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

Abstract

The application is related to purposes of the CDC GAP-associated protein GAP 2 in diagnosis of pancreatic cancer or prognosis.Specifically, this application discloses purposes of the reagent of the expression of CDC GAP-associated protein GAP 2 in subject's sample in diagnosis of pancreatic cancer or prognosis kit is prepared is determined, preferably cancer of pancreas is ductal adenocarcinoma of pancreas.The application reports expression of the CDCA2 albumen in ductal adenocarcinoma of pancreas tissue and its cancer beside organism first, has directive significance to the development of follow-up clinical research.

Description

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

Technical field

The application is related to medical science, clinical diagnosis field.Make in particular to a kind of CDC GAP-associated protein GAP 2 For purposes of the label in terms of cancer diagnosis or prognosis.

Background technology

Cancer of pancreas is that a kind of grade malignancy is very high, and diagnoses and treat all highly difficult malignant tumor of digestive tract.About 90% cancer of pancreas originates from the duct adenocarcinoma of glandular tube epithelium.

According to the position of pancreas carcinogenesis, cancer of pancreas classification has：1) carcinoma of head of pancreas；2) body of pancreas tail cancer；3) full cancer of pancreas.

According to cancer of pancreas histological type, cancer of pancreas classification has：

1) duct adenocarcinoma, duct adenocarcinoma account for the 80% to 90% of cancer of pancreas, mainly by breaking up different degrees of conduit sample knot The body of gland of structure is formed；

2) cancer of the ductal origin of specific type, have following several：Pleomorphic carcinoma, adenosquamous carcinoma, mucous carcinoma, mucoepidermoid Cancer and signet ring cell cancer, ciliated cell's cancer；

3) acinar cell carcinoma, 1% is accounted for, tumour cell is in polygonal, circle or doll shape；

4) glandula cancer；

5) small cell carcinoma, it is similar to small cell carcinoma of lung in the small cell carcinoma form of pancreas, account for the 1% to 3% of cancer of pancreas.

Ductal adenocarcinoma of pancreas incidence of occult, progress are fast, poor prognosis.Update shows, the morbidity of ductal adenocarcinoma of pancreas Rate rises year by year, and the incidence of disease and case fatality rate occupy American population the 4th, 5 annual survival rates about 8% [1], the patient more than 50% Operative chance has been lost during first visit.Therefore, the target spot for finding ductal adenocarcinoma of pancreas molecular therapy is current study hotspot, and then There is very big social benefit [2-3] to the therapeutic effect for improving ductal adenocarcinoma of pancreas patient.

At present, the genomics related on ductal adenocarcinoma of pancreas report is less, this and the distinctive disease of ductal adenocarcinoma of pancreas Reason feature is relevant, and most of compositions in its tumor tissues are mesenchyma stroma of tumors, it is difficult to which the DNA for directly extracting tumour is sequenced.

CDC GAP-associated protein GAP 2 (Cell division cycle associated 2, CDCA2) is also known as Repo-Man (Recruits PP1onto mitotic chromatin at anaphase), it is one of protein phosphatase 1 Binding subunit, participate in the process [4] of histone H 3 dephosphorylation at the end of mitosis.

In the cell, CDCA2 albumen has two important function.First, participating in mitosis, repaiied second, participating in DNA damage It is multiple.During cell mitogen, CDCA2 albumen exist with nucleus.Due to by Cyclin B-CDK1 complex phosphorus Acidifying, prevents CDCA2 albumen from being combined with PP1 γ, and can not can not play its effect with chromosome to combination.There is silk point The later stage is split, as protein phosphatase 1 γ regulation subunit, PP1 γ are raised onto histone H 3, promoted to replicate by CDCA2 albumen Good chromosome separation.This process will also be adjusted by protein kinase A urora B, to ensure what is do not replicated also in DNA When, CDCA2 albumen not can be incorporated on histone H 3 and can not play a role [5].In addition, CDCA2 albumen also passes through coordination The reconstruction of nucleus when the remodeling of nuclear membrane is to participate in mitosis.CDCA2 albumen in cell is reduced by RNA perturbation techniques It during expression, chromatinic replicate exception is occurred, there is also abnormal lopsided nucleus [6-7].

