representing cells.
  • early diagnosis refers to the possibility of discovering cancer before metastasis, preferably before morphological changes of tissues or cells can be observed.
  • Diagnosis in the present disclosure includes, in addition to the early diagnosis of lung cancer, the diagnosis of intermediate and advanced lung cancer, and also includes lung cancer screening, risk assessment, prognosis, disease identification, diagnosis of the stage of the disease, and selection of therapeutic targets.
  • diagnosis can be made by measuring the degree of methylation of HOXA7 obtained from a sample during the progression of lung cancer at different stages or stages.
  • degree of methylation of the HOXA7 gene of a nucleic acid isolated from a sample at each stage of lung cancer By comparing the degree of methylation of the HOXA7 gene of a nucleic acid isolated from a sample at each stage of lung cancer with the HOXA7 gene methylation of one or more nucleic acids isolated from a sample of lung tissue without abnormal cell proliferation It can detect the specific stage of lung cancer in the sample.
  • a "normal" sample refers to a sample of the same type isolated from an individual known to be free of the cancer or tumor.
  • the "subject” is a mammal, such as a human.
  • Samples for methylation detection in the present disclosure include, but are not limited to, DNA, or RNA, or mRNA- and DNA-containing samples, or DNA-RNA hybrids.
  • the DNA or RNA may be single-stranded or double-stranded.
  • methylation detection methods for methylation detection are well known, such as methylation-specific polymerase chain reaction, real-time fluorescence quantitative methylation-specific polymerase chain reaction, pyrosequencing, and the use of methylated DNA-specific binding proteins.
  • PCR quantitative PCR, and DNA chips, differential methylation detection-methylation-sensitive restriction enzymes, differential methylation detection-sulfite sequencing, etc.
  • the methylation detection method can be introduced by the patent US62007687.
  • methylation level is the same as “degree of methylation” and can usually be expressed as the percentage of methylated cytosines, which is the number of methylated cytosines divided by the number of methylated cytosines and The sum of the number of methylated cytosines; and the method of dividing the number of methylated target genes by the number of reference genes is currently commonly used to indicate the level of methylation; and other methods of indicating the level of methylation in the prior art.
  • Methylated DNA has obvious advantages as a detection target. Compared with protein-based markers, DNA can be amplified and easily detected. Compared with mutant markers, the methylated sites of DNA are located in genes. The specific part of the gene is usually in the promoter region, making detection easier and more convenient.
  • the inventors screened hundreds of genes and selected better HOXA7, SHOX2, PCDHGA12, HOXD8, and GATA3 as candidate detection genes, and ⁇ -actin gene as an internal reference gene to study the methylation of each gene Site distribution, primer probes designed for detection were used for real-time fluorescence quantitative methylation-specific PCR (qMSP) detection.
  • the primer probes for each gene are as follows:
  • HOXA7 detection primers and probes are:
  • SEQ ID NO: 3HOXA7-P2 Probe FAM-AGGGCGCGTTGTATGGCGC-BQ1
  • SHOX2 detection primers and probes are:
  • SEQ ID NO: 4SHOX2 Primer F TTTAAAGGGTTCGTCGTTTAAGTC
  • SEQ ID NO: 5SHOX2 Primer R AAACGATTACTTTCGCCCG
  • SEQ ID NO: 6SHOX2 Probe FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1
  • the detection primers and probes for PCDHGA12 are:
  • HOXD8's detection primers and probes are:
  • SEQ ID NO: 12HOXD8 Probe FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
  • GATA3's detection primers and probes are:
  • SEQ ID NO: 15GATA3 Probe FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1
  • the detection primers and probes for ⁇ -actin are:
  • SEQ ID NO: 16 ⁇ -actin primer F TTTTGGATTGTGAATTTGTG
  • SEQ ID NO: 17 ⁇ -actin primer R AAAACCTACTCCTCCCTTAAA
  • SEQ ID NO: 18 ⁇ -actin probe FAM-TTGTGTGTTGTGGGTGGTGGTT-BQ1
  • Specimens from lung cancer patients and non-lung cancer patients were collected, including paraffin tissue samples, sputum samples, and lavage fluid samples. After the samples were pretreated and the cells were isolated, DNA extraction was performed according to the instructions of the US Biotech Kit HiPure FFPE DNA Kit (D3126-03).
