WO2020063903A1 - Réactif de détection de méthylation du hoxa9 - Google Patents

Réactif de détection de méthylation du hoxa9 Download PDF

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WO2020063903A1
WO2020063903A1 PCT/CN2019/108651 CN2019108651W WO2020063903A1 WO 2020063903 A1 WO2020063903 A1 WO 2020063903A1 CN 2019108651 W CN2019108651 W CN 2019108651W WO 2020063903 A1 WO2020063903 A1 WO 2020063903A1
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seq
reagent
detection
lung cancer
hoxa9
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Chinese (zh)
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牛智通
赵荣淞
李仕良
黄龙武
吴幽治
邹鸿志
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广州市康立明生物科技有限责任公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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 gene diagnosis, and more particularly, the present disclosure relates to a lung cancer diagnosis reagent or a kit containing the same based on human HOXA9 gene methylation.
  • 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.
  • the purpose of the present disclosure is to provide an application of a nucleic acid fragment of the HOXA9 gene in the diagnosis of lung cancer.
  • Another object of the present disclosure is to provide a primer and its application in preparing a lung cancer diagnostic reagent or kit.
  • Another object of the present disclosure is to provide a probe and its application in preparing a lung cancer diagnostic reagent or kit.
  • Another object of the present disclosure is to provide a reagent, a kit and a method for diagnosing human HOXA9 gene methylation.
  • Another object of the present disclosure is to provide a lung cancer diagnosis reagent and a kit with high specificity and sensitivity.
  • a further object of the present disclosure is to provide a lung cancer diagnosis reagent and a kit with high sensitivity and specificity for lung adenocarcinoma.
  • Another object of the present disclosure is to provide a lung cancer diagnostic reagent and a kit having a wide application range for lung cancer.
  • Another object of the present disclosure is to provide a non-invasive diagnostic reagent and kit for lung cancer.
  • the inventors provided a detection reagent for HOXA9 gene methylation.
  • the inventors not only verified that the detection reagent has high specificity and sensitivity for 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.
  • the HOXA9 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.
  • HOXA9 is a coding sequence-specific transcriptional regulatory factor. It plays an important role in the development of spatiotemporal space in embryos, cell differentiation, proliferation, and migration, malignant evolution of tumors, and inducing apoptosis. At present, more researches are concerned that the abnormal expression of HOXA9 plays an important role in the occurrence and development of leukemia. There are also reports of HOXA9 gene related to lung cancer, human glioma and other cancers.
  • a first aspect of the present disclosure provides an application of a nucleic acid fragment in preparing a diagnostic reagent or kit for lung cancer; wherein the nucleic acid fragment is derived from the HOXA9 gene.
  • the nucleic acid fragment includes a sequence shown in SEQ ID NO: 22, SEQ ID NO: 24, or SEQ ID NO: 26; as a preferred embodiment, the nucleic acid fragment includes a sequence shown in SEQ ID NO: 22 .
  • the present disclosure also provides a primer comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46 , SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID ID NO: 58 Any one is shown; as a preferred embodiment in the present disclosure, the primer includes the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the present disclosure also provides a probe comprising SEQ ID NO: 3, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ IDNO: 47, SEQ IDNO: 50, SEQ IDNO: 53, SEQ IDNO: 56, SEQ IDNO: 59.
  • the nucleic acid probe comprises a sequence shown in SEQ ID NO: 3.
  • the present disclosure also provides the application of the above-mentioned primers or nucleic acid probes in the preparation of a lung cancer diagnostic reagent or kit.
  • the present disclosure also provides a diagnostic reagent for lung cancer, the reagent containing a detection reagent for methylation of the HOXA9 gene.
  • the detection reagent for methylation of the HOXA9 gene detects a sequence of the HOXA9 gene modified by a transformation reagent.
  • the transformation reagent refers to a reagent that deaminates cytosine in DNA into uracil, and at the same time makes 5-MeC substantially unaffected.