It is existing it is experimentally confirmed that CDCA2 albumen breast cancer, colon cancer, oophoroma, oral squamous cell carcinomas cancerous tissue in it is high Expression, expression are higher than cancer beside organism.In oral squamous cell carcinomas, the expression of Showed by immune group result CDCA2 albumen is by stages and swollen with T Knurl is by stages related [8], and this is consistent with the biological function of CDCA2 albumen.Not yet find on CDCA2 in pancreatic duct gland at present The correlative study report acted in cancer.

In view of cancer of pancreas incidence of occult, progress is fast and poor prognosis, and this area still needs to a kind of accurate, sensitive, special Label is used for the diagnosis or prognosis for aiding in cancer of pancreas.

The content of the invention

In view of the demand in this area, some embodiments of the disclosure, which provide, to be determined in subject's sample Purposes of the reagent of CDCA2 expressions in diagnosis of pancreatic cancer or prognosis kit is prepared.

In some embodiments, cancer of pancreas is selected from：Carcinoma of head of pancreas, carcinoma of body of pancreas, carcinoma of tail of pancreas and full cancer of pancreas.In other realities Apply in mode, cancer of pancreas is selected from：Ductal adenocarcinoma of pancreas, polymorphy cancer of pancreas, adenosquamous carcinoma, pancreas mucous carcinoma, pancreas mucus epidermis Sample cancer and signet ring cell cancer, pancreas ciliated cell cancer, acinar cell carcinoma, pancreas glandula cancer and pancreas small cell carcinoma.

In a particular embodiment, there is provided determine that the reagent of CDCA2 expressions in subject's sample is preparing pancreas Purposes in the diagnosis of gland duct adenocarcinoma or prognosis kit.

In some embodiments, CDCA2 expression is determined in gene level.When determining CDCA2 in gene level Expression when, available reagent is probe or primer pair.Technical staff can voluntarily make according to CDCA2 nucleotide sequence Standby specific probe or primer pair.

In other embodiments, CDCA2 expression is determined in protein level.Determined when in protein level During CDCA2 expression, available reagent is anti-CDCA2 antibody.Polyclonal antibody or monoclonal antibody are used equally for implementing The technical scheme of the disclosure.In a particular embodiment, using the principle of SABC, using anti-CDCA2 monoclonal antibodies Determine CDCA2 expression.

In some embodiments, subject's sample is cancerous tissue sample.In some embodiments, subject's sample is Pancreatic Adenocarcinoma.In a particular embodiment, Pancreatic Adenocarcinoma is obtained by way of biopsy, or surgical excision Pancreatic Adenocarcinoma.In other specific embodiments, when the diagnosis for cancer of pancreas, sample also includes cancer beside organism. Cancer beside organism refers to the pancreatic tissue beyond the cancerous tissue edge 3cm.When the expression of CDCA2 in the cancerous tissue When being significantly higher than CDCA2 expression in the cancer beside organism, indicate that the subject suffers from the cancer of pancreas.

In the context of the present specification, prognosis refers to the possibility course of disease and final result for predicting cancer of pancreas.Prognosis both includes sentencing The specific consequence (such as rehabilitation, certain symptom, sign, complication) of disconnected disease, also include providing time cue (when such as predicting certain section The interior possibility that certain final result occurs).When from the point of view of disease evolution process, prognosis includes such as remission rate, answered Hair rate, disability rate.When from the point of view of disease terminal state, prognosis includes such as cure rate, survival rate, case fatality rate.When When considering from the prognosis time, prognosis includes such as Near-term mortality rate, case fatality rate at a specified future date (referring to Liu Zhenhua chief editor's《Tumor prognosis Learn》).

