WO2022199570A1 - Marker combination for diagnosis of basal-type pancreatic ductal adenocarcinoma (pdac) and application thereof - Google Patents
Marker combination for diagnosis of basal-type pancreatic ductal adenocarcinoma (pdac) and application thereof Download PDFInfo
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- WO2022199570A1 WO2022199570A1 PCT/CN2022/082209 CN2022082209W WO2022199570A1 WO 2022199570 A1 WO2022199570 A1 WO 2022199570A1 CN 2022082209 W CN2022082209 W CN 2022082209W WO 2022199570 A1 WO2022199570 A1 WO 2022199570A1
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- pdac
- ductal adenocarcinoma
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Definitions
- the present invention relates to the field of biomedicine, in particular to a marker combination for the diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) and its application.
- PDAC Basal-type pancreatic ductal adenocarcinoma
- Pancreatic ductal adenocarcinoma is the most common type of pancreatic cancer and ranks third in cancer mortality worldwide. The prognosis for most PDAC patients is extremely poor, with a five-year survival rate of only 9%. Surgical resection is still the main treatment for PDAC, however, the postoperative recurrence rate is high, and most patients eventually die of tumor metastasis. Despite the generally high lethality of PDAC, different patients still exhibit clinical heterogeneity. Among patients whose tumors are surgically removed, some progress to advanced stages in just a few months, while some maintain a stable quality of life for years.
- Pancreatic cancer has been considered an inflammation-induced cancer for nearly two decades, and patients with pancreatitis have a 13-fold higher than normal risk of developing pancreatic cancer.
- the human microbiome plays an important role in activating immune responses and inducing cancer-related inflammatory responses.
- Past research has mainly focused on the role of gut microbes, and found that gut microbes have a certain impact on esophageal cancer, gastric cancer, and colon cancer.
- pancreatic tumor microbes So far, the composition and structure of pancreatic tumor microbes have not been fully elucidated, and the role of microbes in tumor development also needs further research and exploration.
- the purpose of the present invention is to focus on the microbial factors in the heterogeneity of PDAC subtypes, to reveal the unique structural characteristics of tumor microbes in different PDAC subtypes, and to develop new tumor microbes that predict the clinical phenotype of PDAC patients.
- a tumor microorganism or a detection reagent thereof for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma ( PDAC) or assessing the risk of having a Basal-type pancreatic ductal adenocarcinoma (PDAC); or for preparing a reagent or kit for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and /or (b) diagnosing or assessing the risk of developing Basal-type pancreatic ductal adenocarcinoma (PDAC), wherein the tumor microorganism is selected from the group consisting of: Acinetobacter, Pseudomonas Pseudomonas, Sphingopyxis, or a combination thereof.
- pancreatic ductal adenocarcinoma (PDAC) classification includes Basal-type pancreatic ductal adenocarcinoma (PDAC) and UnBasal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC pancreatic ductal adenocarcinoma
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- the reagent or kit is also used to distinguish Basal type pancreatic ductal adenocarcinoma (PDAC) from UnBasal type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal type pancreatic ductal adenocarcinoma
- PDAC UnBasal type pancreatic ductal adenocarcinoma
- the diagnosis includes early diagnosis, auxiliary diagnosis, or a combination thereof.
- the evaluation or diagnosis comprises the steps of:
- step (2) comparing the level (such as abundance) measured in step (1) with a reference data set or a reference value (such as the reference value of healthy controls);
- the reference data set includes the levels of each tumor microorganism in the tumor microorganisms (such as abundance) from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls. Spend).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- the level (eg abundance) of each tumor microorganism in the tumor microorganism increases, indicating that the subject to be tested has Basal type pancreatic ductal adenocarcinoma (PDAC). risk or have Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal type pancreatic ductal adenocarcinoma
- the level measured in step (1) is compared with a reference data set, and further includes constructing a classification model for predicting whether the patient is Basal-type pancreatic ductal adenocarcinoma, preferably , the classification model is a random forest model.
- the subject is determined to have the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) or suffer from There is a Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- the sample is selected from tumor tissue samples.
- the level (eg abundance) of each tumor microorganism in the tumor microorganism is detected by one or more methods of the following group: sequencing, PCR, protein quantitative detection.
- the PCR includes qPCR.
- the method for detecting the level of tumor microorganisms further includes one or more methods selected from the group consisting of quantitative PCR for characteristic genes, metagenomic analysis, 16s rRNA sequencing, mass spectrometry analysis, and western blotting .
- the method before step (1), further includes the step of processing the sample.
- a second aspect of the present invention provides a marker combination comprising two or more selected from the group consisting of Acinetobacter, Pseudomonas, Sphingosine Sphingopyxis, or a combination thereof.
- the marker combination includes Acinetobacter, Pseudomonas and Sphingopyxis.
- the marker combination is used for (a) pancreatic ductal adenocarcinoma (PDAC) classification; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) risk of developing cancer (PDAC).
- PDAC pancreatic ductal adenocarcinoma
- the marker combination is also used to distinguish Basal-type pancreatic ductal adenocarcinoma (PDAC) from UnBasal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- the markers are derived from tumor tissue samples, preferably from PDAC tumor tissue samples.
- the level (eg abundance) of each marker in the marker combination is detected by one or more methods of the following group: sequencing, PCR, protein quantitative detection.
- the PCR includes qPCR.
- the method for detecting the level of the marker group further includes one or more methods selected from the group consisting of quantitative PCR for characteristic genes, metagenomics analysis, 16s rRNA sequencing, mass spectrometry analysis, protein immunity blot.
- the level (eg abundance) of each marker in the marker combination increases, indicating that the subject to be tested has Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- a third aspect of the present invention provides a method for (a) classification of pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ), the reagent combination comprising the reagents for detecting each marker in the marker combination according to the second aspect of the present invention.
- PDAC pancreatic ductal adenocarcinoma
- the reagent includes a substance for detecting the level of each marker in the marker combination described in the second aspect of the present invention by one or more methods selected from the group consisting of sequencing, PCR, protein Quantitative detection.
- the reagent also includes a substance for detecting the level of each marker in the marker combination described in the second aspect of the present invention by one or more methods selected from the group consisting of: quantitative PCR for characteristic genes , metagenomics analysis, 16s rRNA sequencing, mass spectrometry analysis, western blotting.
- the reagent is used to detect the level (eg abundance) of each marker.
- a fourth aspect of the present invention provides a kit comprising the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention.
- each marker in the combination described in the second aspect of the present invention is used as a standard.
- the kit further includes an instruction manual, which describes the data obtained from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and/or UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls A reference dataset of the levels of each marker in the combination as described in the second aspect of the invention.
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- a fifth aspect of the present invention provides a method for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ) method for risk of disease, comprising the steps of:
- step (2) comparing the level measured in step (1) with a reference data set or a reference value (such as a reference value for healthy controls);
- the reference data set includes the respective markers in the combination according to the second aspect of the present invention derived from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls. Level.
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- a sixth aspect of the present invention provides a method for screening candidate compounds for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC), characterized in that it comprises the steps of:
- test group administer the test compound to the subject to be tested, and detect the level V1 of each marker in the combination according to the second aspect of the present invention in the sample derived from the subject in the test group; in the control group, to The subject to be tested is administered a blank control (including a vehicle), and the level V2 of each marker in the combination according to the second aspect of the present invention in the sample derived from the subject in the control group is detected;
- test object is a Basal-type pancreatic ductal adenocarcinoma (PDAC) patient.
- PDAC pancreatic ductal adenocarcinoma
- the test compound is a candidate compound for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- the "significantly lower than” refers to the ratio of level V1/level V2 ⁇ 0.7, preferably ⁇ 0.6.
- the seventh aspect of the present invention provides the use of the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention for screening and treating Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- An eighth aspect of the present invention provides a method for establishing a model for assessing the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) or for diagnosing Basal-type pancreatic ductal adenocarcinoma (PDAC), the method comprising identifying the Basal-type pancreatic ductal adenocarcinoma (PDAC)
- PDAC Basal-type pancreatic ductal adenocarcinoma
- the tissue sample includes a pancreatic tissue sample.
- a ninth aspect of the present invention provides a method for (a) pancreatic ductal adenocarcinoma (PDAC) classification; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ) system at risk of disease, the system comprising:
- a feature input module is used to input the features of a tumor tissue sample of a certain object
- the characteristics of the tumor tissue sample include tumor microorganisms selected from the group consisting of Acinetobacter, Pseudomonas, Sphingopyxis, or a combination thereof.
- a discrimination processing module performs scoring processing according to a predetermined judgment standard for the characteristics of the input tumor tissue samples, thereby obtaining a risk score; and compares the risk score with the Basal type pancreatic ductal adenocarcinoma ( PDAC) risk thresholds are compared to obtain an auxiliary screening result, wherein, when the risk score is higher than the risk threshold, the subject is at risk of suffering from Basal-type pancreatic ductal adenocarcinoma (PDAC). higher than the normal population; when the risk score is lower than the risk threshold, it is suggested that the subject has a higher risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) than the normal population; and
- PDAC Basal type pancreatic ductal adenocarcinoma
- an auxiliary screening result output module the output module is used for outputting the auxiliary screening result.
- the subject is a human.
- the subject includes infants, adolescents or adults.
- the score includes (a) the score of a single feature; and/or (b) the sum of the scores of multiple features.
- the feature input module is selected from the following group: a sample acquisition instrument, a feature signal input terminal, a data preprocessing module, a feature building module, and a feature visualization module.
- the discrimination processing module includes a processor and a storage, wherein the storage stores the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) based on the characteristics of the tumor tissue sample degree threshold data.
- PDAC Basal-type pancreatic ductal adenocarcinoma
- the output module includes a reporting system.
- Figure 1 shows the abundance distribution of Pseudomonas in different PDAC subtypes.
- FIG. 2 shows the abundance distribution of Acinetobacter in different PDAC subtypes.
- FIG. 3 shows the abundance distribution of Sphingopyxis in different PDAC subtypes.
- Figure 4 shows the clustering results of PDAC patients based on microbial markers.
- Figure 5 shows microbial genera significantly enriched in Basal-type PDAC.
- Figure 6 shows that candidate microbial markers can indicate patient prognosis and survival.
- Figure 7 shows the predictive performance of the constructed classifier model in evaluating Basal-type PDAC.
- Acinetobacter, Pseudomonas, and/or Sphingopyxis can be used for (a) pancreatic ductal adenocarcinoma ( PDAC) classification; and/or (b) diagnose Basal-type pancreatic ductal adenocarcinoma (PDAC) or assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC), and the present invention also discovers a new marker for the first time Combination: Acinetobacter, Pseudomonas and Sphingopyxis.
- the marker combination of the present invention can be used to (a) classify pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnose Basal-type pancreatic ductal adenocarcinoma (PDAC) or assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC). It has the advantages of high sensitivity and high specificity, and has important application value. On this basis, the inventors have completed the present invention.
- the term "marker combination” refers to one biomarker, or a combination of two or more biomarkers.