  • Exemplary transformation reagents include hydrazine, bisulfite (such as sodium bisulfite, etc.), bisulfite (such as sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite, etc.), Or, under appropriate reaction conditions, one or more compounds can be generated, such as hydrazine, bisulfite, and bisulfite.
  • the detection reagent for methylation of the HOXA9 gene detects a bisulfite-modified sequence.
  • Methylation occurs when there is an additional methyl group on cytosine.
  • 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.
  • MSP methylation-specific PCR
  • the detection region of the HOXA9 gene targeted by the reagent is a CG-enriched region or a non-CG-enriched region or a CTCF (CTCF-binding sites) region of the HOXA9 gene.
  • the detection region of the reagent is a CG-enriched region or a CTCF (CTCF-binding sites) region of the HOXA9 gene.
  • the detection region targeted by the reagent is the genome of the HOXA9 gene or its promoter region.
  • the detection region of the reagent includes a sequence represented by SEQ ID NO: 22 (Region 1), SEQ ID NO: 24 (Region 2), or SEQ ID NO: 26 (Region 3).
  • the detection region of the reagent includes a sequence shown in SEQ ID NO: 22.
  • the inventors have found through experiments that the selection of the HOXA9 gene detection region will affect the detection efficiency of the tumor. Primers designed according to the CG-rich region of the HOXA9 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 have found through experiments and comparisons that the detection results of GC-enriched regions or CTCF (CTCF-binding sites) regions are significantly better than non-hypermethylated regions.
  • CTCF CTCF-binding sites
  • the diagnostic reagent of the present disclosure includes an amplification primer.
  • the primers include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO : 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 48 At least one of SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 58.
  • the primers include primer pairs as shown in SEQ ID NO: 1 and SEQ ID NO: 2.
  • the primers are used to amplify a specific region of the HOXA9 gene. It is well known in the art that the successful design of primers is critical for PCR. Compared to ordinary PCR, the design of primers is more critical in the methylation detection of genes.
  • the selection of the amplified fragment targeted by the primer such as the length and position of the amplified fragment, and the selection of the primer, etc.
  • the inventors also found through experiments that different amplification target fragments and primers have different detection effects. Many times, it is found that certain genes or nucleic acid fragments have differential expression in tumors and non-tumors, but their distance is converted into tumor markers, and there is still a long distance for clinical application. The most important reason is that the detection sensitivity and specificity of this potential tumor marker are difficult to meet the detection requirements due to the limitation of detection reagents, or the detection method is complicated and expensive to operate, and it is difficult to apply it in clinical practice on a large scale.
  • the diagnostic reagent of the present disclosure further contains a probe.
  • the probe comprises SEQ ID NO: 3, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56 or SEQ ID NO: 59.
  • the probe is shown in SEQ ID NO: 3.
  • the detection probe-labeled fluorescent group includes VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, Texas Red, etc .;
  • the quenching group includes TAMRA, BHQ, MGB, Dabcyl, etc., are applicable to multi-channel PCR detection systems commonly used in clinical detection at present, and realize multi-color fluorescence detection in one reaction tube.
  • the diagnostic reagent 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 reagents of the present disclosure further comprise bisulfite, bisulfite or hydrazine salts for modifying methylated cytosines to thymine.
  • bisulfite, bisulfite or hydrazine salts for modifying methylated cytosines to thymine may not be included in the reagent of the present disclosure. It can be purchased separately when used.
  • the present disclosure further comprises a DNA polymerase reagents, one or more dNTPs, Mg 2+ ions, buffer; preferably, comprising a DNA polymerase, dNTPs, Mg 2+ ions and buffer Solution for the amplification of HOXA9 gene.
  • the reagents of the present disclosure further include 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 shown by SEQ ID NO: 60 (TTTTGGATTTAAGGGGAAGATAAA) and SEQ ID NO: 61 (TTTTTCCTTCTCTACATCTTTCTACCT), and a probe of SEQ ID NO: 62 (AAGGGAAATTGAGAAATGAGAGAGAAGGGA).
