CN105785004B - Purposes of the CDC GAP-associated protein GAP 2 in diagnosis of pancreatic cancer or prognosis - Google Patents

Abstract

This application relates to the use of cell division cycle-associated protein 2 in the diagnosis or prognosis of pancreatic cancer. Specifically, the present application discloses the use of a reagent for determining the expression level of cell division cycle-associated protein 2 in a sample of a subject in the preparation of a diagnostic or prognostic kit for pancreatic cancer, preferably pancreatic ductal adenocarcinoma. This application is the first to report the expression of CDCA2 protein in pancreatic ductal adenocarcinoma tissue and its adjacent tissues, which has guiding significance for the development of subsequent clinical research.

Description

Use of cell division cycle-associated protein 2 in the diagnosis or prognosis of pancreatic cancer

technical field

This application relates to the fields of medicine and clinical diagnosis. Specifically, it relates to the use of a cell division cycle-associated protein 2 as a marker in cancer diagnosis or prognosis.

Background technique

Pancreatic cancer is a highly malignant tumor of the digestive tract that is difficult to diagnose and treat. About 90% of pancreatic cancers originate from ductal adenocarcinoma of the ductal epithelium.

According to the location of pancreatic cancer, pancreatic cancer is classified into: 1) cancer of the head of the pancreas; 2) cancer of the body and tail of the pancreas; 3) cancer of the whole pancreas.

According to the pathological type of pancreatic cancer, pancreatic cancer is classified into:

1) Ductal adenocarcinoma, ductal adenocarcinoma accounts for 80% to 90% of pancreatic cancers, and is mainly composed of glands with different degrees of differentiation of duct-like structures;

2) Special types of carcinomas of ductal origin, including the following: pleomorphic carcinoma, adenosquamous carcinoma, mucinous carcinoma, mucoepidermoid carcinoma, signet ring cell carcinoma, and ciliated cell carcinoma;

3) Acinar cell carcinoma, accounting for 1%, the tumor cells are polygonal, round or short columnar;

4) small glandular carcinoma;

5) Small cell carcinoma, the small cell carcinoma of the pancreas is morphologically similar to the small cell carcinoma of the lung, accounting for about 1% to 3% of pancreatic cancer.

Pancreatic ductal adenocarcinoma has an insidious onset, rapid progression, and extremely poor prognosis. The latest data show that the incidence of pancreatic ductal adenocarcinoma is increasing year by year, and its morbidity and mortality both rank fourth in the American population. The 5-year survival rate is about 8% [1], and more than 50% of patients have lost the opportunity for surgery when they are first diagnosed. Therefore, finding the target for molecular therapy of pancreatic ductal adenocarcinoma is a current research hotspot, which in turn has great social benefits for improving the treatment effect of patients with pancreatic ductal adenocarcinoma [2-3].

At present, there are few genomics reports related to pancreatic ductal adenocarcinoma, which is related to the unique pathological characteristics of pancreatic ductal adenocarcinoma. Most of the components in the tumor tissue are tumor stroma, and it is difficult to directly extract tumor DNA for sequencing.

Cell division cycle associated 2 (CDCA2), also known as Repo-Man (Recruits PP1onto mitotic chromatin at anaphase), is a binding subunit of protein phosphatase 1, involved in the dephosphorylation of histone H3 at the end of mitosis The process of action [4].

Inside cells, the CDCA2 protein has two important roles. One is involved in mitosis, and the other is involved in DNA damage repair. During cell mitosis, CDCA2 protein exists in the nucleus. Due to being phosphorylated by the Cyclin B-CDK1 complex, the CDCA2 protein cannot combine with PP1γ, and cannot combine with chromosomes, so it cannot play its role. During anaphase, as a regulatory subunit of protein phosphatase 1γ, the CDCA2 protein recruits PP1γ to histone H3, prompting segregation of replicated chromosomes. This process is also regulated by the protein kinase Aurora B to ensure that when the DNA has not been replicated, the CDCA2 protein cannot bind to histone H3 and cannot function [5]. In addition, the CDCA2 protein is also involved in the remodeling of the nucleus during mitosis by coordinating the remodeling of the nuclear membrane. When RNA interference technology is used to reduce the expression of CDCA2 protein in cells, not only will the replication of chromatin be abnormal, but also abnormally deformed nuclei will appear [6-7].

