Genome sleeve capable of identifying and diagnosing pulmonary nodules

Abstract

The invention provides a genome kit capable of identifying and diagnosing pulmonary nodules, which comprises the following steps: taking 21-gene panel as a molecular marker, detecting and calculating tumor mutation load of 21-gene in peripheral blood of a pulmonary nodule patient by adopting EV DNA in the peripheral blood, and calculating the tumor mutation load of 21-gene in the peripheral blood of the pulmonary nodule patient. A benign and malignant pulmonary nodule diagnosis scoring system is established by combining four clinical factors (gender, smoking condition, age and nodule diameter), and malignant pulmonary nodules are identified and diagnosed according to diagnosis scores. The 21-gene panel is used as the molecular marker for judging benign and malignant pulmonary nodules, and compared with CT and tissue biopsy, the 21-gene panel has more remarkable recognition and prediction value on malignant pulmonary nodules and has important guiding significance for judging the progression of tumors to invasive adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a method for differential diagnosis of pulmonary nodules and a genomic panel by 21-gene panel. Background Art

[0002] Pulmonary nodules (PN) are discrete, well-defined opacities less than 3 cm in diameter, surrounded by aerated lung tissue and not associated with lymphadenoma, atelectasis or enlarged lymph nodes. It is a non-specific finding in approximately 50% of chest computed tomography scans. Pulmonary nodules may be caused by benign or malignant etiologies. Most pulmonary nodules are early-stage lung cancers, which can be classified into 3 categories according to pathological subtypes: adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), and invasive adenocarcinoma (IAC). According to the size of the nodule and other risk factors, the incidence of malignant pulmonary nodules (MPN) is approximately 40%. The 5-year survival rate can be as high as 100%, indicating that timely and definitive diagnosis and prompt intervention are the best opportunities for curing MPN. At the same time, it is also crucial to prevent unnecessary morbidity and mortality associated with patients with benign pulmonary nodules (BPN). Nevertheless, the differential diagnosis of pulmonary nodules remains challenging in terms of accuracy, efficiency, and convenience. Although MPN has fairly typical radiological features, such as spiculated margins and heterogeneous density, some benign lesions are extremely similar to malignant lesions. Therefore, the effectiveness of imaging-based differential diagnosis is moderate and does not fully meet the ideal expectations. According to the size and growth rate of PN, some guidelines recommend follow-up CT scans at 3, 6, or 12 months until a diagnosis is established. However, this long interval and extended follow-up have, to some extent, led to delayed diagnosis and treatment, increased medical costs, and potentially increased the risk of metastasis. Tissue biopsy may be helpful for the diagnosis and treatment strategy of PN, but its use is controversial. There is currently no consensus on the optimal size threshold or technique for tissue sampling. In addition, factors such as the small size, multiplicity, difficult localization, and sampling bias of PN limit the clinical application of tissue biopsy. In summary, there is an obvious lack of molecular diagnosis that can detect MPN in a timely and definitive manner, forcing us to use liquid biopsy to clarify the pathological information of MPN.

[0003] As a new member of liquid biopsy, extracellular vesicles (EVs) may hold promise in the identification of MPN. EVs are submicron-sized lipid bilayer-enclosed vesicles released by cells, containing tissue-specific markers that include various proteins and nucleic acids. Recent studies have confirmed that EVs can be used for non-invasive cancer (early) diagnosis, staging, and treatment monitoring. Previous studies have confirmed the existence of EV DNA and found that EV DNA represents the entire genome, reflects the mutation status, and has the same copy number variations as the parental cells. Compared with cell-free DNA (cfDNA, with an average of 160 bp), EV DNA is relatively more intact (with an average of 15 kbp) due to its protective lipid envelope. Previous studies have also demonstrated that EV DNA is superior to cfDNA in detecting mutations in early cancers such as early lung cancer, pancreatic cancer, and colon cancer. Moreover, the vast majority of cfDNA mutations in cancer patients are highly correlated with white blood cells. Therefore, in the field of molecular diagnosis of early cancers, EV DNA may have more application prospects than cfDNA. And compared with protein or RNA biomarkers, DNA mutations are more definitive for cancer diagnosis. For decades, there has still been a lack of significant protein and / or RNA biomarkers in cancer patients, indicating that protein and / or RNA expression analysis may not immediately become a viable goal for EV-based cancer diagnosis. Instead, state-of-the-art nucleic acid analysis systems can easily detect DNA mutations at extremely low allele frequencies and quantify the impact of gene mutations in the form of Tumor Mutation Burden (TMB). In summary, highly sensitive and phylogenetic analysis of EV DNA is well-suited for characterizing MPN and is expected to have an immediate impact on clinical practice.