  • Another aspect of the present disclosure provides a kit for the above-mentioned primers, probes, and diagnostic reagents for lung cancer.
  • the kit of the present disclosure includes one or more containers divided into receiving reagents therein, provided that they contain at least one of the primers or probes of the present disclosure or other detection components.
  • the kit may further include nucleic acid extraction reagents and materials.
  • the kit may further include reagents and materials commonly used for amplifying nucleic acids, such as DNA polymerases, dNTPs, Mg 2+ ions, buffers, and the like.
  • the kit may further include a conversion reagent that sensitively converts unmethylated cytosine.
  • the kit includes a first container containing a reagent that sensitively converts unmethylated cytosine; a second container containing amplification primers; and a third container containing a probe.
  • the kit further comprises instructions.
  • the kit further comprises a sampling device.
  • Another aspect of the present disclosure provides a method for detecting DNA methylation of the HOXA9 gene, which includes the following steps:
  • 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
  • Another aspect of the present disclosure provides a diagnostic system for lung cancer, the system comprising:
  • the DNA methylation detecting member of the HOXA9 gene includes the above-mentioned reagent or kit.
  • the methylation detection member includes one or more of a quantitative PCR instrument, a PCR instrument, and a sequencer.
  • the result determining 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 HOXA9 gene detected by the detecting component.
  • the diagnosis result is obtained by comparing a methylation level of the sample to be tested with a normal sample by a result judgment component, and based on a deviation between the methylation level of the sample to be tested and the normal sample.
  • 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 2.3%, the sample to be tested is a lung cancer sample, and when the methylation level is less than or equal to 2.3%, the sample to be tested is determined to be a non-lung cancer sample. .
  • the test sample when the methylation level of the test lavage sample is greater than 0.5%, it is determined that the test sample is a lung cancer sample, and when the methylation level is 0.5% or less, the test sample 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 reagent / kit / method test sample includes sputum, lung lavage fluid, lung tissue, pleural fluid, blood, serum, plasma, urine, prostate fluid, tear fluid, or feces.
  • the detection sample of the diagnostic / detection reagent includes sputum, tissue, or lung lavage fluid.
  • the detection sample of the diagnostic / detection reagent contains sputum.
  • the methylation level of HOXA9 gene in tissues is highly correlated with the incidence of lung cancer.
  • the HOXA9 gene normal group compared with all lung cancer groups has a specificity of 95% and a sensitivity of 78.6%, although the sensitivity is lower than that of the other tumor marker SHOX2 (sensitivity 80.6%) gene in the experiment disclosed, and It was surprisingly found that the methylation level of HOXA9 gene detected in sputum and lung lavage fluid also maintained a high correlation with the incidence of lung cancer.
  • the sensitivity of HOXA9 was 74.3%, specificity 95%; in lung lavage fluid, the sensitivity was 61.9%, specificity 95%.
  • the detection sensitivity or specificity of sputum samples is significantly lower than that of tissue samples.
  • SHOX2, PCDHGA12, HOXD8, and GATA3 have been reported as genes related to lung cancer.
  • the detection sensitivity of SHOX2 in tissues is 80.6%, which is higher than the sensitivity of the HOXA9 gene.
  • the sensitivity is large.
  • the amplitude was reduced to 51.4%, which was significantly lower than 74.3% of the HOXA9 gene (Note, comparison between normal group and all cancer groups). Therefore, when sputum is used as a test sample, the HOXA9 gene is particularly suitable as a tumor marker.
  • the HOXA9 gene also maintains high sensitivity in sputum and lung lavage samples, and maintains specificity as high as 95%, which makes this gene extremely useful as sputum and lung lavage samples. Reliable lung cancer markers. Among them, one reason is that the present disclosure is optimized for the detection area, primers, probes, etc. of HOXA9.
  • the lung cancer is selected from small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC); further, the non-small cell lung cancer is selected from squamous cells Cancer, adenocarcinoma or large cell carcinoma. As a preferred embodiment, the lung cancer is selected from adenocarcinoma.