Existing experiments have confirmed that CDCA2 protein is highly expressed in cancer tissues of breast cancer, colon cancer, ovarian cancer, and oral squamous cell carcinoma, and the expression is higher than that in paracancerous tissues. In oral squamous cell carcinoma, immunohistochemical results showed that the expression of CDCA2 protein was correlated with T stage and tumor stage [8], which was consistent with the biological function of CDCA2 protein. So far, there is no relevant research report on the role of CDCA2 in pancreatic ductal adenocarcinoma.

Considering the insidious onset, rapid progression and extremely poor prognosis of pancreatic cancer, an accurate, sensitive and specific marker is still needed in this field to assist in the diagnosis or prognosis of pancreatic cancer.

Contents of the invention

In view of the above needs in the art, some embodiments of the present disclosure provide the use of a reagent for determining the expression level of CDCA2 in a subject sample in the preparation of a pancreatic cancer diagnosis or prognosis kit.

In some embodiments, the pancreatic cancer is selected from: cancer of the head of the pancreas, cancer of the body of the pancreas, cancer of the tail of the pancreas, and cancer of the whole pancreas. In other embodiments, the pancreatic cancer is selected from the group consisting of: pancreatic ductal adenocarcinoma, pleomorphic pancreatic carcinoma, adenosquamous carcinoma, pancreatic mucinous carcinoma, pancreatic mucoepidermoid carcinoma and signet ring cell carcinoma, pancreatic ciliated cell carcinoma, acinar cell carcinoma carcinoma, small glandular carcinoma of the pancreas, and small cell carcinoma of the pancreas.

In a specific embodiment, use of a reagent for determining the expression level of CDCA2 in a subject sample in the preparation of a diagnostic or prognostic kit for pancreatic ductal adenocarcinoma is provided.

In some embodiments, the expression level of CDCA2 is determined at the gene level. When determining the expression level of CDCA2 at the gene level, useful reagents are probes or primer pairs. According to the nucleotide sequence of CDCA2, skilled artisans can prepare specific probes or primer pairs by themselves.

In other embodiments, the expression level of CDCA2 is determined at the protein level. When determining the expression level of CDCA2 at the protein level, a useful reagent is an anti-CDCA2 antibody. Both polyclonal antibodies and monoclonal antibodies can be used to implement the technical solutions of the present disclosure. In a specific embodiment, the principle of immunohistochemistry is used to determine the expression level of CDCA2 with an anti-CDCA2 monoclonal antibody.

In some embodiments, the subject sample is a cancer tissue sample. In some embodiments, the subject sample is pancreatic cancer tissue. In a specific embodiment, the pancreatic cancer tissue is obtained by biopsy, or surgically removed pancreatic cancer tissue. In other specific embodiments, when used for the diagnosis of pancreatic cancer, the sample also includes paracancerous tissue. Paracancerous tissue refers to pancreatic tissue beyond 3 cm from the edge of the cancer tissue. When the expression level of CDCA2 in the cancer tissue is significantly higher than the expression level of CDCA2 in the paracancerous tissue, it indicates that the subject has the pancreatic cancer.

In the context of this specification, prognosis refers to the prediction of the likely course and outcome of pancreatic cancer. Prognosis not only includes judging the specific consequences of the disease (such as recovery, certain symptoms, signs, complications), but also provides time clues (such as predicting the possibility of a certain outcome within a certain period of time). When considered in terms of disease progression, prognosis includes, for example, remission rate, relapse rate, and disability rate. Prognosis includes, for example, cure rate, survival rate, and case fatality rate when considered in terms of the terminal state of the disease. When considered in terms of prognostic time, prognosis includes, for example, short-term mortality and long-term mortality (see "Tumor Prognosis" edited by Liu Zhenhua).

In some embodiments, prognosis refers specifically to prognosis of overall survival.

In some embodiments, high expression of CDCA2 indicates that the subject has a poor prognosis. In the present application, poor prognosis means that the patient's postoperative survival time is short.

In some embodiments, the high expression of CDCA2 refers to the expression of an immunohistochemical score of 4 to 12, and the immunohistochemical score adopts a scoring system known in the art, such as but not limited to Fang L et al. Oncotarget.2014 The scoring system described in .