- the level of marker substances is determined by the presence or abundance of microorganisms.
- the term "individual” refers to animals, especially mammals, such as primates, preferably humans.
- the term “about” means that the value may vary by no more than 1% from the recited value.
- the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
- the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
- the reference set refers to the training set.
- the training set and the validation set have the same meaning.
- the training set refers to the set of marker levels in pancreatic ductal adenocarcinoma (PDAC) patients of Basal type and control biological samples of pancreatic ductal adenocarcinoma (PDAC) UnBasal type.
- the validation set refers to a data set used to test the performance of the training set.
- the level of the marker can be represented as an absolute value or a relative value according to the method of determination.
- the intensity of the peak can represent the marker level, which is a relative level
- the number of copies of a gene or copies of gene fragments Numbers can represent the level of the marker.
- the reference value refers to the reference value or normal value of UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls.
- PDAC UnBasal-type pancreatic ductal adenocarcinoma
- the range of the normal value (absolute value) of each biomarker can be obtained by a test and calculation method when the number of samples is sufficiently large. Therefore, when biomarker levels are measured using methods other than mass spectrometry, the absolute value of these biomarker levels can be directly compared with normal values to assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC). , and the diagnosis or early diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC). Optionally, statistical methods can also be used.
- PDAC Pancreatic Ductal Adenocarcinoma
- Pancreatic ductal adenocarcinoma is the most common type of pancreatic cancer and ranks third in cancer mortality worldwide. The prognosis for most PDAC patients is extremely poor, with a five-year survival rate of only 9%. Surgical resection is still the main treatment for PDAC, however, the postoperative recurrence rate is high, and most patients eventually die of tumor metastasis. At present, there is no effective method for screening high-risk patients with pancreatic cancer. Traditional treatment options such as surgical resection, radiotherapy, chemotherapy and combination therapy have little effect on pancreatic cancer. Therefore, it is particularly important to find scientific and effective diagnosis and treatment options. .
- pancreatic cancer Despite the generally high lethality of PDAC, different patients still exhibit clinical heterogeneity. Among patients whose tumors are surgically removed, some progress to advanced stages in just a few months, while some maintain a stable quality of life for years. Many genomics studies have demonstrated that the genetic mutations of pancreatic cancer at different stages of progression are homogeneous, mainly frequent mutations of KRAS, CDKN2A, TP53 and SMAD4 genes, and the reasons for the clinical heterogeneity of pancreatic cancer are still unknown. The phenotypic heterogeneity of pancreatic cancer is also reflected in mouse models, and even genetically engineered mouse models with identical genotypes show differences in the rate and extent of tumor progression. We speculate that certain factors in the tumor microenvironment, such as the tumor microbiome, may be responsible for this heterogeneity.
- Basal type through transcriptome analysis a subtype of PDAC called Basal type through transcriptome analysis, which has a more aggressive clinical phenotype and a worse prognosis.
- the molecular typing features of pancreatic cancer reveal the molecular basis of tumor clinical phenotype heterogeneity, which will help us to further explore the functional mechanism behind it.
- pancreatic ductal adenocarcinoma can be further classified into 'Classical', 'Hybrid' and 'Basal'.
- Basal tumors exhibit extremely high DNA replication activity, epithelial-mesenchymal transition activity, and activation of cancer-related signaling pathways, all of which indicate active cancer progression and a highly aggressive phenotype in the Basal subtype .
- Basal tumors exhibited significantly activated pathogen-recognition immune and inflammatory processes, suggesting that excessive immune activation and pathogen-induced inflammatory responses may play an important role in Basal subtype-specific cancer progression.
- Basal-type tumors are associated with squamous malignant transformation of pancreatic duct epithelial cells, which may be induced by the accumulation of lesions in the tumor microenvironment, and this tumor subtype has a worse tumor progression rate. It is considered to be the development of classic pancreatic cancer, and its tumor progression is more stable than that of Basal type.
- Hybrid type may be an intermediate state of the transition from Classical type to Basal type, and both Basal type and Classical type tumor cells exist in tumor tissue.
- Acinetobacter is a common opportunistic pathogen. When the body's resistance is reduced, it is easy to cause infection, which can lead to respiratory tract infection, sepsis, meningitis, skin infection, etc.
- the bacterium is widely distributed in the environment, and also exists in the skin, pharynx, gastrointestinal tract and other parts of ordinary people, and it is easy to cause infection when the body's immunity is low.
- Pseudomonas is widely distributed in water, air and food, and can cause infectious diseases in humans. It is more common in nosocomial infections. Notably, the bacterium has also been shown to be present in human pancreatic organs, but its role in pancreatic cancer development is not well defined.
- Sphingosineum is a Gram-negative bacterium, and its presence has been detected in human fecal samples, but the abundance is not prominent, and the bacterium has not been found to show clear pathogenicity.
- the present invention deeply excavates the microbial composition characteristics in pancreatic cancer tumors. Significant differences were found in the composition of microbes detected in the three PDAC tumor subtypes. Three indicators were used to assess species diversity, including Shannon index, Bray-Curtis dissimilarity, and species richness, to compare the differences in microbial characteristics between different PDAC subtypes. The results showed that microorganisms in the Basal subtype exhibited higher species richness and lower Shannon index, and the Bray-Curtis dissimilarity between the Basal subtype samples was significantly increased. The results of the present invention indicate that the Basal subtype has a relatively unique microbial composition compared to other PDAC subtypes, which may be related to the specific cancer progression in the Basal subtype.
- results of the present invention also show that some cancer-related functions, such as Kras signaling pathway, epithelial-mesenchymal transition process, MAPK signaling pathway, etc., are also significantly related to the above-mentioned bacterial abundance.
- cancer-related functions such as Kras signaling pathway, epithelial-mesenchymal transition process, MAPK signaling pathway, etc.
- the present invention assesses the genetic similarity between individual patients. The results of the present invention show that the closer the genetic variation among individuals, the more similar their tumor microbial composition, which supports our hypothesis that host genetic factors play a key role in shaping the microbial community.
- the present invention deeply explores the relationship between host genetics and microorganisms, and through QTL analysis, multiple genetic loci that are significantly related to microbial abundance are identified, and mutations at these loci may cause loss of functions related to host immunity, including IFN- ⁇ signaling activation, presentation of foreign antigens, etc.
- the kit of the present invention includes the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention.
- each marker in the combination described in the second aspect of the present invention is used as a standard.
- the present invention finds for the first time that Acinetobacter, Pseudomonas, and/or Sphingopyxis can be used for (a) pancreatic ductal adenocarcinoma (PDAC) typing and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of risk for Basal-type pancreatic ductal adenocarcinoma (PDAC).
- PDAC pancreatic ductal adenocarcinoma
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC Basal-type pancreatic ductal adenocarcinoma
- the present invention finds for the first time that Acinetobacter, Pseudomonas, and/or Sphingopyxis can also be used to distinguish Basal-type pancreatic ductal adenocarcinoma (PDAC) from UnBasal type pancreatic ductal adenocarcinoma (PDAC).
- PDAC Basal-type pancreatic ductal adenocarcinoma
- PDAC UnBasal type pancreatic ductal adenocarcinoma
- the present invention focuses on the microbial factors in the heterogeneity of PDAC subtypes for the first time, reveals the unique structural characteristics of tumor microbes in different PDAC subtypes, discovers several types of microorganisms closely related to cancer progression, and functionally The inflammation-inducing potential of these microorganisms is shown.
- the present invention provides a new PDAC typing method based on microbial composition, and provides a new means for the prognosis and diagnosis of patients.
- the present invention provides a novel therapeutic scheme for intervening or treating PDAC by targeting microorganisms or blocking the pro-inflammatory process of microorganisms.
- the present invention reveals the role of microorganisms in the development of PDAC tumors, and provides a new understanding of the tumorigenesis mechanism.
- All tumor samples were obtained from Shanghai Changhai Hospital, and the following requirements should be met: 1.
- the patient was diagnosed with pancreatic ductal adenocarcinoma (PDAC) by imaging or histology; 2.
- the patient had not received antibiotic treatment in the last month; 3. .
- the patient has a primary, resectable tumor.
- a total of 62 patients were included in the study cohort, and relevant clinical information was documented in detail.
- the surgically removed PDAC tumor tissue samples were immediately transferred to liquid nitrogen for storage, and cryopreserved for subsequent nucleic acid extraction within one month.
- Bacterial cell walls were lysed using QIAGEN Pathogen Lysis Tube, and DNA was extracted using the kit.
- the nucleic acid concentration and quality are tested to ensure that the quality of the samples is qualified, and then library construction and next-generation sequencing are performed.
- the off-machine data was processed for quality control, and an average of about 80GB of high-quality DNA sequencing data was obtained for each sample.
- Quality-controlled high-quality DNA sequences are first aligned to the human reference genome to remove host sequence interference from the sequencing data. Then, based on the microbial reference genome database in the NCBI database, the sequence alignment was performed, and each sequenced read was assigned to a known species classification using Kraken2 software, and the Bayesian probability estimation provided by Bracken software was used to evaluate the alignment to each species. The theoretical sequence number on the taxonomy, based on which the abundance results of each microorganism are calculated. Based on the abundance map of the tumor microbiome, further statistical optimization analysis was performed to calculate microbial community diversity indicators and to evaluate the differences in microbial composition among different tumor subtypes.
- the 62 cases of pancreatic ductal adenocarcinoma PDAC tumor samples collected by the present invention include 17 cases of Basal type PDAC, 23 cases of Hybrid type PDAC and 22 cases of Classical type PDAC.
- Kruskal-Wallis test to screen microbial genera with significantly different abundances between different PDAC molecular subtypes, we found that three bacterial genera, Acinetobacter, Pseudomonas and Sphingopyxis, were The abundance of PDAC was significantly enriched in Basal-type PDAC tumors, as shown in Figures 1-3. Boxplot shows the distribution of Acinetobacter, Pseudomonas, and Sphingopyxis in different tumor subtypes of PDAC.
- the y-axis represents the normalized abundance value
- the Kurskal-Wallis test results show that The p value and its significance indicate that there is a significant difference in the abundance of the bacteria among the groups, and the figure shows that the abundance of the bacteria is significantly increased in the Basal subtype.
- the clustering heat map shows the distribution of these three genera among different subtypes of PDAC, the column represents each sample (column annotation information is grouping information), the row represents each genus, and the abundance is normalized by row , the value is uniformly converted to the interval of -2 to 2, and the reddish color indicates the higher the abundance value.
- the figure shows that these three genera are mostly enriched in the Basal subtype.
- the present invention uses LDA (linear discriminant analysis) linear discriminant analysis to screen the flora markers of Basal type PDAC, and the default logarithmic LDA score greater than 2 is considered to be significantly different, and the analysis results indicate that these three types of bacteria are in the Basal subgroup. were significantly enriched in the type ( Figure 5).
- LDA linear discriminant analysis
- the Kaplan-Meier survival curve showed that the high abundance of the three genera could have a significant predictive effect on the poor survival of patients.
