TWI789551B - Application of HOXA9 methylation detection reagent in the preparation of lung cancer diagnostic reagent - Google Patents

Abstract

The present disclosure relates to the use of a detection reagent whose detection object is methylation of HOXA9 in the preparation of a lung cancer diagnostic reagent. In this disclosure, HOXA9 is used as a tumor marker for detecting lung cancer. Its sensitivity in sputum is as high as 74.3%, and its specificity is as high as 95%. The detection sensitivity is higher than that of existing lung cancer markers, and its sensitivity in lavage fluid is as high as 61.9%. , the specificity is as high as 95%, especially for adenocarcinoma, the sensitivity has been greatly improved, and it has great application value for the detection of adenocarcinoma.

Description

Application of HOXA9 methylation detection reagent in the preparation of lung cancer diagnostic reagent

The disclosure belongs to the field of gene diagnosis, and more specifically, the disclosure relates to the application of a detection reagent for diagnosing human HOXA9 gene methylation in the preparation of lung cancer diagnostic reagents, and a method for detecting human HOXA9 gene methylation.

Lung cancer is a malignant tumor of the lung originating 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 obvious tendency of distant metastasis and poor prognosis, but most patients are sensitive to radiotherapy and chemotherapy. 2. Non-small cell lung cancer (NSCLC): In addition to small cell lung cancer, other pathological types of lung cancer include squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. There are certain differences in biological behavior and clinical course. According to the location of occurrence, it can be divided into: 1. Central lung cancer (central lung cancer): lung cancer that grows in the bronchial opening and above of the lung segment. 2. Peripheral lung cancer: Lung cancer that grows far from the opening of the segmental bronchi.

Early diagnosis and early operation of lung cancer are one of the most effective ways to improve the 5-year survival rate and reduce the mortality rate of lung cancer.

The current clinical auxiliary diagnosis of lung cancer mainly includes the following types, but none of them can fully achieve early detection and early diagnosis:

1. Blood biochemical examination: For primary lung cancer, there is currently no specific blood biochemical examination. Elevated blood alkaline phosphatase or blood calcium in patients with lung cancer may consider the possibility of bone metastasis, and elevated blood alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase or bilirubin may consider the possibility of liver metastasis.

2. Tumor marker examination: (1) CEA: 30% to 70% of lung cancer patients have abnormally high levels of CEA in serum, but it is mainly seen in patients with advanced lung cancer. At present, the examination of CEA in serum is mainly used to estimate the prognosis of lung cancer and monitor the course of treatment. (2) NSE: It is the preferred marker for small cell lung cancer, and it is used for the diagnosis and monitoring of treatment response of small cell lung cancer. The reference value is different according to the different detection methods and reagents used. (3) CYFRA21-1: It is the preferred marker for non-small cell lung cancer, and its sensitivity to the diagnosis of squamous cell carcinoma of the lung can reach 60%. The reference value is different according to the different detection methods and reagents used.

3. Imaging examination: (1) Chest X-ray examination: it should include frontal and lateral chest films. In primary hospitals, frontal and lateral chest radiographs are 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. (2) CT examination: Chest CT is the most commonly used and most important examination method for lung cancer. It is used for the diagnosis and differential diagnosis, staging and follow-up after treatment of lung cancer. Hospitals with conditions should include the adrenal glands when performing chest CT scans for lung cancer patients. Enhanced scanning should be used as much as possible, especially in patients with lung-centered lesions. CT is the basic examination method for displaying brain metastases. Patients with clinical symptoms or advanced stage should undergo brain CT scanning, and enhanced scanning should be used as much as possible. CT-guided lung biopsy is an important diagnostic technique for lung cancer. Hospitals with conditions can use it to diagnose intrapulmonary lesions that are difficult to characterize, and for clinical diagnosis of lung cancer that needs to be confirmed by cytology and histology, but other methods are difficult to obtain. cases. (3) Ultrasonography: It is mainly used to detect the metastasis of important abdominal organs and abdominal cavity and retroperitoneal lymph nodes, and it is also used for the examination of cervical lymph nodes. For intrapulmonary lesions or chest wall lesions adjacent to the chest wall, cystic and solid can be identified and ultrasound-guided biopsy can be performed; ultrasound is also commonly used for pleural effusion extraction and positioning. (4) Bone scan: the sensitivity of detecting bone metastases of lung cancer is high, but there is a certain false positive rate. It can be used in the following situations: preoperative examination of lung cancer; patients with local symptoms.

4. Other examinations: (1) Sputum cytology examination: It is a simple and convenient non-invasive diagnosis method for lung cancer at present. Continuous smear examination can increase the positive rate by about 60%. It is a routine diagnosis method for suspicious lung cancer cases. (2) Fiberoptic bronchoscopy: one of the most important methods in the diagnosis of lung cancer, it plays an important role in the qualitative diagnosis of lung cancer and the selection of surgical options. It is a necessary routine inspection item for patients who are going to undergo surgical treatment. However, transbronchoscopic needle biopsy (TBNA) is beneficial for staging before treatment, but due to technical difficulty and high risk, those who need it should be transferred to a higher-level hospital for further examination. (3) Others: such as percutaneous lung biopsy, thoracoscopy biopsy, mediastinoscopy biopsy, pleural effusion cytology, etc., if there are indications, can be used according to the existing conditions to assist in diagnosis.

Multi-slice spiral CT and low-dose CT (LDCT) in imaging examinations are effective screening tools for detecting early lung cancer and reducing mortality. The National Lung Cancer Screening Study (NLST) in the United States has shown that LDCT can reduce lung cancer compared with chest X-ray screening. 20% of lung cancer mortality. It has been proved in clinical practice that the success or failure of any lung cancer screening program depends on the identification of high-risk groups, and the risk prediction model integrating multiple high-risk factors has been recognized worldwide as one of the methods for identifying high-risk groups of lung cancer. Risk models can further improve outcomes for lung cancer patients by assisting clinicians in improving interventions or treatments. Although the world has recognized 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 to solve. In order to maximize the benefit-harm ratio of lung cancer screening, the key issue is how to define the high-risk population; the second is how to screen the population, including the definition of high-risk factors and overall risk. Quantitative summarization and selection of screening benefit cutoffs.

The existing lung cancer detection technologies mainly have low sensitivity, high false positives, and invasiveness, and it is difficult to detect early lung cancer with conventional detection technologies.

The non-invasive detection of lung cancer, for example, sputum detection, is more difficult. Although some researchers have also studied tumor markers in sputum of lung cancer patients, the success rate of sputum samples is very low compared with the detection and evaluation of tumor markers in blood samples of other cancer patients. This is mainly due to the following reasons: ① the composition of sputum is relatively complex, and the composition and viscosity of sputum vary greatly among different groups of people in different diseases or environments; ② sputum contains more tracheal epithelial cells and bacteria, oral cavity Non-lung cancer cell components such as mucosal cells, the general sample processing method cannot effectively enrich a sufficient number of DNA derived from lung cancer; ③ There are many smoking patients who do not show sputum. A J Hubers et al. studied the past 10 literatures in "Molecular sputum analysis for the diagnosis of lung cancer" and showed that the median methylation degree of markers in lung cancer tissue was 48%, while the median The degree of methylation was 38%, and the results showed that the detection rate of methylation markers in tissues was significantly higher than that in sputum. At the same time, Rosalia Cirincione (Methylation profile in tumor and sputum samples of lung cancer patients detected by spiral computed tomography: A nested case–contro) reported that the detection rates of RARbeta2, P16, and RASSF1A in lung cancer tissues reached 65.5%, 41.4%, and 51.7%, respectively. %, and only 44.4%, 5%, and 5% in sputum.

Currently, the missed detection rate of lung cancer is high. Especially for this type of adenocarcinoma, the non-invasive detection of sputum is even more difficult, and the detection rate is extremely low. This is because most adenocarcinomas originate from the smaller bronchi, which is a peripheral type of lung cancer, and the exfoliated cells in the deep lung are more difficult to cough up through sputum. Therefore, the current sputum detection methods for adenocarcinoma are almost zero.

It is especially important to reduce the missed detection rate in early tumor screening. If an early tumor screening product cannot screen out all or most of the patients, those who miss the test will not be able to receive sufficient risk warnings, thus delaying the timing of treatment, which is a huge loss for patients loss.

Although some tumor markers related to lung cancer have been found in the prior art, the sensitivity and specificity of these tumor markers cannot meet the requirements due to the limitation of detection reagents or detection means for these tumor markers. Further research is needed on screening methods that can be practically applied to lung cancer. Although non-invasive screening has unique advantages in sampling, it also has some limitations in other aspects. For example, adenocarcinoma in lung cancer is difficult to cough up through sputum because of the exfoliated cells in the deep lung. Speaking of which, those skilled in the art would think that this type of lung cancer is not suitable for non-invasive screening. On the other hand, even for other types of lung cancer, the currently reported non-invasive screening methods are difficult to meet the requirements of clinical use. Although relevant research has been progressing for many years, there is still no non-invasive screening method for lung cancer that can be promoted to the clinic.