[0004] There have been no reports on any studies related to the liquid biopsy of specific genomic sets for pulmonary nodules, especially the lack of the application of specific genomic sets in the differential diagnosis of pulmonary nodules in peripheral blood EV DNA. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a method and a genomic set for the differential diagnosis of pulmonary nodules.

[0006] Technical Solution: The present invention provides a method for the differential diagnosis of pulmonary nodules, comprising the following steps:

[0007] Using a 21-gene panel as a molecular marker, extracting EV DNA from peripheral blood, detecting the Tumor Mutation Burden (TMB) of the 21 genes in the peripheral blood of pulmonary nodule patients, and establishing a diagnostic scoring system for benign and malignant nodules by combining 4 clinical factors (smoking status, gender, age, and nodule diameter), and differentiating and diagnosing MPN according to the diagnostic score.

[0008] Preferably, the DNA to be detected is peripheral blood EV DNA.

[0009] Preferably, the method for differential diagnosis of MPN by genomic panel includes the following steps:

[0010] (a) Isolate and extract EV DNA from the peripheral blood of patients with lung nodules;

[0011] (b) Perform gene mutation detection using a 21-gene panel on an Illumina NextSeq 2000 sequencer;

[0012] (c) Calculate the TMB of the patient's peripheral blood EV DNA, and collect information such as gender, smoking status, age, and the diameter of the lung nodule;

[0013] (d) Incorporate the five factors into a diagnostic scoring system, set the threshold at 70 points. When the score is less than or equal to the threshold, the risk that the lung nodule has progressed to MPN is less than or equal to 50%, and it is considered not to resect the lymph nodes and perform regular follow-up; when the score is higher than the threshold, the risk that the lung nodule has progressed to MPN is greater than 50%, and it is considered to resect the lymph nodes and closely monitor for recurrence.

[0014] More preferably, in step (d), the scoring rule is as follows:

[0015] Risk score = 0.0122 * (gender * 5.08 + smoking * 6.35 + age * 0.44735 + tumor diameter * 28.57 + tumor mutation burden of 21 genes * 383.627692) - 0.3719

[0016] (Note: male = 0, female = 1; no smoking history = 0, smoking = 1; the unit of tumor diameter is cm)

[0017] The present invention also provides a genomic panel for differential diagnosis of MPN, and the genomic panel includes the following:

[0018] Customize the 21-gene panel using a standard next-generation sequencing platform.

[0019] Preferably, the 21-gene panel includes the following:

[0020] EGFR, TP53, MSH3, NOTCH3, KRAS, ATR, EP300, NOTCH1, PTPRB, APC, ARID1A, BAP1, BLM, BRCA2, BRIP1, MSH2, PTCH1, RAD50, RAD51D, DBI, FLT4.

[0021] The present invention finally also provides the application of the genomic set in the differential diagnosis of pulmonary nodules.

[0022] Preferably, the application includes the following steps:

[0023] Using the 21-gene panel as a molecular marker, detecting the TMB of peripheral blood EV DNA by means of the genomic set, establishing a diagnostic scoring system, and differentiating and diagnosing MPN according to the diagnostic score.

[0024] Preferably, the DNA to be detected is peripheral blood EV DNA.