- the abundance values of the above three types of microorganisms are used as input features, and the random forest algorithm is used to construct a classification model, and the constructed model is used to predict the validation set. in accordance with.
- Table 1 shows the importance index of each feature in the prediction model and the individual discriminant performance of each marker. It can be seen that these three types of markers have good prediction performance respectively, and the combination of the three can play the best role prediction performance. In addition, it can also be inferred from Table 1 and Figure 7 that the pairwise combinations of the three types of microorganisms of the present invention also have good predictive performance.
- the abundance of the three types of bacteria (and the integrated model) as a feature factor predicts the ROC curve of the sample tumor type, and the legend in the lower right corner annotates the value of each type of characteristic AUC.
- the figure shows the three types of bacteria in the validation set of 18 cases. And its integrated model has excellent performance in predicting tumor type.
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Abstract
The present invention provides a marker combination for the diagnosis of basal-type pancreatic ductal adenocarcinoma (PDAC) and an application thereof. Specifically, the present invention provides uses of tumor microorganisms or detection reagents and kits thereof for (a) PDAC typing; and/or (b) diagnosis of basal-type PDAC or assessment of risk for basal-type PDAC, the tumor microorganisms being selected from the group consisting of Acinetobacter, Pseudomonas, Sphingopyxis, or a combination thereof.
Description
本发明涉及生物医药领域，具体地涉及用于Basal型胰腺导管腺癌(PDAC)诊断的标志物组合及其应用。The present invention relates to the field of biomedicine, in particular to a marker combination for the diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) and its application.
胰腺导管腺癌(PDAC)是胰腺癌中最常见的类型，在全世界的癌症死亡率中排列第三。大多数PDAC患者的预后极差，五年生存率仅为9％。手术切除仍是PDAC治疗的主要手段，然而术后复发率较高，大多数患者最终死于肿瘤转移。尽管PDAC有着普遍的高致死率，不同患者仍然表现出临床上的异质性。在手术切除肿瘤的患者中，一部分在短短数月内发展至晚期，而某些患者则能保持长达数年的稳定生存质量。近些年的研究通过转录组分析鉴定出一类称为Basal型的PDAC亚型，该肿瘤亚型有着更为侵略性的临床表型和更差的预后。科学家们试图去揭示不同PDAC亚型的遗传学机制，然而不同的肿瘤亚型间并没有发现显著的基因突变差异。导致PDAC亚型临床异质性的原因至今尚不清楚。Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and ranks third in cancer mortality worldwide. The prognosis for most PDAC patients is extremely poor, with a five-year survival rate of only 9%. Surgical resection is still the main treatment for PDAC, however, the postoperative recurrence rate is high, and most patients eventually die of tumor metastasis. Despite the generally high lethality of PDAC, different patients still exhibit clinical heterogeneity. Among patients whose tumors are surgically removed, some progress to advanced stages in just a few months, while some maintain a stable quality of life for years. Studies in recent years have identified a subtype of PDAC called Basal type through transcriptome analysis, which has a more aggressive clinical phenotype and a worse prognosis. Scientists have tried to uncover the genetic mechanism of different PDAC subtypes, but no significant gene mutation differences have been found between different tumor subtypes. The reasons for the clinical heterogeneity of PDAC subtypes are still unclear.
近二十年来，胰腺癌一直被认为是一种炎症诱导的癌症，患有胰腺炎的病人发展至胰腺癌的风险比正常情况高出13倍。有研究表明炎症间质反应对PDAC的进展有着重要影响，然而炎症是如何导致PDAC进展的详细分子机制尚未阐明。越来越多的证据表明人体微生物组在激活免疫应答以及诱导癌症相关的炎症反应过程中扮演重要角色。过去的研究主要关注肠道微生物的作用，并且发现肠道微生物对食管癌、胃癌、结肠癌都造成一定影响。Pancreatic cancer has been considered an inflammation-induced cancer for nearly two decades, and patients with pancreatitis have a 13-fold higher than normal risk of developing pancreatic cancer. Studies have shown that the inflammatory interstitial response has an important impact on the progression of PDAC, however, the detailed molecular mechanism of how inflammation leads to the progression of PDAC has not been elucidated. There is increasing evidence that the human microbiome plays an important role in activating immune responses and inducing cancer-related inflammatory responses. Past research has mainly focused on the role of gut microbes, and found that gut microbes have a certain impact on esophageal cancer, gastric cancer, and colon cancer.
目前为止，胰腺肿瘤微生物的组成结构还未完全阐明，微生物对肿瘤发展的作用也需要进一步的研究探索。So far, the composition and structure of pancreatic tumor microbes have not been fully elucidated, and the role of microbes in tumor development also needs further research and exploration.
因此，本领域迫切需要着眼于PDAC亚型异质性中的微生物因素，揭示了不同PDAC亚型中独特的肿瘤微生物组成结构特征，开发新的预测PDAC患者临床表型的肿瘤微生物。Therefore, there is an urgent need in the field to focus on the microbial factors in the heterogeneity of PDAC subtypes, reveal the unique structural characteristics of tumor microbes in different PDAC subtypes, and develop new tumor microbes that predict the clinical phenotype of PDAC patients.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于着眼于PDAC亚型异质性中的微生物因素，揭示了不同PDAC亚型中独特的肿瘤微生物组成结构特征，开发新的预测PDAC患者临床表 型的肿瘤微生物。The purpose of the present invention is to focus on the microbial factors in the heterogeneity of PDAC subtypes, to reveal the unique structural characteristics of tumor microbes in different PDAC subtypes, and to develop new tumor microbes that predict the clinical phenotype of PDAC patients.
在本发明的第一方面，提供了一种肿瘤微生物或其检测试剂的用途，用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险；或用于制备一试剂或试剂盒，所述试剂或试剂盒用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险，其中所述肿瘤微生物选自下组：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。In a first aspect of the present invention, use of a tumor microorganism or a detection reagent thereof is provided for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma ( PDAC) or assessing the risk of having a Basal-type pancreatic ductal adenocarcinoma (PDAC); or for preparing a reagent or kit for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and /or (b) diagnosing or assessing the risk of developing Basal-type pancreatic ductal adenocarcinoma (PDAC), wherein the tumor microorganism is selected from the group consisting of: Acinetobacter, Pseudomonas Pseudomonas, Sphingopyxis, or a combination thereof.
在另一优选例中，所述胰腺导管腺癌(PDAC)分型包括Basal型胰腺导管腺癌(PDAC)和UnBasal型胰腺导管腺癌(PDAC)。In another preferred embodiment, the pancreatic ductal adenocarcinoma (PDAC) classification includes Basal-type pancreatic ductal adenocarcinoma (PDAC) and UnBasal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述试剂或试剂盒还用于区分Basal型胰腺导管腺癌(PDAC)和UnBasal型胰腺导管腺癌(PDAC)。In another preferred embodiment, the reagent or kit is also used to distinguish Basal type pancreatic ductal adenocarcinoma (PDAC) from UnBasal type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述的诊断包括早期诊断、辅助性诊断、或其组合。In another preferred embodiment, the diagnosis includes early diagnosis, auxiliary diagnosis, or a combination thereof.
在另一优选例中，所述评估或诊断包括步骤：In another preferred embodiment, the evaluation or diagnosis comprises the steps of:
(1)提供一来源于待测对象的样品，对样品中所述肿瘤微生物中各个肿瘤微生物的水平(如丰度)进行检测；(1) providing a sample derived from the object to be tested, and detecting the level (such as abundance) of each tumor microorganism in the tumor microorganism in the sample;
(2)将步骤(1)测得的水平(如丰度)与一参考数据集或一参考值(如健康对照者的参考值)进行比较；(2) comparing the level (such as abundance) measured in step (1) with a reference data set or a reference value (such as the reference value of healthy controls);
较佳地，所述的参考数据集包括来源于Basal型胰腺导管腺癌(PDAC)患者和UnBasal型胰腺导管腺癌(PDAC)对照者的如所述肿瘤微生物中各个肿瘤微生物的水平(如丰度)。Preferably, the reference data set includes the levels of each tumor microorganism in the tumor microorganisms (such as abundance) from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls. Spend).
在另一优选例中，当与一参考值进行比较时，所述肿瘤微生物中的各肿瘤微生物的水平(如丰度)增加，则表明待测对象具有Basal型胰腺导管腺癌(PDAC)患病风险或患有Basal型胰腺导管腺癌(PDAC)。In another preferred example, when compared with a reference value, the level (eg abundance) of each tumor microorganism in the tumor microorganism increases, indicating that the subject to be tested has Basal type pancreatic ductal adenocarcinoma (PDAC). risk or have Basal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述的将步骤(1)测得的水平与一参考数据集进行比较，还包括构建一分类模型，用于预测患者是否为Basal型胰腺导管腺癌，较佳地，所述的分类模型为随机森林模型。In another preferred embodiment, the level measured in step (1) is compared with a reference data set, and further includes constructing a classification model for predicting whether the patient is Basal-type pancreatic ductal adenocarcinoma, preferably , the classification model is a random forest model.
在另一优选例中，如果所述的预测为Basal型胰腺导管腺癌(PDAC)的可能性≥0.5，所述的对象被判定为具有Basal型胰腺导管腺癌(PDAC)患病风险或 患有Basal型胰腺导管腺癌(PDAC)。In another preferred embodiment, if the predicted probability of Basal-type pancreatic ductal adenocarcinoma (PDAC) is greater than or equal to 0.5, the subject is determined to have the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) or suffer from There is a Basal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述的样品选自肿瘤组织样品。In another preferred embodiment, the sample is selected from tumor tissue samples.
在另一优选例中，通过下组的一种或多种方法对所述肿瘤微生物中的各肿瘤微生物的水平(如丰度)进行检测：测序、PCR、蛋白定量检测。In another preferred embodiment, the level (eg abundance) of each tumor microorganism in the tumor microorganism is detected by one or more methods of the following group: sequencing, PCR, protein quantitative detection.
在另一优选例中，所述PCR包括qPCR。In another preferred embodiment, the PCR includes qPCR.
在另一优选例中，对所述肿瘤微生物水平检测的方法还包括选自下组的一种或多种方法：特征基因定量PCR、宏基因组学分析、16s rRNA测序、质谱分析、蛋白质免疫印迹。In another preferred embodiment, the method for detecting the level of tumor microorganisms further includes one or more methods selected from the group consisting of quantitative PCR for characteristic genes, metagenomic analysis, 16s rRNA sequencing, mass spectrometry analysis, and western blotting .
在另一优选例中，在步骤(1)之前，所述的方法还包括对样品进行处理的步骤。In another preferred embodiment, before step (1), the method further includes the step of processing the sample.
本发明第二方面提供了一种标志物组合，所述标志物组合包括选自下组的两种或多种：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。A second aspect of the present invention provides a marker combination comprising two or more selected from the group consisting of Acinetobacter, Pseudomonas, Sphingosine Sphingopyxis, or a combination thereof.