The purpose of the present disclosure is to provide a use of a lung cancer tumor marker methylation detection reagent in the preparation of a lung cancer diagnostic reagent.

Another object of the present disclosure is to provide a use of a methylation detection reagent whose detection sensitivity in sputum and lavage fluid is not significantly lower than that of lung cancer tumor markers in tissues in the preparation of lung cancer diagnostic reagents.

Another object of the present disclosure is to provide a detection reagent for the methylation of a tumor marker with high sensitivity and specificity for adenocarcinoma in sputum and lavage fluid in the preparation of a diagnostic reagent for lung cancer.

Another object of the present disclosure is to provide a method for detecting HOXA9 gene methylation.

The above-mentioned purpose of the present disclosure is achieved by the following technical means:

After in-depth research, the inventor revealed a method for detecting gene methylation to increase the detection rate of lung cancer, and the methylation gene is the human HOXA9 gene. The inventor not only verified that the detection of HOXA9 methylation has high specificity and sensitivity for the detection of lung cancer in tissue samples, but also verified that it has the same high specificity and sensitivity in sputum samples and lavage fluid samples sex.

The first aspect of the present disclosure provides the use of the detection reagent for HOXA9 gene methylation in the preparation of a lung cancer diagnostic reagent.

The HOXA9 gene is a member of the HOX (homebox homology box) gene family and belongs to the HOXA cluster gene on chromosome 7p15-p14. Like other HOX genes, it contains a 180bp DNA fragment, and the transcription consists of 60 amino acids. homology domain. HOXA9 is a coding sequence-specific transcriptional regulatory factor, which plays an important role in the spatiotemporal development of embryos, cell differentiation, proliferation and migration, malignant evolution of tumors and induction of apoptosis. At present, there are many studies that the abnormal expression of HOXA9 plays an important role in the occurrence and development of leukemia. There are also some reports that the HOXA9 gene is related to lung cancer, human glioma and other cancers.

Wherein, the HOXA9 gene methylation detection reagent detects the sequence of the HOXA9 gene modified by the transformation reagent. Wherein, the conversion reagent refers to a reagent that deaminates cytosine in DNA to uracil, while leaving 5-MeC substantially unaffected. Exemplary transformation reagents include hydrazine salts, bisulfites (such as sodium bisulfite, etc.), bisulfites (such as sodium metabisulfite, potassium bisulfite, cesium bisulfite, ammonium bisulfite etc.), or one or several compounds that can produce hydrazine salt, bisulfite, and bisulfite under appropriate reaction conditions. As an exemplary embodiment, the HOXA9 gene methylation detection reagent detects the sequence modified by bisulfite.

The occurrence of methylation is that there is an extra methyl group on cytosine. After treatment with bisulfite or bisulfite or hydrazine salt, cytosine will become uracil, because uracil and thymus Pyrimidine is similar and will be recognized as thymine, which is reflected in the PCR amplification sequence that unmethylated cytosine becomes thymine (C becomes T), and methylated cytosine (C) does not change . The technology for detecting methylated genes by PCR is usually methylation-specific PCR (Methylmion Specific PCR, MSP), and primers are designed for the processed methylated fragments (that is, the unchanged C in the fragments) for PCR amplification. Amplification indicates that methylation has occurred, and no amplification indicates no methylation.

As an optional embodiment, the detection region of the HOXA9 gene targeted by the HOXA9 gene methylation detection reagent includes the CG-rich region or non-CG-rich region or CTCF (CTCF-binding sites) region of the HOXA9 gene. As a preferred embodiment, the detection region targeted by the HOXA9 gene methylation detection reagent includes the CG-rich region or CTCF (CTCF-binding sites) region of the HOXA9 gene.

Alternatively, the detection region of the HOXA9 gene targeted by the HOXA9 gene methylation detection reagent includes the HOXA9 gene body or its promoter region.

In some embodiments, the detection reagent for HOXA9 gene methylation comprises SEQ ID NO: 22 (region 1), SEQ ID NO: 24 (region 2) or SEQ ID NO: 26 (region 3) for the detection region sequence shown. As a more preferred embodiment, the reagent includes the sequence shown in SEQ ID NO: 22 for the detection region.

The inventors have found through experiments that the selection of the detection region of the HOXA9 gene will have an impact on the detection efficiency of the tumor. The primers designed according to the CG-enriched region of the HOXA9 gene have obvious differences in the detection results of the primers designed for different regions. The detection rate of tumor is 50%~60% higher than that of poor area. The inventors found through experimental comparison that the detection results of GC-rich regions or CTCF (CTCF-binding sites) regions are significantly better than those of non-hypermethylated regions.

The detection reagent for HOXA9 gene methylation of the present disclosure comprises amplification primers. As an optional embodiment, the primer comprises 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 NO: 58 at least any one. As a preferred embodiment, the primers comprise a pair of primers shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The detection reagent for HOXA9 gene methylation of the present disclosure also includes a probe. As an optional embodiment, the probe is such as 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 Shown in NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56 or SEQ ID NO: 59. As a preferred embodiment, the probe is shown in SEQ ID NO: 3. As a preferred mode of the present disclosure, for the convenience of clinical use, the fluorescent groups labeled by the detection probes include but are not limited to VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, Texas Red, etc.; the quenching groups include But not limited to TAMRA, BHQ, MGB, Dabcyl, etc., it is suitable for the multi-channel PCR detection system commonly used in clinical detection at present, and realizes multi-color fluorescence detection in one reaction tube.

As a preferred embodiment, the HOXA9 gene methylation detection reagent of the present disclosure comprises the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2 and the probe shown in SEQ ID NO: 3.

As an optional embodiment, the detection of HOXA9 gene methylation in the present disclosure further includes bisulfite, bisulfite or hydrazine salt for modifying methylated cytosine in HOXA9 into thymine. Of course, it may not be included in the reagent of the present disclosure. It can be purchased independently when using.

As an optional embodiment, the detection of HOXA9 gene methylation in the present disclosure also includes one or more of DNA polymerase, dNTPs, Mg²⁺ ions, and buffer; preferably, DNA polymerase, dNTPs, Mg²⁺ Ions and buffers for amplification reactions of the HOXA9 gene.

As an optional embodiment, the detection of HOXA9 gene methylation in the present disclosure also includes a detection reagent for a reference gene. Preferably, the reference gene comprises β-actin or COL2A1. In addition to these two reference genes, other reference genes for methylation detection in the prior art can also be used. Further, the detection reagent for the reference gene β-actin includes primers and probes for the reference gene. As a preferred embodiment, the detection reagent of the reference gene comprises the primer pair shown in SEQ ID NO: 16, SEQ ID NO: 17, and the probe of SEQ ID NO: 18. The detection reagent of the reference gene COL2A1 comprises a primer pair shown in SEQ ID NO: 60 (TTTTGGATTTAAGGGGAAGATAAA), SEQ ID NO: 61 (TTTTTCCTTCTCTAC​ATCTTTCTACCT), and a probe shown in SEQ ID NO: 62 (AAGGGAAATTGAGAAATGAGAGAAG​GGA) .

Another aspect of the disclosure provides a method for detecting DNA methylation of the HOXA9 gene, comprising the following steps: (1) Treat the test sample with bisulfite, bisulfite or hydrazine salt to obtain a modified test sample; (2) Using the above-mentioned detection of HOXA9 gene methylation, detect the HOXA9 gene methylation of the modified detection sample in step (1).

As an optional way, use methylation-specific polymerase chain reaction (MSP) or real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) for detection. Other DNA methylation detection methods reported in the prior art can also be applied in the present disclosure. A prior art methylation detection method is incorporated into this disclosure by patent USSN 62/175,916.

Another aspect of the present disclosure also provides a detection/diagnosis/prognosis system for lung cancer, the system comprising: a DNA methylation detection component of the HOXA9 gene, and, Result judgment component.

In some embodiments, the HOXA9 gene DNA methylation detection component contains the above-mentioned HOXA9 gene methylation detection reagent.

In some embodiments, the methylation detection component includes one or more of a fluorescent quantitative PCR instrument, a PCR instrument, and a sequencer. In some embodiments, the result judging means is used to output diagnostic results such as the risk of lung cancer and/or the type of lung cancer according to the DNA methylation level of the HOXA9 gene detected by the detection means;

In some embodiments, the disease risk is determined based on the deviation between the methylation levels of the test sample and the normal sample by comparing the methylation levels of the test sample and the normal sample through the result judgment component.

In some embodiments, the outcome determination means comprises a data processing machine.

In some embodiments, the data processing machine includes any equipment or instrument or device capable of data processing that can be used by those skilled in the art.

In some embodiments, the data processing machine includes one or more of a computer and a computer. The computer is attached with any software or program that can be used by those skilled in the art for data processing or statistical analysis. In some embodiments, the computer includes a computer loaded with one or more software of SPSS, SAS, and Excel.

In some embodiments, the result judging component further includes a result outputter. The outputter includes any device or instrument or device that can display data processing results as readable content. In some embodiments, the result outputter comprises one or more of a screen, a paper report.