In some embodiments, prognosis refers in particular to the prognosis to overall survival.

In some embodiments, CDCA2 high expression indicates this described subject's prognosis mala.In this application, in advance Afterwards it is bad refer to patient the post-operative survival rates time it is short.

In some embodiments, CDCA2 high expression refers to the expression that immunohistochemistry scoring is 4 to 12, described Immunohistochemistry scoring employ points-scoring system well known in the art, such as, but not limited to Fang L et al. Points-scoring system described in Oncotarget.2014.

In a particular embodiment, 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 point according to non-coloring, faint yellow, brown color, sepia respectively Value；

- positive cell the ratio is by 0% to 24%, 25% to 49%, 50% to 74%, >=75% be determined as 1 respectively, 2nd, 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 expression, 4 to 12 points are high expression.

It will be appreciated by those skilled in the art that although the statement of specific faint yellow, brown color, sepia is employed herein.But It is, however it is not limited to the lightness change of yellow form and aspect, because this is relevant with used developer form and aspect during dyeing.

In some embodiments, in the situation of diagnosis, subject is any mankind, can not suffer from or with pancreas The subject of gland cancer.In the situation of prognosis, subject is just to suffer from the subject of cancer of pancreas or once suffering from cancer of pancreas Subject's (regardless of whether healing).

In a particular embodiment, subject is that male subject either impassivity infringement (is also invaded as peripheral nerve Violate, PNI) subject.It is further preferred that subject is the subject for just suffering from or once suffering from cancer of pancreas of male；It is or tested Person is the subject for just suffering from or once suffering from cancer of pancreas that impassivity invades (also making peripheral nerve infringement, PNI).

In a particular embodiment, when carrying out prognostic evaluation to subject, further relate to carry out the N of subject by stages really It is fixed.In the context of the disclosure, described N is based on International Union Against Cancer (UICC) and tumour joint committee of the U.S. by stages (AJCC) TNM stage system.Alphabetical N suffix numeral 0 to 1, represents no lymph node carcinoma, has lymphatic metastasis respectively Cancer.In a preferred embodiment, when the expression of the CDCA2 is high expression and N is by stages N1, instruction is described tested Person's prognosis mala.

According to another aspect of the present disclosure, there is provided both are combined for cancer of pancreas prognosis by stages by CDCA2 expressions and N Method.

According to another aspect of the present disclosure, there is provided one kind is used for diagnosis of pancreatic cancer or prognosis kit, and it, which is included, is used for really Determine the reagent of CDCA2 expressions.

In a particular embodiment, anti-CDCA2 antibody is included for diagnosis of pancreatic cancer or prognosis kit.Another In a little specific embodiments, the probe or primer pair for being specific to CDCA2 are included for diagnosis of pancreatic cancer or prognosis kit. In some embodiments, kit can be prepared into the form of chip.

Brief description of the drawings

Fig. 1 is the comparison that CDCA2 is expressed in ductal adenocarcinoma of pancreas tissue and cancer beside organism.

Fig. 2 .CDCA2 protein expressions and follow-up patient's Kaplan-Meier survival analysises.

Fig. 3 A. are in male patient, CDCA2 protein expressions and follow-up patient's Kaplan-Meier survival analysises.

Fig. 3 B. are in the patient that impassivity is invaded, CDCA2 protein expressions and follow-up patient Kaplan-Meier existence point Analysis.

Embodiment

Tests below is audited by Ethics Committee of Chinese Academy of Medical Sciences Beijing Union Medical College Hospital, and is declared in accordance with Helsinki Speech, all patients are informed the purpose of sample collection and storage and sign written consent book in detail.

1. material

1.1 histopathology samples

Collect the 183 ductal adenocarcinoma of pancreas patient tissue samples for surgically cutting off and being made a definite diagnosis through pathological examination (pathological diagnosis and staging scale be American Cancer Society's Cancer Staging Handbook the 7th edition).Patient age 34-85 year, average year 60.6 ± 10.64 years old age, the median age 61 years old.There are 155 there are preferable Follow-up results in 183 patients.Life span defines Terminate to death or follow-up to end for operation.