  • Sample information A total of 185 lung tissue samples, of which 87 were normal tissue samples, 98 were cancer tissue samples, 15 were squamous cell carcinoma in the 98 cancer group samples, 81 were adenocarcinomas, and 2 were lung cancers that were not clearly classified. And 73 pairs of adjacent cancer control samples.
  • ROC curves of HOXA7, SHOX2, PCDHGA12, HOXD8, and GATA3 in all tissue specimens are shown in Figure 1.
  • the statistical results of the detection of each gene in the tissue are shown in Table 3.
  • the inventors further screened the five markers HOXA7, SHOX2, PCDHGA12, HOXD8, and GATA3 in sputum because sputum is used as a non-invasive test The sample is even more significant.
  • Sample information A total of 90 sputum samples were tested, of which 55 were in the normal control group, 35 were in the cancer control group, 12 were squamous cell carcinoma, 6 were small cell cancer, 9 were adenocarcinoma, and 35 were large in the 35 cancer group. There were 2 cases of cell carcinoma and 6 cases of lung cancer without a clear classification.
  • the dosing system is as follows:
  • the amplification system is as follows:
  • ROC curves of HOXA7, SHOX2, PCDHGA12, HOXD8, and GATA3 in sputum samples are shown in Figure 2, and the statistical results are shown in Table 6. From the above results, it can be seen that in the sputum samples, the five genes are detected at the same time. For comparison, no matter whether the lung cancer as a whole is compared or analyzed according to the lung cancer subtype, the detection effect of HOXA7 is better than that of the other four genes. Especially for the detection of adenocarcinoma, the detection rate of HOXA7 is 88.9%, which is much higher than other genes. Adenocarcinoma is generally peripheral.
  • SHOX2 which has the highest sensitivity to adenocarcinoma in tissues, has a sensitivity in sputum. Significantly reduced to 11.1%, so the detection of this part is more difficult and meaningful.
  • SHOX2 can be used as a marker for detecting lung cancer
  • patents showing [CN201510203539-method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene-application publication] SHOX2 is used in alveolar lavage fluid and lesions Tissue, pleural fluid, sputum and other samples have higher detection rates.
  • the inventors simultaneously detected the detection efficiency of HOXA7 and SHOX2 genes.
  • the detection efficiency of SHOX2 gene uses the primer and probe sequences disclosed in patent CN201510203539, and the SHOX2 gene It is expressed as SHOX2_n3 to distinguish it from the SHOX2 gene detected by self-designed primers and probes in Examples 1 and 2 of the present disclosure.
  • the primer probes for each gene are as follows:
  • HOXA7 detection primers and probes are:
  • SEQ ID NO: 3HOXA7-P2 Probe FAM-AGGGCGCGTTGTATGGCGC-BQ1
  • the detection primers and probes for SHOX2_n3 are:
  • the dosing system is as follows:
  • the amplification system is as follows:
  • the threshold value of HOXA7 is 0.77.
  • the threshold of SHOX2_n3 is 1.3.
  • Non-lung cancer control 0.2 0.0 - - 28 Non-lung cancer control 0.0 0.0 - - 29
  • Non-lung cancer control 0.0 0.0 - - 31 Non-lung cancer control 0.0 0.4 - - 32
  • Non-lung cancer control 0.0 0.5 - - 34 Non-lung cancer control 0.4 0.7 - - 35
  • Non-lung cancer control 0.0 0.1 - - 38 Non-lung cancer control 2.2 0.6 + - 39
  • Non-lung cancer control 0.0 0.2 - - 41 Non-lung cancer control 0.6 1.7 - + 42
  • Non-lung cancer control 0.0 0.0 - - 43 Non-lung cancer control 0.0 0.6 - - 44
  • the ROC curve detected by HOXA7 and SHOX2_n3 in the sputum sample is shown in Figure 3, the amplification curve is shown in Figure 4, and the statistical results are shown in Table 11. From the above results, it can be seen that the lung cancer as a whole is compared and analyzed. Compared with the subtypes, the detection effect of HOXA7 is better than that of SHOX2 gene. It is not obvious that the detection sensitivity of HOXA7 in sputum is higher than that in tissue. Especially for the detection of adenocarcinoma, the detection rate of HOXA7 was 88.9%, which was much higher than the SHOX2 gene of 33.3%. Adenocarcinoma is generally peripheral.
  • the present disclosure has discovered a marker that can detect adenocarcinoma with sputum as a sample and can greatly increase the sensitivity to 88.9%. This breakthrough has great significance for the detection of adenocarcinoma.