Another aspect of the present disclosure provides a method for diagnosing lung cancer, the method comprising the following steps: Detect the HOXA9 gene methylation level of the test sample from the test subject; Comparing the HOXA9 gene methylation level of the sample to be tested with the normal sample; Lung cancer is diagnosed based on the deviation of the methylation level between the test sample and the normal sample.

In some embodiments, when the methylation level of the sputum sample to be tested is greater than 2.3%, it is determined that the sample to be tested is a lung cancer sample, and when the methylation level is less than or equal to 2.3%, it is determined that the sample to be tested is a non-lung cancer sample .

In some embodiments, when the methylation level of the lavage fluid sample to be tested is greater than 0.5%, it is determined that the sample to be tested is a lung cancer sample, and when the methylation level is less than or equal to 0.5%, it is determined that the sample to be tested is non-cancerous Lung cancer samples.

The diagnostic method of the present disclosure can be used before and after the treatment of lung cancer or in combination with the treatment of lung cancer, and used after treatment such as evaluating the success of treatment or monitoring 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 in the art to patients diagnosed as lung cancer by the above diagnostic method. Any other types of lung cancer treatments used and combinations of these treatments. In the present disclosure, the detection samples of the HOXA9 gene methylation detection reagent include sputum, lung lavage fluid, lung tissue, pleural effusion, blood, serum, plasma, urine, prostatic fluid, tears or feces, etc. . As a preferred embodiment, the detection sample of the HOXA9 gene methylation detection reagent includes sputum, tissue or lung lavage fluid. As a more preferred embodiment, the detection sample of the HOXA9 gene methylation detection reagent includes sputum or lung lavage fluid.

The present disclosure finds that the methylation level of the HOXA9 gene in tissues is highly correlated with the incidence of lung cancer. Among the 185 tissues, the specificity of HOXA9 gene normal group was as high as 95% and the sensitivity was 78.6% compared with the whole lung cancer group, although the sensitivity was lower than that of another tumor marker SHOX2 (sensitivity 80.6%) gene in the disclosed experiment, and let Surprisingly, it was found that the methylation level of HOXA9 gene detected in sputum and lung lavage fluid also maintained a high degree of correlation with the incidence of lung cancer. In sputum, the sensitivity of HOXA9 was 74.3 %, specificity 95%; in lung lavage fluid, sensitivity was 61.9%, specificity 95%.

Among the various molecular markers studied by the inventors, the detection sensitivity or specificity of sputum samples is significantly lower than that of tissue samples. For example, SHOX2, PCDHGA12, HOXD8, and GATA3 are reported to be related to lung cancer. Among them, the detection sensitivity of SHOX2 in tissue is 80.6%, which is higher than that of HOXA9 gene, while in sputum, the sensitivity is higher. 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 detection sample, it is especially suitable to use HOXA9 gene as a tumor marker.

The study found that a variety of lung cancer markers only showed good detection sensitivity and specificity in tissues; however, in sputum and lung lavage fluid samples, no matter how the detection area, detection primers, probes, etc. were designed and optimized, the sensitivity However, the sex still dropped significantly, seriously affecting the diagnosis of lung cancer.

The HOXA9 gene also maintains a high sensitivity in sputum and lung lavage fluid samples, and maintains a specificity as high as 95%, which makes this gene extremely special as a sputum and lung lavage fluid sample reliable lung cancer markers.

In the present disclosure, the lung cancer is selected from small cell lung cancer (small cell lung cancer, SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC); further, the non-small cell lung cancer is selected from squamous cell lung cancer Carcinoma, adenocarcinoma, or large cell carcinoma. As a preferred embodiment, the lung cancer is selected from adenocarcinoma.

It has been confirmed by experiments that the HOXA9 gene has high specificity and high sensitivity in many different types of lung cancer, even in lung adenocarcinoma, it has higher sensitivity than other tumor markers, such as in sputum, The detection sensitivity of HOXA9 is 55.6%, while the sensitivity of SHOX2 is 11.1% (see Table 6 of Example 2), which further shows that HOXA9 is especially suitable for the detection of sputum, especially for the detection of lung adenocarcinoma. At present, the missed detection rate of lung adenocarcinoma is high. On the one hand, because lung adenocarcinoma is more likely to occur in women and non-smokers, the incidence rate is lower than that of squamous cell carcinoma and undifferentiated carcinoma, and the age of onset is younger, and women are relatively more common; on the other hand, most adenocarcinomas originate from small Bronchi is a peripheral type of lung cancer, and the exfoliated cells in the deep lung are more difficult to cough up through sputum; on the other hand, lung adenocarcinoma generally has no obvious clinical symptoms in the early stage. Therefore, the detection of lung adenocarcinoma is more difficult and valuable.

The beneficial effect of one of the above-mentioned technical solutions is: not only can tissue be used as a detection sample, but also it has higher sensitivity in sputum and lung lavage fluid, and can be easily used in sputum and lung lavage fluid. Lung lavage fluid was used as a test sample for reliable diagnosis of lung cancer. Sputum samples are obtained very easily without causing any pain and inconvenience to the patient. The amount of sample used is very small, the sampling process is very convenient and has no impact on patients. At the same time, the sample is convenient to mail or take to the hospital for examination.

The beneficial effect of one of the technical solutions above is that it can detect multiple types of lung cancer, and it also has higher sensitivity than other markers for difficult-to-detect adenocarcinoma.

The beneficial effect of one of the above technical solutions is that it does not need to consider the detected object and age, and has a wide range of applications.

The beneficial effect of one of the above-mentioned technical solutions is: detecting and diagnosing cancer through the level of methylation. More and more studies have confirmed that methylation changes are early events in the process of tumorigenesis, and it is easier to detect abnormal methylation detect early lesions.

The technical solution of the present disclosure is further described through specific examples below, and the specific examples do not represent limitations on the protection scope of the present disclosure. Some non-essential modifications and adjustments made by others based on the concepts of the present disclosure still fall within the protection scope of the present disclosure.

A "primer" or "probe" in the present disclosure refers to an oligonucleotide comprising a region complementary to a 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 primer or probe sequence 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 A region of sequence complementarity of consecutive or discontinuous block nucleotides. When a primer or probe comprises a region "complementary to at least x contiguous nucleotides of a target molecule", said primer or probe is at least 95% complementary to at least x contiguous nucleotides of a target molecule. In some embodiments, the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.

"Diagnosis" in the present disclosure includes not only early diagnosis of lung cancer, but also intermediate and late diagnosis of lung cancer, and also includes lung cancer screening, risk assessment, prognosis, disease identification, diagnosis of disease stage and selection of therapeutic targets.

The application of lung cancer marker HOXA9 makes the early diagnosis of lung cancer possible. When a gene that is determined to be methylated in a cancer cell is methylated in a clinically or morphologically normal-appearing cell, this indicates that the normal-appearing cell has progressed to cancer. Thus, lung cancer can be diagnosed at an early stage by the methylation of the lung cancer-specific gene HOXA9 in cells with normal appearance.

As an optional implementation of the diagnosis of disease stages, the progression of lung cancer at different stages or periods can be diagnosed by measuring the degree of methylation of HOXA9 obtained from samples. By comparing the degree of HOXA9 gene methylation of nucleic acids isolated from samples of each stage of lung cancer with the HOXA9 gene methylation of one or more nucleic acids isolated from samples of lung tissue without cell proliferation abnormalities The degree of differentiation can detect the specific stage of lung cancer in the sample.

In this disclosure, a "normal" sample refers to a sample of the same type isolated from an individual known to be free of said cancer or tumor.

In the present disclosure, 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 DNA and RNA samples containing mRNA, or DNA-RNA hybrids. Wherein DNA or RNA can be single-stranded or double-stranded.

Methods for methylation detection are well known, such as methylation-specific polymerase chain reaction, real-time fluorescent quantitative methylation-specific polymerase chain reaction, pyrosequencing, 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., in addition, other The methylation detection method can be introduced through the patent US62007687.

In the present disclosure, "methylation level" and "degree of methylation" can usually be expressed as the percentage of methylated cytosine, which is the number of methylated cytosine divided by the number of methylated cytosine divided by the number of unmethylated cytosine. The sum of the number of methylated cytosines; and the current method of dividing the number of methylated target genes by the number of reference genes to express the methylation level; and other methods of expressing the methylation level in the prior art.