在另一优选例中，所述标志物组合包括不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)和鞘氨醇菌属(Sphingopyxis)。In another preferred embodiment, the marker combination includes Acinetobacter, Pseudomonas and Sphingopyxis.
在另一优选例中，所述标志物组合用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险。In another preferred embodiment, the marker combination is used for (a) pancreatic ductal adenocarcinoma (PDAC) classification; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) risk of developing cancer (PDAC).
在另一优选例中，所述标志物组合还用于区分Basal型胰腺导管腺癌(PDAC)和UnBasal型胰腺导管腺癌(PDAC)。In another preferred example, the marker combination is also used to distinguish Basal-type pancreatic ductal adenocarcinoma (PDAC) from UnBasal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述的标志物来源于肿瘤组织样品，较佳地，来源于PDAC肿瘤组织样品。In another preferred embodiment, the markers are derived from tumor tissue samples, preferably from PDAC tumor tissue samples.
在另一优选例中，通过下组的一种或多种方法对所述标志物组合中的各个标志物的水平(如丰度)进行检测：测序、PCR、蛋白定量检测。In another preferred embodiment, the level (eg abundance) of each marker in the marker combination is detected by one or more methods of the following group: sequencing, PCR, protein quantitative detection.
在另一优选例中，所述PCR包括qPCR。In another preferred embodiment, the PCR includes qPCR.
在另一优选例中，对所述标志物组水平检测的方法还包括选自下组的一种或多种方法：特征基因定量PCR、宏基因组学分析、16s rRNA测序、质谱分析、蛋白质免疫印迹。In another preferred embodiment, the method for detecting the level of the marker group further includes one or more methods selected from the group consisting of quantitative PCR for characteristic genes, metagenomics analysis, 16s rRNA sequencing, mass spectrometry analysis, protein immunity blot.
在另一优选例中，当与一参考值进行比较时，所述标志物组合中的各标志物的水平(如丰度)增加，则表明待测对象具有Basal型胰腺导管腺癌(PDAC)患病风险 或患有Basal型胰腺导管腺癌(PDAC)。In another preferred example, when compared with a reference value, the level (eg abundance) of each marker in the marker combination increases, indicating that the subject to be tested has Basal-type pancreatic ductal adenocarcinoma (PDAC). At risk of developing or having Basal-type pancreatic ductal adenocarcinoma (PDAC).
本发明第三方面提供了一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的试剂组合，所述试剂组合包括用于检测本发明第二方面所述的标志物组合中各个标志物的试剂。A third aspect of the present invention provides a method for (a) classification of pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ), the reagent combination comprising the reagents for detecting each marker in the marker combination according to the second aspect of the present invention.
在另一优选例中，所述的试剂包括用选自下组的一种或多种方法检测本发明第二方面所述的标志物组合中各个标志物的水平的物质：测序、PCR、蛋白定量检测。In another preferred embodiment, the reagent includes a substance for detecting the level of each marker in the marker combination described in the second aspect of the present invention by one or more methods selected from the group consisting of sequencing, PCR, protein Quantitative detection.
在另一优选例中，所述的试剂还包括用选自下组的一种或多种方法检测本发明第二方面所述的标志物组合中各个标志物的水平的物质：特征基因定量PCR、宏基因组学分析、16s rRNA测序、质谱分析、蛋白质免疫印迹。In another preferred embodiment, the reagent also includes a substance for detecting the level of each marker in the marker combination described in the second aspect of the present invention by one or more methods selected from the group consisting of: quantitative PCR for characteristic genes , metagenomics analysis, 16s rRNA sequencing, mass spectrometry analysis, western blotting.
在另一优选例中，所述试剂用于检测各个标志物的水平(如丰度)。In another preferred embodiment, the reagent is used to detect the level (eg abundance) of each marker.
本发明第四方面提供了一种试剂盒，所述的试剂盒包括本发明第二方面所述的标志物组合和/或本发明第三方面所述的试剂组合。A fourth aspect of the present invention provides a kit comprising the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention.
在另一优选例中，本发明第二方面所述的组合中各个标志物用作标准品。In another preferred embodiment, each marker in the combination described in the second aspect of the present invention is used as a standard.
在另一优选例中，所述的试剂盒还包括一说明书，所述的说明书记载了来源于Basal型胰腺导管腺癌(PDAC)患者和/或UnBasal型胰腺导管腺癌(PDAC)对照者的如本发明第二方面所述组合中各个标志物的水平的参考数据集。In another preferred embodiment, the kit further includes an instruction manual, which describes the data obtained from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and/or UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls A reference dataset of the levels of each marker in the combination as described in the second aspect of the invention.
本发明第五方面提供了一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的方法，包括步骤：A fifth aspect of the present invention provides a method for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ) method for risk of disease, comprising the steps of:
(1)提供一来源于待测对象的样品，对样品中本发明第二方面所述组合中各个标志物的水平进行检测；(1) providing a sample derived from the object to be tested, and detecting the level of each marker in the combination according to the second aspect of the present invention in the sample;
(2)将步骤(1)测得的水平与一参考数据集或一参考值(如健康对照者的参考值)进行比较；(2) comparing the level measured in step (1) with a reference data set or a reference value (such as a reference value for healthy controls);
较佳地，所述的参考数据集包括来源于Basal型胰腺导管腺癌(PDAC)患者和UnBasal型胰腺导管腺癌(PDAC)对照者的如本发明第二方面所述组合中各个标志物的水平。Preferably, the reference data set includes the respective markers in the combination according to the second aspect of the present invention derived from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls. Level.
本发明第六方面提供了一种筛选治疗Basal型胰腺导管腺癌(PDAC)的候选化合物的方法，其特征在于，包括步骤：A sixth aspect of the present invention provides a method for screening candidate compounds for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC), characterized in that it comprises the steps of:
(1)在测试组中，向待测对象施用测试化合物，检测测试组中来源于所述对象 的样品中本发明第二方面所述组合中各个标志物的水平V1；在对照组中，向待测对象施用空白对照(包括溶媒)，检测对照组中来源于所述对象的样品中本发明第二方面所述组合中各个标志物的水平V2；(1) In the test group, administer the test compound to the subject to be tested, and detect the level V1 of each marker in the combination according to the second aspect of the present invention in the sample derived from the subject in the test group; in the control group, to The subject to be tested is administered a blank control (including a vehicle), and the level V2 of each marker in the combination according to the second aspect of the present invention in the sample derived from the subject in the control group is detected;
(2)比较上一步骤检测得到的水平V1和水平V2进行比较，从而确定所述测试化合物是否是治疗Basal型胰腺导管腺癌(PDAC)的候选化合物。(2) Comparing the level V1 and the level V2 detected in the previous step to determine whether the test compound is a candidate compound for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述的待测对象为Basal型胰腺导管腺癌(PDAC)患者。In another preferred embodiment, the test object is a Basal-type pancreatic ductal adenocarcinoma (PDAC) patient.
在另一优选例中，如果所述组合中各个标志物的水平V1显著低于水平V2，表明测试化合物为治疗Basal型胰腺导管腺癌(PDAC)的候选化合物。In another preferred example, if the level V1 of each marker in the combination is significantly lower than the level V2, it indicates that the test compound is a candidate compound for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC).
在另一优选例中，所述“显著低于”指水平V1/水平V2的比值≤0.7，较佳地≤0.6。In another preferred embodiment, the "significantly lower than" refers to the ratio of level V1/level V2≤0.7, preferably≤0.6.
本发明第七方面提供了一种本发明第二方面所述的标志物组合和/或本发明第三方面所述的试剂组合的用途，用于筛选治疗Basal型胰腺导管腺癌(PDAC)的候选化合物和/或用于评估候选化合物对Basal型胰腺导管腺癌(PDAC)的治疗效果。The seventh aspect of the present invention provides the use of the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention for screening and treating Basal-type pancreatic ductal adenocarcinoma (PDAC). Candidate compounds and/or for evaluating the therapeutic effect of candidate compounds on Basal-type pancreatic ductal adenocarcinoma (PDAC).
本发明第八方面提供了一种建立评估Basal型胰腺导管腺癌(PDAC)患病风险或用于Basal型胰腺导管腺癌(PDAC)诊断的模型的方法，所述的方法包括识别Basal型胰腺导管腺癌患者和UnBasal型胰腺导管腺癌对照者之间，组织样品中差异表达物质的步骤,其中，所述的差异表达物质包括一种或多种本发明第二方面所述组合中的标志物。An eighth aspect of the present invention provides a method for establishing a model for assessing the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) or for diagnosing Basal-type pancreatic ductal adenocarcinoma (PDAC), the method comprising identifying the Basal-type pancreatic ductal adenocarcinoma (PDAC) The step of differentially expressing substances in tissue samples between ductal adenocarcinoma patients and UnBasal-type pancreatic ductal adenocarcinoma controls, wherein the differentially expressed substances include one or more markers in the combination of the second aspect of the present invention thing.
在另一优选例中，所述组织样品包括胰腺组织样品。In another preferred embodiment, the tissue sample includes a pancreatic tissue sample.
本发明第九方面提供了一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的系统，所述系统包括：A ninth aspect of the present invention provides a method for (a) pancreatic ductal adenocarcinoma (PDAC) classification; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type pancreatic ductal adenocarcinoma (PDAC) ) system at risk of disease, the system comprising:
(a)特征输入模块，所述特征输入模块用于输入某一对象的肿瘤组织样本的特征；(a) a feature input module, the feature input module is used to input the features of a tumor tissue sample of a certain object;
其中所述的肿瘤组织样本的特征包括选自下组的肿瘤微生物：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。The characteristics of the tumor tissue sample include tumor microorganisms selected from the group consisting of Acinetobacter, Pseudomonas, Sphingopyxis, or a combination thereof.
(b)判别处理模块，所述处理模块对于输入的肿瘤组织样本的特征，按预定的判断标准进行评分处理，从而获得风险度评分；并且将所述风险度评分与Basal型胰腺导管腺癌(PDAC)的风险度阈值进行比较，从而得出辅助筛查结果，其中，当所述风险度评分高于所述风险度阈值时，则提示该对象患Basal型胰腺导管腺癌(PDAC)的风险高于正常人群；当所述风险度评分低于所述风险度阈值时，则提示该对象患Basal型胰腺导管腺癌(PDAC)的风险高于正常人群；和(b) a discrimination processing module, the processing module performs scoring processing according to a predetermined judgment standard for the characteristics of the input tumor tissue samples, thereby obtaining a risk score; and compares the risk score with the Basal type pancreatic ductal adenocarcinoma ( PDAC) risk thresholds are compared to obtain an auxiliary screening result, wherein, when the risk score is higher than the risk threshold, the subject is at risk of suffering from Basal-type pancreatic ductal adenocarcinoma (PDAC). higher than the normal population; when the risk score is lower than the risk threshold, it is suggested that the subject has a higher risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) than the normal population; and
(c)辅助筛查结果输出模块，所述输出模块用于输出所述的辅助筛查结果。(c) an auxiliary screening result output module, the output module is used for outputting the auxiliary screening result.