In a specific embodiment, the immunohistochemical scoring is carried out according to the following method:

- Cells with stained nuclei are regarded as positive cells;

- The staining intensity of the nucleus is judged as 0, 1, 2, and 3 according to no coloring, light yellow, brownish yellow, and tan;

- The ratio of positive cells is judged to be 1, 2, 3, or 4 according to 0% to 24%, 25% to 49%, 50% to 74%, and ≥75%, respectively;

- The score of staining intensity and the score of positive cell ratio are multiplied to obtain a score of 0 to 12, wherein 0 to 3 is low expression, and 4 to 12 is high expression.

Those skilled in the art understand that although the specific expressions of light yellow, brownish yellow and tan are used here. However, it is not limited to the lightness change of the yellow hue, because it is related to the hue of the color developer used for dyeing.

In some embodiments, in the case of diagnosis, the subject is any human being, which may be a subject without or with pancreatic cancer. In the case of prognosis, the subject is a subject who is suffering from pancreatic cancer, or a subject who has had pancreatic cancer (whether cured or not).

In a specific embodiment, the subject is a male subject or a subject without nerve invasion (also known as peripheral nerve invasion, PNI). More preferably, the subject is a male subject who has or has had pancreatic cancer; or, the subject is a subject who has or has had pancreatic cancer without nerve invasion (also known as peripheral nerve invasion, PNI). cancer subjects.

In a specific embodiment, when evaluating the prognosis of the subject, it also involves determining the N stage of the subject. In the context of this disclosure, the N staging described is based on the TNM staging system of the Union Internationale Cancer (UICC) and the American Joint Committee on Cancer (AJCC). The letters N suffixed with numbers 0 to 1 represent no lymph node metastases and lymph node metastases respectively. In a preferred embodiment, when the expression level of CDCA2 is high and the N stage is N1, it indicates that the prognosis of the subject is poor.

According to another aspect of the present disclosure, there is provided a method of combining both CDCA2 expression level and N stage for pancreatic cancer prognosis.

According to another aspect of the present disclosure, there is provided a diagnostic or prognostic kit for pancreatic cancer, which includes reagents for determining the expression level of CDCA2.

In a specific embodiment, the kit for the diagnosis or prognosis of pancreatic cancer comprises an anti-CDCA2 antibody. In other specific embodiments, the diagnostic or prognostic kit for pancreatic cancer comprises a probe or primer pair specific to CDCA2. In some embodiments, the kit can be prepared in the form of a chip.

Description of drawings

Figure 1 is a comparison of the expression of CDCA2 in pancreatic ductal adenocarcinoma tissue and adjacent tissue.

Figure 2. CDCA2 protein expression and Kaplan-Meier survival analysis of follow-up patients.

Figure 3A. In male patients, CDCA2 protein expression and follow-up patient Kaplan-Meier survival analysis.

Figure 3B. In patients without neural invasion, CDCA2 protein expression and follow-up patient Kaplan-Meier survival analysis.

detailed description

The following experiments were reviewed by the Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, and complied with the Declaration of Helsinki. All patients were informed of the purpose of sample collection and storage in detail and signed a written consent form.

1. Materials

1.1 Histopathological specimens

Tissue samples from 183 patients with pancreatic ductal adenocarcinoma who were surgically resected and confirmed by pathological examination were collected (the pathological diagnosis and staging standards are based on the seventh edition of the American Cancer Society Cancer Staging Manual). The patients were 34-85 years old, with an average age of 60.6±10.64 years and a median age of 61 years. 155 of 183 patients had good follow-up results. Survival time was defined as the end of surgery to death of the patient or the end of follow-up.

1.2 The inclusion criteria are as follows:

- Cases undergoing surgical resection;

-Pancreatic ductal adenocarcinoma diagnosed by pathology after surgery with corresponding paracancerous tissue, paracancerous tissue is defined as pancreatic tissue beyond 3cm from the tumor edge;

- None of the patients received preoperative neoadjuvant therapy.

2. Test method

2.1 See Table 1 for the reagents used in the test.

Table 1. Reagents required for immunohistochemical staining

2.2 The preparation of the tissue chip and the related equipment and instruments required for immunohistochemistry are shown in Table 2.

Table 2. Relevant equipment and instruments required for the preparation of tissue chips and immunohistochemistry

2.3 Preparation of immunohistochemically stained sections required for the experiment

The pathologists read the slides to screen out the pathological tissue sections of cancer tissue and paracancerous tissue with typical pancreatic cancer characteristics, search for the patient information according to the number, and call out the corresponding paraffin block of the specimen stored in the pathological specimen bank. Technicians assisted in the preparation of tissue chips required for immunohistochemical staining, with each spot 1.5 mm in diameter and 5 μm in thickness.