Example 1 : Selection of detection target genes

Using methylated DNA as the detection target has obvious advantages. Compared with protein markers, DNA can be amplified and easily detected; compared with mutation markers, the DNA methylation sites are all located in the gene Specific site, generally in the promoter region, which makes the detection easier and more convenient. In order to complete this disclosure, the inventors screened out hundreds of candidate genes from hundreds of candidate genes as candidate detection genes, β-actin gene as a reference gene, and studied each The distribution of gene methylation sites, the primer probes designed for detection were used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescent quantitative methylation-specific PCR, qMSP) detection. The primer probes for each gene detection are as follows:

The detection primers and probes of HOXA9 are: SEQ ID NO: 1 HOXA9-F2 Primer F: TTAGTTTTTTTCGGTAGGCGGC SEQ ID NO: 2 HOXA9-R2 Primer R: AAACGCCAAACACCGTCG SEQ ID NO: 3 HOXA9-P2 probe: FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1

The detection primers and probes of SHOX2 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 of PCDHGA12 are: SEQ ID NO: 7 PCDHGA12 Primer F: TTGGTTTTTACGGTTTTCGAC SEQ ID NO: 8 PCDHGA12 Primer R: AAATTCTCCGAAACGCTCG SEQ ID NO: 9 PCDHGA12 Probe: FAM-ATTCGGTGCGTATAGGTATCGCGC-BQ1

The detection primers and probes of HOXD8 are: SEQ ID NO: 10 HOXD8 Primer F: TTAGTTTCGGCGCGTAGC SEQ ID NO: 11 HOXD8 Primer R: CCTAAAACCGACGCGATCTA SEQ ID NO: 12 HOXD8 probe: FAM-AAAACTTACGATCGTCTACCCTCCG-BQ1

The detection primers and probes of GATA3 are: SEQ ID NO: 13 GATA3 Primer F: TTTCGGTAGCGGGTATTGC SEQ ID NO: 14 GATA3 Primer R: AAAATAACGACGAACCAACCG SEQ ID NO: 15 GATA3 probe: FAM-CGCGTTTATGTAGGAGTGGTTGAGGTTC-BQ1

The detection primers and probes for β-actin are: SEQ ID NO: 16 β-actin Primer F: TTTTGGATTGTGAATTTGTG SEQ ID NO: 17 β-actin Primer R: AAAACCTACTCCCTCCCTTAAA SEQ ID NO: 18 β-actin probe: FAM-TTGTGTGTTGGGTGGTGGTT-BQ1

experiment procedure:

1 ,extract dna

Collect specimens from confirmed lung cancer patients and non-tumor patients, including paraffin tissue specimens, sputum specimens, and lavage fluid specimens, sample pretreatment and cell separation, according to the kit HiPure FFPE DNA Kit (D3126-03) of Meiji Biotechnology Co., Ltd. Instructions for DNA extraction.

2 , dna bisulfite treatment

Bisulfite modification was carried out according to the instruction manual of EZ DNA Methylation™ KIT (D5002) from ZYMO RESEARCH Bio Company.

3. Amplification and detection [Table 1] Liquid preparation system HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin Reactive components Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Upstream Primer (100 µM) 0.125 0.05 0.125 0.05 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 Magnesium ion (25 mM) 4 5 5 5 3 5 dNTPs (10 mM) 1 1 1 1 1 1 Taq Polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 5× buffer 5 5 5 5 5 5 Sterilized water 13.2 12.275 12.2 12.275 14.2 12.2 template DNA 1 1 1 1 1 1 total capacity 25 25 25 25 25 25

Amplification system: [Table 2] PCR reaction process HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin step temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time number of cycles pre-denatured 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 55°C 60 seconds 55°C 60 seconds 55°C 60 seconds 55°C 60 seconds 54°C 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1

4 ,Test results

4.1. Detection results in paraffin tissue

Sample information: A total of 185 lung tissue samples, including 87 normal tissue samples, 98 cancer tissue samples, 15 squamous cell carcinoma samples, 81 adenocarcinoma samples, and 2 lung cancer samples that were not clearly classified. And 73 pairs of paracancerous control samples.

The ROC curves of HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 detected in all tissue samples are shown in Figure 1. The statistical results of the detection of each gene in the tissue are shown in Table 3. [Table 3] Detection results in tissues Analysis group index HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 78.6% 80.6% 69.4% 40.8% 67.3% Comparison between normal group and all squamous cell carcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 93.3% 93.3% 53.3% 60.0% 73.3% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 75.3% 79.0% 71.6% 38.3% 65.4%

From the above results, it can be seen that in tissue samples, SHOX2 and HOXA9 have the best detection results, followed by PCDHGA12 and GATA3, whether comparing lung cancer as a whole or comparing lung cancer subtypes. , the detection effect of HOXD8 was the worst, with a sensitivity of only 40.8%.

According to the above results, in order to study the detection of different genes in sputum, the invention further screens the five markers HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 in sputum, because sputum is used as a non-invasive detection sample, more significant.

Example 2 : HOXA9 , SHOX2 , PCDHGA12 , HOXD8 and GATA3 Gene detection in sputum

Sample information: A total of 90 cases of sputum samples were tested, including 55 cases of normal control group samples, 35 cases of cancer group control samples, 12 cases of squamous cell carcinoma, 6 cases of small cell carcinoma, 9 cases of adenocarcinoma, and large There were 2 cases of cell carcinoma and 6 cases of unclassified lung cancer.

Experimental procedure:

a. Collect sputum samples from patients diagnosed with lung cancer and non-lung cancer patients, thicken with DTT, centrifuge to separate the pellet, wash twice with PBS, and then use Magen’s DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.

b. Use the DNA transformation kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Bio Company to carry out the bisulfite modification of DNA.

c. The liquid preparation system is as follows: [Table 4] Liquid preparation system HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin Reactive components Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Amount added (µl) Upstream Primer (100 µM) 0.125 0.05 0.125 0.05 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 0.05 0.05 0.05 Magnesium ion (25 mM) 4 5 5 5 3 5 dNTPs (10 mM) 1 1 1 1 1 1 Taq Polymerase (5 unit/µl) 0.5 0.5 0.5 0.5 0.5 0.5 5× buffer 5 5 5 5 5 5 Sterilized water 13.2 12.275 12.2 12.275 14.2 12.2 template DNA 1 1 1 1 1 1 total capacity 25 25 25 25 25 25

d. The amplification system is as follows: [Table 5] Amplification system HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 β-actin step temperature and time temperature and time temperature and time temperature and time temperature and time temperature and time number of cycles pre-denatured 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 60°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 55°C 60 seconds 55°C 60 seconds 55°C 60 seconds 55°C 60 seconds 54°C 60 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 40°C for 30 seconds 1

e. The test results are as follows: [Table 6] Test results in sputum Analysis group index HOXA9 SHOX2 PCDHGA12 HOXD8 GATA3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 74.3% 51.4% 20.0% 42.9% 17.1% Comparison between normal group and all squamous cell carcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 75.0% 66.7% 25.0% 66.7% 25.0% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 55.6% 11.1% 33.3% 11.1% 0.0% Comparison of normal group and all small cell carcinoma group specificity 95.0% 95.0% 95.0% 95.0% 95.0% sensitivity 100% 83.3% 0.0% 50.0% 33.3%

f. The ROC curves of 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 sputum samples, the simultaneous detection of these five genes In comparison, the detection effect of HOXA9 is better than the detection effect of the other four genes, no matter whether the comparative analysis is performed on lung cancer as a whole or according to the subtype of lung cancer. Especially for the detection effect of adenocarcinoma, the detection rate of HOXA9 is much higher than that of other genes. Adenocarcinoma is generally of the peripheral type. Due to the tree-like physiological structure of the bronchi, it is more difficult for the exfoliated cells in the deep lung to be coughed up through sputum. In the present disclosure, SHOX2, which has the highest sensitivity to adenocarcinoma in tissue, has a greatly reduced sensitivity to 11.1% in sputum, so the detection of this part is more difficult and meaningful.

Example 3 : HOXA9 and SHOX2 Gene detection in sputum

A large number of documents show that SHOX2 can be used as a marker for the detection of lung cancer, and a patent shows [CN201510203539-method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene-application disclosure], SHOX2 can be used in alveolar lavage fluid, lesion tissue , pleural effusion, sputum and other samples have a high detection rate. In order to verify the detection effect of HOXA9, in this example, 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 ones in Examples 1 and 2 of the present disclosure. The SHOX2 gene detected by self-designed primers and probes.