1.2 inclusive criterias are as follows：

- receive the case of surgical resection；

- postoperative pathological is diagnosed as ductal adenocarcinoma of pancreas and has corresponding cancer beside organism, and cancer beside organism is defined as away from tumour side Pancreatic tissue beyond edge 3cm；

- patient did not received preoperative neoadjuvant treatment.

2. test method

2.1 experiment agents useful for same are shown in Table 1.

Reagent needed for the immunohistochemical staining of table 1.

Relevant device and instrument are shown in Table 2 needed for the preparation of 2.2 organization chips and SABC.

Relevant device and instrument needed for the preparation of the organization chip of table 2. and SABC

2.3 prepare the immunohistochemical staining section needed for experiment

The cancerous tissue for possessing typical cancer of pancreas feature and the pathological tissue of cancer beside organism are filtered out by pathologists diagosis Section, patient data is searched according to numbering, and recall the corresponding sample paraffin mass preserved in Pathologic specimen storehouse.By technology people Prepared by the organization chip that member assists to complete needed for immunohistochemical staining, per spot diameter 1.5mm, 5 μm of thickness.

2.4 immunohistochemical staining experimental procedures

(1) flake is prevented：Organization chip is toasted 1.5 hours at 68 DEG C；(2) section dewaxing aquation：By organization chip Soaked in dimethylbenzene 3 times, every time 10 minutes；Then it is soaked in successively in 100%, 95%, 80%, 70% alcohol and PBS, often Secondary 2 minutes, examine tissue samples and stay water effect, it is determined that dewaxing successfully；(3) antigen retrieval：By the immuning tissue after dewaxing Chemistry section handstand is put into the citrate buffer boiled, is adjusted to 800 watts, and 130 DEG C are repaired 4 minutes.After natural cooling, PBS It is middle to clean 3 times, 2 minutes every time, gentle manipulation, prevent flake；(4) endogenous peroxydase is removed：After section dries, it is placed in Add 3%H in culture dish₂O₂Submergence, insert PBS after wet box lucifuge is incubated 15 minutes and wash 3 times, 2 minutes/time；(5) it is incubated CDCA2 mono- It is anti-：By 1：400 ratios dilute CDCA2 monoclonal antibodies with antibody diluent, are prepared into CDCA2 primary antibody working solutions, dilute in proportion Nonimmune rabbit anteserum is released as negative control.The μ L of CDCA2 primary antibodies working solution about 100 are added dropwise in section after drying, ensure tissue specimen Covered by primary antibody, uniformly pave completely, prevent edge effect, be placed in 4 DEG C of refrigerator overnights.Take out to be put into PBS afterwards and wash 3 It is secondary, 2 minutes/time；(6) it is incubated secondary antibody：The μ L of secondary antibody working solution 100 are added dropwise to section, section is put into 37 DEG C of incubations of insulating box 30 minutes, washed 3 times with PBS after taking-up, 2 minutes/time；(7) DAB develops the color：The DABI examinations of DAB dilution II reagent 1ml enrichings contracting The drop of agent 1 is mixed with DAB nitrite ions, is kept in dark place, uses as early as possible, and DAB development steps are completed in half an hour.After chip drying, 50 μ L DAB colour developing mixed liquors are added dropwise, uniformly spread out, placement is observed under the microscope, colour developing degree according to the observation, works as background Section is put in into running water in time during coloring to rinse；(8) haematoxylin is redyed：Pathological section is placed in haematoxylin and dyed 2 minutes, Background is washed off repeatedly with water；(9) break up, return indigo plant：Pathological section is put into differentiation liquid to remove uncombined haematoxylin, steamed Distilled water is rinsed.Then section is placed in and returns blue liquid 2 minutes, distilled water flushing；(10) section dehydration, transparent and mounting：Will section It is positioned in 70%, 80%, 95% and 100% alcohol each 2 minutes；Xylene soak 3 times, 5 minutes/time, taking-up is dried.It is added dropwise Neutral Instant cement will cut into slices mounting, Microscopic observation.

