WO2020063901A1 - Hoxa7和hoxa9甲基化检测试剂在制备肺癌诊断试剂中的用途 - Google Patents

Hoxa7和hoxa9甲基化检测试剂在制备肺癌诊断试剂中的用途 Download PDF

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WO2020063901A1
WO2020063901A1 PCT/CN2019/108648 CN2019108648W WO2020063901A1 WO 2020063901 A1 WO2020063901 A1 WO 2020063901A1 CN 2019108648 W CN2019108648 W CN 2019108648W WO 2020063901 A1 WO2020063901 A1 WO 2020063901A1
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seq
gene
hoxa7
detection
hoxa9
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牛智通
李仕良
黄龙武
赵荣淞
吴幽治
赵霞
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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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

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  • the present disclosure belongs to the field of gene diagnosis, and more particularly, the present disclosure relates to an application of human HOXA7 and human HOXA9 gene methylation detection reagents in the preparation of a diagnostic reagent for lung cancer, and a method of human HOXA7 gene and HOXA9 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 missed detection rate of lung cancer is generally 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.
  • Another object of the present disclosure is to provide a lung tumor marker capable of non-invasive detection.
  • Another object of the present disclosure is to provide a lung tumor marker that can reduce the rate of missed detection.
  • Another object of the present disclosure is to provide an application of the HOXA7 and HOXA9 genes or their nucleic acid fragments in the preparation of a tumor diagnostic reagent.
  • Another object of the present disclosure is to provide a tumor marker having high sensitivity and specificity for adenocarcinoma in a non-invasive manner.
  • Another object of the present disclosure is to provide a reagent / kit and method for detecting methylation of HOXA7 and HOXA9 genes.
  • An object of the present disclosure is to provide a lung tumor detection reagent / kit for a wide range of applications to lung cancer.
  • the present disclosure provides a HOXA7 gene or a nucleic acid fragment thereof, and an application of the HOXA9 gene or a nucleic acid fragment thereof in preparing a tumor diagnostic reagent.
  • the inventors revealed for the first time a method for simultaneously detecting the methylation of the HOXA7 gene and the HOXA9 gene or their nucleic acid fragments to improve the detection rate of lung cancer.
  • the combined methylation detection of HOXA7 and HOXA9 genes can achieve a non-invasive method that greatly improves the detection rate.
  • the missed detection rate is 0, which is the effect that no technology can compare to so far.
  • the methylated genes are human HOXA7 gene and human HOXA9 gene.
  • the inventors not only verified that the detection of HOXA7 and HOXA9 gene 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 in sputum samples and lavage fluid samples. Sex and sensitivity.
  • lung cancer markers such as CHRDL1, JAK1, p-EphB1, FABP1, p-LCK, SOST, p-ZAP70, BARD1, UHRF1, MiRNA-4731-3p, miRNA-6729-5p, AKAP4, ABCB1, AKT1, ALK, APC, ATIC, etc.
  • the effect of joint detection between markers is unexpected.
  • the combined detection of several tumor markers is more significant for the diagnosis of tumors.
  • the clinical detection of tumors is basically based on multi-marker combined detection for auxiliary diagnosis.
  • liver cancer uses AFP, CEA, free-b-hCG, CA199, CA242 combined detection
  • colorectal cancer uses CEA, CA242, CA199 combined detection .
  • HOXA7 and HOXA9 genes are members of the HOX (homebox homology box) gene family. They belong to the HOXA cluster gene on chromosome 7p15-p14. Like other HOX genes, they contain a 180bp DNA fragment and are transcribed by 60 amino acids. Composition of homologous domains.
  • 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.
  • HOXA9 is a coding sequence-specific transcriptional regulatory factor.
  • HOXA9 plays an important role in the development of spatiotemporal space in embryos, cell differentiation, proliferation, and migration, malignant evolution of tumors, and inducing apoptosis.
  • HOXA9 gene related to lung cancer, human glioma and other cancers are also reports.
  • HOXA7 and HOXA9 genes have been reported as a tumor marker for lung cancer.
  • the present disclosure detects lung cancer for the first time based on the HOXA7 and HOXA9 genes, or their nucleic acid fragments. When the two tests are combined, the detection rate of lung cancer is greatly improved.
  • the present disclosure provides application of detection reagents for methylation of HOXA7 and HOXA9 genes in the preparation of a diagnostic reagent for lung cancer.
  • the methylated detection reagent contains a non-invasive sample as a detection object, such as sputum, lung lavage fluid, tear fluid, or feces.
  • the detection of methylation is a diagnostic reagent for small cell carcinoma or adenocarcinoma; in particular, it is a diagnostic reagent for small cell carcinoma.
  • the detection reagent for methylation of the HOXA7 gene and the HOXA9 gene detects sequences modified by the transformation reagent of the HOXA7 and HOXA9 genes.
  • the conversion reagent refers to an agent that deaminates cytosine in DNA into uracil, and at the same time makes 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 and HOXA9 genes detects a bisulfite-modified sequence.
  • 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 Methylmion Specific PCR (MSP). Primers are designed for the methylated fragments after processing (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 Methylmion Specific PCR
  • the detection region of H targeted by the detection reagents for methylation of the HOXA7 and HOXA9 genes includes a CG-enriched region or a non-CG-enriched region or a CTCF (CTCF-binding sites) region of the gene.
  • the detection region of the reagent includes a CG-enriched region or a CTCF (CTCF-binding sites) region of a gene.
  • the detection region targeted by the detection reagents for methylation of the HOXA7 and HOXA9 genes includes a gene body or a promoter region thereof.
  • the detection region of the methylated detection reagent for HOXA7 includes the sequence shown in SEQ ID NO: 25 or SEQ ID ID: 27; the detection region of the reagent for HOXA9 includes SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33.
  • the detection region of the reagent for HOXA7 includes the sequence shown in SEQ ID NO: 27, and the detection region of the reagent for HOXA9 includes the sequence shown in SEQ ID NO: 29.
  • the methylation detection reagent of the present disclosure further contains a primer pair for detecting the HOXA7 gene and the HOXA9 gene.
  • the primer pair for detecting the HOXA7 gene is selected from the group consisting of SEQ ID NO: 1-2, SEQ ID NO: 37-38, SEQ ID NO: 40-41, SEQ ID NO: 43-44, or SEQ ID
  • the primer pair for detecting HOXA9 gene is selected from SEQ ID NO: 4-5, SEQ ID NO: 64-65, SEQ ID NO: 67-68, SEQ ID NO: 70-71, SEQ ID NO: 73-74, SEQ ID NO: 76-77, SEQ ID NO: 79-80, SEQ ID NO: 82-83, SEQ ID
  • the primer pair for detecting the HOXA7 gene is selected from the primer pair shown in SEQ ID NO: 1-2; the primer pair for detecting the HOXA9 gene is selected from the primer pair shown in SEQ ID NO: 4-5.
  • the methylation detection reagent of the present disclosure further contains a probe for detecting the HOXA7 gene and the HOXA9 gene.
  • the probe for detecting the HOXA7 gene is selected from SEQ ID NO: 3, SEQ ID NO: 39, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 51 SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 60 or SEQ ID NO: 63;
  • the probe for detecting the HOXA9 gene is selected from SEQ ID NO: 6, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 84, SEQ ID NO: 87, SEQ ID NO: 90, or SEQ ID NO : Sequence shown by 93.
  • the probe for detecting the HOXA7 gene is selected from the sequence shown in SEQ ID NO: 3; the probe for detecting the HOXA9 gene is selected from the sequence shown in SEQ ID NO: 6.
  • the fluorescent group labeled by the detection probe may be VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, Texas Red, etc .; the quenching group may be 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 methylated detection reagent of the present disclosure contains the primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2; the primer pairs shown in SEQ ID NO: 4 and SEQ ID NO: 5; The probes are shown in SEQ ID NO: 3 and SEQ ID ID NO: 6.
  • the detection of methylation in the present disclosure further includes a detection reagent for an internal reference gene.
  • the internal reference gene comprises ⁇ -actin or COL2A1.
  • 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: 19, SEQ ID NO: 20, and a probe of SEQ ID NO: 21.
  • the detection reagent for the internal reference gene COL2A1 contains a primer pair represented by SEQ ID NO: 94 (TTTTGGATTTAAGGGGAAGATAAA), a primer pair represented by SEQ ID NO: 95 (TTTTTCCTTCTCTCTACATCTTTCTACCT), and a probe represented by SEQ ID ID NO: 96 (AAGGGAAATTGAGAAATGAGAGAGAAGGGA).
  • the methylated detection reagent of the present disclosure further comprises bisulfite, bisulfite or hydrazine salt for modifying methylated cytosine to thymine.
  • bisulfite, bisulfite or hydrazine salt 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 methylation detection reagent of the present disclosure further comprises one or more of a DNA polymerase, dNTPs, Mg 2+ ions, and a buffer solution; preferably, it includes a DNA polymerase, dNTPs, Mg 2+ ions and buffers for amplification of HOXA7 and HOXA9 genes
  • the detection sample of the methylated detection reagent includes sputum, lung lavage fluid, lung tissue, pleural fluid, blood, serum, plasma, urine, prostate fluid, tear fluid, or feces, etc. Wait.
  • the detection sample includes sputum, lung tissue, or lung lavage fluid.
  • the detection sample includes sputum or lung lavage fluid.
  • the present disclosure also provides a method for detecting DNA methylation of the HOXA7 gene and the HOXA9 gene, including 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
  • the present disclosure also provides a diagnostic system for lung cancer, the system including:
  • the DNA methylation detection member of the HOXA7 gene and the HOXA9 gene includes the above-mentioned methylation detection reagent.
  • the methylation detection member includes one or more of a quantitative PCR instrument, a PCR instrument, and a sequencer.
  • the result judging component is used to detect DNA methylation levels of the HOXA7 gene and the HOXA9 gene according to a detection component, and output a diagnosis result such as a risk of lung cancer and / or a type of lung cancer.
  • the risk of disease is obtained by comparing a methylation level of a sample to be tested with a normal sample through 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 HOXA7 gene of the sputum sample to be tested is greater than 0.77% or the methylation level of the HOXA9 gene is greater than 2.3%, the sample to be tested is a lung cancer sample.
  • the sample to be tested is determined to be a non-lung cancer sample.