The primer probes for each gene detection are as follows:

The detection primers and probes of HOXA9 are: SEQ ID NO: 1 HOXA9-F2 Primer F: TTAGTTTTTTTCGGTAGGCGGC SEQ ID NO: 2 HOXA9-R2 Primer R: AAACGCCAAACACCGTCG SEQ ID NO: 3 HOXA9-P2 probe: FAM-ACGTTGGTCGAGTATTTCGATTTTAGTTC-BQ1

The detection primers and probes of SHOX2_n3 are: SEQ ID NO: 19 SHOX2_n3 Primer F: TTTGGATAGTTAGGTAATTTTCG SEQ ID NO: 20 SHOX2_n3 Primer R: CGTACACGCCTATACTCGTACG SEQ ID NO: 21 SHOX2_n3.2 Probe: FAM-CCCCGATCGAACAAACGAAAC-BQ1

a. The liquid preparation system is as follows: [Table 7] Liquid preparation system HOXA9 SHOX2_n3 β-actin Reactive components Amount added (µl) Amount added (µl) Amount added (µl) Upstream Primer (100 µM) 0.125 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 Magnesium ion (25 mM) 4 5 5 dNTPs (10 mM) 1 1 1 Taq Polymerase (5 unit/µl) 0.5 0.5 0.5 5× buffer 5 5 5 Sterilized water 13.2 12.2 12.2 template DNA 1 1 1 total capacity 25 25 25

b. The amplification system is as follows: [Table 8] Amplification system HOXA9 β-actin step temperature and time temperature and time number of cycles pre-denatured 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 54°C 60 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 1 [Table 9] SHOX2_n3 reaction program step temperature and time number of cycles pre-denatured 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 45 60°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 1

c. The test results are as follows:

Use the standard curve to calculate the methylation copy number of each gene in the sample, use the ratio = target gene copy number/ACTB copy number*100 to judge the degree of methylation of the two groups of samples, and finally select the threshold of HOXA9 as 2.3, The threshold of SHOX2_n3 is 1.3, which is used as a criterion for judging the cancer group and the control group. The converted ratio can be judged as positive "+" if it exceeds the set threshold, and it can be judged as negative "-" if it is equal to or less than the set threshold. According to this standard, the test results of 90 cases of sputum samples are as follows: [Table 10] Test results HOXA9 SHOX2_n3 HOXA9 SHOX2_n3 serial number sample type Methylation rate Methylation rate Detection Detection 1 non-lung cancer control 0.1 0.2 − − 2 non-lung cancer control 0.5 0.0 − − 3 non-lung cancer control 0.5 0.0 − − 4 non-lung cancer control 0.8 0.2 − − 5 non-lung cancer control 0.5 0.1 − − 6 non-lung cancer control 0.6 0.3 − − 7 non-lung cancer control 1.6 0.2 − − 8 non-lung cancer control 0.6 0.3 − − 9 non-lung cancer control 0.3 0.0 − − 10 non-lung cancer control 7.1 4.0 + + 11 non-lung cancer control 0.9 0.4 − − 12 non-lung cancer control 0.0 0.2 − − 13 non-lung cancer control 0.3 0.0 − − 14 non-lung cancer control 0.4 0.1 − − 15 non-lung cancer control 0.0 0.0 − − 16 non-lung cancer control 0.3 0.3 − − 17 non-lung cancer control 0.4 0.0 − − 18 non-lung cancer control 0.4 0.0 − − 19 non-lung cancer control 0.0 0.0 − − 20 non-lung cancer control 2.3 1.1 − − twenty one non-lung cancer control 1.3 0.4 − − twenty two non-lung cancer control 0.8 2.0 − + twenty three non-lung cancer control 0.3 0.1 − − twenty four non-lung cancer control 0.5 0.0 − − 25 non-lung cancer control 1.2 0.5 − − 26 non-lung cancer control 0.5 0.3 − − 27 non-lung cancer control 0.2 0.0 − − 28 non-lung cancer control 0.4 0.0 − − 29 non-lung cancer control 0.3 0.0 − − 30 non-lung cancer control 0.0 0.0 − − 31 non-lung cancer control 0.0 0.4 − − 32 non-lung cancer control 0.0 0.0 − − 33 non-lung cancer control 0.3 0.5 − − 34 non-lung cancer control 0.0 0.7 − − 35 non-lung cancer control 0.2 0.3 − − 36 non-lung cancer control 0.7 0.8 − − 37 non-lung cancer control 0.0 0.1 − − 38 non-lung cancer control 0.8 0.6 − − 39 non-lung cancer control 0.2 0.3 − − 40 non-lung cancer control 0.0 0.2 − − 41 non-lung cancer control 3.7 1.7 + + 42 non-lung cancer control 0.4 0.0 − − 43 non-lung cancer control 0.6 0.6 − − 44 non-lung cancer control 0.0 1.1 − − 45 non-lung cancer control 0.0 0.2 − − 46 non-lung cancer control 0.1 0.2 − − 47 non-lung cancer control 0.0 0.2 − − 48 non-lung cancer control 0.1 0.0 − − 49 non-lung cancer control 0.1 0.4 − − 50 non-lung cancer control 0.1 0.2 − − 51 non-lung cancer control 0.0 0.4 − − 52 non-lung cancer control 0.0 0.2 − − 53 non-lung cancer control 0.5 0.6 − − 54 non-lung cancer control 0.0 0.0 − − 55 non-lung cancer control 2.3 0.7 + − 56 squamous cell carcinoma 4.9 3.5 + + 57 squamous cell carcinoma 46.5 22.7 + + 58 squamous cell carcinoma 27.4 8.3 + + 59 squamous cell carcinoma 73.8 23.6 + + 60 squamous cell carcinoma 2.2 9.5 − + 61 squamous cell carcinoma 4.7 1.2 + − 62 squamous cell carcinoma 7.2 7.1 + + 63 squamous cell carcinoma 98.0 96.8 + + 64 squamous cell carcinoma 2.1 0.9 − − 65 squamous cell carcinoma 22.3 23.0 + + 66 squamous cell carcinoma 66.7 94.2 + + 67 squamous cell carcinoma 0.9 0.0 − − 68 Adenocarcinoma 2.5 2.9 + + 69 Adenocarcinoma 1.0 0.2 − − 70 Adenocarcinoma 0.7 0.0 − − 71 Adenocarcinoma 1.7 1.6 − + 72 Adenocarcinoma 6.0 0.0 + − 73 Adenocarcinoma 2.8 0.4 + − 74 Adenocarcinoma 3.5 2.7 + + 75 Adenocarcinoma 0.8 0.1 − − 76 Adenocarcinoma 2.5 0.0 + − 77 small cell carcinoma 29.3 17.3 + + 78 small cell carcinoma 34.1 16.8 + + 79 small cell carcinoma 29.9 20.4 + + 80 small cell carcinoma 14.8 16.5 + + 81 small cell carcinoma 21.3 40.1 + + 82 small cell carcinoma 5.9 0.8 + − 83 large cell carcinoma 9.9 7.7 + + 84 large cell carcinoma 1.1 0.1 − − 85 poorly differentiated carcinoma 0.3 0.5 − − 86 poorly differentiated carcinoma 9.0 11.6 + + 87 lung cancer 29.9 6.3 + + 88 lung cancer 49.4 88.7 + + 89 lung cancer 48.6 48.6 + + 90 lung cancer 4.4 1.1 + −

d. Result analysis [Table 11] Statistical results Analysis group index HOXA9 SHOX2_n3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% sensitivity 74.3% 62.9% Comparison between normal group and all squamous cell carcinoma group specificity 95.0% 95.0% sensitivity 75.0% 75.0% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% sensitivity 55.6% 33.3% Comparison of normal group and all small cell carcinoma group specificity 95.0% 95.0% sensitivity 100% 83.3%

e. The ROC curves of HOXA9 and SHOX2_n3 in all sputum samples are shown in Figure 3, the amplification curves of HOXA9 and SHOX2_n3 in sputum samples are shown in Figure 4, and the statistical results are shown in Table 11. From the above results, it can be seen that in sputum In the liquid samples, the detection effect of HOXA9 is better than that of SHOX2 gene, no matter whether lung cancer is compared and analyzed as a whole, or according to the subtype of lung cancer. Especially for the detection of adenocarcinoma, the detection rate of HOXA9 is much higher than that of SHOX2 gene by 33.3%. Adenocarcinoma is generally of the peripheral type. Due to the tree-like physiological structure of the bronchi, the exfoliated cells in the deep lung are more difficult to cough up through sputum. This disclosure is the first to discover a marker that can detect adenocarcinoma using sputum as a sample, and can greatly increase the sensitivity to 55.6%. This breakthrough is of great significance to the detection of adenocarcinoma.

Example 4 : HOXA9 as well as SHOX2 Detection of genes in lavage fluid

Sample information: A total of 79 cases of alveolar lavage fluid samples were tested, including 58 cases of normal control group samples, 21 cases of cancer control samples, and 21 cases of cancer group samples, including 6 cases of squamous cell carcinoma, 4 cases of small cell carcinoma, and 11 cases of adenocarcinoma .