3. immunohistochemical staining result is observed and interpretation

The result of immunohistochemical staining is distinguished sentence read result by two pathologists and scored, and is finally averaged Value.

Cancerous tissue and cancer beside organism to tissue specimen are evaluated respectively, each Positive Cell Counts random selection of cutting into slices 5 high power fields.

There is mark of the dyeing as positive expression using nucleus.Positive cell stains intensity by non-coloring, faint yellow, brown Yellow, sepia are determined as 0,1,2,3 point respectively, and positive cell ratio presses 0-24%, and 25-49%, 50-74%, ＞ 75% divides It is not determined as 1,2,3,4 point, according to two integrated product results as last comprehensive grading standard [9].

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

4. collecting the clinical and pathological data for including case, particular content is as follows：

(1) essential information of case：Patient name, medical record numbering, patient age, patient gender, contact method；

(2) clinical case information：Tumor locus and size, operation concrete mode, the type of surgery excision, excision extension；

(3) pathological information：Surgical resection margins situation, histological typing, tumor differentiation degree, neoplasm staging, lymph are carried down Shift one's love condition, peripheral nerve Infiltrating；

(4) staging scale：The cancer of pancreas TNM stage of case is included according to International Union Against Cancer (UICC) and U.S.'s tumour connection The conjunction committee TNM stage system (the 7th edition) announced in 2009；

(5) final result：Follow-up is carried out to including case, and inquires about the patient medical history of case.Collect the final result of case, total existence Temporal information.Finally collect effective patient's prognosis information 155.

5. statistical analysis

Using statistics software SPSS 17.0, statistical significant difference is defined as P ＜ 0.05.Examined using McNemar The difference for comparing CDCA2 Normal Pancreas vessel cell expression by pancreatic cancer cell and cancer is examined with Mann-Whitney U.Meter Number logging data application card side or the accurate test evaluation CDCA2 of Fisher ' s expression and each variable of clinical pathology of cancer of pancreas case With the presence or absence of correlation.Analysis of Survival Time is carried out with Kaplan-meier methods, total time-survivor curve is fitted, using Log- Rank examines the survivorship curve difference carried out between single factor analysis difference group；Using Cox proportional hazards regression modelses carry out it is more because Plain survival analysis.

6. result

6.1 SABCs detect expression of the CDCA2 in ductal adenocarcinoma of pancreas and cancer beside organism

CDCA2 is expressed in nucleus, is expressed as faint yellow to dark-brown, also has CDCA2 eggs in the cancer beside organism of part White expression (Fig. 1).According to standards of grading, overall expression levels of the CDCA2 in ductal adenocarcinoma of pancreas tissue is higher than cancer beside organism, P ＜ 0.001 (Fig. 1).Prompting CDCA2 albumen may have good additive diagnostic value.It is common in clinical position to arrive iconography The patient for finding the swollen thing of pancreas is checked, raises unobvious or the less patient of swollen thing for wherein tumor marker, diagnosis is relative Difficulty, successive treatment way choice are more intractable.The technologies such as existing EUS aspiration biopsy sometimes fail to obtain very well Tissue carry out Pathomorphology inspection, it may be considered that add and do SABC, by being expressed in the malignant tumours such as CDCA2 albumen Higher albumen to provide reference to good, pernicious diagnosis.

The relation of CDCA2 expressions and clinical pathologic characteristic in 6.2 ductal adenocarcinoma of pancreas tissues

The comprehensive grading carried out according to positive cell percentage and staining power, low expression group is allocated as by 0 to 3,4 to 12 points Make high expression group, immunohistochemical staining is scored and statistical analysis.