  • the test sample when the methylation level of the HOXA7 gene of the test lavage fluid sample is greater than 0.7% or the methylation level of the HOXA9 gene is greater than 0.5%, the test sample is determined to be a lung cancer sample. If the methylation level of the HOXA7 gene is less than or equal to 0.7% and the methylation level of the HOXA9 gene is less than or equal to 0.5%, the sample to be tested is a 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 lung cancer is selected from small cell lung cancer (SCL) or non-small cell lung cancer (NSCL); 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 small cell lung cancer or adenocarcinoma.
  • methylation of the HOXA7 and HOXA9 genes has been reported to be methylated in lung cancer, respectively.
  • the present disclosure is the first to combine the HOXA7 and HOXA9 genes as the tumor markers for lung cancer. Increased the detection rate of lung cancer.
  • HOXA7 and HOXA9 genes for lung cancer were 95%, and the sensitivity of HOXA7 and HOXA9 genes for lung cancer was 88.6% and 74.3%, respectively.
  • the sensitivity to all lung cancers is increased to 97.1%.
  • high sensitivity is very rare in existing lung cancer markers.
  • high sensitivity is critical.
  • the sensitivity reported so far is generally around 45% -75%.
  • the combined detection of HOXA7 gene and HOXA9 gene has high specificity and sensitivity for different types of lung cancer, including squamous cell carcinoma, large cell carcinoma, and adenocarcinoma in small cell lung cancer and non-small cell lung cancer. It can be used as a tumor marker for almost all lung cancers.
  • the existing lung cancer markers for clinical use are generally only applicable to the detection of one type of lung cancer, such as NSE for the diagnosis and monitoring of response to small cell lung cancer, and CYFRA21-1 is the first choice marker for non-small cell lung cancer. .
  • lung adenocarcinoma In lung adenocarcinoma, the combined detection of HOXA7 and HOXA9 genes has a particularly improved sensitivity compared to other tumor markers, reaching 100% sensitivity. 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.
  • the inventors have found through experiments that the combined detection of HOXA7 and HOXA9 genes in sputum has a detection sensitivity of 100% for adenocarcinoma, which is already the highest level of sensitivity in the field of adenocarcinoma detection (for comparison, SHOX2 Gene sensitivity to adenocarcinoma 33.3%).
  • Existing tumor markers have high sensitivity to adenocarcinoma in tissues, but when sputum is used as a detection sample, the sensitivity is greatly reduced, and the role of tumor markers is lost.
  • the SHOX2 gene has a sensitivity of 79.0% to adenocarcinoma in tissue samples, which is the gene most sensitive to adenocarcinoma among the tumor markers studied in this disclosure. 33.3%.
  • the sensitivity of the combined detection of HOXA7 and HOXA9 genes to adenocarcinoma is also as high as 81.8%, which is even higher than its sensitivity in tissue samples, which is very rare.
  • the combined detection of HOXA7 and HOXA9 genes has outstanding advantages, and the preferred test samples are sputum and lung lavage fluid.
  • sputum and lung lavage fluid as detection samples, it has a more prominent sensitivity than other markers.
  • the detection sensitivity for squamous cell carcinoma, adenocarcinoma, and small cell cancer is all When it reaches 100%, it is helpful for timely detection of patient abnormalities, and further combined with other detection methods to determine whether it is adenocarcinoma, and it greatly reduces the ease of clinical tumor screening.
  • the non-invasive acquisition of lung lavage and sputum samples makes the combined detection of HOXA7 and HOXA9 extremely useful for the detection of adenocarcinoma and other types of lung cancer.
  • tissue can be used as a detection sample, but also prominent, it also has high sensitivity in sputum and lung lavage fluid.
  • sputum The sensitivity of liquid and lung lavage fluid is even higher than that of tissues. Therefore, sputum and lung lavage fluid can be simply used as test samples to reliably diagnose lung cancer. Sputum and lung lavage samples are easily obtained without causing 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 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 technical solution of the above technical solution has the beneficial effects that it 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 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.
  • Figure 4 Amplification curves of HOXA7, HOXA9, and SHOX2_n3 in sputum samples (A is the amplification map of HOXA7, B is the amplification map of HOXA9, and C is the amplification map of SHOX2_n3);
  • FIG. 6 Amplification curves of HOXA7, HOXA9, and SHOX2_n3 in lavage fluid samples (A is the amplification map of HOXA7, B is the amplification map of HOXA9, and C 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 in which 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 the target molecule's at least x consecutive or discontinuous block nucleotides 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% 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 markers HOXA7 and 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 lung cancer-specific genes HOXA7 and HOXA9 in normal-represented cells.
  • early diagnosis refers to the possibility of discovering cancer before metastasis, preferably before morphological changes of tissues or cells can be observed.
  • diagnosis can be made by measuring the degree of methylation of HOXA7 and HOXA9 obtained from a sample during the progression of lung cancer at different stages or stages.
  • degree of methylation of the HOXA7 and HOXA9 genes of nucleic acids isolated from samples from each stage of lung cancer with HOXA7 of one or more nucleic acids isolated from samples in lung tissue without abnormal cell proliferation
  • the degree of methylation of genes and HOXA9 genes can detect specific stages of lung cancer in samples.
  • 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 represent the methylation level; and other methods of representing methylation levels 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 methylation sites of DNA are located in the gene. Specific parts, usually in the promoter region, make detection easier and more convenient.
  • the inventors screened a large number of genes, selected representative HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 as candidate detection genes, and ⁇ -actin gene as an internal reference gene to study the methylation sites of each gene 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:
  • HOXA9 detection primers and probes are:
  • SHOX2 detection primers and probes are:
  • SEQ ID NO: 7 SHOX2 Primer F: TTTAAAGGGTTCGTCGTTTAAGTC
  • SEQ ID NO: 9 SHOX2 probe: FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1
  • the detection primers and probes for PCDHGA12 are:
  • HOXD8's detection primers and probes are:
  • GATA3's detection primers and probes are:
  • GATA3 primer F TTTCGGTAGCGGGTATTGC
  • SEQ ID NO: 18 GATA3 probe FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1
  • the detection primers and probes for ⁇ -actin are:
  • SEQ ID NO: 19 ⁇ -actin primer F TTTTGGATTGTGAATTTGTG
  • SEQ ID NO: 20 ⁇ -actin primer R AAAACCTACTCCTCCCTTAAA
  • 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 detected by HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3, and HOXA7 and HOXA9 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.
  • HOXA7, HOXA9, and HOXD8 which are both of the HOXA family, are combined in pairs, only the combination of HOXA7 & HOXA9, whether in all cancers, squamous cell carcinomas, or lung cancers that are more difficult to detect. Higher sensitivity than other combinations, while combinations of other HOXA families have no significant enhancement.
  • the inventors further screened the six markers HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 in sputum.
  • Example 2 Detection of HOXA7, HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3, and the combination of HOXA7 and HOXA9 in sputum
  • 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 in the 35 cancer group, 6 were small cell carcinoma, 9 were adenocarcinoma, and large cell There were 2 cases of cancer and 6 cases of lung cancer without a clear classification.
  • the dosing system is as follows:
  • the amplification system is as follows:
  • the combination of HOXA7 and HOXA9 has a better detection rate than other genes, and the sensitivity even reaches 100%.
  • Such a high sensitivity can basically achieve zero missed detection of early screening for adenocarcinoma. Rate, so that patients can get accurate risk tips, early treatment, and reduce mortality from adenocarcinoma.
  • the existing tumor markers in lung cancer have a sensitivity of only 14.3% to adenocarcinoma, and the sensitivity of adenocarcinoma to HOXA7 or HOXA9 alone is only 88.9%. The sensitivity is increased to 100%, and the two are synergistic.
  • Adenocarcinoma is generally of the peripheral type. Due to the tree-like physiological structure of the bronchus, exfoliated cells in the deep lungs are more difficult to cough through sputum, so the detection of this part is more difficult and meaningful.
  • SHOX2 can be used as a marker for detecting lung cancer
  • patents that show that [CN201510203539 method and kit for diagnosing human SHOX2 gene and human RASSF1A gene methylation-application publication], SHOX2 in alveolar lavage fluid, lesion tissue , Pleural fluid, sputum and other samples have a higher detection rate.
  • the inventors In order to verify the detection effect of HOXA7 and HOXA9, the inventors simultaneously detected HOXA7 and HOXA9SHOX2_n3 genes.
  • 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 self-designed primers and probes used in Examples 1 and 2 of the present disclosure. Needle detection of the SHOX2 gene.
  • the primer probes for each gene are as follows:
  • HOXA7 detection primers and probes are:
  • HOXA9 detection primers and probes are:
  • HOXA9-P2 probe FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1SHOX2_n3
  • the detection primers and probes are:
  • SEQ ID NO: 22 SHOX2_n3 Primer F: TTTGGATAGTTAGGTAATTTTCG
  • the dosing system is as follows:
  • the amplification system is as follows:
  • the threshold value of HOXA7 is 0.77.
  • the threshold value of HOXA9 is 2.3, and the threshold value of SHOX2_n3 is 1.3.
  • the ratio after conversion exceeds the set threshold value can be judged as positive “+”, and equal to or less than the set threshold value can be judged as negative “- ".
  • the test results of 90 sputum samples were as follows:
  • the positive detection rate was 97.1%, and for squamous cell carcinoma, adenocarcinoma, and small cell cancer, the detection sensitivity reached 100%. In many types of lung cancer, such high sensitivity can be achieved, and when used in clinical detection, it is undoubtedly more versatile.
  • the sensitivity of HOXA7 and HOXA9 in detecting adenocarcinoma is as high as 100%, while the sensitivity of existing primers and probes for SHOX2 detection to adenocarcinoma is only 33.3%.
  • Adenocarcinoma is generally of the peripheral type.
  • a sputum can be used as a sample to detect adenocarcinoma, and the sensitivity can be improved. A significant increase to 100% markers, this breakthrough is of 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 HOXA9 is 0.5, and the threshold value of SHOX2_n3 is 0.6.
  • the converted ratio exceeding the set threshold value can be judged as a positive "+", and equal to or less than the set threshold value can be judged as a negative "- ".