  • the reagents / kits of the present disclosure have high specificity and high sensitivity in many different types of lung cancer. Even in lung adenocarcinoma, they have higher sensitivity than other tumor markers. For example, in sputum, the detection sensitivity of HOXA9 is 55.6%, while the sensitivity of SHOX2 is 11.1% (see Table 6 in the Example), which further illustrates that HOXA9 is particularly suitable for using sputum as a test sample, especially for lungs. Detection of adenocarcinoma. At present, the rate of missed detection of lung adenocarcinoma is high.
  • 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.
  • a beneficial effect of one of the above technical solutions is that the lung cancer diagnostic reagent / kit can not only use tissue as a detection sample, but also prominently, it has higher sensitivity in sputum and lung lavage fluid, Sputum and lung lavage fluid can be easily used as test samples to reliably diagnose 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. At the same time, samples are easy to mail or take to the hospital for examination.
  • a beneficial effect of one of the above technical solutions is that the lung cancer diagnostic reagent / kit can detect multiple types of lung cancer, and it also has higher sensitivity than other markers for difficult-to-detect adenocarcinomas.
  • a technical solution of the above technical solution has the beneficial effect that the lung cancer diagnosis reagent / kit does not need to consider the detection object and age, and has a wide application range.
  • the beneficial effect of one of the above technical solutions is that the reagents / kits described in the present disclosure detect and diagnose cancer by methylation level, and more and more studies have confirmed that the methylation changes are in the process of tumorigenesis. Early events, detection of abnormal methylation are more likely to detect early lesions.
  • Figure 4 Amplification curve of HOXA9 and SHOX2_n3 in sputum samples (A is the amplification map of HOXA9, B is the amplification map of SHOX2_n3;
  • Figure 6 Amplification curves of HOXA9 and SHOX2_n3 in lavage fluid samples (A is the amplification map of HOXA9, 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.
  • 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.
  • lung cancer marker HOXA9 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 HOXA9 in normal-representing cells.
  • the reagents / kits of the present disclosure also have the potential to be used for lung cancer screening, risk assessment, prognosis, disease identification, diagnosis of stage of disease, and selection of therapeutic targets.
  • diagnosis can be made by measuring the degree of methylation of HOXA9 obtained from the sample during the progression of lung cancer at different stages or periods.
  • degree of methylation of the HOXA9 gene of a nucleic acid isolated from a sample of each stage of lung cancer with the HOXA9 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 methylation-targeted genes by the number of internal reference genes is currently commonly used to represent the methylation level; and other methylation levels commonly used in the prior art.
  • methylated DNA as a detection target has obvious advantages. Compared to protein-based markers, DNA can be amplified and easily detected; compared to mutant markers, the sites where DNA methylation occurs are all located Specific parts of the gene, usually in the promoter region, make detection easier and more convenient.
  • the inventors have selected HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 as candidate detection genes from hundreds of candidate genes through several rounds of screening.
  • ⁇ -actin gene is used as an internal reference gene. Distribution of gene methylation sites.
  • Primer probes designed for detection were used for real-time fluorescent methylation-specific PCR (qMSP) detection. The primer probes for each gene are as follows:
  • HOXA9 detection primers and probes are:
  • SHOX2 detection primers and probes are:
  • SEQ ID NO: 4 SHOX2 Primer F: TTTAAAGGGTTCGTCGTTTAAGTC
  • SEQ ID NO: 5 SHOX2 Primer R AAACGATTACTTTCGCCCG
  • SEQ ID NO: 6 SHOX2 probe: FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1
  • the detection primers and probes for PCDHGA12 are:
  • HOXD8's detection primers and probes are:
  • SEQ ID NO: 12 HOXD8 Probe: FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
  • GATA3's detection primers and probes are:
  • GATA3 primer F TTTCGGTAGCGGGTATTGC
  • GATA3 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
  • the bisulfite modification was performed using the ZYMO RESEARCH Biologicals kit EZ DNA Methylation TM KIT (D5002) instructions.