在另一优选例中，所述的对象是人。In another preferred embodiment, the subject is a human.
在另一优选例中，所述的对象包括婴幼儿、青少年或成年人。In another preferred embodiment, the subject includes infants, adolescents or adults.
在另一优选例中，所述的评分包括(a)单个特征的评分；和/或(b)多个特征的评分之和。In another preferred embodiment, the score includes (a) the score of a single feature; and/or (b) the sum of the scores of multiple features.
在另一优选例中，所述的特征输入模块选自下组：样本采集仪、特征信号输入端、数据预处理模块、特征构建模块、特征可视化模块。In another preferred embodiment, the feature input module is selected from the following group: a sample acquisition instrument, a feature signal input terminal, a data preprocessing module, a feature building module, and a feature visualization module.
在另一优选例中，所述的判别处理模块包括一处理器，以及一储存器，其中所述的储存器中存储有基于肿瘤组织样本的特征的Basal型胰腺导管腺癌(PDAC)的风险度阈值数据。In another preferred embodiment, the discrimination processing module includes a processor and a storage, wherein the storage stores the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) based on the characteristics of the tumor tissue sample degree threshold data.
在另一优选例中，所述的输出模块包括报告系统。In another preferred embodiment, the output module includes a reporting system.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
图1显示了假单胞菌属(Pseudomonas)在不同PDAC亚型中的丰度分布情况。Figure 1 shows the abundance distribution of Pseudomonas in different PDAC subtypes.
图2显示了不动杆菌属(Acinetobacter)在不同PDAC亚型中的丰度分布情况。Figure 2 shows the abundance distribution of Acinetobacter in different PDAC subtypes.
图3显示了鞘氨醇菌属(Sphingopyxis)在不同PDAC亚型中的丰度分布情况。Figure 3 shows the abundance distribution of Sphingopyxis in different PDAC subtypes.
图4显示了基于微生物标志物对PDAC患者的聚类结果。Figure 4 shows the clustering results of PDAC patients based on microbial markers.
图5显示了在Basal型PDAC中显著富集的微生物属。Figure 5 shows microbial genera significantly enriched in Basal-type PDAC.
图6显示了候选的微生物标志物能够指示患者预后生存情况。Figure 6 shows that candidate microbial markers can indicate patient prognosis and survival.
图7显示了构建的分类器模型在评估Basal型PDAC中的预测效能。Figure 7 shows the predictive performance of the constructed classifier model in evaluating Basal-type PDAC.
本发明人经过广泛而深入地研究，首次发现不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、和/或鞘氨醇菌属(Sphingopyxis)可用于(a)胰腺导 管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险，并且本发明还首次发现了一种新的标志物组合：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)和鞘氨醇菌属(Sphingopyxis)。本发明的标志物组合可用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险，并且具有高灵敏性、高特异性的优点，具有重要的应用价值。在此基础上，发明人完成了本发明。After extensive and in-depth research, the present inventors found for the first time that Acinetobacter, Pseudomonas, and/or Sphingopyxis can be used for (a) pancreatic ductal adenocarcinoma ( PDAC) classification; and/or (b) diagnose Basal-type pancreatic ductal adenocarcinoma (PDAC) or assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC), and the present invention also discovers a new marker for the first time Combination: Acinetobacter, Pseudomonas and Sphingopyxis. The marker combination of the present invention can be used to (a) classify pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnose Basal-type pancreatic ductal adenocarcinoma (PDAC) or assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC). It has the advantages of high sensitivity and high specificity, and has important application value. On this basis, the inventors have completed the present invention.
术语the term
本发明所用术语具有相关领域普通技术人员通常理解的含义。然而，为了更好地理解本发明，对一些定义和相关术语的解释如下：Terms used herein have the meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:
根据本发明，术语“标志物组合”是指一种生物标志物、或两种及两种以上生物标志物的组合。According to the present invention, the term "marker combination" refers to one biomarker, or a combination of two or more biomarkers.
根据本发明，标志物质的水平通过微生物的存在含量或丰度确定。根据本发明，术语“个体”指动物，特别是哺乳动物，如灵长类动物，最好是人。According to the present invention, the level of marker substances is determined by the presence or abundance of microorganisms. According to the present invention, the term "individual" refers to animals, especially mammals, such as primates, preferably humans.
根据本发明，术语如“一”、“一个”和“这”不仅指单数的个体，而是包括可以用来说明特定实施方式的通常的一类。In accordance with the present invention, terms such as "a," "an," and "the" do not refer only to the singular, but include the general class that may be used to describe particular embodiments.
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。As used herein, when used in reference to a specifically recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."
需要说明的是，在此提供术语的解释仅为了使本领域技术人员更好地理解本发明，并非对本发明限制。It should be noted that the explanations of terms provided here are only for the purpose of enabling those skilled in the art to better understand the present invention, rather than limiting the present invention.
根据本发明，参考集指训练集。According to the present invention, the reference set refers to the training set.
根据本发明，由现有技术可知，训练集和验证集具有相同的含义。在本发明的一个实施方式中，训练集指Basal型胰腺导管腺癌(PDAC)患者和UnBasal型胰腺导管腺癌(PDAC)对照生物样品中的标志物水平的集。在本发明的一个实施方式中，验证集是指用于测试训练集性能的数据集。在本发明的一个实施方式中，标志物的水平可以根据测定的方法代表为绝对值或相对值。例如，当用质谱来测定标志物的水平时，峰的强度可以代表标志物水平，这是一个相对值的 水平；当用PCR来测定标志物的水平时，基因的拷贝数或基因片段的拷贝数可以代表的标志物的水平。According to the present invention, as known from the prior art, the training set and the validation set have the same meaning. In one embodiment of the invention, the training set refers to the set of marker levels in pancreatic ductal adenocarcinoma (PDAC) patients of Basal type and control biological samples of pancreatic ductal adenocarcinoma (PDAC) UnBasal type. In one embodiment of the present invention, the validation set refers to a data set used to test the performance of the training set. In one embodiment of the present invention, the level of the marker can be represented as an absolute value or a relative value according to the method of determination. For example, when mass spectrometry is used to measure marker levels, the intensity of the peak can represent the marker level, which is a relative level; when PCR is used to measure marker levels, the number of copies of a gene or copies of gene fragments Numbers can represent the level of the marker.
在本发明的一个实施方式中，参照值是指UnBasal型胰腺导管腺癌(PDAC)对照的参考值或正常值。本领域的技术人员清楚，在样品数量足够多情况下，每个每个生物标记的正常值(绝对值)的范围可以通过检验和计算方法得到。因此，当利用除质谱以外的其他方法来检测生物标志物的水平时，这些生物标志物水平的绝对值，可以直接与正常值比较，从而评价患有Basal型胰腺导管腺癌(PDAC)的风险，以及诊断或早期诊断Basal型胰腺导管腺癌(PDAC)。任选地，还可以使用统计方法。In one embodiment of the present invention, the reference value refers to the reference value or normal value of UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls. It is clear to those skilled in the art that the range of the normal value (absolute value) of each biomarker can be obtained by a test and calculation method when the number of samples is sufficiently large. Therefore, when biomarker levels are measured using methods other than mass spectrometry, the absolute value of these biomarker levels can be directly compared with normal values to assess the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC). , and the diagnosis or early diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC). Optionally, statistical methods can also be used.
胰腺导管腺癌(PDAC)Pancreatic Ductal Adenocarcinoma (PDAC)
胰腺导管腺癌(PDAC)是胰腺癌中最常见的类型，在全世界的癌症死亡率中排列第三。大多数PDAC患者的预后极差，五年生存率仅为9％。手术切除仍是PDAC治疗的主要手段，然而术后复发率较高，大多数患者最终死于肿瘤转移。目前还没有筛查胰腺癌高危患者的有效手段，传统的治疗方案比如手术切除、放疗，化疗以及联合疗法等，都对胰腺癌收效甚微，因此，寻找科学有效的诊断及治疗方案显得尤为重要。Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and ranks third in cancer mortality worldwide. The prognosis for most PDAC patients is extremely poor, with a five-year survival rate of only 9%. Surgical resection is still the main treatment for PDAC, however, the postoperative recurrence rate is high, and most patients eventually die of tumor metastasis. At present, there is no effective method for screening high-risk patients with pancreatic cancer. Traditional treatment options such as surgical resection, radiotherapy, chemotherapy and combination therapy have little effect on pancreatic cancer. Therefore, it is particularly important to find scientific and effective diagnosis and treatment options. .
尽管PDAC有着普遍的高致死率，不同患者仍然表现出临床上的异质性。在手术切除肿瘤的患者中，一部分在短短数月内发展至晚期，而某些患者则能保持长达数年的稳定生存质量。许多基因组学研究已经证明不同进展阶段的胰腺癌的遗传突变是同质的，主要为KRAS、CDKN2A、TP53和SMAD4基因的频发突变，导致胰腺癌临床异质性的原因目前依然未知。胰腺癌的表型异质性同样体现在小鼠模型中，即便是经基因工程改造的基因型完全一致的小鼠模型，在肿瘤进展的速度和程度上依然表现出表别。我们推测肿瘤微环境中的某些因素，比如肿瘤微生物组，可能是造成该异质性的原因。Despite the generally high lethality of PDAC, different patients still exhibit clinical heterogeneity. Among patients whose tumors are surgically removed, some progress to advanced stages in just a few months, while some maintain a stable quality of life for years. Many genomics studies have demonstrated that the genetic mutations of pancreatic cancer at different stages of progression are homogeneous, mainly frequent mutations of KRAS, CDKN2A, TP53 and SMAD4 genes, and the reasons for the clinical heterogeneity of pancreatic cancer are still unknown. The phenotypic heterogeneity of pancreatic cancer is also reflected in mouse models, and even genetically engineered mouse models with identical genotypes show differences in the rate and extent of tumor progression. We speculate that certain factors in the tumor microenvironment, such as the tumor microbiome, may be responsible for this heterogeneity.
近些年的研究通过转录组分析鉴定出一类称为Basal型的PDAC亚型，该肿瘤亚型有着更为侵略性的临床表型和更差的预后。胰腺癌的分子分型特征揭示了肿瘤临床表型异质性的分子基础，这将有助于我们进一步挖掘其背后的功能机制。Studies in recent years have identified a subtype of PDAC called Basal type through transcriptome analysis, which has a more aggressive clinical phenotype and a worse prognosis. The molecular typing features of pancreatic cancer reveal the molecular basis of tumor clinical phenotype heterogeneity, which will help us to further explore the functional mechanism behind it.
胰腺导管腺癌(PDAC)分型Pancreatic ductal adenocarcinoma (PDAC) classification
在本发明中，胰腺导管腺癌(PDAC)可进一步分型为’Classical’、’Hybrid’ 和’Basal’。In the present invention, pancreatic ductal adenocarcinoma (PDAC) can be further classified into 'Classical', 'Hybrid' and 'Basal'.