  • the test results of 79 lavage fluid samples were as follows:
  • the ROC curve detected by HOXA7 and HOXA9 in combination with SHOX2_n3 in all lavage fluid samples is shown in Figure 5, and the amplification curves of HOXA7, HOXA9 and SHOX2_n3 in lavage fluid samples are shown in Figure 6, and the statistical results are shown in Table 16.
  • the results show that, in the lavage fluid sample, whether the comparative analysis of lung cancer as a whole, or the comparative analysis of lung cancer subtypes, the detection effect of HOXA7 and HOXA9 is better than that of the SHOX2 gene.
  • the combined detection of samples with HOXA9 can effectively improve the positive detection rate. In all samples, the positive detection rate is 85.7%.
  • the sensitivity of HOXA7 and HOXA9 to detect adenocarcinoma is as high as 81.8%, much higher than 36.4% of SHOX2, and also higher than the sensitivity of HOXA7 and HOXA9 genes to adenocarcinoma in tissues. This is Very rare and rare.
  • Adenocarcinoma is generally of the peripheral type. Due to the tree-like physiological structure of the bronchus, exfoliated cells in the deep lungs are more difficult to cough through sputum, so the detection of this part is more difficult and meaningful.
  • the sensitivity to small cell carcinoma reached 100%, while whether it was HOXA7 or HOXA9 or SHOX2, its sensitivity to small cell carcinoma was only 75%. Such a zero miss rate is of great significance for the early screening of small cell carcinoma.
  • HOXA7 and HOXA9 has better detection effect on the detection and diagnosis of lung cancer, especially the application of biological samples such as sputum and alveolar lavage fluid. Can be more easily applied to large-scale population screening. It has more superior social economic value.
  • group 8, group 9, and group 10 are methylated primers and probes designed according to region 1; group 1, group 2, group 3, group 4, group 5, group 6, and group 7 are based on region 2 Designed methylation primers and probes.
  • group 1, group 2, group 3, group 4 and group 5 are methylated primers and probes designed according to region 1; group 6, group 7, and group 8 are methylated primers designed according to region 2 And probes; groups 9, 10 and 11 are methylated primers and probes designed according to region 3.
  • the following 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 carcinomas, and 21 were adenocarcinomas in the 25 cancer group samples.
  • the test results are shown in Table 18 below.
  • HOXA9 no matter how primers and probes are designed for regions 2 and 3, the detection sensitivity for these two regions can only reach a maximum of 32%, and regardless of the primers and probes designed by the present disclosure, region 1 has the lowest detection sensitivity Can also reach 40%, up to 76%. Therefore, the detection rate of HOXA7 area 1 is significantly higher than that of 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 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: 37 GCGTTTGCGATAAGACGGAC HOXA7 gene upstream primer H7-R3 SEQ ID NO: 38 CCAACCTAACCCGCCTAACG HOXA7 gene downstream primer H7-P3 SEQ ID NO: 39 FAM-CGTTGTATGGCGCGGTTGAGG-BQ1 HOXA7 gene detection probe H7-F4 SEQ ID NO: 40 CGTTCGGTTACGGTTTGGGC HOXA7 gene upstream primer H7-R4 SEQ ID NO: 41 GCCCTCGTCCGTCTTATC
  • H7-P11 SEQ ID NO: 63 FAM-CGGATTTTTTGGTCGTATATTTGAGT-BQ1 HOXA7 gene detection probe
  • H7-F2, H7-R2, H7-P2 100% 80%
  • 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.
  • HOXA7 had better detection rates in groups 1, 2, 4, 5, and 6.
  • HOXA9 has a good detection rate in group 1, group 2, group 4, and group 5.
  • 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 H9-F2 SEQ ID NO: 4 TTAGTTTTTTCGGTAGGCGGC HOXA9 gene upstream primer H9-R2 SEQ ID NO: 5 AAACGCCAAACACCGTCG HOXA9 gene downstream primer H9-P2 SEQ ID NO: 6 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9 gene detection probe A3-TqMF SEQ ID NO: 19 TTTTGGATTGTGAATTTGTG ⁇ -actin gene upstream primer A3-TqMR SEQ ID NO: 20 AAAACCTACTCCTC

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Abstract

一种以痰液为检测样本，且以HOXA7和HOXA9的甲基化为检测对象的检测试剂在制备肺癌诊断试剂中的应用。首次在痰液中，共同以HOXA7和HOXA9作为检测肺癌的标志物，其在痰液中的敏感度高达97.1％，特异性高达90.9％，检测敏感度高于现有的肺癌标志物，尤其是对腺癌，敏感度甚至达到了100％，对于腺癌的检测有极大的应用价值。

Description

HOXA7和HOXA9甲基化检测试剂在制备肺癌诊断试剂中的用途 技术领域
本公开属于基因诊断领域，更具体地，本公开涉及一种人HOXA7和人HOXA9基因甲基化检测试剂在制备肺癌诊断试剂中的应用，以及人HOXA7基因和HOXA9基因甲基化检测的方法。
技术背景
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。
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。有条件的医院在肺癌病人行胸部CT扫描时范围应包括肾上腺。应尽量采用增强扫描，尤其是肺中心型病变的患者。CT是显示脑转移瘤的基本检查方法，有临床症状者或进展期病人应行脑CT扫描，并尽可能采用增强扫描。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。(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，在有适应症的情况下，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。
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 profile 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％。
目前肺癌的漏检率普遍较高，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。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。