As a result find, in addition to the age (p=0.041), CDCA2 expressions and sex, tumor locus, tumor size, Tumor differentiation degree, T by stages, N there is no correlation (table 3) by stages.

Table 3.CDCA2 protein expression levels and clinical pathology information

In the above results, do not find that the height difference of CDCA2 protein expressions is by stages related to tumour T, this may be with disease The acquisition source for managing tissue is relevant.Ductal adenocarcinoma of pancreas patient's poor prognosis, has often lost operative chance in first visit.This public affairs The Pathologic specimen included in opening is surgery excision sample.Therefore, the patient included in the disclosure be the state of an illness relatively earlier Ductal adenocarcinoma of pancreas patient, so may have bias in patient chooses, this is inevitable in ductal adenocarcinoma of pancreas research , thus fail to find CDCA2 albumen and the correlation of neoplasm staging.

In the above results, it is found that CDCA2 protein expressions are age-related, inventors believe that this is also inclined with the selection of patient Move it is relevant because most of advanced age ductal adenocarcinoma of pancreas patient will not select radical surgery to cut off.

The single factor analysis that 6.3 each indexs influence on Overall survival

To there is the 155 of Follow-up Data ductal adenocarcinoma of pancreas patients to carry out overall survival correlation analysis.According to cancerous tissue CDCA2 ImmunohistochemistryResults Results, age, sex, tumor locus, tumor size, tumor differentiation degree, T by stages, N by stages, nerve leaching The indexs such as profit are grouped, and compare the correlation between each group index and overall survival phase using Kaplan-Meier.

As a result show：Corresponding ductal adenocarcinoma of pancreas patient is total by stages by the height of CDCA2 protein expression levels, different N There is significant difference (p ＜ 0.05) life cycle, is the hazards (table 4 and Fig. 2) for influenceing prognosis.

The survival analysis of table 4.

The multiplicity that 6.4 each indexs influence on Overall survival

For the single factor analysis index significant to Overall survival, including the height of CDCA2 protein expression levels, N points Phase includes Cox regression models and carries out multiplicity, counts influence of each factor to Overall survival, and determine relative risk. As a result show, N is the independent hazard factor for influenceing prognosis by stages, has statistical significance (p ＜ 0.05).The wherein danger of N1 phases Degree is 1.667 times of N0 phases.CDCA2 protein expression levels are not independent hazard factor (table 4).

The survival analysis of 6.5 different subgroups

Patient is divided into different subgroups and carries out multifactor survival analysis.

As a result it is shown in male patient (p=0.003) (Fig. 3 A), impassivity infringement patient (p=0.012) (Fig. 3 B), Prognosis and the height expression of CDCA2 albumen in cancer cell are significantly correlated.This shows that the expression of CDCA2 albumen is especially male Property and impassivity invade (PNI) colony in, can have statistical significance as the independent hazard factor of prognosis.

7. discuss

(referring to 6.4 sections) in single factor analysis, CDCA2 protein expressions, N are by stages related to patient's prognosis.CDCA2 albumen High patient is expressed, prognosis is poor.The patient's prognosis that lymphatic metastasis be present is poor.In subgroup analysis, male patient and without god Through invading in patient, the high person's prognosis of CDCA2 protein expressions is poor (referring to 6.5 sections).

Although CDCA2 protein expressions are not independent hazard factor in multiplicity, it is contemplated that pre- as judging Reference index afterwards, add when ductal adenocarcinoma of pancreas postoperative patient carries out pathological diagnosis and do SABC inspection, to help clinic Doctor preferably selects postoperative adjuvant therapy mode.

In summary, CDCA2 albumen high expression in ductal adenocarcinoma of pancreas, may be by promoting tumor cell proliferation, supporting The modes such as anti-apoptotic promote ductal adenocarcinoma of pancreas malignant progression.Help to face as the reference index of diagnosis and judging prognosis Bed instructs diagnosis and treatment.

Bibliography

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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.