CN113777309A - Application of autoantibody in preparation of pancreatic ductal adenocarcinoma diagnostic kit - Google Patents
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Abstract
The invention relates to the technical field of medical biological detection, and provides 10 autoantibodies: the invention further provides a kit for diagnosing pancreatic ductal adenocarcinoma by detecting the 10 autoantibodies. The kit and the detection method are simple, convenient, reliable, short in period, high in specificity and easy to popularize clinically.
Description
Technical Field
The invention relates to the technical field of medical biological detection, and relates to application of 10 autoantibodies in diagnosis of pancreatic ductal adenocarcinoma.
Background
The Pancreatic Ductal Adenocarcinoma (PDAC) is one of the most common malignant tumors in China, about 90 percent of Pancreatic cancers are ductal adenocarcinoma originated from glandular epithelium, and the Pancreatic ductal adenocarcinoma has the characteristics of high morbidity, difficult early diagnosis, early recurrence and metastasis, poor prognosis and the like, and the 5-year survival rate is only 9 percent[1,2]. Surgical resection is the first choice and the only curative method for early-stage PDAC, but about 85 percent of PDAC diagnosis belongs to local advanced stage or is accompanied by distant metastasis, and surgical resection cannot be carried out, and chemotherapy is the main treatment measure for the part of patients, but is easy to resist drugs and poor in curative effect[3]. Therefore, the search for a diagnostic marker with high sensitivity and high specificity and the improvement of the early diagnosis rate still remain the problems to be solved urgently at present.
CA19-9 is a PDAC diagnostic marker which is widely used at present. The results of the series of studies showed that the median sensitivity and specificity of CA19-9 diagnostic PDAC were 79% and 82%, respectively[4,5]. The sensitivity of the PDAC with the tumor size less than or equal to 2cm diagnosed by CA19-9 is worse and is only 48.4 percent[6]. In addition, the increase of CA19-9 is also commonly seen in biliary tract malignant tumor, pancreatitis, jaundice, etc., and false positive appears, and over 15% of PDACs appear false negative due to Lewis antigen negative, etc[7-8]. There is therefore a need to combine or find new diagnostic markers.
The tumor immune microenvironment is one of ten characteristics of tumors, and the immune system of the body can generate autoantibodies against tumor antigens when recognizing the tumor as a 'foreign body'. When the tumor antigen level is very low and cannot be detected by using a conventional protein detection method, the immune system can monitor the existence of the protein, initiate immune reaction, generate a large amount of antibodies and play a certain amplification role in antigen signals. Therefore, the detection of the autoantibody is more sensitive than the tumor antigen, and a new way for early diagnosis of the tumor is possibly opened. It was found that several autoantibodies (e.g., anti-Ezrin, p53, p16, p62, survivin, Koc, IMP1, etc.) were involved in the development of PDAC[9-10]However, there is no systematic study on the sensitivity and specificity of these autoantibodies and their combinations for PDAC diagnosis, and there is no high throughput screening study on PDAC-related autoantibodies.
Disclosure of Invention
The present invention is made to solve the above technical problems, and an object of the present invention is to provide a diagnostic marker for pancreatic cancer, particularly ductal pancreatic adenocarcinoma, and an object of the present invention is to provide a novel use of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, and LDLRAD4, that is, an application in the preparation of a ductal pancreatic adenocarcinoma diagnostic kit.
The 10 autoantibodies of the invention adopt HuProtTMScreening of human proteome chip, HuprotTMThe human proteome chip was originally developed by john hopkins university in usa, and through continuous upgrade, currently contains more than 20000 personal recombinant proteins, and is the highest-throughput human recombinant proteome chip so far. We are the first timeThis high throughput protein chip was used for the diagnosis of PDAC.
A preliminary screening was performed, and serum samples were collected from 30 PDAC patients and 30 healthy persons, each sample being tested using 1 HuProtTM protein chip and analyzed using the same standard as follows: for each protein, Fold Change (FC) (PDAC group mean/healthy group mean) and P value (t-test) were calculated, and autoantibodies meeting the conditions <0.05, FC >1.2 were selected, and a total of 165 were selected, wherein the type of response antibody was 83 IgG and the type of response antibody was 82 IgM.