发明内容
本公开的目的在于提供肺肿瘤标记物。
本公开的另一个目的在于提供一种可进行无创检测的肺肿瘤标记物。
本公开的另一个目的在于提供一种可降低漏检率的肺肿瘤标记物
本公开的另一个目的在于提供一种HOXA7和HOXA9基因或它们的核酸片段在制备肿瘤诊断试剂的应用。
本公开的另一个目的还在于提供一种在无创中，针对腺癌具有高敏感性和特异性的肿瘤标志物。
本公开的另一个目的还在于提供一种检测HOXA7和HOXA9基因甲基化的试剂/试剂盒和方法。
本公开的目的还在于提供一种特异性强、敏感度高的肺肿瘤检测试剂/试剂盒。
本公开的目的还在于提供一种对肺癌应用范围广的肺肿瘤检测试剂/试剂盒。
本公开提供了如下方案：
一方面，本公开提供了HOXA7基因或其核酸片段，以及HOXA9基因或其核酸片段在制备肿瘤诊断试剂中的应用。
发明人经过深入的研究，首次揭示一种同时检测HOXA7基因和HOXA9基因或它们的核酸片段的甲基化来提高肺癌检出率的方法。尤其是首次揭示了HOXA7基因和HOXA9基因的联合甲基化检测可以实现无创的、极大提高检出率的方法。尤其是对于小细胞癌来说其漏检率为0，这是截至目前从未有过任何技术可以比拟的效果。
所述甲基化基因是人HOXA7基因和人HOXA9基因。发明人不仅在组织样本中验证了检测HOXA7和HOXA9基因甲基化对肺癌的检出具有较高的特异性和灵敏性，同时验证了在痰液样本和灌洗液样本中具有同样高的特异性和灵敏性。
已知的肺癌标志物有非常多种，如CHRDL1，JAK1，p-EphB1，FABP1，p-LCK，SOST，p-ZAP70，BARD1，UHRF1，MiRNA-4731-3p，miRNA-6729-5p，AKAP4，ABCB1、AKT1、ALK、APC、ATIC等等。标志物之间的联合检测效果是不可预期的。因为大部分肿瘤标志物对肿瘤的诊断仅有相关性，而无特异性，所以数种肿瘤标志物联合检测对肿瘤的诊断意义更大。目前临床上对肿瘤的检测基本都采用多标志物联合检测进行辅助诊断，例如肝癌采用AFP、CEA、free-b-hCG、CA199、CA242联合检测；结直肠癌采用CEA、CA242、CA199等联合检测。
HOXA7和HOXA9基因均是HOX(homebox同源盒)基因家族的一员，属于染色体7p15-p14上的HOXA簇基因，与其他的HOX基因一样，均含有一段180bp的DNA片段，转录由60个氨基酸组成的同源结构域。HOXA7在正常造血细胞的增殖分化中发挥调节作用，目前研究比较多的是HOXA7的异常表达在白血病的发生和发展中起着重要的作用，也有部分报道HOXA7基因的甲基化与肺癌相关。HOXA9是编码序列特异性转录调控因子，在胚胎的时空发育，细胞的分化、增殖与迁移，肿瘤的恶性演变和诱发凋亡都有着重要作用。目前研究比较多的是HOXA9的异常表达在白血病的发生和发展中起着重要的作用，也有部分报道HOXA9基因的肺癌、人细胞胶质瘤等癌症相关。
目前，尚未有报道将HOXA7和HOXA9基因联合作为肺癌的肿瘤标志物。本公开首次基于HOXA7和HOXA9基因，或它们核酸片段，检测肺癌。二者联合检测时，极大的提高肺癌的检出率。
另一方面，本公开提供了HOXA7和HOXA9基因甲基化的检测试剂在制备肺癌诊断试剂中的应用。
作为优选的实施方式，所述甲基化的检测试剂含有并以无创式的样本为检测对象，例如，痰液、肺部灌洗液、泪液或粪便等等。
作为优选的实施方式，本公开提供的甲基化的检测为小细胞癌或腺癌的诊断试剂；尤其地，为小细胞癌的诊断试剂。
进一步地，所述HOXA7基因和HOXA9基因甲基化的检测试剂检测HOXA7和HOXA9基因经转化试剂修饰后的序列。其中，转 化试剂是指使DNA中胞嘧啶脱氨基成为尿嘧啶，同时使5-MeC基本上不受影响的试剂。示例性的转化试剂包括肼盐、重亚硫酸氢盐(例如重亚硫酸氢钠等)、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)、或在适当的反应条件下可产生肼盐、重亚硫酸氢盐、亚硫酸氢盐的化合物中的一种或几种。作为一种示范性的实施方式，所述HOXA7和HOXA9基因甲基化的检测试剂检测经重亚硫酸氢盐修饰后的序列。
甲基化的发生是胞嘧啶上多了一个甲基，经过亚硫酸氢盐或重亚硫酸氢盐或肼盐处理后，胞嘧啶会变成脲嘧啶，因为在进行PCR扩增时尿嘧啶与胸腺嘧啶相似而会被识别为胸腺嘧啶，体现在PCR扩增序列上就是没有发生甲基化的胞嘧啶变成了胸腺嘧啶(C变成T)，甲基化的胞嘧啶(C)则不会发生变化。PCR检测甲基化基因的技术通常为甲基化特异性PCR(Methylmion Specific PCR，MSP)，针对处理后的甲基化片段(即片段中未改变的C)设计引物，进行PCR扩增，如果有扩增则说明发生了甲基化，无扩增则没有甲基化。
作为可选的实施方式，HOXA7和HOXA9基因甲基化的检测试剂所针对H的检测区域包含基因的CG富集区域或非CG富集区域或CTCF(CTCF-binding sites)区域。作为优选的实施方式，所述试剂的检测区域包含基因的CG富集区域或CTCF(CTCF-binding sites)区域。
或者，HOXA7和HOXA9基因甲基化的检测试剂所针对的检测区域包含基因体(gene body)或者其启动子区域。
作为本公开中优选的实施方式，所述甲基化的检测试剂针对HOXA7的检测区域包含SEQ ID NO：25或SEQ ID NO：27所示的序列；所述试剂针对HOXA9的检测区域包含SEQ ID NO：29、SEQ ID NO：31或SEQ ID NO：33所示的序列。作为更优选的实施方式，所述试剂针对HOXA7的检测区域包含SEQ ID NO：27所示的序列，所述试剂针对HOXA9的检测区域包含SEQ ID NO：29所示的序列。
本公开甲基化的检测试剂，还含有检测HOXA7基因和HOXA9基因的引物对。作为可选的实施方式，检测HOXA7基因的引物对选自SEQ ID NO：1-2，SEQ ID NO：37-38，SEQ ID NO：40-41，SEQ ID NO：43-44，或SEQ ID NO：46-47，SEQ ID NO：49-50，SEQ ID NO：52-53，SEQ ID NO：55-56，SEQ ID NO：58-59或SEQ ID NO：61-62所示的引物对；检测HOXA9基因的引物对选自SEQ ID NO：4-5，SEQ ID NO：64-65，SEQ ID NO：67-68，SEQ ID NO：70-71，SEQ ID NO：73-74，SEQ ID NO：76-77，SEQ ID NO：79-80，SEQ ID NO：82-83，SEQ ID NO：85-86，SEQ ID NO：88-89或SEQ ID NO：91-92所示的引物对。
作为优选的实施方式，检测HOXA7基因的引物对选自SEQ ID NO：1-2所示的引物对；检测HOXA9基因的引物对选自SEQ ID NO：4-5所示的引物对。
本公开甲基化的检测试剂，还含有检测HOXA7基因和HOXA9基因的探针。作为可选的实施方式，检测HOXA7基因的探针选自SEQ ID NO：3、SEQ ID NO：39、SEQ ID NO：42、SEQ ID NO：45、SEQ ID NO：48、SEQ ID NO：51、SEQ ID NO：54、SEQ ID NO：57、SEQ ID NO：60或SEQ ID NO：63所示的序列；检测HOXA9基因的探针选自SEQ ID NO：6、SEQ ID NO：66、SEQ ID NO：69、SEQ ID NO：72、SEQ ID NO：75、SEQ ID NO：78、SEQ ID NO：81、SEQ ID NO：84、SEQ ID NO：87、SEQ ID NO：90或SEQ ID NO：93所示的序列。作为优选的实施方式，检测HOXA7基因的探针选自SEQ ID NO：3所示的序列；检测HOXA9基因的探针选自SEQ ID NO：6所示的序列。作为本公开的优选方式，为方便临床使用，检测探针标记的荧光基团可以是VIC、ROX、FAM、Cy5、HEX、TET、JOE、NED、Texas Red等；淬灭基团可以是TAMRA、BHQ、MGB、Dabcyl等等，以适用于目前临床检测常用的多通道PCR检测系统，实现在一个反应管中进行多色荧光检测。
作为优选的实施方式，本公开甲基化的检测试剂含有SEQ ID NO：1和SEQ ID NO：2所示引物对、SEQ ID NO：4和SEQ ID NO：5所示的引物对，以及如SEQ ID NO：3所示和SEQ ID NO：6所示的探针。
作为可选的实施方式，本公开甲基化的检测还含有内参基因的检测试剂。
优选地，所述内参基因包含β-actin或COL2A1，除了这两个内参基因，也可以采用现有技术的其他的甲基化检测的内参基因。进一步的，所述内参基因的检测试剂包含针对内参基因的引物和探针。作为优选的实施方式，所述内参基因β-actin的检测试剂包含SEQ ID NO：19、SEQ ID NO：20所示的引物对，以及SEQ ID NO：21的探针。所述内参基因COL2A1的检测试剂含有SEQ ID NO：94(TTTTGGATTTAAGGGGAAGATAAA)、SEQ ID NO：95(TTTTTCCTTCTCTACATCTTTCTACCT)所示的引物对，以及SEQ ID NO：96(AAGGGAAATTGAGAAATGAGAGAAGGGA)所示的探针。
作为可选的实施方式，本公开甲基化的检测试剂还包含亚硫酸氢盐、重亚硫酸氢盐或肼盐，用于将甲基化的胞嘧啶修饰成胸腺嘧啶。当然，也可以不包含在本公开的试剂中，使用时独立购买即可。
作为可选的实施方式，本公开甲基化的检测试剂还包含DNA聚合酶、dNTPs、Mg 2+离子、缓冲液中的一种或几种；优选地，包含有DNA聚合酶、dNTPs、Mg 2+离子和缓冲液，用于对HOXA7和HOXA9基因进行扩增反应。
在优选的实施方案中，所述甲基化的检测试剂的检测样本包含痰液、肺部灌洗液、肺部组织、胸水，血液、血清、血浆、尿液、前列腺液、泪液或粪便等等。作为优选的实施方式，所述检测样本包含痰液、肺部组织或肺部灌洗液。作为更优选的实施方式，所述检测样本包含痰液或肺部灌洗液。
另一方面，本公开还提供了一种检测HOXA7基因和HOXA9基因的DNA甲基化的方法，包括以下步骤：
(1)将检测样本进行亚硫酸氢盐或重亚硫酸氢盐或肼盐处理，获得经修饰的检测样本；
(2)利用上述甲基化的检测或试剂盒对步骤(1)经修饰的检测样本进行测HOXA7基因和HOXA9基因甲基化情况检测；
作为可选的方式，采用甲基化特异性聚合酶链反应(MSP)或实时荧光定量甲基化特异性聚合酶链反应(real-time fluorescent quantitative methylation-specific PCR,qMSP)进行检测。其余现有技术中报道的DNA甲基化检测方法也可以应用于本公开中。现有技术的甲基化检测方法可通过专利USSN62/175,916引入本公开。
另一方面，本公开还提供了一种肺癌的诊断系统，所述系统包含：
a.HOXA7基因和HOXA9基因的DNA甲基化检测构件，以及，
b.结果判断构件；
在一些实施方式中，所述HOXA7基因和HOXA9基因的DNA甲基化检测构件包含上述甲基化的检测试剂。
在一些实施方式中，所述甲基化检测构件包含荧光定量PCR仪、PCR仪、测序仪中的一种或多种。
在一些实施方式中，所述结果判断构件用于根据检测构件检测HOXA7基因和HOXA9基因的DNA甲基化水平，输出诊断结果如肺癌的患病风险和/或肺癌类型等。
在一些实施方式中，所述患病风险是通过结果判断构件比较待测样本与正常样本的甲基化水平，基于待测样本与正常样本的甲基化水平的偏离得出的。
在一些实施方式中，所述结果判断构件包含数据处理机器。