2.4 Immunohistochemical staining experimental steps

(1) Prevention of detachment: Bake the tissue chip at 68°C for 1.5 hours; (2) Dewaxing and hydration of the slice: Soak the tissue chip in xylene for 3 times, each time for 10 minutes; then soak in 100% , 95%, 80%, 70% alcohol and PBS, each time for 2 minutes, carefully observe the water retention effect of the tissue sample, and determine the success of dewaxing; (3) Antigen retrieval: put the immunohistochemical section upside down after dewaxing Boil citrate buffer, turn to 800 W, fix at 130 °C for 4 min. After natural cooling, wash in PBS for 3 times, 2 minutes each time, and operate gently to prevent detachment; (4) Eliminate endogenous peroxidase: After the slices are dried, place them in a petri dish and add 3% H ₂ O ₂ Submerged, placed in a wet box to incubate in the dark for 15 minutes, then washed 3 times with PBS, 2 minutes/time; (5) Incubate CDCA2 primary antibody: dilute CDCA2 monoclonal antibody with antibody diluent at a ratio of 1:400 to prepare CDCA2- Anti-working solution, dilute non-immune rabbit serum in the same proportion as negative control. After the slices were dried, about 100 μL of CDCA2 primary antibody working solution was added dropwise to ensure that the tissue specimens were completely covered by the primary antibody, spread evenly to prevent edge effects, and placed in a 4°C refrigerator overnight. Then take it out and wash it in PBS for 3 times, 2 minutes each time; (6) Secondary antibody incubation: add 100 μL of the secondary antibody working solution dropwise to the slice, put the slice in an incubator at 37°C and incubate for 30 minutes, take it out and wash it with PBS 3 times, 2 minutes/time; (7) DAB color development: 1ml of DAB diluent II reagent and 1 drop of concentrated DABI reagent were mixed to prepare DAB color development solution, kept away from light, used as soon as possible, and the DAB color development step was completed within half an hour . After the slices are dried, add 50 μL of DAB chromogenic mixture dropwise, spread them evenly, and place them under a microscope for observation. According to the degree of color development observed, when the background is colored, rinse the slices in tap water in time; (8) Hematoxylin counterstaining: put The pathological sections were stained in hematoxylin for 2 minutes, and washed repeatedly with water to remove the background; (9) Differentiation and bluing: put the pathological sections in the differentiation solution to remove unbound hematoxylin, and rinse with distilled water. Then place the slices in the blue solution for 2 minutes, and rinse with distilled water; (10) Dehydration, transparency and sealing of the slices: place the slices in 70%, 80%, 95% and 100% alcohol for 2 minutes each; soak in xylene for 3 minutes times, 5 minutes/time, take it out and let it dry. Add neutral quick-drying glue dropwise to seal the slices and observe under the microscope.

3. Observation and interpretation of immunohistochemical staining results

The results of immunohistochemical staining were interpreted and scored by two pathologists, and the average value was finally taken.

The cancer tissue and paracancerous tissue of the tissue samples were evaluated separately, and 5 high-power fields were randomly selected for counting positive cells in each section.

The staining of cell nuclei was used as a sign of positive expression. The coloring intensity of positive cells is judged as 0, 1, 2, and 3 points according to no coloring, light yellow, brown yellow, and brown, respectively, and the ratio of positive cells is 0-24%, 25-49%, 50-74%, and >75% They are judged as 1, 2, 3, and 4 points respectively, and the final comprehensive scoring standard is based on the product of the two points [9].

0-3 points are judged as low expression; 4-12 points are judged as high expression.

4. Collect the clinicopathological data of the included cases, the details are as follows:

(1) Basic information of the case: patient's name, medical record number, patient's age, patient's gender, and contact information;

(2) Clinical case information: tumor location and size, specific surgical method, type of surgical resection, and resection range;

(3) Pathological information: surgical margin, pathological type, degree of tumor differentiation, tumor stage, lymph node metastasis, and peripheral nerve invasion;

(4) Staging standard: The TNM staging system of pancreatic cancer included in the cases was based on the TNM staging system (version 7) published by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer in 2009;

(5) Outcome: The included cases were followed up, and the outpatient medical records of the cases were inquired. The outcome and overall survival time information of cases were collected. Finally, 155 cases of valid patient prognosis information were collected.