Experimental procedure:

a. Collect alveolar lavage fluid samples from lung cancer patients and non-lung cancer patients, centrifuge the cells, and then use Magen’s DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.

b. Use the DNA transformation kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Bio Company to carry out the bisulfite modification of DNA.

c. The amplification detection system is as follows: [Table 12] Amplification system HOXA9 SHOX2_n3 β-actin Reactive components Amount added (µl) Amount added (µl) Amount added (µl) Upstream Primer (100 µM) 0.125 0.125 0.125 Downstream Primer (100 µM) 0.125 0.125 0.125 Probe (100 µM) 0.05 0.05 0.05 Magnesium ion (25 mM) 4 5 5 dNTPs (10 mM) 1 1 1 Taq Polymerase (5 unit/µl) 0.5 0.5 0.5 5× buffer 5 5 5 Sterilized water 13.2 12.2 12.2 template DNA 1 1 1 total capacity 25 25 25

d. The detection system is as follows: [Table 13] Reaction program of HOXA9 and β-actin HOXA9 β-actin step temperature and time temperature and time number of cycles pre-denatured 95°C for 5 minutes 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 95°C for 20 seconds 10 63°C for 30 seconds 62°C for 30 seconds 70°C for 30 seconds 70°C for 30 seconds Amplification 2 95°C for 20 seconds 95°C for 20 seconds 45 60°C for 30 seconds 54°C 60 seconds 72°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 40°C for 30 seconds 1 [Table 14] SHOX2_n3 reaction program step temperature and time number of cycles pre-denatured 95°C for 5 minutes 1 Amplification 1 95°C for 20 seconds 45 60°C for 30 seconds 72°C for 30 seconds cool down 40°C for 30 seconds 1

e. The test results are as follows:

Use the standard curve to calculate the methylation copy number of each gene in the sample, use the ratio = target gene copy number/ACTB copy number*100 to judge the degree of methylation of the two groups of samples, and finally select the threshold value of HOXA9 as 0.5, The threshold of SHOX2_n3 is 0.6, which is used as a criterion for judging the cancer group and the control group. The converted ratio can be judged as positive "+" if it exceeds the set threshold, and it can be judged as negative "−" if it is equal to or less than the set threshold. According to this standard, the test results of 79 cases of lavage fluid samples are as follows: [Table 15] Test results HOXA9 SHOX2_n3 HOXA9 SHOX2_n3 serial number organization type Methylation rate Methylation rate Detection Detection 1 non-lung cancer control 0.2 0.3 − − 2 non-lung cancer control 0.1 0.7 − + 3 non-lung cancer control 0.2 0.1 − − 4 non-lung cancer control 0.5 0.6 − − 5 non-lung cancer control 0.1 0.2 − − 6 non-lung cancer control 0.2 0.1 − − 7 non-lung cancer control 0.0 0.0 − − 8 non-lung cancer control 0.1 0.0 − − 9 non-lung cancer control 0.0 0.0 − − 10 non-lung cancer control 0.0 0.0 − − 11 non-lung cancer control 0.0 0.1 − − 12 non-lung cancer control 0.0 0.1 − − 13 non-lung cancer control 0.0 0.1 − − 14 non-lung cancer control 0.3 0.1 − − 15 non-lung cancer control 0.3 0.5 − − 16 non-lung cancer control 0.1 0.1 − − 17 non-lung cancer control 0.0 0.0 − − 18 non-lung cancer control 0.4 0.2 − − 19 non-lung cancer control 0.1 0.2 − − 20 non-lung cancer control 0.0 0.3 − − twenty one non-lung cancer control 0.0 0.0 − − twenty two non-lung cancer control 0.0 0.2 − − twenty three non-lung cancer control 0.0 0.1 − − twenty four non-lung cancer control 0.0 0.1 − − 25 non-lung cancer control 0.0 0.0 − − 26 non-lung cancer control 0.1 0.2 − − 27 non-lung cancer control 0.1 0.1 − − 28 non-lung cancer control 0.1 0.3 − − 29 non-lung cancer control 0.1 0.1 − − 30 non-lung cancer control 0.5 0.5 − − 31 non-lung cancer control 0.0 0.2 − − 32 non-lung cancer control 0.0 0.4 − − 33 non-lung cancer control 0.1 0.1 − − 34 non-lung cancer control 0.5 0.5 − − 35 non-lung cancer control 0.1 0.2 − − 36 non-lung cancer control 0.8 0.8 + + 37 non-lung cancer control 0.1 0.4 − − 38 non-lung cancer control 0.1 0.1 − − 39 non-lung cancer control 0.2 0.2 − − 40 non-lung cancer control 0.3 0.3 − − 41 non-lung cancer control 0.4 0.3 − − 42 non-lung cancer control 0.1 0.1 − − 43 non-lung cancer control 0.8 0.6 + + 44 non-lung cancer control 0.0 0.1 − − 45 non-lung cancer control 0.2 0.2 − − 46 non-lung cancer control 0.0 0.1 − − 47 non-lung cancer control 0.1 0.1 − − 48 non-lung cancer control 0.3 0.2 − − 49 non-lung cancer control 0.1 0.4 − − 50 non-lung cancer control 0.0 0.0 − − 51 non-lung cancer control 0.2 0.0 − − 52 non-lung cancer control 0.5 0.5 − − 53 non-lung cancer control 0.0 0.0 − − 54 non-lung cancer control 1.1 0.1 + − 55 non-lung cancer control 0.4 0.0 − − 56 non-lung cancer control 0.0 0.0 − − 57 non-lung cancer control 0.2 0.0 − − 58 non-lung cancer control 0.0 0.0 − − 59 squamous cell carcinoma 0.5 0.3 − − 60 squamous cell carcinoma 76.9 321.9 + + 61 squamous cell carcinoma 60.9 56.7 + + 62 squamous cell carcinoma 0.3 0.0 − − 63 squamous cell carcinoma 17.7 7.6 + + 64 squamous cell carcinoma 1.5 0.6 + + 65 Adenocarcinoma 0.5 0.6 − − 66 Adenocarcinoma 0.3 0.0 − − 67 Adenocarcinoma 0.3 0.4 − − 68 Adenocarcinoma 3.7 33.5 + + 69 Adenocarcinoma 2.9 2.3 + + 70 Adenocarcinoma 0.1 0.0 − − 71 Adenocarcinoma 0.3 0.3 − − 72 Adenocarcinoma 0.0 1.4 + + 73 Adenocarcinoma 20.8 8.6 + + 74 Adenocarcinoma 5.3 0.3 + − 75 Adenocarcinoma 0.9 0.2 + − 76 small cell carcinoma 0.8 0.3 + − 77 small cell carcinoma 26.4 97.7 + + 78 small cell carcinoma 1.8 7.6 + + 79 small cell carcinoma 0.6 2.4 + + [Table 16] Analysis results Analysis group index HOXA9 SHOX2_n3 Comparison of normal group and all cancer groups specificity 95.0% 95.0% sensitivity 61.9% 52.4% Comparison between normal group and all squamous cell carcinoma group specificity 95.0% 95.0% sensitivity 66.7% 66.7% Comparison of normal group and all adenocarcinoma group specificity 95.0% 95.0% sensitivity 45.5% 36.4% Comparison of normal group and all small cell carcinoma group specificity 95.0% 95.0% sensitivity 100% 75.0%

f. The ROC curves of HOXA9 and SHOX2_n3 detected in all lavage fluid samples are shown in Figure 5, the amplification curves are shown in Figure 6, and the statistical results are shown in Table 16. From the above results, it can be seen that in the lavage fluid samples, HOXA9 and SHOX2 were detected at the same time, and the lung cancer as a whole was compared and analyzed. The detection rate of HOXA9 was 61.9%, which was higher than the 52.4% of SHOX2. According to the subtype of lung cancer For comparative analysis, the detection rate of HOXA9 in adenocarcinoma and small cell carcinoma is higher than that of SHOX2_n3. Because adenocarcinoma is generally of the peripheral type, and due to the tree-like physiological structure of the bronchi, the alveolar lavage fluid is not easy to contact the alveoli or the deep lung. Cancer tissue, so the detection of this part is more difficult and meaningful. In addition, for small cell carcinoma, the sensitivity of HOXA9 was as high as 100%, which was significantly higher than that of SHOX2 at 75.0%.

Based on the above four examples, it can be fully demonstrated that HOXA9 has a better detection effect in the detection and diagnosis of lung cancer, especially in biological samples such as sputum and alveolar lavage fluid. It can be more easily applied to large-scale population screening. It has more superior socioeconomic value.

Example 5 : HOXA9 The influence of the detection area, primers and probes on the detection effect

1. The influence of the detection area on the detection effect

Various research data show that the methylation status and distribution of the same gene are not uniform. Therefore, for the same gene, the methylation primers and probe detection systems designed in different regions can be used for the diagnosis of the same sample and the same tumor. The detection efficiency is not the same, and sometimes the selected area is not suitable, resulting in no diagnostic effect on the tumor at all. After repeated research and comparison of multiple detection areas, some exemplary detection areas are shown in Table 17. [Table 17] Sequences to be detected sequence name sequence SEQ ID NO: 22 HOXA9 region 1 reference sequence AATTTCCGTGGGTCGGGCCGGGCGGGCCAGGCGCTGGGCACGGTGATGGCCACCACTGGGGCCCTGGGCAACTACTACGTGGACTCGTTCCTGCTGGGCGCCGACGCCGCGGATGAGCTGAGCGTTGGCCGCTATGCGCCGGGGACCCTGGGCCAGCCTCCCCGGCAGGCGGCGACGCTGCCGAGCACC CCGACTTCAGCCCGTGCAGCTTCCAGTCCAAGGCGACGGTGTTTGGCGCCTCGTGGAACCCAGTGCACGCGGCGGGCGCCAACGCTGTACCCGCTGCGGTGTACCACCATCACCACCACCCCTACGTGCACCCCCAGGCGCCCGTGGCGGCG SEQ ID NO: 23 The converted sequence of HOXA9 region 1 sequence after bisulfite treatment AATTTTCGTGGGTCGGGTCGGGCGGGTTAGGCGTTGGGTACGGTGATGGTTATTATTGGGGTTTTGGGGTAATTATTACGTGGATTCGTTTTTTGTTGGGCGTCGACGTCGCGGATGAGTTGAGCGTTGGTCGTTATGCGTCGGGGATTTTGGGTTAGTTTTTTGGGTAGGCGGCGACGTTGGTCGAGTATT TCGATTTTAGTTCGTGTAGTTTTTAGTTTAAGGCGACGGTGTTTGGCGTTTCGTGGAATTTAGTGTACGCGGCGGGCGTTAACGTTGTATTCGTTGCGGTGTATTATTATTATTATTATTATTTTTACGTGTATTTTTAGGCGTTCGTGGCGGCG SEQ ID NO: 24 HOXA9 region 2 reference sequence SEQ ID NO: 25 The converted sequence of HOXA9 region 2 reference sequence after bisulfite treatment SEQ ID NO: 26 HOXA9 region 3 reference sequence SEQ ID NO: 27 The converted sequence of HOXA9 region 3 reference sequence after bisulfite treatment SEQ ID NO: 28 β-actin region reference sequence SEQ ID NO: 29 β-actin sequence converted after bisulfite treatment