通过基因功能富集分析发现Basal肿瘤表现出极高的DNA复制活性、上皮间质转化活性以及癌症相关信号通路的激活，这些都表明了Basal亚型中活跃的癌症进展和高度侵略性的表型。同时我们还发现Basal肿瘤呈现显著激活的病原体识别的免疫反应和炎症反应过程，这些结果表明过度的免疫激活和病原体诱导的炎症反应可能对Basal亚型特异性的癌症进展发挥重要的作用。Gene functional enrichment analysis revealed that Basal tumors exhibit extremely high DNA replication activity, epithelial-mesenchymal transition activity, and activation of cancer-related signaling pathways, all of which indicate active cancer progression and a highly aggressive phenotype in the Basal subtype . We also found that Basal tumors exhibited significantly activated pathogen-recognition immune and inflammatory processes, suggesting that excessive immune activation and pathogen-induced inflammatory responses may play an important role in Basal subtype-specific cancer progression.
已有的研究认为，Basal型肿瘤与胰管上皮细胞的鳞状恶变有关，可能由肿瘤微环境中的病变积累所诱导，该肿瘤亚型有着更恶劣的肿瘤进展速度；相对的，Classical型被认为是经典的胰腺癌发展，相较于Basal型其肿瘤进展更稳定；而Hybrid型或许是Classical型向Basal型转变的中间状态，肿瘤组织中同时存在Basal型和Classical型的肿瘤细胞。Existing studies suggest that Basal-type tumors are associated with squamous malignant transformation of pancreatic duct epithelial cells, which may be induced by the accumulation of lesions in the tumor microenvironment, and this tumor subtype has a worse tumor progression rate. It is considered to be the development of classic pancreatic cancer, and its tumor progression is more stable than that of Basal type. Hybrid type may be an intermediate state of the transition from Classical type to Basal type, and both Basal type and Classical type tumor cells exist in tumor tissue.
不动杆菌属(Acinetobacter)Acinetobacter
不动杆菌是常见的条件致病菌，当机体抵抗力降低时容易引起感染，可导致呼吸道感染、败血症、脑膜炎、皮肤感染等。该细菌广泛分布于环境中，也存在于普通人的皮肤、咽部、胃肠道等部位，在机体免疫力低下时易导致感染。Acinetobacter is a common opportunistic pathogen. When the body's resistance is reduced, it is easy to cause infection, which can lead to respiratory tract infection, sepsis, meningitis, skin infection, etc. The bacterium is widely distributed in the environment, and also exists in the skin, pharynx, gastrointestinal tract and other parts of ordinary people, and it is easy to cause infection when the body's immunity is low.
假单胞菌属(Pseudomonas)Pseudomonas
假单胞菌广泛分布于水、空气和食物中，可引起人类感染疾病，在医院感染中较为常见。值得注意的是，该细菌也被证实存在于人类胰腺器官中，但其对胰腺癌发展的作用并未明确。Pseudomonas is widely distributed in water, air and food, and can cause infectious diseases in humans. It is more common in nosocomial infections. Notably, the bacterium has also been shown to be present in human pancreatic organs, but its role in pancreatic cancer development is not well defined.
鞘氨醇菌属(Sphingopyxis)Sphingopyxis
鞘氨醇菌是革兰氏阴性细菌，在人的粪便样本中检测到了其存在，然而丰度并不突出，目前并未发现该细菌表现出明确致病性。有研究发现该细菌外膜中的抗原物质-糖基神经酰胺，能够有效刺激机体免疫激活，表明该细菌外膜有着典型的病原体相关分子模式。Sphingosineum is a Gram-negative bacterium, and its presence has been detected in human fecal samples, but the abundance is not prominent, and the bacterium has not been found to show clear pathogenicity. Some studies have found that the antigenic substance-glycosylceramide in the bacterial outer membrane can effectively stimulate the immune activation of the body, indicating that the bacterial outer membrane has a typical pathogen-related molecular pattern.
本发明深入挖掘胰腺癌肿瘤中的微生物组成特征。发现3种PDAC肿瘤亚型中检出微生物的组成结构存在显著差别。利用三项指标来评估物种多样性，包括Shannon指数、Bray-Curtis相异度以及物种丰富度，进而比较不同PDAC亚型之间的微生物特征差别。结果显示Basal亚型中的微生物表现更高的物种丰富度 和较低的Shannon指数，此外Basal亚型样本间的Bray-Curtis相异度显著升高。本发明的结果表明，相较于其他PDAC亚型，Basal亚型有着一个相对独特的微生物组成结构，这可能与Basal亚型中特殊的癌症进展有关。The present invention deeply excavates the microbial composition characteristics in pancreatic cancer tumors. Significant differences were found in the composition of microbes detected in the three PDAC tumor subtypes. Three indicators were used to assess species diversity, including Shannon index, Bray-Curtis dissimilarity, and species richness, to compare the differences in microbial characteristics between different PDAC subtypes. The results showed that microorganisms in the Basal subtype exhibited higher species richness and lower Shannon index, and the Bray-Curtis dissimilarity between the Basal subtype samples was significantly increased. The results of the present invention indicate that the Basal subtype has a relatively unique microbial composition compared to other PDAC subtypes, which may be related to the specific cancer progression in the Basal subtype.
进一步的，本研究发现了一群在Basal肿瘤中丰度显著升高的微生物，其中不动杆菌属Acinetobacter、假单胞菌属Pseudomonas和鞘氨醇菌属Sphingopyxis这三类细菌最为明显。这三类细菌的丰度指标对PDAC的预后有着潜在的预测价值。此外，通过微生物基因功能分析，Basal亚型中的微生物表现出更高的代谢活性、细胞运动能力、复制效率、抗生素耐药性以及细胞膜合成活性，这些功能反映了微生物更高的病原性和炎症诱导潜力。这些结果揭示了Basal亚型中微生物对癌症发展的作用，靶向这些微生物或阻断微生物促炎过程的治疗策略将会有效的干预PDAC的进展。Further, this study found a group of microorganisms with significantly increased abundance in Basal tumors, among which the three types of bacteria, Acinetobacter, Pseudomonas, and Sphingopyxis, were the most obvious. The abundance indicators of these three types of bacteria have potential predictive value for the prognosis of PDAC. In addition, by microbial gene function analysis, microorganisms in the Basal subtype exhibited higher metabolic activity, cell motility, replication efficiency, antibiotic resistance, and cell membrane synthesis activity, which reflected higher pathogenicity and inflammation of the microorganisms induction potential. These results reveal the role of microbes in the Basal subtype on cancer development, and therapeutic strategies that target these microbes or block microbial pro-inflammatory processes will effectively interfere with PDAC progression.
随后，进一步研究肿瘤微生物与宿主细胞功能的相互作用，通过肿瘤细胞基因表达和微生物丰度的关联分析，发现某些细菌丰度，包括前面提到的不动杆菌属Acinetobacter、假单胞菌属Pseudomonas和鞘氨醇菌属Sphingopyxis，与宿主的抗原识别、脂多糖应答反应、补体系统激活等功能呈显著正相关，这些结果反映了宿主对外源病原体的应答激活。此外，本发明的结果还显示一些癌症相关的功能，比如Kras信号通路、上皮间质转化过程、MAPK信号通路等，也跟上述的细菌丰度显著相关。这些数据从基因功能水平上揭示了肿瘤微生物对宿主细胞功能造成的影响，同时也证明了某些细菌在肿瘤发生中的重要作用。Subsequently, the interaction between tumor microorganisms and host cell functions was further studied. Through the correlation analysis of tumor cell gene expression and microbial abundance, it was found that certain bacterial abundances, including the aforementioned Acinetobacter, Pseudomonas Pseudomonas and Sphingopyxis were significantly positively correlated with the host's antigen recognition, lipopolysaccharide response, complement system activation and other functions. These results reflect the host's response to exogenous pathogens. In addition, the results of the present invention also show that some cancer-related functions, such as Kras signaling pathway, epithelial-mesenchymal transition process, MAPK signaling pathway, etc., are also significantly related to the above-mentioned bacterial abundance. These data reveal the impact of tumor microbes on host cell function at the level of gene function, and also demonstrate the important role of certain bacteria in tumorigenesis.
为何不同PDAC亚型中的微生物组成存在差异，什么因素导致了人群中多样性的肿瘤微生物结构呢。本研究试图去探究宿主的遗传因素是否在其中发挥一定作用。基于患者的基因型信息，本发明评估患者个体之间的遗传相似度。本发明的结果表明，个体之间的遗传变异情况越相近，他们的肿瘤微生物组成越相似，这支持了我们的猜想——宿主遗传因素对微生物群落的塑造有着关键作用。本发明深入探究宿主遗传与微生物间的关联，通过QTL分析鉴定出了多个与微生物丰度显著相关的遗传位点，这些位点的突变可能引起宿主免疫相关的功能缺失，包括IFN-γ信号激活、外源抗原的递呈等。这些发现表明了宿主基因突变与微生物间的关联，同时提出了一项可能的理论假设——宿主的某些免疫功能缺失可能导致微生物结构失衡，从而提高特定病原菌在胰腺中的定殖风险以及炎症诱导，最终导致严重的胰腺癌进展。Why does the microbial composition differ among different PDAC subtypes, and what factors contribute to the diversity of tumor microbial structures in the population. This study attempts to explore whether the genetic factors of the host play a role. Based on the patient's genotype information, the present invention assesses the genetic similarity between individual patients. The results of the present invention show that the closer the genetic variation among individuals, the more similar their tumor microbial composition, which supports our hypothesis that host genetic factors play a key role in shaping the microbial community. The present invention deeply explores the relationship between host genetics and microorganisms, and through QTL analysis, multiple genetic loci that are significantly related to microbial abundance are identified, and mutations at these loci may cause loss of functions related to host immunity, including IFN-γ signaling activation, presentation of foreign antigens, etc. These findings suggest a link between host genetic mutations and the microbe, and suggest a possible hypothesis that some loss of immune function in the host could lead to an imbalance in microbial architecture that increases the risk of colonization of the pancreas by specific pathogens and inflammation induced, eventually leading to severe pancreatic cancer progression.
试剂盒Reagent test kit
在本发明中，本发明的试剂盒包括本发明第二方面所述的标志物组合和/或本发明第三方面所述的试剂组合。In the present invention, the kit of the present invention includes the marker combination described in the second aspect of the present invention and/or the reagent combination described in the third aspect of the present invention.
在另一优选例中，本发明第二方面所述的组合中各个标志物用作标准品。In another preferred embodiment, each marker in the combination described in the second aspect of the present invention is used as a standard.
本发明的主要优点包括：The main advantages of the present invention include:
(1)本发明首次发现，不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、和/或鞘氨醇菌属(Sphingopyxis)可用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险。(1) The present invention finds for the first time that Acinetobacter, Pseudomonas, and/or Sphingopyxis can be used for (a) pancreatic ductal adenocarcinoma (PDAC) typing and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of risk for Basal-type pancreatic ductal adenocarcinoma (PDAC).