5. Statistical Analysis

Statistical software SPSS 17.0 was used, and a statistically significant difference was defined as P<0.05. McNemar test and Mann–Whitney U test were used to compare the expression of CDCA2 in pancreatic cancer cells and adjacent normal pancreatic ductal cells. Chi-square or Fisher's exact test was used to evaluate the correlation between the expression of CDCA2 and the clinicopathological variables of pancreatic cancer cases. Kaplan-meier method was used for survival time analysis, and the overall survival time curve was fitted. Log-rank test was used for univariate analysis of differences in survival curves between different groups; Cox proportional hazards regression model was used for multivariate survival analysis.

6. Results

6.1 Immunohistochemical detection of CDCA2 expression in pancreatic ductal adenocarcinoma and adjacent tissues

CDCA2 is expressed in the nucleus, and the expression is light yellow to dark brown, and CDCA2 protein is also expressed in some paracancerous tissues (Figure 1). According to the scoring standard, the overall expression level of CDCA2 in pancreatic ductal adenocarcinoma tissue was higher than that in adjacent tissue, p<0.001 (Figure 1). It suggested that CDCA2 protein may have good auxiliary diagnostic value. In clinical work, it is common to see patients with pancreatic tumors found in imaging examinations. For patients with insignificant tumor markers or small tumors, the diagnosis is relatively difficult, and the choice of subsequent treatment methods is even more difficult. Existing techniques such as endoscopic ultrasonographic biopsy sometimes fail to obtain good tissue for pathological and morphological examination. Immunohistochemistry can be considered to detect benign and malignant tumors through CDCA2 protein and other highly expressed proteins in malignant tumors. Diagnosis provides reference.

6.2 Relationship between CDCA2 expression level and clinicopathological features in pancreatic ductal adenocarcinoma

According to the comprehensive score of positive cell number ratio and staining intensity, 0 to 3 points were regarded as the low expression group, and 4 to 12 points were regarded as the high expression group, and immunohistochemical staining was scored and statistically analyzed.

It was found that, except for age (p=0.041), the expression level of CDCA2 was not correlated with gender, tumor location, tumor size, tumor differentiation, T stage, and N stage (Table 3).

Table 3. CDCA2 protein expression level and clinicopathological information

In the above results, no difference in CDCA2 protein expression was found to be related to tumor T stage, which may be related to the source of pathological tissue. The prognosis of patients with pancreatic ductal adenocarcinoma is poor, and they often lose the chance of surgery when they are first diagnosed. The pathological specimens included in this disclosure are surgical resection specimens. Therefore, the patients included in this disclosure are all patients with pancreatic ductal adenocarcinoma with relatively early disease, so there may be bias in patient selection, which is inevitable in the study of pancreatic ductal adenocarcinoma, so it is impossible to find the relationship between CDCA2 protein and tumor Staging relevance.

In the above results, it was found that the expression of CDCA2 protein was related to age, and the inventors believed that this was also related to the selection bias of patients, because most elderly patients with pancreatic ductal adenocarcinoma would not choose radical surgical resection.

6.3 Univariate analysis of the impact of each indicator on overall survival

The correlation analysis of overall survival was performed on 155 patients with pancreatic ductal adenocarcinoma with follow-up data. According to the CDCA2 immunohistochemical results of cancer tissue, age, gender, tumor location, tumor size, tumor differentiation degree, T stage, N stage, nerve invasion and other indicators, the groups were grouped, and Kaplan-Meier was used to compare the indicators of each group with the overall survival period. relevance.

The results showed that the level of CDCA2 protein expression and the overall survival of patients with pancreatic ductal adenocarcinoma corresponding to different N stages were significantly different (p<0.05), which were risk factors affecting prognosis (Table 4 and Figure 2).

Table 4. Survival Analysis

6.4 Multivariate analysis of the impact of each indicator on overall survival

For indicators that are meaningful to overall survival in univariate analysis, including the level of CDCA2 protein expression level and N stage, the Cox regression model is used for multivariate analysis, and the impact of each factor on overall survival is counted, and the relative risk is determined. The results showed that N stage was an independent risk factor affecting the prognosis, which was statistically significant (p<0.05). Among them, the risk of stage N1 is 1.667 times that of stage N0. CDCA2 protein expression level was not an independent risk factor (Table 4).