  • 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 HOXA9, 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.
  • 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 detected by HOXA9, 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 simultaneously In comparison, no matter whether the lung cancer as a whole is compared or analyzed according to the lung cancer subtype, the detection effect of HOXA9 is better than that of the other four genes. Especially for the detection of adenocarcinoma, the detection rate of HOXA9 is much higher than other genes. Adenocarcinoma is generally of the peripheral type.
  • SHOX2 which has the highest sensitivity to adenocarcinoma in tissues, has a greatly reduced sensitivity to 11.1% in sputum, so the detection of this part is more difficult and meaningful.
  • SHOX2 can be used as a marker for detecting lung cancer
  • SHOX2 is used in alveolar lavage fluid and lesions Tissue, pleural fluid, sputum and other samples have higher detection rates.
  • the detection efficiency of the SHOX2 gene uses the primer and probe sequences disclosed in the patent CN201510203539, and the SHOX2 gene is expressed as SHOX2_n3 to distinguish it from the examples 1 and 2 of the present disclosure.
  • the SHOX2 gene was detected by self-designed primers and probes.
  • the primer probes for each gene are as follows:
  • HOXA9 detection primers and probes are:
  • the detection primers and probes for SHOX2_n3 are:
  • SEQ ID NO: 19 SHOX2_n3 Primer F: TTTGGATAGTTAGGTAATTTTCG
  • the dosing system is as follows:
  • the amplification system is as follows:
  • the HOXA9 threshold is 2.3.
  • the threshold value of SHOX2_n3 is 1.3.
  • ROC curves detected by HOXA9 and SHOX2_n3 in all sputum samples are shown in Figure 3, and the amplification curves of HOXA9 and SHOX2_n3 in sputum samples are shown in Figure 4. From the above results, it can be seen that in the sputum samples, whether For lung cancer as a whole for comparative analysis, or for lung cancer subtypes, the detection effect of HOXA9 is better than that of SHOX2 gene. Especially for the detection of adenocarcinoma, the detection rate of HOXA9 is much higher than 33.3% of the SHOX2 gene. Adenocarcinoma is generally peripheral.
  • 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 HOXA9 is 0.5.
  • the threshold value of SHOX2_n3 is 0.6.
  • HOXA9 is as high as 100%, which is significantly higher than 75.0% of SHOX2.
  • HOXA9 has a better detection effect on lung cancer detection and diagnosis, especially on biological samples such as sputum and 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 HOXA9 on the detection effect
  • methylation primers and probe detection systems designed in different regions are selected for the same sample and the same tumor.
  • the diagnostic detection efficiency is not the same, and sometimes the selected area is not suitable to cause no diagnostic effect on the tumor at all.
  • the inventor has repeatedly researched and compared multiple detection areas. Some exemplary detection areas are shown in Table 17 below.
  • the above 11 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 the table below.
  • Region 1 has the lowest detection sensitivity Can also reach 40%, up to 76%. Therefore, the detection rate in area 1 is significantly higher than in areas 2 and 3 (see Table 18).
  • 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 detection probes are shown in Table 20 below, and the detection results are shown in Table 21. All primers and probes were synthesized by Invejet (Shanghai) Trading Co., Ltd.
  • H9-F2 Serial number sequence effect H9-F2 SEQ ID NO: 1 TTAGTTTTTTCGGTAGGCGGC HOXA9 gene upstream primer H9-R2 SEQ ID NO: 2 AAACGCCAAACACCGTCG HOXA9 gene downstream primer H9-P2 SEQ ID NO: 3 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9 gene detection probe

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Abstract

L'invention concerne un réactif et un kit de détection de la méthylation du gène HOXA9, ces derniers étant utilisés pour la détection et le diagnostic du cancer du poumon.
PCT/CN2019/108651 2018-09-29 2019-09-27 Réactif de détection de méthylation du hoxa9 WO2020063903A1 (fr)

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