  • Sample information A total of 79 alveolar lavage fluid samples were tested, including 58 normal control samples, 21 cancer control samples, 21 cancer group samples including squamous cell carcinoma, 4 small cell carcinomas, and 11 adenocarcinomas. .
  • the amplification detection system is as follows:
  • the detection system is as follows:
  • the threshold value of HOXA7 is 0.7.
  • the threshold value of SHOX2_n3 is 0.6.
  • Non-lung cancer control 0.3 0.3 - - 2 Non-lung cancer control 0.6 0.7 - + 3
  • Non-lung cancer control 0.3 0.6 - - 5 Non-lung cancer control 0.0 0.2 - - 6
  • Non-lung cancer control 0.0 0.0 - - 8 Non-lung cancer control 0.2 0.0 - - 9
  • Non-lung cancer control 0.0 0.0 - - 10 Non-lung cancer control 0.0 0.0 - - 11
  • Non-lung cancer control 0.0 0.1 - - 12 Non-lung cancer control 0.3 0.1 - - 13
  • Non-lung cancer control 0.70 0.5 + - 16
  • Non-lung cancer control 0.2 0.1 - - 47 Non-lung cancer control 0.2 0.1 - - 48
  • Non-lung cancer control 0.2 0.4 - - 50 Non-lung cancer control 0.0 0.0 - - 51
  • Non-lung cancer control 0.2 0.0 - - 52 Non-lung cancer control 0.4 0.5 - - 53
  • Non-lung cancer control 0.2 0.0 - - 56 Non-lung cancer control 0.0 0.0 - - 57
  • Non-lung cancer control 0.0 0.0 - - 59 Squamous cell carcinoma 0.2 0.3 - - 60
  • the ROC curves detected by HOXA7 and SHOX2_n3 in all lavage fluid samples are shown in Figure 5, the amplification curve is shown in Figure 6, and the statistical results are shown in Table 16. From the above results, it can be seen that when HOXA7 and SHOX2 are detected at the same time, the lung cancer as a whole is compared and analyzed.
  • the detection rate of HOXA7 is 76.2%, which is much higher than 52.4% of SHOX2, and also higher than the sensitivity of HOXA7 in tissues. .
  • the detection result of HOXA7 in squamous cell carcinoma group was 16.6% higher than that in SHOX2.
  • adenocarcinoma small ncRNA, pair, novel, biomarkers, early-stage lungadenocarcinoma, screening
  • the diagnostic sensitivity of adenocarcinoma is 70.4% and the specificity is 72.7%, although the sensitivity of this disclosure is not significantly higher than
  • the detection of the adenocarcinoma of the present disclosure can maintain a sensitivity as high as 95%, and has a higher accuracy rate.
  • adenocarcinoma is generally of the peripheral type, due to the tree-like physiological structure of the bronchi, alveolar lavage fluid cannot easily contact the alveoli or cancerous tissues in the deep lung.
  • the present disclosure finds that a sputum sample can be used to detect adenocarcinoma, and while maintaining high specificity, it can greatly increase the sensitivity to 72.7%. This breakthrough has great significance for the detection of adenocarcinoma.
  • HOXA7 has a better detection effect on lung cancer detection and diagnosis, especially on biological samples such as sputum, alveolar lavage fluid. It can be more easily applied to large-scale population screening and has more superior socioeconomic value.
  • Example 5 The effect of the detection area, primers, and probes of HOXA7 on the detection effect
  • each primer probe is shown in Table 18, where Group 8, Group 9, and Group 10 are based on Methylated primers and probes designed in region 1; group 1, group 2, group 3, group 4, group 5, group 6, group 7 are methylated primers and probes (primers and probes) designed according to region 2 (See Table 19).
  • the above 10 sets of primer probe combinations were detected in 36 lung tissue samples, of which 11 were normal tissue samples, 25 were cancer tissue samples, 4 were squamous cell carcinoma, and 21 were adenocarcinoma in the 25 cancer group samples.
  • the test results are shown in Table 18 below.
  • primers and probes In addition to the detection area that will affect the detection effect, primers and probes also have a great impact on the detection effect of tumor markers.
  • the inventor designed multiple pairs of primers and their corresponding probes to find as much as possible Probes and primers that improve detection sensitivity and specificity, so that the detection reagents of the present disclosure can be practically applied to clinical detection.