在一些实施方式中，所述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。
在一些实施方式中，所述数据处理机器包含计算器、计算机中的一种或多种。所述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。在一些实施方式中，所述计算机包含附载有SPSS、SAS、Excel中一种或多种软件的计算机。
在一些实施方式中，所述结果判断构件还包含结果输出器。所述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。在一些实施方式中，所述结果输出器包含屏幕、纸质报告中的一种或多种。
本公开另一方面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HOXA7基因和HOXA9基因的甲基化水平；
(2)将待测样本与正常样本的HOXA7基因和HOXA9基因水平比较
(3)基于待测样本与正常样本的甲基化水平的偏离，诊断肺癌；
在一些实施方式中，当所述待测痰液样本HOXA7基因的甲基化水平大于0.77％或HOXA9基因的甲基化水平大于2.3％时，则判定待测样本为肺癌样本，若待测痰液样本HOXA7基因的甲基化水平小于等于0.77％且HOXA9基因的甲基化水平小于等于2.3％时，则判定待测样本为非肺癌样本。
在一些实施方式种，当所述待测灌洗液样本HOXA7基因的甲基化水平大于0.7％或HOXA9基因的甲基化水平大于0.5％时，则判定待测样本为肺癌样本。若待测灌洗液样本HOXA7基因的甲基化水平小于等于0.7％且HOXA9基因的甲基化水平小于等于0.5％时，则判定待测样本为非肺癌样本。
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)。
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。
本公开中，所述肺癌选自小细胞肺癌(small cell lung cancer，SCL)或非小细胞肺癌(non-small cell lung cancer,NSCL)；进一步地，所述非小细胞肺癌选自鳞状细胞癌、腺癌或大细胞癌。作为优选的实施方式，所述肺癌选自小细胞肺癌或腺癌。
虽然，现有技术中，HOXA7和HOXA9基因的甲基化分别被报道了在肺癌中有甲基化。然而，有关肺癌的肿瘤标志物的报道，不计其数，本公开在众多被报道的，跟肺癌可能相关甲基化基因中，首次将HOXA7和HOXA9基因联合，作为肺癌的肿瘤标志物，很大程度地提高了肺癌检出率。
针对痰液样本，HOXA7和HOXA9基因的单独检测对全部肺癌的特异性达到了95％，HOXA7基因和HOXA9基因单独检测对肺癌的敏感性分别为88.6％和74.3％。通过联合HOXA7和HOXA9检测，对全部肺癌的敏感性提升到97.1％，如此高的灵敏度在现有的肺癌标志物是非常少见的。而在临床中，作为早期筛查的工具，高的灵敏度是非常关键的。目前肺癌早期筛查，尤其是无创筛查领域的技术严重缺失，最主要的原因就是缺少具有高的灵敏度的检测方法。目前已报道的灵敏度一般在45％-75％左右。
此外，HOXA7基因和HOXA9基因联合检测针对不同类型的肺癌，包括小细胞肺癌和非小细胞肺癌中的鳞癌、大细胞癌、腺癌均具有很高的特异性和灵敏度，其适用范围广，基本上能作为所有肺癌的肿瘤标志物。而现有的用于临床的肺癌标志物，其一般仅能适用于一类肺癌的检测，如NSE用于小细胞肺癌的诊断和监测治疗反应，而CYFRA21-1是非小细胞肺癌的首选标记物。
在小细胞癌中，不管是在痰液中还是在灌洗液样本的检测中，采用HOXA7基因和HOXA9基因联合检测的无创筛查方式，竟都获得了100％的灵敏度。这样的0漏检率在本领域是一个巨大的突破。
在肺腺癌中，HOXA7基因和HOXA9基因联合检测比其他肿瘤标志物具有尤其提高的灵敏度，达到100％的灵敏度。目前，肺腺癌的漏检率较高。一方面，由于肺腺癌较容易发生于女性及不抽烟者，发病率比鳞癌和未分化癌低，发病年龄较小，女性相对多见；另一方面，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；再一方面，肺腺癌早期一般没有明显的临床症状。因此，对肺腺癌的检测更具困难性和价值性。
发明人经实验发现，在痰液中，HOXA7和HOXA9基因联合检测，对腺癌的检测灵敏度达到了100％，在腺癌检测领域已经是目前绝无仅有的极高灵敏度的水平了(作为对比，SHOX2基因对腺癌灵敏度33.3％)。现有的肿瘤标志物，即便是在组织中对腺癌有很高的灵敏度，但是当以痰液为检测样本时，灵敏度却大幅下降，失去肿瘤标志物的作用。如SHOX2基因，在组织样本中，对腺癌灵敏度79.0％，为本公开研究的肿瘤标志物中对腺癌灵敏度最高的基因，但是在痰液样本中，SHOX2基因对腺癌灵敏度大幅度下降到33.3％。
在肺部灌洗液中，HOXA7和HOXA9基因联合检测对腺癌的敏感度也高达81.8％，该灵敏度甚至高于其在组织样本中的灵敏度，这是十分难得的。
因此，对于腺癌来说，HOXA7和HOXA9基因联合检测具有突出的优势，且优选的检测样本为痰液和肺部灌洗液。以痰液和肺部灌洗液作为检测样本，其具有相对比其他标志物更为突出的灵敏度，尤其是以痰液作为检测样本时，对于鳞癌、腺癌和小细胞癌的检测灵敏度均到了100％，有利于及时发现患者异常，进一步结合其他检测手段确诊是否为腺癌，且极大的降低了临床上肿瘤筛查的简便性，无需针对特定类型的肺癌使用特定的肿瘤标志物，再者，肺部灌洗液和痰液样品的获取无创，使得HOXA7和HOXA9联合检测对于腺癌等多种类型的肺癌的检测来说具有极大的应用意义。
上述技术方案中的一个技术方案的有益效果为：不仅可以以组织作为检测样本，且突出的，其在痰液和肺部灌洗液中也具有很高的灵敏度，对于某些肺癌类型，痰液和肺部灌洗液的灵敏度甚至高于组织，因此，可以简便地以痰液和肺部灌洗液作为检测样本，对肺癌进行可靠的诊断。痰液和肺部灌洗液样品获得非常容易，而且不会对病人造成任何的痛苦和不便。使用样本量极少，取样过程非常方便且对病人无任何影响。同时样品便于邮寄或者是随身带到医院做检查。
上述技术方案中的一个技术方案的有益效果为：可以检测多种类型的肺癌，且针对难检测的腺癌，其也具有相对其他标志物更高的灵敏度。
上述技术方案中的一个技术方案的有益效果为：无需考虑检测的对象和年龄，适用范围广。
上述技术方案中的一个技术方案的有益效果为：通过甲基化水平来检测和诊断癌症，越来越多的研究证实甲基化改变是肿瘤发生过程中的早期事件，检测甲基化异常更易发现早期病变。
附图说明
图1 HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9组合在所有组织标本中检测的ROC曲线；
图2 HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9组合在所有痰液标本中检测的ROC曲线；
图3 HOXA7、HOXA9、与SHOX2_n3、以及HOXA7和HOXA9组合在所有痰液标本中检测的ROC曲线；
图4 HOXA7、HOXA9与SHOX2_n3在痰液标本中的扩增曲线(A为HOXA7的扩增图，B为HOXA9的扩增图，C为SHOX2_n3的扩增图)；
图5 HOXA7、HOXA9、SHOX2_n3、以及HOXA7和HOXA9组合在所有灌洗液标本中检测的ROC曲线；
图6 HOXA7、HOXA9与SHOX2_n3在灌洗液标本中的扩增曲线(A为HOXA7的扩增图，B为HOXA9的扩增图，C为SHOX2_n3的扩增图)。
具体实施方式
本公开中的“引物”或“探针”是指一种寡核苷酸，其包含与靶核酸分子(例如靶基因)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。
本公开中的“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HOXA7和HOXA9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基因HOXA7和HOXA9的甲基化而诊断。
其中，早期诊断指的是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。
作为病症阶段可选的实施方式，可在肺癌在不同阶段或时期的进展，通过从样品中获取的HOXA7和HOXA9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HOXA7基因和HOXA9基因甲基化程度与从没有细胞增殖性异常的肺部组织中的样品中分离出的一个或多个核酸的HOXA7基因和HOXA9基因甲基化程度，可检测样品中肺癌的具体阶段。
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。
本公开中，所述“受试者”是哺乳动物，例如是人。
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。
用于甲基化检测的方法是公知的，如甲基化特异性聚合酶链反应、实时荧光定量甲基化特异性聚合酶链反应、焦磷酸测序、使用甲基化DNA特异性结合蛋白的PCR，定量PCR，以及DNA芯片、差别化甲基化检测—甲基化敏感的限制性内切酶、差别化甲基化检测--亚硫酸盐测序法等等，除此之外，其他的甲基化检测方法可以通过专利US62007687引入。
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。
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。
实施例1：检测靶基因的选择
甲基化DNA作为检测靶标具有明显的优点，相比蛋白质类标志物，DNA是可以扩增的，并且很容易检测到；与突变类标志物相比，DNA甲基化的部位都位于基因的特定部位，一般在启动子区，使检测变得更容易和方便。为了完成本公开，发明人筛选了大量基因，选择有代表性的HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3作为候选的检测基因，β-actin基因作为内参基因，研究各个基因甲基化位点分布情况，设计检测的引物探针分别用于实时荧光定量甲基化特异性聚合酶链反应(real-time fluorescent quantitative methylation-specific PCR,qMSP)检测。各基因检测引物探针如下：
HOXA7的检测引物和探针为：
SEQ ID NO：1 HOXA7-F2引物F：TAAAGGCGTTTGCGATAAGAC
SEQ ID NO：2 HOXA7-R2引物R：TAACCCGCCTAACGACTACG
SEQ ID NO：3 HOXA7-P2探针：FAM-AGGGCGCGTTGTATGGCGC-BQ1
HOXA9的检测引物和探针为：
SEQ ID NO：4 HOXA9-F2引物F：TTAGTTTTTTCGGTAGGCGGC
SEQ ID NO：5 HOXA9-R2引物R：AAACGCCAAACACCGTCG
SEQ ID NO：6 HOXA9-P2探针：FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1
SHOX2的检测引物和探针为：
SEQ ID NO：7 SHOX2引物F：TTTAAAGGGTTCGTCGTTTAAGTC
SEQ ID NO：8 SHOX2引物R：AAACGATTACTTTCGCCCG
SEQ ID NO：9 SHOX2探针：FAM-TTAGAAGGTAGGAGGCGGAAAATTAG-BQ1
PCDHGA12的检测引物和探针为：