We designed different methylation primers and probes according to sequence region 1, region 2 and region 3, and the information of each primer probe is shown in Table 18, wherein group 1, group 2, group 3, group 4 and group 5 are based on region 1 Designed methylation primers and probes; group 6, group 7, and group 8 are methylation primers and probes designed according to region 2; groups 9, 10, and group 11 are methylation primers designed according to region 3 and probes (see Table 20 for the sequences of primers and probes).

The above 11 primer-probe combinations were detected in 36 lung tissue samples, including 11 normal tissue samples, 25 cancer tissue samples, 4 squamous cell carcinoma samples and 21 adenocarcinoma samples among the 25 cancer samples. The test results are shown in the table below. [Table 18] Detection results in tissues the area group Primer Probe Assemblies specificity sensitivity area 1 group 1 H9-F2, H9-R2, H9-P2 100% 76% area 1 group 2 H9-F3, H9-R3, H9-P3 100% 76% area 1 group 3 H9-F4, H9-R4, H9-P4 100% 40% area 1 group 4 H9-F5, H9-R5, H9-P5 100% 60% area 1 Group 5 H9-F6, H9-R6, H9-P6 100% 68% area 2 Group 6 H9-F7, H9-R7, H9-P7 100% 32% area 2 Group 7 H9-F8, H9-R8, H9-P8 100% 20% area 2 Group 8 H9-F9, H9-R9, H9-P9 100% 12% area 3 Group 9 H9-F10, H9-R10, H9-P10 100% 16% area 3 Group 10 H9-F11, H9-R11, H9-P11 100% twenty four% area 3 Group 11 H9-F12, H9-R12, H9-P12 100% twenty four%

The results show that no matter how primers and probes are designed in regions 2 and 3, the highest detection sensitivity for these two regions can only reach 32%, and no matter what kind of primers and probes designed in the present disclosure are used, the detection sensitivity of region 1 is the lowest It can also reach 40%, up to 76%. Therefore, the detection rate of area 1 is significantly higher than that of areas 2 and 3 (see Table 18).

According to the detection results of each set of primer probes, the preferred detection sequences are shown in Table 19 below. [Table 19] Optimized detection sequence sequence name sequence SEQ ID NO: 22 HOXA9 region 1 reference sequence AATTTCCGTGGGTCGGGCCGGGCGGGCCAGGCGCTGGGCACGGTGATGGCCACCACTGGGGCCCTGGGCAACTACTACGTGGACTCGTTCCTGCTGGGCGCCGACGCCGCGGATGAGCTGAGCGTTGGCCGCTATGCGCCGGGGACCCTGGGCCAGCCTCCCCGGCAGGCGGCGACGCTGCCGAGCACC CCGACTTCAGCCCGTGCAGCTTCCAGTCCAAGGCGACGGTGTTTGGCGCCTCGTGGAACCCAGTGCACGCGGCGGGCGCCAACGCTGTACCCGCTGCGGTGTACCACCATCACCACCACCCCTACGTGCACCCCCAGGCGCCCGTGGCGGCG SEQ ID NO: 23 The converted sequence of HOXA9 region 1 sequence after bisulfite treatment AATTTTCGTGGGTCGGGTCGGGCGGGTTAGGCGTTGGGTACGGTGATGGTTATTATTGGGGTTTTGGGGTAATTATTACGTGGATTCGTTTTTTGTTGGGCGTCGACGTCGCGGATGAGTTGAGCGTTGGTCGTTATGCGTCGGGGATTTTGGGTTAGTTTTTTGGGTAGGCGGCGACGTTGGTCGAGTATT TCGATTTTAGTTCGTGTAGTTTTTAGTTTAAGGCGACGGTGTTTGGCGTTTCGTGGAATTTAGTGTACGCGGCGGGCGTTAACGTTGTATTCGTTGCGGTGTATTATTATTATTATTATTATTTTTACGTGTATTTTTAGGCGTTCGTGGCGGCG

2. Influence of primers and probes on the detection effect

In addition to the detection area will affect the detection effect, primers and probes also have a great impact on the detection effect of tumor markers. Sensitive and specific probes and primers enable the detection reagents of the present disclosure to 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 Yingwei Jieji (Shanghai) Trading Co., Ltd. [Table 20] Primers and probes name serial number sequence effect H9-F2 SEQ ID NO: 1 TTAGTTTTTTTCGGTAGGCGGC 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 H9-F3 SEQ ID NO: 30 AATTTT CG TGGGT CG GGT C HOXA9 gene upstream primer H9-R3 SEQ ID NO: 31 CCAAACACCGTCGCCTTAA HOXA9 gene downstream primer H9-P3 SEQ ID NO: 32 FAM-ACGTGGATTCGTTTTTGTTGGGC-BQ1 HOXA9 gene detection probe H9-F4 SEQ ID NO: 33 CGTCGCGGATGAGTTGAGC HOXA9 gene upstream primer H9-R4 SEQ ID NO: 34 CACGAACGCCTAAAAATACACG HOXA9 gene downstream primer H9-P4 SEQ ID NO: 35 FAM-TGGTCGTTATGCGTCGGGGATT-BQ1 HOXA9 gene detection probe H9-F5 SEQ ID NO: 36 AGCGTTGGTCGTTATGCGTC HOXA9 gene upstream primer H9-R5 SEQ ID NO: 37 CCAAACACCGTCGCCTTAAAC HOXA9 gene downstream primer H9-P5 SEQ ID NO: 38 FAM-GACGTTGGTCGAGTATTTCGATTTTAG-BQ1 HOXA9 gene detection probe H9-F6 SEQ ID NO: 39 CGACGTTGGTCGAGTATTTC HOXA9 gene upstream primer H9-R6 SEQ ID NO: 40 GCCACGAACGCCTAAAAAT HOXA9 gene downstream primer H9-P6 SEQ ID NO: 41 FAM-TTAGTTTAAGGCGACGGTGTTTGG-BQ1 HOXA9 gene detection probe H9-F7 SEQ ID NO: 42 CGTTTTGGTGGCGGTTGGTC HOXA9 gene upstream primer H9-R7 SEQ ID NO: 43 AATAATCCCTACGATCCCCGA HOXA9 gene downstream primer H9-P7 SEQ ID NO: 44 FAM-GGGCGTTTTTCGTTTTAGGCGG-BQ1 HOXA9 gene detection probe H9-F8 SEQ ID NO: 45 TCGTTTGGGACGGATTTTGC HOXA9 gene upstream primer H9-R8 SEQ ID NO: 46 ACAAATTCGTTTACCTCGCCG HOXA9 gene downstream primer H9-P8 SEQ ID NO: 47 FAM-ATTCGTTTTTTTCGGGGATCGTAGG-BQ1 HOXA9 gene detection probe H9-F9 SEQ ID NO: 48 AATAG CG TG CG GAGTGATTTA C HOXA9 gene upstream primer H9-R9 SEQ ID NO: 49 CGACCCGACCCACGAAAAT HOXA9 gene downstream primer H9-P9 SEQ ID NO: 50 FAM-CGTTATTGTTTTGTTGGACGGGTACG-BQ1 HOXA9 gene detection probe H9-F10 SEQ ID NO: 51 TTTAGGCGTTCGTGGCGGC HOXA9 gene upstream primer H9-R10 SEQ ID NO: 52 CCCTTCTAACCGACAACGATTC HOXA9 gene downstream primer H9-P10 SEQ ID NO: 53 FAM-CGGACGGTAGGTATATGCGTTTTTGG-BQ1 HOXA9 gene detection probe H9-F11 SEQ ID NO: 54 AATTCGTCGTCGTTTTTACGTC HOXA9 gene upstream primer H9-R11 SEQ ID NO: 55 TCCCGTAACCCCTACGAACCG HOXA9 gene downstream primer H9-P11 SEQ ID NO: 56 FAM-TCGGATTTGTTGGTTCGTTAGGTTTTTTTC-BQ1 HOXA9 gene detection probe H9-F12 SEQ ID NO: 57 TTAGATTGTTCGTGAGCGGC HOXA9 gene upstream primer H9-R12 SEQ ID NO: 58 TTATAACCCGAATCGCCCG HOXA9 gene downstream primer H9-P12 SEQ ID NO: 59 FAM-TTGGGACGTTGGGGGAGACGGT-BQ1 HOXA9 gene detection probe A3-TqMF SEQ ID NO: 16 TTTTGGATTGTGAATTTGTG β-actin gene upstream primer A3-TqMR SEQ ID NO: 17 AAAACCTACTCCTCCCCTTAAA β-actin gene downstream primer A3-TqP SEQ ID NO: 18 FAM-TTGTGTGTTGGGTGGTGGTT-BQ1 β-actin gene detection probe [Table 21] Detection results in tissues group Primer Probe Assemblies specificity sensitivity group 1 H9-F2, H9-R2, H9-P2 100% 76% group 2 H9-F3, H9-R3, H9-P3 100% 76% group 3 H9-F4, H9-R4, H9-P4 100% 40% group 4 H9-F5, H9-R5, H9-P5 100% 60% Group 5 H9-F6, H9-R6, H9-P6 100% 68% Group 6 H9-F7, H9-R7, H9-P7 100% 32% Group 7 H9-F8, H9-R8, H9-P8 100% 20% Group 8 H9-F9, H9-R9, H9-P9 100% 12% Group 9 H9-F10, H9-R10, H9-P10 100% 16% Group 10 H9-F11, H9-R11, H9-P11 100% twenty four% Group 11 H9-F12, H9-R12, H9-P12 100% twenty four%