(2)本发明首次发现，不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、和/或鞘氨醇菌属(Sphingopyxis)还可用于区分Basal型胰腺导管腺癌(PDAC)和UnBasal型胰腺导管腺癌(PDAC)。(2) The present invention finds for the first time that Acinetobacter, Pseudomonas, and/or Sphingopyxis can also be used to distinguish Basal-type pancreatic ductal adenocarcinoma (PDAC) from UnBasal type pancreatic ductal adenocarcinoma (PDAC).
(3)本发明首次着眼于PDAC亚型异质性中的微生物因素，揭示了不同PDAC亚型中独特的肿瘤微生物组成结构特征，发现了与癌症进展密切相关的几类微生物，并且从功能上表明了这些微生物的炎症诱导潜能。(3) The present invention focuses on the microbial factors in the heterogeneity of PDAC subtypes for the first time, reveals the unique structural characteristics of tumor microbes in different PDAC subtypes, discovers several types of microorganisms closely related to cancer progression, and functionally The inflammation-inducing potential of these microorganisms is shown.
(4)本发明的研究结果首次表明了肿瘤微生物在预测PDAC患者临床表型中的应用价值，同时也提示微生物可以作为胰腺癌干预和治疗的有效靶点。(4) The research results of the present invention show for the first time the application value of tumor microorganisms in predicting the clinical phenotype of PDAC patients, and also suggest that microorganisms can be used as effective targets for pancreatic cancer intervention and treatment.
(5)本发明提供了一种新的基于微生物组成结构的PDAC分型方法，对患者的预后诊断提供新的手段。(5) The present invention provides a new PDAC typing method based on microbial composition, and provides a new means for the prognosis and diagnosis of patients.
(6)本发明提供了一种新的靶向微生物或阻断微生物促炎过程的干预或治疗PDAC的治疗方案。(6) The present invention provides a novel therapeutic scheme for intervening or treating PDAC by targeting microorganisms or blocking the pro-inflammatory process of microorganisms.
(7)本发明揭示了微生物在PDAC肿瘤发展中的作用，提供了对肿瘤发生机制的新的理解。(7) The present invention reveals the role of microorganisms in the development of PDAC tumors, and provides a new understanding of the tumorigenesis mechanism.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
如无特别说明，本发明实施例中所用的试剂和材料均为市售产品。Unless otherwise specified, the reagents and materials used in the examples of the present invention are all commercially available products.
通用方法general approach
1.1临床样本收集和宏基因组测序：1.1 Clinical sample collection and metagenomic sequencing:
所有肿瘤样本均来源于上海长海医院，入组病例需满足以下要求：1.经过影像学或组织病例学诊断为胰腺导管腺癌PDAC；2.患者在最近一个月内未接受过抗生素治疗；3.患者为原发性的、可切除型肿瘤。总共62例患者被纳入研究队列中，相关临床信息被详细记录。All tumor samples were obtained from Shanghai Changhai Hospital, and the following requirements should be met: 1. The patient was diagnosed with pancreatic ductal adenocarcinoma (PDAC) by imaging or histology; 2. The patient had not received antibiotic treatment in the last month; 3. . The patient has a primary, resectable tumor. A total of 62 patients were included in the study cohort, and relevant clinical information was documented in detail.
通过外科手术切除的PDAC肿瘤组织样本立刻转移至液氮保存，冻存一个月内进行后续的核酸抽提处理。使用QIAGEN Pathogen Lysis Tube对细菌细胞壁进行裂解，使用试剂盒进行DNA抽提。对核酸浓度和质量进行检测，确保样本的质量合格随后进行建库和二代测序。下机数据进行质控处理，平均每个样本得到了大约80GB的高质量DNA测序数据。The surgically removed PDAC tumor tissue samples were immediately transferred to liquid nitrogen for storage, and cryopreserved for subsequent nucleic acid extraction within one month. Bacterial cell walls were lysed using QIAGEN Pathogen Lysis Tube, and DNA was extracted using the kit. The nucleic acid concentration and quality are tested to ensure that the quality of the samples is qualified, and then library construction and next-generation sequencing are performed. The off-machine data was processed for quality control, and an average of about 80GB of high-quality DNA sequencing data was obtained for each sample.
1.2微生物分类学鉴定及丰度计算：1.2 Microbial taxonomic identification and abundance calculation:
经过质控处理的高质量DNA序列首先比对至人类参考基因组中，以去除测序数据中的宿主序列干扰。随后基于NCBI数据库中的微生物参考基因组数据库进行序列比对，使用Kraken2软件将每一段测序读段分配至已知的物种分类上，结合Bracken软件提供的贝叶斯概率估计来评估比对到各个物种分类上的理论序列数，据此计算每种微生物的丰度结果。基于肿瘤微生物组的丰度图谱，开展进一步的统计优化分析，计算微生物群落多样性指标，评估不同肿瘤亚型间的微生物组成区别。Quality-controlled high-quality DNA sequences are first aligned to the human reference genome to remove host sequence interference from the sequencing data. Then, based on the microbial reference genome database in the NCBI database, the sequence alignment was performed, and each sequenced read was assigned to a known species classification using Kraken2 software, and the Bayesian probability estimation provided by Bracken software was used to evaluate the alignment to each species. The theoretical sequence number on the taxonomy, based on which the abundance results of each microorganism are calculated. Based on the abundance map of the tumor microbiome, further statistical optimization analysis was performed to calculate microbial community diversity indicators and to evaluate the differences in microbial composition among different tumor subtypes.
实施例1筛选与Basal型胰腺导管腺癌相关的菌群标志物Example 1 Screening of bacterial markers associated with Basal-type pancreatic ductal adenocarcinoma
本发明收集的62例胰腺导管腺癌PDAC肿瘤样本包含17例Basal型PDAC、23例Hybrid型PDAC以及22例Classical型PDAC。使用Kruskal-Wallis检验来筛选不同PDAC分子亚型间存在显著差异丰度的微生物属，我们发现三类细菌属，不动杆菌属Acinetobacter、假单胞菌属Pseudomonas和鞘氨醇菌属Sphingopyxis，它们的丰度在Basal型PDAC肿瘤中显著富集，如图1-图3所示。Boxplot展示了不动杆菌属Acinetobacter、假单胞菌属Pseudomonas和鞘氨醇菌属Sphingopyxis在PDAC不同肿瘤亚型中的分布情况，y轴代表经过标准化后的丰度值，Kurskal-Wallis检验结果显示p值及其显著，表示该菌丰度在组 间存在显著差异，该图显示在Basal亚型中该菌丰度显著升高。The 62 cases of pancreatic ductal adenocarcinoma PDAC tumor samples collected by the present invention include 17 cases of Basal type PDAC, 23 cases of Hybrid type PDAC and 22 cases of Classical type PDAC. Using the Kruskal-Wallis test to screen microbial genera with significantly different abundances between different PDAC molecular subtypes, we found that three bacterial genera, Acinetobacter, Pseudomonas and Sphingopyxis, were The abundance of PDAC was significantly enriched in Basal-type PDAC tumors, as shown in Figures 1-3. Boxplot shows the distribution of Acinetobacter, Pseudomonas, and Sphingopyxis in different tumor subtypes of PDAC. The y-axis represents the normalized abundance value, and the Kurskal-Wallis test results show that The p value and its significance indicate that there is a significant difference in the abundance of the bacteria among the groups, and the figure shows that the abundance of the bacteria is significantly increased in the Basal subtype.
随后我们使用以上三类菌属的丰度来对所有肿瘤样本进行层次聚类分析，结果显示这三类菌属能够很好的将Basal型PDAC样本区分开(图4)。聚类热图展示了这三类菌属在PDAC不同亚型间的分布情况，列代表每个样本(列注释信息为分组信息)，行表示每个菌属，丰度按行进行归一化，数值统一转换为-2～2的区间，颜色偏红表示丰度值越高。该图表示这三个菌属大多富集在Basal亚型中。We then used the abundance of the above three genera to perform hierarchical clustering analysis on all tumor samples, and the results showed that these three genera could well distinguish Basal-type PDAC samples (Figure 4). The clustering heat map shows the distribution of these three genera among different subtypes of PDAC, the column represents each sample (column annotation information is grouping information), the row represents each genus, and the abundance is normalized by row , the value is uniformly converted to the interval of -2 to 2, and the reddish color indicates the higher the abundance value. The figure shows that these three genera are mostly enriched in the Basal subtype.
进一步的，本发明使用LDA(linear discriminant analysis)线性判别分析筛选Basal型PDAC的菌群标志物，，默认大于2的logarithmic LDA score被认为存在显著差异，分析结果表示这三类菌属在Basal亚型中显著性富集(图5).Further, the present invention uses LDA (linear discriminant analysis) linear discriminant analysis to screen the flora markers of Basal type PDAC, and the default logarithmic LDA score greater than 2 is considered to be significantly different, and the analysis results indicate that these three types of bacteria are in the Basal subgroup. were significantly enriched in the type (Figure 5).
我们根据以上三类菌属的丰度水平将患者进行分组，随后开展生存分析来比较患者的生存情况。如图6所示，结果表明这三类菌属的高丰度预示着PDAC患者更短的生存期，提示它们能够作为PDAC预后的预测指标。We grouped the patients according to the abundance levels of the above three genera, and then performed a survival analysis to compare the survival of the patients. As shown in Figure 6, the results indicated that the high abundance of these three genera predicted shorter survival in PDAC patients, suggesting that they could be used as predictors of PDAC prognosis.
Kaplan-Meier生存曲线显示三类菌属的高丰度能够对患者的低生存期有显著的预测效果。我们使用Cox比例风险回归模型计算三个菌属的危险率HR值，结果表明三类菌属的HR值均大于1，提示它们都是胰腺癌发展中的危险因素。The Kaplan-Meier survival curve showed that the high abundance of the three genera could have a significant predictive effect on the poor survival of patients. We used the Cox proportional hazards regression model to calculate the hazard rate HR values of the three genera, and the results showed that the HR values of the three genera were all greater than 1, suggesting that they are all risk factors for the development of pancreatic cancer.
实施例2构建基于肿瘤微生物的PDAC预测模型Example 2 Construction of a PDAC prediction model based on tumor microorganisms
将62例PDAC样本随机抽样，取出70％的样本作为训练集(n＝44,其中Basal型12例,unBasal型30例)，剩余30％的样本作为验证集(n＝18,其中Basal型5例,unBasal型13例)。62 PDAC samples were randomly sampled, 70% of the samples were taken as the training set (n=44, including 12 cases of Basal type, 30 cases of unBasal type), and the remaining 30% of the samples were used as the validation set (n=18, of which Basal type 5 Example, 13 cases of unBasal type).