SEQ ID NO：10 PCDHGA12引物F：TTGGTTTTTACGGTTTTCGAC
SEQ ID NO：11 PCDHGA12引物R：AAATTCTCCGAAACGCTCG
SEQ ID NO：12 PCDHGA12探针：FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1
HOXD8的检测引物和探针为：
SEQ ID NO：13 HOXD8引物F：TTAGTTTCGGCGCGTAGC
SEQ ID NO：14 HOXD8引物R：CCTAAAACCGACGCGATCTA
SEQ ID NO：15 HOXD8探针：FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1
GATA3的检测引物和探针为：
SEQ ID NO：16 GATA3引物F：TTTCGGTAGCGGGTATTGC
SEQ ID NO：17 GATA3引物R：AAAATAACGACGAACCAACCG
SEQ ID NO：18 GATA3探针：FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1
β-actin的检测引物和探针为：
SEQ ID NO：19β-actin引物F：TTTTGGATTGTGAATTTGTG
SEQ ID NO：20β-actin引物R：AAAACCTACTCCTCCCTTAAA
SEQ ID NO：21β-actin探针：FAM-TTGTGTGTTGGGTGGTGGTT-BQ1
实验过程：
1、提取DNA
收集确诊肺癌患者的标本和非肿瘤患者标本，分别包括石蜡组织标本、痰液标本、灌洗液标本，样品预处理及分离细胞，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)说明书进行重亚硫酸氢盐修饰。
3、扩增与检测
配液体系
表1配液体系
Figure PCTCN2019108648-appb-000001
扩增体系：
表2 PCR反应过程
Figure PCTCN2019108648-appb-000002
Figure PCTCN2019108648-appb-000003
4、检测结果
4.1、石蜡组织中的检测结果
样本信息：肺组织样本共计185例，其中正常组织样本87例，癌组织样本98例，98例癌症组样本中有鳞癌15例，腺癌81例，未明确分类的肺癌2例，其中癌和癌旁对照样本73对。
HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3、以及HOXA7和HOXA9组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 PCTCN2019108648-appb-000004
续表3组织中的检测结果
Figure PCTCN2019108648-appb-000005
从以上结果可以看出，在组织样本中，无论是将肺癌作为一个整体进行比较分析，还是按照肺癌的亚型进行比较分析，SHOX2和HOXA9的检测效果最好，HOXA7、PCDHGA12和GATA3的检测效果次之，HOXD8的检测效果最差，灵敏度只达到40.8％。
尽管联合检测已经在现有技术中有所应用，然而，并非联合检测就具有协同性，如Hubers等人(DNA hypermethylation analysis in sputum forthe diagnosis of lung cancer:training validation set approach)研究RASSF1A,3OST2and PHACTR3单独在痰液中检测肺癌的灵敏度分别为42.5％、31.5％和28.8％，3者联合检测灵敏度只能达到67.1％，特异性由96.5％降至89.5％，结果表明肺癌标志物的组合检测，效果并没有很协同性的增强。同样地，本公开中，同为HOXA家族的HOXA7、HOXA9、HOXD8三者两两组合，仅HOXA7&HOXA9的组合，无论是在全部癌症中，还是在鳞癌中或者是较难检测的肺癌中，具有比其他组合更高的灵敏度，而其他HOXA家族的联合则无显著增强效果。
根据以上结果，为了研究痰液中的不同基因的检测情况，发明人对HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8和GATA3这6个标志物在痰液中进行进一步的筛选。
实施例2：HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8和GATA3、以及HOXA7和HOXA9组合在痰液中的检测
样本信息：测试痰样本共计90例，其中正常对照组样本55例，癌症组对照样本35例，35例癌症组样本中有鳞癌12例，小细胞癌6例，腺癌9例，大细胞癌2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DTT解稠后，离心取沉淀分离细胞，使用PBS洗涤2遍，然后使用美基生物公司(Magen)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的亚硫酸氢盐修饰。
c.配液体系如下：
表4配液体系
Figure PCTCN2019108648-appb-000006
d.扩增体系如下：
表5扩增体系
Figure PCTCN2019108648-appb-000007
e.检测结果如下：
表6痰液中的检测结果
Figure PCTCN2019108648-appb-000008
Figure PCTCN2019108648-appb-000009
续表6痰液中的检测结果
Figure PCTCN2019108648-appb-000010
f.HOXA7、HOXA9、SHOX2、PCDHGA12、HOXD8、GATA3以及HOXA7和HOXA9组合在所有痰液标本中检测的ROC曲线见图2，统计结果见表6，从以上结果可以看出，在痰液样本中，同时检测这6个基因进行比较，无论是将肺癌作为一个整体进行比较分析，还是按照肺癌的亚型进行比较分析，HOXA7和HOXA9的联合检测效果都要优于其他4个基因或者HOXA7和HOXA9分别单独检测的效果；同时也优于HOXA7&HOXD8和HOXA9&HOXD8的组合。
特别是对腺癌的检测效果，HOXA7和HOXA9的组合相比其他基因的检出率更加好，敏感度甚至达到100％，如此高的灵敏度基本上可实现对腺癌早期筛查的0漏检率，从而使患者可以得到准确的风险提示，及早治疗，降低腺癌的死亡率。而现有的肺癌中的肿瘤标志物其对腺癌的敏感性仅为14.3％，而HOXA7或HOXA9单独检测时，对腺癌的敏感性也仅有88.9％，二者联合检测时，对肺癌的灵敏度提升到100％，二者具有协同性。
比较各基因的检测结果，发现最优的两个基因HOXA7和HOXA9对提高体系检测阳性率具有较明显的协同作用，具体表现为明显提高了腺癌的检出率。腺癌一般为周围型，由于支气管的树状生理结构，肺深部的脱落细胞更加难以通过痰液咳出，因此对这部分的检测更加困难和有意义。
实施例3：HOXA7、HOXA9以及SHOX2基因在痰液中的检测
大量文献显示SHOX2可用作检测肺癌的标志物，并且有专利显示[CN201510203539诊断人SHOX2基因和人RASSF1A基因甲基化的方法和试剂盒-申请公开]，SHOX2在肺泡灌洗液、病变部位组织、胸水、痰液等样本中具有较高的检出率。为了验证HOXA7和HOXA9的检测效果，发明人同时检测HOXA7、HOXA9SHOX2_n3基因。在该实施例中，SHOX2基因的检出效率采用专利CN201510203539中公开的引物和探针序列，并将SHOX2基因表述为SHOX2_n3，以区别于本公开实施例1和2中采用自行设计的引 物和探针检测的SHOX2基因。
的检出结果。
各基因检测引物探针如下：
HOXA7的检测引物和探针为：
SEQ ID NO：1 HOXA7-F2引物F：TAAAGGCGTTTGCGATAAGAC
SEQ ID NO：2 HOXA7-R2引物R：TAACCCGCCTAACGACTACG
SEQ ID NO：3 HOXA7-P2探针：FAM-AGGGCGCGTTGTATGGCGC-BQ1
HOXA9的检测引物和探针为：
SEQ ID NO：4 HOXA9-F2引物F：TTAGTTTTTTCGGTAGGCGGC
SEQ ID NO：5 HOXA9-R2引物R：AAACGCCAAACACCGTCG
SEQ ID NO：6 HOXA9-P2探针：FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1SHOX2_n3的检测引物和探针为：
SEQ ID NO：22 SHOX2_n3引物F：TTTGGATAGTTAGGTAATTTTCG
SEQ ID NO：23 SHOX2_n3引物R：CGTACACGCCTATACTCGTACG
SEQ ID NO：24 SHOX2_n3.2探针：FAM-CCCCGATCGAACAAACGAAAC-BQ1
a.配液体系如下：
表7配液体系
  HOXA7 HOXA9 SHOX2_n3 β-actin
反应组份 加入量(ul) 加入量(ul) 加入量(ul) 加入量(ul)
上游引物(100uM) 0.125 0.125 0.125 0.125
下游引物(100uM) 0.125 0.125 0.125 0.125
探针(100uM) 0.05 0.05 0.05 0.05
镁离子(25mM) 5 4 5 5
dNTPs(10mM) 1 1 1 1
Taq聚合酶(5unit/ul) 0.5 0.5 0.5 0.5
5X缓冲液 5 5 5 5
灭菌水 12.2 13.2 12.2 12.2
模板DNA 1 1 1 1
总体积 25 25 25 25
b.扩增体系如下：
表8扩增体系
Figure PCTCN2019108648-appb-000011
表9 SHOX2_n3反应程序
Figure PCTCN2019108648-appb-000012
c.检测结果如下：
利用标准曲线计算各基因在标本中的甲基化拷贝数，采用比值＝靶向基因拷贝数/ACTB拷贝数*100来进行判断两组样本的甲基化程度，最后选取HOXA7的阈值为0.77，HOXA9的阈值为2.3，SHOX2_n3的阈值为1.3，作为判断癌症组和对照组的标准，换算后的比值超过设定阈值可判断为阳性“+”，等于或小于设定阈值可判断为阴性“-”。根据此标准，90例痰液标本的检测结果如下：
表10检测结果
Figure PCTCN2019108648-appb-000013
Figure PCTCN2019108648-appb-000014
Figure PCTCN2019108648-appb-000015
注意：“+”为甲基化DNA检测阳性，“-”为甲基化DNA检测阴性；在协同检测标本时，两者为阴性则判为阴性，一阴一阳或两者均为阳性则判为阳性。
d.结果分析
表11统计结果
Figure PCTCN2019108648-appb-000016
e.HOXA7与HOXA9组合与SHOX2_n3在所有痰液标本中检测的ROC曲线见图3，HOXA7、HOXA9与SHOX2_n3在痰液标本中的扩增曲线见图4，统计结果见表11，从以上结果可以看出，在痰液样本中，将肺癌作为一个整体进行比较分析，还是按照肺癌的亚型进行比较分析，HOXA7和HOXA9的检测效果都要优于SHOX2基因的检测效果，通过HOXA7和HOXA9组合检测样本，可以有效提高阳性检出率，在所有样本中，阳性检出率为97.1％，其中对于鳞癌、腺癌和小细胞癌，检测灵敏度均达到了100％。在多种类型的肺癌中，均能达到如此高的灵敏度，当用于临床检测时，无疑具有更大的通用性。特别是对腺癌的检测效果，HOXA7和HOXA9联合检出腺癌的灵敏性高达100％，而现有的针对SHOX2检测的引物和探针对腺癌的敏感度仅为33.3％。腺癌一般为周围型，由于支气管的树状生理结构，肺深部的脱落细胞更加难以通过痰液咳出，而本公开首次发现了一种能以痰液作为样本检测腺癌，并可将灵敏度大幅提升至100％的标志物，这一突破对腺癌的检测具有重要意义。