The above 11 primer-probe combinations were detected in 36 lung tissue samples, including 11 normal tissue samples, 25 cancer tissue samples, 4 squamous cell carcinoma samples and 21 adenocarcinoma samples among the 25 cancer samples. The results showed that Group 1, Group 2, Group 4, and Group 5 all had good detection rates.

In order to further verify its detection rate in sputum, we selected 22 cases of sputum samples for verification, including 7 cases of normal controls, 15 cases of lung cancer controls, 7 cases of squamous cell carcinoma and 7 cases of adenocarcinoma among the 15 cases of lung cancer , 1 case of large cell carcinoma, the test results are shown in Table 22. [Table 22] Test results in sputum group Primer Probe Assemblies specificity sensitivity group 1 H9-F2, H9-R2, H9-P2 100% 66.7% group 2 H9-F3, H9-R3, H9-P3 100% 44.6% group 4 H9-F5, H9-R5, H9-P5 100% 33.3% Group 5 H9-F6, H9-R6, H9-P6 100% 40.0%

The detection results from 22 cases of sputum samples showed that the detection rate of group 1: H9-F2, H9-R2, H9-P2 was the highest, reaching 66.7%. Although in tissue samples, the sensitivity of group 1 and group 2 can both reach 76%, but for sputum test samples, the sensitivity of group 2 drops sharply to 44.6%, which also confirms on the one hand that for sputum samples, the design Detection reagents with high sensitivity are especially difficult.

Finally, according to the detection results of each group of primer probes, the most preferred primer probe sequences are shown in Table 23 below. [Table 23] Optimized primers name serial number sequence effect H9-F2 SEQ ID NO: 1 TTAGTTTTTTTCGGTAGGCGGC 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

none.

[Figure 1] ROC curves of HOXA9, SHOX2, PCDHGA12, HOXD8, GATA3 detected in all tissue samples; [Figure 2] ROC curves for the detection of HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 in sputum samples; [Figure 3] ROC curves for the detection of HOXA9 and SHOX2-n3 in all sputum samples; [Figure 4] Amplification curves of HOXA9 and SHOX2_n3 in sputum samples (A is the amplification graph of HOXA9, B is the amplification graph of SHOX2_n3; [Figure 5] ROC curves of HOXA9 and SHOX2_n3 detected in all lavage fluid samples; [Figure 6] Amplification curves of HOXA9 and SHOX2_n3 in lavage fluid samples (A is the amplification graph of HOXA9, B is the amplification graph of SHOX2_n3).

<110> Continental Guangzhou Kangliming Biotechnology Co., Ltd.

<120> Use of HOXA9 Methylation Detection Reagent in Preparation of Lung Cancer Diagnostic Reagent

<150> CN 201811152029.6

<151> 2018-09-29

<160> 62

<170> PatentIn version 3.3

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Claims (21)

A use of a HOXA9 gene methylation detection reagent in the preparation of lung cancer diagnostic reagents; wherein, the HOXA9 gene methylation detection reagent has a pair of primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 and SEQ ID NO: The probe shown in 3; or the HOXA9 gene methylation detection reagent has the primer pair shown in SEQ ID NO: 30 and SEQ ID NO: 31 and the probe shown in SEQ ID NO: 32; or the HOXA9 gene methylation The detection reagent has the primer pair shown in SEQ ID NO: 33 and SEQ ID NO: 34 and the probe shown in SEQ ID NO: 35; or the detection reagent for the HOXA9 gene methylation has SEQ ID NO: 36 and SEQ ID NO The primer pair shown in: 37 and the probe shown in SEQ ID NO: 38; or the HOXA9 gene methylation detection reagent has the primer pair shown in SEQ ID NO: 39 and SEQ ID NO: 40 and the primer pair shown in SEQ ID NO: 41 display probe. According to the use described in Claim 1, wherein the test sample is sputum. According to the use described in claim 1, wherein the HOXA9 gene methylation detection reagent detects the sequence of the HOXA9 gene modified by a transformation reagent. According to the use described in Claim 1, the conversion reagent is selected from at least one of hydrazine salt, bisulfite and bisulfite. According to the purposes described in claim 4, the conversion reagent is selected from bisulfite. According to the use described in claim 1, wherein the detection reagent for HOXA9 gene methylation further comprises bisulfite, bisulfite or hydrazine salt. According to the use described in Claim 1, wherein the HOXA9 gene methylation detection reagent further comprises at least one of DNA polymerase, dNTPs, Mg ²⁺ ions and buffer. According to the use described in claim 1, wherein the detection reagent for HOXA9 gene methylation comprises DNA polymerase, dNTPs, Mg ²⁺ ions and buffer. According to the use described in claim 1, wherein the HOXA9 gene methylation detection reagent further includes a reference gene detection reagent. The use according to claim 9, wherein the reference gene comprises β-actin or COL2A1. According to the use described in Claim 9, wherein the detection reagent for the reference gene comprises primers and probes for the reference gene. According to the use described in Claim 10, wherein the detection reagent for the reference gene β-actin has a pair of primers shown in SEQ ID NO: 16 and SEQ ID NO: 17, and a probe shown in SEQ ID NO: 18. According to the use described in claim 10, wherein the detection reagent for the reference gene COL2A1 has a pair of primers shown in SEQ ID NO:60 and SEQ ID NO:61, and a probe shown in SEQ ID NO:62. A method for detecting DNA methylation of the HOXA9 gene, comprising the following steps: (1) extracting DNA from a test sample and then treating it with bisulfite, bisulfite or hydrazine salt to obtain a modified test sample; (2 ) using the HOXA9 gene methylation detection reagent described in any one of claim items 1-13 to detect HOXA9 gene methylation on the modified detection sample in step (1); Wherein the detection sample is sputum. According to the method described in claim item 14, wherein in the step (2), detection is performed by methylation-specific polymerase chain reaction or real-time fluorescence quantitative methylation-specific polymerase chain reaction. A diagnostic system for lung cancer, comprising: a. a DNA methylation detection component of the HOXA9 gene; and b. a result judgment component; the DNA methylation detection component of the HOXA9 gene includes HOXA9 described in any one of claims 1-13 A detection reagent for gene methylation; wherein the detection sample for the HOXA9 gene methylation detection reagent is sputum. The diagnostic system according to claim 16, wherein the methylation detection component includes one or more of a fluorescent quantitative PCR instrument, a PCR instrument, and a sequencer. The diagnostic system according to claim 16, wherein the result judging means includes a data processing machine. The diagnostic system according to claim 16, wherein the result judgment means is used to output a diagnosis result according to the DNA methylation level of the HOXA9 gene detected by the detection means. The diagnosis system according to claim 16, wherein the diagnosis result is obtained based on the deviation between the methylation levels of the test sample and the normal sample by comparing the methylation levels of the test sample and the normal sample through the result judging means. A method for diagnosing lung cancer, comprising the following steps: (1) detecting the methylation level of the HOXA9 gene in a test sample derived from a subject; (2) compare the methylation level of the HOXA9 gene between the sample to be tested and the normal sample; (3) diagnose lung cancer based on the deviation of the methylation level between the sample to be tested and the normal sample; in step (1), use request item 1 The detection reagent for detecting HOXA9 gene methylation in any one of -13 is used to detect the methylation level of the HOXA9 gene in the sample to be tested; wherein, the sample to be tested is sputum.

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