将上述三类微生物的丰度值(用SizeFactor标准化过的)作为输入特征，使用随机森林算法构建分类模型，利用该构建好的模型对验证集进行预测，预测结果绘制AUC曲线作为评判模型效能的依据。这3类微生物构建的分类器对18例验证集样本的判别效能为：AUC＝96.9％(这3类微生物的组合)，95％置信区间CI＝89.6-100％。(图7)The abundance values of the above three types of microorganisms (standardized by SizeFactor) are used as input features, and the random forest algorithm is used to construct a classification model, and the constructed model is used to predict the validation set. in accordance with. The discriminative efficiency of the classifier constructed by these three types of microorganisms for 18 samples in the validation set is: AUC=96.9% (combination of these three types of microorganisms), 95% confidence interval CI=89.6-100%. (Figure 7)
表1展示了预测模型中每个特征的重要性指数，以及每个标志物单独的判别效能，可以看出这三类标志物分别都具备良好的预测效能，并且三者的组合能够发挥最佳的预测效能。此外，从表1和图7中还可以推断，本发明的三类微生物的两两组合也具备良好的预测效能。Table 1 shows the importance index of each feature in the prediction model and the individual discriminant performance of each marker. It can be seen that these three types of markers have good prediction performance respectively, and the combination of the three can play the best role prediction performance. In addition, it can also be inferred from Table 1 and Figure 7 that the pairwise combinations of the three types of microorganisms of the present invention also have good predictive performance.
表1Table 1
| 微生物标志物Microbial markers | 重要性importance | 判别AUCDiscriminate AUC |
| AcinetobacterAcinetobacter | 7.3027.302 | 0.9230.923 |
| PseudomonasPseudomonas | 7.0387.038 | 0.9540.954 |
| SphingopyxisSphingopyxis | 2.6992.699 | 0.9380.938 |
3类菌属的丰度(以及整合模型)作为特征因素预测样本肿瘤分型所绘ROC曲线，右下角图例标注了每类特征AUC的值.该图显示在18例验证集中这3类菌属及其整合模型在预测肿瘤分型上有着优秀的效能。The abundance of the three types of bacteria (and the integrated model) as a feature factor predicts the ROC curve of the sample tumor type, and the legend in the lower right corner annotates the value of each type of characteristic AUC. The figure shows the three types of bacteria in the validation set of 18 cases. And its integrated model has excellent performance in predicting tumor type.
分类模型(这3类微生物的组合)对18例验证样本的具体预测结果如表2所示，概率≥0.5预测样本为Basal型PDAC肿瘤。The specific prediction results of the classification model (combination of these three types of microorganisms) for the 18 validation samples are shown in Table 2, and the probability ≥ 0.5 predicts that the samples are Basal type PDAC tumors.
表2Table 2
结果表明，我们筛选得到的微生物标志物以及构建完成的预测模型具有较高的准确度和特异性，能够准确预测Basal型的PDAC患者，具有良好的市场开发前景。The results show that the microbial markers we screened and the prediction model constructed have high accuracy and specificity, can accurately predict Basal-type PDAC patients, and have good market development prospects.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (10)
- 一种肿瘤微生物或其检测试剂的用途，其特征在于，用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险；或用于制备一试剂或试剂盒，所述试剂或试剂盒用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险，其中所述肿瘤微生物选自下组：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。Use of a tumor microorganism or a detection reagent thereof, characterized in that it is used for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of Basal-type risk of pancreatic ductal adenocarcinoma (PDAC); or for preparing a reagent or kit for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of risk for Basal-type pancreatic ductal adenocarcinoma (PDAC), wherein the tumor microorganism is selected from the group consisting of Acinetobacter, Pseudomonas ), Sphingopyxis, or a combination thereof.
- 如权利要求1所述的用途，其特征在于，所述胰腺导管腺癌(PDAC)分型包括Basal型胰腺导管腺癌(PDAC)和UnBasal型胰腺导管腺癌(PDAC)。The use of claim 1, wherein the pancreatic ductal adenocarcinoma (PDAC) classification includes Basal-type pancreatic ductal adenocarcinoma (PDAC) and UnBasal-type pancreatic ductal adenocarcinoma (PDAC).
- 一种标志物组合，其特征在于，所述标志物组合包括选自下组的两种或多种：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。A marker combination, characterized in that the marker combination comprises two or more selected from the group consisting of Acinetobacter, Pseudomonas, Sphingosine ( Sphingopyxis), or a combination thereof.
- 一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的试剂组合，其特征在于，所述试剂组合包括用于检测权利要求3所述的标志物组合中各个标志物的试剂。A reagent for (a) pancreatic ductal adenocarcinoma (PDAC) typing; and/or (b) diagnosis of Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessment of risk for Basal-type pancreatic ductal adenocarcinoma (PDAC) The combination, characterized in that the reagent combination includes a reagent for detecting each marker in the marker combination of claim 3 .
- 一种试剂盒，其特征在于，所述的试剂盒包括权利要求3所述的标志物组合和/或权利要求4所述的试剂组合。A kit, characterized in that, the kit comprises the marker combination of claim 3 and/or the reagent combination of claim 4.
- 一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的方法，其特征在于，包括步骤：A method for (a) classification of pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnosing or assessing risk of developing Basal ductal adenocarcinoma (PDAC) , is characterized in that, comprises the steps:(1)提供一来源于待测对象的样品，对样品中权利要求3所述组合中各个标志物的水平进行检测；(1) providing a sample derived from the object to be tested, and detecting the level of each marker in the combination described in claim 3 in the sample;(2)将步骤(1)测得的水平与一参考数据集或一参考值(如健康对照者的参考值)进行比较；(2) comparing the level measured in step (1) with a reference data set or a reference value (such as a reference value for healthy controls);较佳地，所述的参考数据集包括来源于Basal型胰腺导管腺癌(PDAC)患者和UnBasal型胰腺导管腺癌(PDAC)对照者的如权利要求3所述组合中各个标志物的水平。Preferably, the reference data set includes the levels of each marker in the combination according to claim 3 derived from Basal-type pancreatic ductal adenocarcinoma (PDAC) patients and UnBasal-type pancreatic ductal adenocarcinoma (PDAC) controls.
- 一种筛选治疗Basal型胰腺导管腺癌(PDAC)的候选化合物的方法，其特征在于，包括步骤：A method for screening candidate compounds for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC), comprising the steps of:(1)在测试组中，向待测对象施用测试化合物，检测测试组中来源于所述对象的样品中权利要求3所述组合中各个标志物的水平V1；在对照组中，向待测对象施用空白对照(包括溶媒)，检测对照组中来源于所述对象的样品中权利要求3所述组合中各个标志物的水平V2；(1) in the test group, administer the test compound to the subject to be tested, and detect the level V1 of each marker in the combination according to claim 3 in the sample derived from the subject in the test group; in the control group, to the subject to be tested The subject is administered a blank control (including a vehicle), and the level V2 of each marker in the combination according to claim 3 in the sample derived from the subject in the control group is detected;(2)比较上一步骤检测得到的水平V1和水平V2进行比较，从而确定所述测试化合物是否是治疗Basal型胰腺导管腺癌(PDAC)的候选化合物。(2) Comparing the level V1 and the level V2 detected in the previous step to determine whether the test compound is a candidate compound for the treatment of Basal-type pancreatic ductal adenocarcinoma (PDAC).
- 一种权利要求3所述的标志物组合和/或权利要求4所述的试剂组合的用途，其特征在于，用于筛选治疗Basal型胰腺导管腺癌(PDAC)的候选化合物和/或用于评估候选化合物对Basal型胰腺导管腺癌(PDAC)的治疗效果。A use of the marker combination according to claim 3 and/or the reagent combination according to claim 4, characterized in that, for screening candidate compounds for the treatment of Basal type pancreatic ductal adenocarcinoma (PDAC) and/or for use in screening To evaluate the therapeutic effect of candidate compounds on Basal-type pancreatic ductal adenocarcinoma (PDAC).
- 一种建立评估Basal型胰腺导管腺癌(PDAC)患病风险或用于Basal型胰腺导管腺癌(PDAC)诊断的模型的方法，其特征在于，所述的方法包括识别Basal型胰腺导管腺癌患者和UnBasal型胰腺导管腺癌对照者之间，组织样品中差异表达物质的步骤,其中，所述的差异表达物质包括一种或多种权利要求3所述组合中的标志物。A method for establishing a model for assessing the risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) or for diagnosing Basal-type pancreatic ductal adenocarcinoma (PDAC), characterized in that the method comprises identifying Basal-type pancreatic ductal adenocarcinoma The step of differentially expressing substances in a tissue sample between patients and UnBasal-type pancreatic ductal adenocarcinoma controls, wherein the differentially expressed substances include one or more markers in the combination of claim 3.
- 一种用于(a)胰腺导管腺癌(PDAC)分型；和/或(b)诊断Basal型胰腺导管腺癌(PDAC)或评估Basal型胰腺导管腺癌(PDAC)的患病风险的系统，其特征在于，所述系统包括：A system for (a) typing pancreatic ductal adenocarcinoma (PDAC); and/or (b) diagnosing Basal-type pancreatic ductal adenocarcinoma (PDAC) or assessing risk of having Basal-type pancreatic ductal adenocarcinoma (PDAC) , characterized in that the system includes:(a)特征输入模块，所述特征输入模块用于输入某一对象的肿瘤组织样本的特征；(a) a feature input module, the feature input module is used to input the features of a tumor tissue sample of a certain object;其中所述的肿瘤组织样本的特征包括选自下组的肿瘤微生物：不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)、鞘氨醇菌属(Sphingopyxis)、或其组合。The characteristics of the tumor tissue sample include tumor microorganisms selected from the group consisting of Acinetobacter, Pseudomonas, Sphingopyxis, or a combination thereof.(b)判别处理模块，所述处理模块对于输入的肿瘤组织样本的特征，按预定的判断标准进行评分处理，从而获得风险度评分；并且将所述风险度评分与Basal型胰腺导管腺癌(PDAC)的风险度阈值进行比较，从而得出辅助筛查结果，其中，当所述风险度评分高于所述风险度阈值时，则提示该对象患Basal型胰腺导管腺癌(PDAC)的风险高于正常人群；当所述风险度评分低于所述风险度阈值时，则提示该对象患Basal型胰腺导管腺癌(PDAC)的风险高于正常人群；和(b) a discrimination processing module, the processing module performs scoring processing according to a predetermined judgment standard for the characteristics of the input tumor tissue samples, thereby obtaining a risk score; and compares the risk score with the Basal type pancreatic ductal adenocarcinoma ( PDAC) risk thresholds are compared to obtain an auxiliary screening result, wherein, when the risk score is higher than the risk threshold, the subject is at risk of suffering from Basal-type pancreatic ductal adenocarcinoma (PDAC). higher than the normal population; when the risk score is lower than the risk threshold, it is suggested that the subject has a higher risk of Basal-type pancreatic ductal adenocarcinoma (PDAC) than the normal population; and(c)辅助筛查结果输出模块，所述输出模块用于输出所述的辅助筛查结果。(c) an auxiliary screening result output module, the output module is used for outputting the auxiliary screening result.
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