Then, screening is carried out continuously, a pancreatic cancer exclusive chip containing the 165 proteins is customized, and detection verification is carried out on 266 cases of PDACs, 248 cases of healthy people and 98 cases of patients with pancreatitis. Screening out the autoantibodies meeting the conditions that FC is greater than 1.2 and P value is less than 0.05; the positive rate is more than 10%; the ROC curve is AUC >0.7, specificity > 70% and sensitivity > 70%. The total number of antibodies satisfying the above conditions is 10, and all antibodies are IgG antibodies: the 10 autoantibodies can be used as serum markers for pancreatic cancer diagnosis, namely anti-TSPAN 18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39A14, SLC7A4 and LDLRAD4 antibodies.
In a first aspect of the present invention, there is provided an application of an autoantibody in preparing a pancreatic ductal adenocarcinoma diagnostic reagent or a diagnostic kit, wherein the autoantibody is any one or a combination of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, and LDLRAD 4.
Preferably, the diagnostic reagent is a reagent for detecting the content of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4 and LDLRAD4 in a biological serum sample.
Preferably, the diagnostic kit comprises reagents for detecting the content of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, LDLRAD4 in a biological serum sample.
Preferably, the biological sample is obtained from peripheral blood of the subject and the serum sample is obtained by centrifugation after blood sample collection.
In the invention, the content or concentration of the antibody is detected by adopting a conventional method, such as a traditional precipitation test, an agglutination test, a complement fixation test and the like, and the enzyme-linked immunosorbent assay, the fluorescence immunity, the radioimmunoassay or the immunoblotting method which are developed in recent years are all suitable.
In a second aspect of the present invention, a pancreatic ductal adenocarcinoma diagnostic kit is provided, which comprises reagents for detecting the content of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, and LDLRAD4 in a biological serum sample, that is, related reagents corresponding to the detection method are included.
In a third aspect of the present invention, there is provided a method for diagnosing pancreatic ductal adenocarcinoma using the above-mentioned kit, comprising:
A. centrifuging a blood sample to be detected at normal temperature to obtain serum;
B. the levels of the above 10 autoantibodies in serum were detected by a conventional method of antibody detection, and whether pancreatic ductal adenocarcinoma was detected or not was judged based on the detection results.
After pairwise comparison between groups by using an LSD method, the pancreatic ductal adenocarcinoma is found to have significant difference in expression level with the chronic pancreatitis and the normal human antibody respectively. The ROC analysis result indicates that when the kit is used for distinguishing pancreatic ductal adenocarcinoma, chronic pancreatitis and a mixture of normal people, the area under AUC is higher than 0.75, and the specificity and the sensitivity are higher than 70%; when the method is used for distinguishing pancreatic ductal adenocarcinoma from chronic pancreatitis, the area under AUC is higher than 0.82, the diagnosis accuracy is higher, and the false positive rate of cancer screening is reduced.
The invention has the following beneficial guarantee and effects:
in terms of technology, the detection of 10 kinds of autoantibodies is essentially the detection of antibody levels or concentrations in serum samples, and the clinical detection method is mature, has the characteristics of simple and convenient operation, sensitive detection, good specificity, high repeatability and the like, and is high in clinical popularization.
In addition, the area under AUC of the 10 autoantibody indexes related by the invention is higher than 0.75, the diagnosis accuracy is at least moderate, the sensitivity of most antibodies is maintained above 71%, the specificity is as high as above 80%, compared with CA19-9, the clinical reference value and the reliability are higher, and compared with pathological section diagnosis, the pain and the harm of a detector are small. Therefore, the detection kit can improve the detection rate and accuracy of the pancreatic ductal adenocarcinoma, and can obtain a detection result only by collecting blood of a detector, so that the compliance of a patient is high.
The invention provides a pancreatic ductal adenocarcinoma assay kit based on antibody level assay, which can diagnose pancreatic cancer by detecting the antibody level in a patient serum sample, has simple and convenient assay method, short period and high sensitivity, and is an effective supplement of the existing assay method.