6.5 Survival analysis of different subgroups

Patients were divided into different subgroups for multivariate survival analysis.

The results showed that in male patients (p=0.003) ( FIG. 3A ), and in patients without nerve invasion (p=0.012) ( FIG. 3B ), the prognosis was significantly correlated with the high expression of CDCA2 protein in cancer cells. This shows that the expression level of CDCA2 protein can be used as an independent risk factor for prognosis especially in males and in the group without neurological invasion (PNI), which has statistical significance.

7. Discussion

In univariate analysis (see Section 6.4), CDCA2 protein expression and N stage were associated with patient prognosis. Patients with high CDCA2 protein expression had a poorer prognosis. Patients with lymph node metastases have a poorer prognosis. In subgroup analysis, among male patients and patients without nerve invasion, those with higher CDCA2 protein expression had a poorer prognosis (see section 6.5).

Although the expression of CDCA2 protein is not an independent risk factor in the multivariate analysis, it can be considered as a reference indicator for judging the prognosis. Immunohistochemical examination can be added to the pathological diagnosis of pancreatic ductal adenocarcinoma patients to help clinicians better Choose postoperative adjuvant therapy.

In summary, CDCA2 protein is highly expressed in pancreatic ductal adenocarcinoma, which may promote the malignant progression of pancreatic ductal adenocarcinoma by promoting tumor cell proliferation and resisting apoptosis. It can be used as a reference index for diagnosis and prognosis to help guide clinical diagnosis and treatment.

references

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Claims (15)

1. determine that the reagent of the expression of CDC GAP-associated protein GAP 2 in subject's sample is preparing diagnosis of pancreatic cancer or pre- Purposes in kit afterwards,

Wherein described cancer of pancreas is ductal adenocarcinoma of pancreas,

The expression of CDC GAP-associated protein GAP 2 is wherein determined in protein level.

2. purposes according to claim 1, wherein described reagent is antibody.

3. purposes according to claim 2, wherein the antibody is monoclonal antibody.

4. purposes according to claim 1, wherein described subject's sample is cancerous tissue sample.

5. purposes according to claim 4, wherein the cancerous tissue sample is biopsy sample.

6. purposes according to claim 4, wherein the cancerous tissue sample is the sample of surgical excision.

7. purposes according to claim 4, wherein described subject's sample also includes cancer beside organism, wherein by the cancer Tissue refers to the pancreatic tissue beyond the cancerous tissue edge 3cm.

8. purposes according to claim 1, described prognosis refers to the prognosis of overall survival.

9. purposes according to claim 7, wherein

When the expression of CDC GAP-associated protein GAP 2 in the cancerous tissue is significantly higher than cell point in the cancer beside organism When splitting the expression of cycle GAP-associated protein GAP 2, indicate that the subject suffers from the ductal adenocarcinoma of pancreas.

10. purposes according to claim 1, when the expression of the CDC GAP-associated protein GAP 2 is expressed to be high When, indicate subject's prognosis mala.

11. purposes according to claim 10, wherein described high expression refers to immunohistochemistry scoring for 4 to 12 Expression；

The immunohistochemistry scoring is carried out as follows：

There is the cell dyed as positive cell in-nucleus；

The staining power of-nucleus is determined as 0,1,2,3 score value according to non-coloring, faint yellow, brown color, sepia respectively；

- positive cell the ratio is by 0% to 24%, 25% to 49%, 50% to 74%, >=75% be determined as 1 respectively, 2,3, 4 score value；

- the score value product of the score value of staining power and positive cell ratio obtained into 0 to 12 score value, wherein 0 to 3 point is low table Reach, 4 to 12 points are high expression.

12. purposes according to claim 8, wherein described subject is the subject for suffering from ductal adenocarcinoma of pancreas Or once suffer from the subject of ductal adenocarcinoma of pancreas.

13. purposes according to claim 8, described subject is male subject or impassivity invade it is tested Person.

14. according to the purposes any one of claim 1-13, wherein it is also described tested including determining to evaluate the prognosis The N of person is by stages.

15. purposes according to claim 14, when the expression of the CDC GAP-associated protein GAP 2 is expressed to be high And N indicates subject's prognosis mala when being N1 by stages.