实施例4：HOXA7、HOXA9以及SHOX2基因在灌洗液中的检测
样本信息：测试肺泡灌洗液样本共计79例，其中正常对照组样本58例，癌症组对照样本21例，21例癌症组样本中有鳞癌6例，小细胞癌4例，腺癌11例。
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Magen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.扩增检测体系如下：
表12扩增体系
  HOXA7 HOXA9 SHOX2_n3 β-actin
反应组份 加入量(ul) 加入量(ul) 加入量(ul) 加入量(ul)
上游引物(100uM) 0.125 0.125 0.125 0.125
下游引物(100uM) 0.125 0.125 0.125 0.125
探针(100uM) 0.05 0.05 0.05 0.05
镁离子(25mM) 5 4 5 5
dNTPs(10mM) 1 1 1 1
Taq聚合酶(5unit/ul) 0.5 0.5 0.5 0.5
5X缓冲液 5 5 5 5
灭菌水 12.2 13.2 12.2 12.2
模板DNA 1 1 1 1
总体积 25 25 25 25
d.检测体系如下：
表13 HOXA7和HOXA9的反应程序
Figure PCTCN2019108648-appb-000017
表14 SHOX2_n3反应程序
Figure PCTCN2019108648-appb-000018
e.检测结果如下：
利用标准曲线计算各基因在标本中的甲基化拷贝数，采用比值＝靶向基因拷贝数/ACTB拷贝数*100来进行判断两组样本的甲基化程度，最后选取HOXA7的阈值为0.7，HOXA9的阈值为0.5，SHOX2_n3的阈值为0.6，作为判断癌症组和对照组的标准，换算后的比值超过设定阈值可判断为阳性“+”，等于或小于设定阈值可判断为阴性“-”。根据此标准，79例灌洗液标本的检测结果如下：
表15检测结果
Figure PCTCN2019108648-appb-000019
Figure PCTCN2019108648-appb-000020
注意：“+”为甲基化DNA检测阳性，“-”为甲基化DNA检测阴性；在协同检测标本时，两者为阴性则判为阴性，一阴一阳或两者均为阳性则判为阳性。
表16统计结果
Figure PCTCN2019108648-appb-000021
Figure PCTCN2019108648-appb-000022
f.HOXA7与HOXA9组合与SHOX2_n3在所有灌洗液标本中检测的ROC曲线见图5，HOXA7、HOXA9与SHOX2_n3在灌洗液标本中的扩增曲线见图6，统计结果见表16，从以上结果可以看出，在灌洗液样本中，无论将肺癌作为一个整体进行比较分析，还是按照肺癌的亚型进行比较分析，HOXA7和HOXA9的检测效果都要优于SHOX2基因的检测效果，通过HOXA7和HOXA9组合检测样本，可以有效提高阳性检出率，在所有样本中，阳性检出率为85.7％。特别是对腺癌的检测效果，HOXA7和HOXA9联合检出腺癌的灵敏性高达81.8％，远高于SHOX2的36.4％，也高于HOXA7和HOXA9基因在组织中对腺癌的灵敏度，这是十分难得且罕见的。腺癌一般为周围型，由于支气管的树状生理结构，肺深部的脱落细胞更加难以通过痰液咳出，因此对这部分的检测更加困难和有意义。
此外，在灌洗液中，针对小细胞癌，其敏感性达到了100％，而无论是HOXA7或HOXA9，或SHOX2，其对小细胞癌的灵敏度仅为75％。这样的0漏检率对小细胞癌的早期筛查具有重要的意义。
综合上述的4个实施例，能够充分的说明HOXA7与HOXA9组合在对肺癌检测诊断，尤其是应用痰液，肺泡灌洗液等生物样本上具有更好的检测效果。能够更加容易的应用于大规模的人群筛查。具有更加优越的社会经济学价值。
实施例5：HOXA7和HOXA9的引物设计和优化
各种研究资料表明，同一个基因的甲基化状态和分布并不均匀，因此对于同一个基因来说，选择不同的区域设计的甲基化引物、探针检测体系对同一样本，同一肿瘤的诊断检测效能并不一样，甚至有时候选择的区域不合适造成对肿瘤完全没有诊断效果，发明人经过反复的研究和比较后，部分HOXA7和HOXA9的区域序列如下：
表17 HOXA7待检测序列
Figure PCTCN2019108648-appb-000023
Figure PCTCN2019108648-appb-000024
续表17 HOXA9待检测序列
Figure PCTCN2019108648-appb-000025
Figure PCTCN2019108648-appb-000026
Figure PCTCN2019108648-appb-000027
Figure PCTCN2019108648-appb-000028
我们根据HOXA7序列的区域1、区域2，以及HOXA9序列区域1、区域2和区域3设计不同的甲基化引物和探针，各引物探针信息见表19。
针对HOXA7序列，其中组8、组9、组10是根据区域1设计的甲基化引物和探针；组1、组2、组3、组4、组5、组6、组7是根据区域2设计的甲基化引物和探针。
针对HOXA9序列其中组1、组2、组3、组4和组5是根据区域1设计的甲基化引物和探针；组6、组7、组8是根据区域2设计的甲基化引物和探针；组9、组10、组11是根据区域3设计的甲基化引物和探针。
在36例肺组织样本检测以下引物探针组合，其中正常组织样本11例，癌组织样本25例，25例癌症组样本中有鳞癌4例，腺癌21例。检测结果如下表18。
表18 HOXA7的引物在组织中的检测结果
所处区域 组别 引物探针组合 特异性 灵敏性
区域2 组1 H7-F2,H7-R2,H7-P2 100％ 80％
区域2 组2 H7-F3,H7-R3,H7-P3 100％ 76％
区域2 组3 H7-F4,H7-R4,H7-P4 100％ 56％
区域2 组4 H7-F5,H7-R5,H7-P5 100％ 72％
区域2 组5 H7-F6,H7-R6,H7-P6 100％ 80％
区域2 组6 H7-F7,H7-R7,H7-P7 100％ 72％
区域2 组7 H7-F8,H7-R8,H7-P8 100％ 56％
区域1 组8 H7-F9,H7-R9,H7-P9 100％ 48％
区域1 组9 H7-F10,H7-R10,H7-P10 100％ 16％
区域1 组10 H7-F11,H7-R11,H7-P11 100％ 24％
Figure PCTCN2019108648-appb-000029
结果显示，针对HOXA7，无论区域1如何设计引物和探针，针对区域1的检测灵敏度最高仅能达到48％，而无论采用本公开设计的何种引物和探针，区域2的检测灵敏度最低也可达到56％，最高达到80％。因此，HOXA7的区域2的检出率明显较区域1高(见表18)。
针对HOXA9，无论区域2和3如何设计引物和探针，针对这两个区域的检测灵敏度最高仅能达到32％，而无论采用本公开设计的何种引物和探针，区域1的检测灵敏度最低也可达到40％，最高达到76％。因此，HOXA7区域1的检出率明显较区域2和3高(见表18)。
二、引物和探针对检测效果的影响
除了检测区域会影响检测效果，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，以使本公开的检测试剂能够实际应用到临床检测中。部分引物和探针如下表19所示，检测结果如表20所示。所有的引物和探针均由英潍捷基(上海)贸易有限公司合成。
表19 HOXA7的引物和探针
名称 序列编号 序列 作用
H7-F2 SEQ ID NO：1 TAAAGGCGTTTGCGATAAGAC HOXA7基因上游引物
H7-R2 SEQ ID NO：2 TAACCCGCCTAACGACTACG HOXA7基因下游引物
H7-P2 SEQ ID NO：3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7基因检测探针
H7-F3 SEQ ID NO：37 GCGTTTGCGATAAGACGGAC HOXA7基因上游引物
H7-R3 SEQ ID NO：38 CCAACCTAACCCGCCTAACG HOXA7基因下游引物
H7-P3 SEQ ID NO：39 FAM-CGTTGTATGGCGCGGTTGAGG-BQ1 HOXA7基因检测探针
H7-F4 SEQ ID NO：40 CGTTCGGTTACGGTTTGGGC HOXA7基因上游引物
H7-R4 SEQ ID NO：41 GCCCTCGTCCGTCTTATCGC HOXA7基因下游引物
H7-P4 SEQ ID NO：42 FAM-TCGACGTTTACGGTAATTTGTTTTGCG-BQ1 HOXA7基因检测探针
H7-F5 SEQ ID NO：43 ATTTCGTTAAAGGCGTTTGC HOXA7基因上游引物
H7-R5 SEQ ID NO：44 TCCGCCAACCTAACCCG HOXA7基因下游引物
H7-P5 SEQ ID NO：45 FAM-CGGACGAGGGCGCGTTGTAT-BQ1 HOXA7基因检测探针
H7-F6 SEQ ID NO：46 CGTTAAAGGCGTTTGCGATAAGAC HOXA7基因上游引物
H7-R6 SEQ ID NO：47 TACGCTCCCCGCTCCCGAT HOXA7基因下游引物
H7-P6 SEQ ID NO：48 FAM-GACGGACGAGGGCGCGTTGTATG-BQ1 HOXA7基因检测探针
H7-F7 SEQ ID NO：49 CAACGCTACGCTCCCCGCT HOXA7基因下游引物
H7-R7 SEQ ID NO：50 GCGATAAGACGGACGAGGGC HOXA7基因上游引物
H7-P7 SEQ ID NO：51 FAM-CCGATCCCGCTCCGCCAACC-BQ1 HOXA7基因检测探针
H7-F8 SEQ ID NO：52 CGTTAGGCGGGTTAGGTTGGC HOXA7基因上游引物
H7-R8 SEQ ID NO：53 GCCGAAAATCACGAAACTACCG HOXA7基因下游引物
H7-P8 SEQ ID NO：54 FAM-AGCGGGATCGGGAGCGGG-BQ1 HOXA7基因检测探针
H7-F9 SEQ ID NO：55 TCGGGTCGTTTGGCGTTC HOXA7基因上游引物
H7-R9 SEQ ID NO：56 AACCTAACGCGTCCCGCG HOXA7基因下游引物
H7-P9 SEQ ID NO：57 FAM-TTGGTCGTTAGTTGGGCGTTTTCGC-BQ1 HOXA7基因检测探针
H7-F10 SEQ ID NO：58 GGCGTTCGTAGATTTTAGTGC HOXA7基因上游引物
H7-R10 SEQ ID NO：59 CAAATAATCTCGCTCCGCA HOXA7基因下游引物
H7-P10 SEQ ID NO：60 FAM-GGTCGTTAGTTGGGCGTTTTCG-BQ1 HOXA7基因检测探针
H7-F11 SEQ ID NO：61 GCGTTAGGTTCGTCGGGC HOXA7基因上游引物
H7-R11 SEQ ID NO：62 AAACTCGCCGCAACACGTAA HOXA7基因下游引物