Drawings
FIG. 1 is HuprotTMA detection schematic diagram of a human proteome chip;
FIG. 2 is a flow chart of the screening of 10 autoantibodies according to the present invention;
FIG. 3 is a ROC curve for diagnosing pancreatic cancer using 10 autoantibodies of the present invention: A. pancreatic cancer vs (healthy + pancreatitis), b. pancreatic cancer vs healthy person, c. pancreatic cancer vs pancreatitis;
FIG. 4 shows the expression level differences of 10 autoantibodies of the present invention in pancreatic cancer, pancreatitis and serum of healthy persons (. about.P <0.05,. about.P <0.01,. about.P <0.001,. about.P <0.0001, ns no statistical difference)
FIG. 5 is a ROC curve of 10 autoantibodies of the invention diagnosing CA199 negative pancreatic cancer: A.CA199-pancreatic cancer vs. (healthy + pancreatitis), B.CA199-pancreatic cancer vs healthy human, C.CA199-pancreatic cancer vs pancreatitis.
Detailed Description
The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.
EXAMPLE one Ten autoantibodies screening and validation
The screening and verification process in this embodiment is shown in fig. 2, and specifically includes the following steps:
1. preliminary screening stage
Serum samples were collected from 30 PDAC patients and 30 healthy persons, and each sample was examined using 1 HuProtTM protein chip according to the following detection principle, as shown in FIG. 1: the specific antibody (including IgG, IgM or other antibodies) of the chip is combined with the protein fixed on the chip, the unbound antibody and other proteins are removed by cleaning, then the anti-human IgM fluorescent labeled secondary antibody (cy5 labeled and shown in red) and the anti-human IgG fluorescent secondary antibody (cy3 labeled and shown in green) are used for detection, the signal is read by a fluorescence scanner, and the strength of the signal is positively correlated with the affinity and the quantity of the antibody. And extracting and normalizing the data, and analyzing the data.
IgG and IgM data were integrated and analyzed on the same standard as follows: for each protein, Fold Change (FC) (PDAC group mean/healthy group mean) and P value (t-test) were calculated, and autoantibodies meeting the conditions <0.05, FC >1.2 were selected, and a total of 165 were selected, wherein the type of response antibody was 83 IgG and the type of response antibody was 82 IgM.
2. Verification phase
A pancreatic cancer-specific chip containing 165 proteins was prepared, and the detection was performed in 266 cases of PDACs, 248 cases of healthy persons, and 98 cases of pancreatitis patients. And extracting and normalizing the data, and analyzing the data.
IgG and IgM data were integrated and analyzed on the same standard as follows: for each protein, Fold Change (FC) (PDAC group mean/healthy + pancreatitis group mean), P value (t test), and positive rate were calculated, i.e., the frequency of autoantibodies appearing in each group of samples was counted under a certain threshold. Firstly, setting a threshold value for the response of each protein, namely the average value of a control group sample of the protein +2 SD; secondly, samples above this threshold are defined as autoantibody positive), ROC curve (AUC, sensitivity, specificity).
Screening out the autoantibodies meeting the conditions that FC is greater than 1.2 and P value is less than 0.05; the positive rate is more than 10%; the ROC curve is AUC >0.7, specificity > 70% and sensitivity > 70%. The total number of the proteins meeting the above conditions is 10, and all the proteins are IgG antibodies: anti-TSPAN 18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, LDLRAD4 antibodies. The 10 autoantibodies can be used as serum markers for early diagnosis of pancreatic cancer.
10 autoantibodies screened according to the above procedure: anti-TSPAN 18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, LDLRAD4 antibodies, AUC, sensitivity, specificity for PDAC diagnosis as shown in table 1 below, ROC curves for 10 autoantibodies to PDAC diagnosis are shown in fig. 3.
Table 110 summary of cut-off, sensitivity, specificity, positive predictive value, negative predictive value for autoantibody diagnostic PDACs
The ROC analysis result indicates that the area under AUC of the PDAC vs. healthy human group is between 0.77 and 0.80, the diagnosis accuracy is moderate, the specificity result is between 66 and 82 percent, and the sensitivity is between 62 and 78 percent; in the PDAC vs. pancreatitis group, the area under AUC is between 0.82 and 0.89, the specificity result is between 72 and 89 percent, the sensitivity is between 64 and 80 percent, and PDAC patients can be effectively distinguished from healthy people and pancreatitis patients.
According to fig. 4, the LSD method was used to compare two-by-two groups, and 10 autoantibodies were found to be significantly higher in serum of patients with pancreatic ductal adenocarcinoma than those of chronic pancreatitis and healthy persons (P <0.001), and it was preliminarily determined that 10 autoantibodies could be used as a diagnosis of pancreatic cancer.