[0025] Beneficial effects: Through extensive and in-depth research, the applicant has first discovered that detecting the TMB of peripheral blood of pulmonary nodule patients by means of the 21-gene panel has excellent sensitivity and specificity for judging the risk of pulmonary nodules progressing to MPN. Based on the correlation between the TMB of the 21-gene panel and the progression of pulmonary nodules, using this genomic set as a molecular marker to detect its mutations can be used for the preoperative differential diagnosis of MPN, with high cost-effectiveness and high timeliness in helping to make medical decisions within five days after the initial consultation. Compared with CT and tissue biopsy, the diagnostic scoring system established with the 21-gene panel has a more significant differential diagnostic value for MPN.

[0026] Based on the above findings, the present invention provides a method and a genomic set for differentiating and diagnosing pulmonary nodules. This method uses the 21-gene panel as a molecular marker, extracts EV DNA from peripheral blood, detects the tumor mutation burden (TMB) of the 21 genes in the peripheral blood of pulmonary nodule patients, and combines 4 clinical factors (smoking status, gender, age, and nodule diameter) to establish a diagnostic scoring system for benign and malignant nodules, and differentiates and diagnoses MPN according to the diagnostic score.

[0027] The discovery of the correlation between the 21-gene panel of the present invention and the progression of pulmonary nodules provides a new biomarker for the clinical differential diagnosis of pulmonary nodules. When the score is less than or equal to 70 points, the risk of pulmonary nodules progressing to MPN is less than or equal to 50%, and it is considered not to excise the lymph nodes and perform regular follow-up; when the score is higher than 70 points, the risk of pulmonary nodules progressing to MPN is greater than 50%, and it is considered to excise the lymph nodes and closely monitor the recurrence situation. Detailed implementation manners

[0028] The following details the specific implementation manners provided by the present invention in combination with the discovery and implementation process of the 21-gene panel:

[0029] Collect 79 cases of CT images and surgical samples taken before surgery from patients with early-stage lung cancer at Wuxi People's Hospital Affiliated to Nanjing Medical University for a discovery study of the 21-gene panel. The specific steps are as follows:

[0030] (1) Two pathologists examined the histological type and evaluated the tumor purity before DNA extraction;

[0031] (2) Tissue DNA was isolated using the DNeasy kit (Qiagen 69504);

[0032] (3) Tissue DNA was analyzed using the 618-gene panel (Agilent), and then the analysis was carried out, including mutational signatures, tumor mutational burden, co-occurrence and mutual exclusivity, clinical relevance, and the construction of a diagnostic scoring system.

[0033] The analysis described in step (3) is as follows:

[0034] (a) Analyze the overall landscape of gene mutations in 79 MPN patients ( Figure 1 );

[0035] (b) Study the co-occurrence and mutual exclusivity of mutations and mutational signatures in MPN ( Figure 2 );

[0036] (c) Correlation between gene mutations and gender and smoking status ( Figure 3 );

[0037] (d) Establish and validate a diagnostic scoring system for predicting the risk of IAC ( Figures 4-5 ).

[0038] The genomic set prepared according to the method of the present invention contains: EGFR, TP53, MSH3, NOTCH3, KRAS, ATR, EP300, NOTCH1, PTPRB, APC, ARID1A, BAP1, BLM, BRCA2, BRIP1, MSH2, PTCH1, RAD50, RAD51D, DBI, FLT4.