  • Some primers and probes are shown in Table 19 below, and the detection results are shown in Table 20. All primers and probes were synthesized by Invejet (Shanghai) Trading Co., Ltd.
  • H7-F2 Serial number sequence effect H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC HOXA7 gene upstream primer H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG HOXA7 gene downstream primer H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7 gene detection probe H7-F3 SEQ ID NO: 28 GCGTTTGCGATAAGACGGAC HOXA7 gene upstream primer H7-R3 SEQ ID NO: 29 CCAACCTAACCCGCCTAACG HOXA7 gene downstream primer H7-P3 SEQ ID NO: 30 FAM-CGTTGTATGGCGCGGTTGAGG-BQ1 HOXA7 gene detection probe H7-F4 SEQ ID NO: 31 CGTTCGGTTACGGTTTGGGC HOXA7 gene upstream primer H7-R4 SEQ ID NO: 32 GCCCTCGTCCGTCTTATC
  • H7-R7 SEQ ID NO: 41 GCGATAAGACGGACGAGGGC HOXA7 gene upstream primer H7-P7 SEQ ID NO: 42 FAM-CCGATCCCGCTCCGCCAACC-BQ1 HOXA7 gene detection probe H7-F8 SEQ ID NO: 43 CGTTAGGCGGGTTAGGTTGGC HOXA7 gene upstream primer H7-R8 SEQ ID NO: 44 GCCGAAAATCACGAAACTACCG HOXA7 gene downstream primer H7-P8 SEQ ID NO: 45 FAM-AGCGGGATCGGGAGCGGG-BQ1 HOXA7 gene detection probe H7-F9 SEQ ID NO: 46 TCGGGTCGTTTGGCGTTC HOXA7 gene upstream primer H7-R9 SEQ ID NO: 47 AACCTAACGCGTCCCGCG HOXA7 gene downstream primer H7-P9 SEQ ID NO: 48 FAM-TTGGTCGTTAGTTGGGCGTTTTCGC-BQ1 HOX
  • H7-F2, H7-R2, H7-P2 100% 80% Group 2 H7-F3, H7-R3, H7-P3 100% 76% Group 3 H7-F4, H7-R4, H7-P4 100% 56% Group 4 H7-F5, H7-R5, H7-P5 100% 72% Group 5 H7-F6, H7-R6, H7-P6 100% 80% Group 6 H7-F7, H7-R7, H7-P7 100% 72% Group 7 H7-F8, H7-R8, H7-P8 100% 56% Group 8 H7-F9, H7-R9, H7-P9 100% 48% Group 9 H7-F10, H7-R10, H7-P10 100% 16% Group 10 H7-F11, H7-R11, H7-P11 100% twenty four%
  • the primers and probes in Table 19 were used to verify the tissue samples.
  • the above 10 sets of primer probe combinations were detected in 36 lung tissue samples, of which 11 were normal tissue samples, 25 were cancer tissue samples, 4 were squamous cell carcinoma, and 21 were adenocarcinoma in the 25 cancer group samples.
  • the test results are shown in Table 20 above. The results show that Group 1, Group 2, Group 4, Group 5, and Group 6 all have good detection rates.
  • H7-F2, H7-R2, H7-P2 100% 73.3%
  • Group 2 H7-F3, H7-R3, H7-P3 100% 53.3%
  • Group 4 H7-F5, H7-R5, H7-P5 100% 60.0%
  • Group 5 H7-F6, H7-R6, H7-P6 100% 53.3%
  • Group 6 H7-F7, H7-R7, H7-P7 100% 46.7%
  • H7-F2 Serial number sequence effect H7-F2 SEQ ID NO: 1 TAAAGGCGTTTGCGATAAGAC HOXA7 gene upstream primer H7-R2 SEQ ID NO: 2 TAACCCGCCTAACGACTACG HOXA7 gene downstream primer H7-P2 SEQ ID NO: 3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7 gene detection probe

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Abstract

La présente invention concerne l'utilisation d'un agent de détection qui fait intervenir la méthylation de HOXA7 en tant qu'objet de détection dans la préparation d'un réactif de diagnostic pour le cancer du poumon.
PCT/CN2019/108645 2018-09-29 2019-09-27 Utilisation de réactif de détection de méthylation hoxa7 dans la préparation d'un réactif de diagnostic pour le cancer du poumon Ceased WO2020063898A1 (fr)

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