3. Diagnostic efficacy in CA 199-negative pancreatic cancer patients
CA199 is a widely used pancreatic cancer diagnostic marker at present, and the diagnostic efficacy of these 10 autoantibodies in CA 199-negative pancreatic cancer patients was further analyzed.
Table 210 Critical values, sensitivity, specificity, Positive predictive value, negative predictive value summary of autoantibody diagnosis CA199 negative PDACs
According to the ROC analysis result of FIG. 5, the area under AUC of the CA199 negative PDAC vs. healthy group is between 0.70 and 0.78, the diagnosis accuracy is moderate, the specificity result is between 65% and 83%, and the sensitivity is between 58% and 79%; in the CA199 negative PDAC vs. pancreatitis group, the area under AUC is between 0.77 and 0.86, the specificity result is between 72 and 90 percent, the sensitivity is between 67 and 79 percent, and CA199 negative PDAC patients can be effectively distinguished from healthy people and pancreatitis patients.
Reference documents:
[1]Siegel RL,Miller KD,Fuchs HE,et al.Cancer Statistics,2021.CA:a cancer journal for clinicians 2021；71.
[2]McGuigan A,Kelly P,Turkington RC,et al.Pancreatic cancer:A review of clinical diagnosis,epidemiology,treatment and outcomes.World journal of gastroenterology 2018；24:4846-4861.
[3]Chari ST,Kelly K,Hollingsworth MA,et al.Early detection of sporadic pancreatic cancer:summative review.Pancreas 2015；44:693-712.
[4]Goonetilleke KS,Siriwardena AK.Systematic review of carbohydrate antigen(CA 19-9)as a biochemical marker in the diagnosis of pancreatic cancer,European journal of surgical oncology 2007；33:266-270.
[5]Luo G,Jin K,Deng S,et al.Roles of CA19-9 in pancreatic cancer:Biomarker,predictor andpromoter.Biochim Biophys Acta Rev Cancer.2021；1875(2):188409.
[6]Egawa S,Takeda K,Fukuyama S,et al.Clinicopathological aspects ofsmall pancreatic cancer.Pancreas 2004；28(3):235–40.
[7]Mann DV,Edwards R,Ho S,et al.Elevated tumour marker CA19-9:clinical interpretation and influence of obstructive jaundice.European journal of surgical oncology 2000；26:474-479.
[8]Kim JE,Lee KT,Lee JK,et al.Clinical usefulness of carbohydrate antigen 19-9 as a screening test for pancreatic cancer in an asymptomatic population.Journal of gastroenterology and hepatology 2004；19:182-186.
[9]Capello M,Cappello P,Linty FC,et al.Autoantibodies to Ezrin are an early sign of pancreatic cancer in humans and in genetically engineered mouse models.Journal of hematology&oncology 2013；6:67.
[10]Li J,Wang LJ,Ying X,et al.Immunodiagnostic value of combined detection of autoantibodies to tumor-associated antigens as biomarkers in pancreatic cancer.Scandinavian journal ofimmunology 2012；75:342-349.
while the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Claims (6)
1. The application of the autoantibody in preparing a pancreatic ductal adenocarcinoma diagnostic reagent or a diagnostic kit is characterized in that any one or more of anti-TSPAN 18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39A14, SLC7A4 and LDLRAD4 antibodies are combined.
2. The use of claim 1, wherein the diagnostic reagent is a reagent for detecting the levels of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, LDLRAD4 in a biological serum sample.
3. The use of claim 1, wherein the diagnostic kit comprises reagents for detecting the level of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39a14, SLC7a4, LDLRAD4 in a biological serum sample.
4. The use of claim 2 or 3, wherein the biological sample is obtained from peripheral blood of the subject.
5. A pancreatic ductal adenocarcinoma diagnostic kit comprises reagents for detecting the content of antibodies against TSPAN18, ENTPD1, IZUMO4, DHRS7C, ATP5G1, SECTM1, CPT1A, SLC39A14, SLC7A4 and LDLRAD4 in a biological serum sample.
6. The pancreatic ductal adenocarcinoma diagnostic kit according to claim 5, characterized in that:
wherein, the reagent is detected by adopting a precipitation test, an agglutination test, a complement fixation test, enzyme-linked immunosorbent assay, fluorescence immunization, radioimmunoassay or an immunoblotting method.
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