[0039] Using a standard next-generation sequencing platform, customize the above 21-gene panel for detecting peripheral blood EV DNA. Use this genomic set to carry out implementation examples:

[0040] Collect 18 cases of peripheral blood and surgical samples taken before surgery from patients with early-stage invasive lung cancer at Wuxi People's Hospital Affiliated to Nanjing Medical University for a study on the implementation process of the 21-gene panel. The specific steps are as follows:

[0041] (1) Two pathologists examined the histological type and evaluated the tumor purity before DNA extraction;

[0042] (2) Withdraw approximately 10 ml of blood sample using a 10-ml EDTA (K2) tube;

[0043] (3) Collect plasma after centrifuging at 2500 g for 15 minutes;

[0044] (4) Further centrifuge the plasma sample at 16500 g for 20 minutes at 4°C and filter it using a 0.22-μm pore size filter;

[0045] (5) Ultracentrifuge the filtered plasma sample at 4°C and 100000 g for 4 hours;

[0046] (6) Suspend the EV precipitate in 100 μl of PBS;

[0047] (7) Extract EV DNA using the QIAamp DNA Micro Kit (Qiagen 56304);

[0048] (8) Detect gene mutations of EV DNA using the 21-gene panel (TE-93316425, Twist) and analyze the mutation landscape ( Figure 6 );

[0049] (9) Calculate the TMB and incorporate it into the diagnostic scoring system for differential diagnosis of benign and malignant nodules. The specific scoring criteria are as follows:

[0050] Risk score = 0.0122 * (gender * 5.08 + smoking * 6.35 + age * 0.44735 + tumor diameter * 28.57 + tumor mutation burden of 21 genes * 383.627692) - 0.3719

[0051] (Note: male = 0, female = 1; no smoking history = 0, smoking = 1; tumor diameter unit is cm)

[0052] Adopt the above methods and genomic sets for differential diagnosis of pulmonary nodules, establish a diagnostic scoring system for risk scoring. When the score is less than or equal to 7, the risk that the pulmonary nodule has progressed to MPN is less than or equal to 50%, and it is considered not to excise the lymph nodes and perform regular follow-up; when the score is higher than 70, the risk that the pulmonary nodule has progressed to MPN is greater than 50%, and it is considered to excise the lymph nodes and closely monitor the recurrence situation. Description of the Drawings

[0053] Figure 1. Overall landscape of gene mutations in biopsy tissues of 79 patients collected by Wuxi People's Hospital Affiliated to Nanjing Medical University. (A) Mutation information of each gene in AIS, MIS, and IAC subtypes. (B-D) Statistical calculations of mutation information based on different variant classifications, variant types, and SNV categories. (E) Variant description and summary of variant classifications for each sample. (F) Top 20 mutated genes in MPN. (G) Domain range and mutation positions of the EGFR protein sequence. (H) The left panel shows the distribution of SNVs. The right panel shows the distribution of the mutation spectra for each sample. (I) TMB in AIS, MIA, and IAC subtypes. (J) Single-gene mutation analysis of genes with statistical significance in AIS, MIA, and IAC subtypes.

[0054] Figure 2 . Co-occurrence and mutual exclusivity of mutations and mutational signatures in MPN. (A-D) Triangular matrices showing co-occurring and mutually exclusive gene pairs. Green indicates co-occurrence, while brown indicates mutual exclusivity (*p < 0.05). (E) Mutational signatures are illustrated using 96 patterns defined by substitution types and the sequence context flanking the mutated base 5' and 3'.

[0055] Figure 3 . Association of mutations with gender and smoking status. (A-B) Mutation information of each gene in female, male, smoker, and non-smoker groups. (C) Mutational signatures shared among the four groups and independent signatures of smokers in MPN.

[0056] Figure 4 . Using tumor tissue DNA to define a 21-gene panel for constructing a diagnostic scoring system to predict the risk of IAC. (A-B) Mutation information of each gene in the training and test sets. (C) The diagnostic nomogram is constructed from gender, smoking status, age, tumor diameter, and the 21-gene panel. (D) Calibration plot. The predicted results (green solid line) are closer to the ideal model (diagonal line). (E) The ROC curve indicates good predictive accuracy. (F) Decision curve analysis of the diagnostic nomogram. The blue solid line represents the IAC diagnostic nomogram. The red solid line represents the assumption that not all patients are diagnosed with IAC. The thin green solid line represents the assumption that no patients are IAC.