H7-P11 SEQ ID NO：63 FAM-CGGATTTTTTGGTCGTATATTTGAGT-BQ1 HOXA7基因检测探针
续表19 HOXA9的引物和探针
Figure PCTCN2019108648-appb-000030
表20 HOXA7在组织中的检测结果
组别 引物探针组合 特异性 灵敏性
组1 H7-F2,H7-R2,H7-P2 100％ 80％
组2 H7-F3,H7-R3,H7-P3 100％ 76％
组3 H7-F4,H7-R4,H7-P4 100％ 56％
组4 H7-F5,H7-R5,H7-P5 100％ 72％
组5 H7-F6,H7-R6,H7-P6 100％ 80％
组6 H7-F7,H7-R7,H7-P7 100％ 72％
组7 H7-F8,H7-R8,H7-P8 100％ 56％
组8 H7-F9,H7-R9,H7-P9 100％ 48％
组9 H7-F10,H7-R10,H7-P10 100％ 16％
组10 H7-F11,H7-R11,H7-P11 100％ 24％
续表20 HOXA9在组织中的检测结果
组别 引物探针组合 特异性 灵敏性
组1 H9-F2,H9-R2,H9-P2 100％ 76％
组2 H9-F3,H9-R3,H9-P3 100％ 76％
组3 H9-F4,H9-R4,H9-P4 100％ 40％
组4 H9-F5,H9-R5,H9-P5 100％ 60％
组5 H9-F6,H9-R6,H9-P6 100％ 68％
组6 H9-F7,H9-R7,H9-P7 100％ 32％
组7 H9-F8,H9-R8,H9-P8 100％ 20％
组8 H9-F9,H9-R9,H9-P9 100％ 12％
组9 H9-F10,H9-R10,H9-P10 100％ 16％
组10 H9-F11,H9-R11,H9-P11 100％ 24％
组11 H9-F12,H9-R12,H9-P12 100％ 24％
本公开中，采用表19中的引物和探针对组织样本的进行了验证。在36例肺组织样本检测以上10组引物探针组合，其中正常组织样本11例，癌组织样本25例，25例癌症组样本中有鳞癌4例，腺癌21例。检测结果如上表20，
结果显示HOXA7的组1、组2、组4、组5、组6均有较好的检出率。HOXA9的组1、组2、组4、组5均有较好的检出率。
为了进一步验证其在痰液中的检出率，我们选取了22例痰液标本，采用表19中的引物和探针进行验证，其中包括7例正常对照，15例肺癌对照，15例肺癌中有鳞癌7例，腺癌7例，大细胞癌1例，检测结果如下表21。
表21 HOXA7在痰液中的检测结果
组别 引物探针组合 特异性 灵敏性
组1 H7-F2,H7-R2,H7-P2 100％ 73.3％
组2 H7-F3,H7-R3,H7-P3 100％ 53.3％
组4 H7-F5,H7-R5,H7-P5 100％ 60.0％
组5 H7-F6,H7-R6,H7-P6 100％ 53.3％
组6 H7-F7,H7-R7,H7-P7 100％ 46.7％
Figure PCTCN2019108648-appb-000031
从22例痰液标本的检测结果显示，HOXA7的组1：H7-F2,H7-R2,H7-P2的检出率最高，达到73.3％。虽然在组织样本中，组1和组5的灵敏度均能达到80％，但针对痰液检测样本，组5的灵敏度却大幅下降至53.3％。
HOXA9的组1：H9-F2,H9-R2,H9-P2的检出率最高，达到66.7％。虽然在组织样本中，组1和组2的灵敏度均能达到76％，但针对痰液检测样本，组2的灵敏度却大幅下降至44.6％。
最终优选的引物如下表22所示。
表22优化后的引物和探针
名称 序列编号 序列 作用
H7-F2 SEQ ID NO：1 TAAAGGCGTTTGCGATAAGAC HOXA7基因上游引物
H7-R2 SEQ ID NO：2 TAACCCGCCTAACGACTACG HOXA7基因下游引物
H7-P2 SEQ ID NO：3 FAM-AGGGCGCGTTGTATGGCGC-BQ1 HOXA7基因检测探针
H9-F2 SEQ ID NO：4 TTAGTTTTTTCGGTAGGCGGC HOXA9基因上游引物
H9-R2 SEQ ID NO：5 AAACGCCAAACACCGTCG HOXA9基因下游引物
H9-P2 SEQ ID NO：6 FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1 HOXA9基因检测探针
A3-TqMF SEQ ID NO：19 TTTTGGATTGTGAATTTGTG β-actin基因上游引物
A3-TqMR SEQ ID NO：20 AAAACCTACTCCTCCCTTAAA β-actin基因下游引物
A3-TqP SEQ ID NO：21 FAM-TTGTGTGTTGGGTGGTGGTT-BQ1 β-actin基因检测探针

Claims (13)

  1. HOXA7基因或其核酸片段，以及HOXA9基因或其核酸片段在制备肺癌诊断试剂或试剂盒中的应用。
  2. HOXA7基因和HOXA9基因甲基化的检测试剂在制备肺癌诊断试剂中的应用。
  3. 根据权利要求2所述应用，所述HOXA7和HOXA9基因甲基化的检测试剂检测HOXA7基因和HOXA9基因经转化试剂修饰后的序列；
    优选地，所述转化试剂选自肼盐、重亚硫酸氢盐和亚硫酸氢盐中的一种或几种；
    优选地，所述转化试剂选自重亚硫酸氢盐。
  4. 根据权利要求2-3任一所述应用，所述甲基化的检测试剂的检测区域包含HOXA7基因和HOXA9基因的基因体或启动子区域；
    优选地，所述试剂针对HOXA7的检测区域包含SEQ ID NO：25或SEQ ID NO：27所示的序列；所述试剂针对HOXA9的检测区域包含SEQ ID NO：29、SEQ ID NO：31或SEQ ID NO：33所示的序列；
    更优选地，所述试剂针对HOXA7的检测区域包含SEQ ID NO：27所示的序列，所述试剂针对HOXA9的检测区域包含SEQ ID NO：29所示的序列。
  5. 根据权利要求2-4任一所述应用，所述甲基化的检测试剂包含检测HOXA7基因和HOXA9基因的引物对；
    优选地，检测HOXA7基因的引物对包含SEQ ID NO：1-2，SEQ ID NO：37-38，SEQ ID NO：40-41，SEQ ID NO：43-44，或SEQ ID NO：46-47，SEQ ID NO：49-50，SEQ ID NO：52-53，SEQ ID NO：55-56，SEQ ID NO：58-59或SEQ ID NO：61-62所示的引物对；
    更优选地，检测HOXA7基因的引物对包含SEQ ID NO：1-2所示的引物对；
    优选地，检测HOXA9基因的引物对包含SEQ ID NO：4-5，SEQ ID NO：64-65，SEQ ID NO：67-68，SEQ ID NO：70-71，SEQ ID NO：73-74，SEQ ID NO：76-77，SEQ ID NO：79-80，SEQ ID NO：82-83，SEQ ID NO：85-86，SEQ ID NO：88-89或SEQ ID NO：91-92所示的引物对；
    更优选地，检测HOXA9基因的引物对包含SEQ ID NO：4-5所示的引物对。
  6. 根据权利要求2-5任一所述应用，所述甲基化的检测试剂包含检测HOXA7基因和HOXA9基因的探针；
    优选地，检测HOXA7基因的探针包含SEQ ID NO：3、SEQ ID NO：39、SEQ ID NO：42、SEQ ID NO：45、SEQ ID NO：48、SEQ ID NO：51、SEQ ID NO：54、SEQ ID NO：57、SEQ ID NO：60或SEQ ID NO：63所示的序列；
    更优选地，检测HOXA7基因的探针包含SEQ ID NO：3所示的序列；
    优选地，检测HOXA9基因的探针包含SEQ ID NO：6、SEQ ID NO：66、SEQ ID NO：69、SEQ ID NO：72、SEQ ID NO：75、SEQ ID NO：78、SEQ ID NO：81、SEQ ID NO：84、SEQ ID NO：87、SEQ ID NO：90或SEQ ID NO：93所示的序列；
    更优选地，检测HOXA9基因的探针包含SEQ ID NO：6所示的序列。
  7. 根据权利要求2-6任一所述应用，所述甲基化的检测试剂还包含内参基因的检测试剂；
    优选地，所述内参基因包含β-actin或COL2A1；
    优选地，所述内参基因的检测试剂含包含内参基因的引物和探针；
    更优选地，所述内参基因β-actin的检测试剂包含SEQ ID NO：19和SEQ ID NO：20所示引物对，以及SEQ ID NO：21所示探针；
    优选地，所述内参基因COL2A1的检测试剂包含SEQ ID NO：94和SEQ ID NO：95所示引物对，以及SEQ ID NO：96所示探针。
  8. 根据权利要求2-7任一所述应用，所述甲基化的检测试剂还包含亚硫酸氢盐、重亚硫酸氢盐或肼盐。
  9. 根据权利要求2-8任一所述应用，所述甲基化的检测试剂还包含DNA聚合酶、dNTPs、Mg2+离子和缓冲液中的一种或几种；
    优选地，包含DNA聚合酶、dNTPs、Mg 2+离子和缓冲液。
  10. 根据权利要求2-9任一所述应用，所述甲基化的检测试剂的检测样本包含痰液、肺部灌洗液、肺部组织、胸水，血液、血清、血 浆、尿液、唾液、前列腺液、泪液或粪便；
    优选地，所述检测样本包含痰液、肺部组织或肺部灌洗液；
    更优选地，所述检测样本包含痰液。
  11. 一种检测HOXA7基因和HOXA9基因的DNA甲基化的方法，包括以下步骤：
    (1)将待测样品进行亚硫酸氢盐或重亚硫酸氢盐或肼盐处理，获得经修饰的检测样本；
    (2)利用权利要求2-10任一所述甲基化的检测试剂对步骤(1)经修饰的检测样本进行HOXA7基因和HOXA9基因甲基化情况检测；
    优选地，步骤(2)中，采用甲基化特异性聚合酶链反应或实时荧光定量甲基化特异性聚合酶链反应进行检测。
  12. 一种肺癌的诊断系统，所述系统包含：
    i.HOXA7基因和HOXA9基因的DNA甲基化检测构件，以及，
    ii.结果判断构件；
    优选地，所述HOXA7基因和HOXA9基因的DNA甲基化检测构件包含权利要求2-10任一所述甲基化的检测试剂；
    优选地，所述甲基化检测构件包含荧光定量PCR仪、PCR仪、测序仪中的一种或多种；
    优选地，所述结果判断构件包含数据处理机器；
    优选地，所述结果判断构件用于根据检测构件检测的HOXA7基因和HOXA9基因的DNA甲基化水平，输出诊断结果；
    更优选地，所述诊断结果通过结果判断构件比较待测样本与正常样本的甲基化水平，基于待测样本与正常样本的甲基化水平的偏离得出的。
  13. 一种肺癌的诊断方法，其中所述方法包括以下步骤：
    a)检测来源于受试者的待测样本HOXA7基因和HOXA9基因的甲基化水平；
    b)将待测样本与正常样本的HOXA7基因和HOXA9基因水平比较
    c)基于待测样本与正常样本的甲基化水平的偏离，诊断肺癌；
    优选地，所述待测样本包含痰液、肺部灌洗液、肺部组织、胸水、血液、血清、血浆、尿液、唾液、前列腺液、泪液或粪便；
    优选地，所述待测样本包含痰液、肺部组织或肺部灌洗液；
    更优选地，所述待测样本包含痰液或肺部灌洗液。
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