[0057] Figure 5.Validate the 21-gene panel using tumor tissue DNA and tumor EV DNA samples. (A) Mutation information for each gene in MPN and BPN samples. (B) Statistical calculations of mutation information based on different variant classifications and variant types in MPN and BPN samples, respectively. (C) TMB in tissue DNA and EV DNA. (D) Variant descriptions for each sample and a summary of variant classifications in 18 samples. (E) Illustration of the mutation counts in 18 samples. (F) TMB at T1a, T1b, and T1c stages in tissue DNA and EV DNA. (G) Mutation gene counts in 18 samples. (H) Average concordance of gene mutations in MPN samples and BPN samples, respectively.

[0058] Figure 6 .Validate the diagnostic nomogram model using paired tissue DNA and EV DNA samples. (A) Prediction points of the diagnostic nomogram model using MPN and BPN samples based on tissue DNA and EV DNA samples, respectively. (B) Prediction points between T1a, T1b, and T1c stages using tissue DNA and EV DNA, respectively. (C) Correlation analysis of TMB scores for tissue DNA and EV DNA samples using the Pearson method.

[0059] Figure 7 .Brief description of the implementation process of the present invention.

Claims

1. A genomic panel for differential diagnosis of pulmonary nodules, characterized in that, Comprising the following steps: Using the 21-gene panel as a molecular marker, extracting EV DNA from peripheral blood, detecting the TMB of the 21 genes in the peripheral blood of patients with lung nodules, combining 4 clinical factors (gender, smoking status, age, and nodule diameter) to establish a diagnostic scoring system for benign and malignant nodules, and differentiating and diagnosing MPN according to the diagnostic score.

2. The method for differential diagnosis of MPN by genomic panel according to claim 1, characterized in that, The DNA detected is peripheral blood EV DNA.

3. The method for differential diagnosis of MPN by genomic panel according to claim 1, characterized in that, Comprising the following steps: (a) Isolating and extracting EV DNA from the peripheral blood of patients with lung nodules; (b) Performing gene mutation detection using the 21-gene panel on an Illumina NextSeq 2000 sequencer; (c) Calculating the TMB of the EV DNA in the peripheral blood of the patient, and collecting information such as gender, smoking status, age, and the diameter of the lung nodule; (d) Incorporating the 5 factors into the diagnostic scoring system, setting the threshold at 70 points. When the score is less than or equal to the threshold, the risk that the lung nodule has progressed to MPN is less than or equal to 50%; when the score is higher than the threshold, the risk that the lung nodule has progressed to MPN is greater than 50%.

4. The method for differential diagnosis of MPN by genomic panel according to claim 3, characterized in that, In step (d), the scoring rule is as follows: Risk score = 0.0122 * (gender * 5.08 + smoking * 6.35 + age * 0.44735 + tumor diameter * 28.57 + tumor mutation burden of 21 genes * 383.627692) - 0.3719 (Note: male = 0, female = 1; no smoking history = 0, smoking = 1).

5. A genomic panel for differential diagnosis of pulmonary nodules, characterized in that, The genomic set comprises the following: Customizing the aforementioned 21-gene panel using a standard next-generation sequencing platform.

6. The genomic set for differential diagnosis of pulmonary nodules according to claim 5, characterized in that, The 21-gene panel comprises the following: EGFR, TP53, MSH3, NOTCH3, KRAS, ATR, EP300, NOTCH1, PTPRB, APC, ARID1A, BAP1, BLM, BRCA2, BRIP1, MSH2, PTCH1, RAD50, RAD51D, DBI, FLT4.

7. Use of the genomic set according to claim 5 in differentiating and diagnosing lung nodules.

8. The application according to claim 7, wherein The use comprises the following steps: Using the 21-gene panel as a molecular marker, extracting peripheral blood EV DNA, detecting and calculating its TMB, incorporating 4 clinical factors into the diagnostic scoring system, and differentiating and diagnosing MPN according to the diagnostic score.

9. The application according to claim 7, wherein The DNA detected is peripheral blood EV DNA.

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