CN112961921B

CN112961921B - Preparation for judging prognosis of early endometrial cancer and recurrence risk model

Info

Publication number: CN112961921B
Application number: CN202110329816.9A
Authority: CN
Inventors: 于双妮; 宗丽菊; 陈杰; 向阳; 卢朝辉; 曹冬焱; 杨隽钧; 常晓燕; 姜英; 李冬梅; 贾丛伟; 周娜
Original assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2022-07-01
Anticipated expiration: 2041-03-26
Also published as: CN112961921A

Abstract

The invention relates to a preparation for judging prognosis of early endometrial cancer and a recurrence risk model. The invention judges the POLE mutation state by sequencing, detects mismatch repair protein, p53 and VISTA in 4 by an immunohistochemical method, and judges the histology grade and immunohistochemical result of tumor by microscopic observation, thereby determining the molecular typing, the tumor grade and the expression of VISTA of the intimal cancer, then endowing a risk score, and carrying out 0 score, 1 score of dMMR and NSMP type and 2 score of p53 type on POLE mutation type in the molecular typing; score 0 for VISTA positive and score 1 for VISTA negative; high grade tumors were assigned a score of 1 and low grade tumors were assigned a score of 0. The total risk score is the sum of the 3 scores, and the total score range is 0-4: 0-1 is low risk, 2 is medium risk, and 3-4 is high risk. The model can effectively predict the recurrence and death risk of early-stage (FIGOI stage and II stage) endometrial cancer, thereby avoiding excessive treatment and insufficient treatment and achieving the aims of accurate treatment and individualized treatment.

Description

Preparation for judging prognosis of early endometrial cancer and recurrence risk model

Technical Field

The invention relates to the field of biomedicine, in particular to the field of judging prognosis of early endometrial cancer.

Background

The endometrial cancer is a common female genital tract malignant tumor in China, and the onset age of the endometrial cancer is in a trend of being younger. In 2015, about 6.3 thousands of new cases of endometrial cancer and about 2.2 thousands of death cases exist in China. Since abnormal vaginal bleeding is the most common symptom of endometrial cancer, 70-80% of patients are diagnosed with early stage endometrial cancer, i.e., FIGO stage I and II. The treatment means of patients with early-stage endometrial cancer is mainly surgery, and patients with high risk of relapse after surgery need combined treatment, chemotherapy and other auxiliary treatments. The selection of the postoperative adjuvant therapy mode is mainly made according to the pathological result of the operation and the operation stage. The most widely used in clinic at present is the ESMO risk stratification standard of European medical oncology institute, which mainly incorporates FIGO stages, histological types, tumor differentiation degree, muscle layer infiltration depth and Lymphatic Vascular Space Infiltration (LVSI), and intimal cancer is divided into low-risk, medium-risk, high-medium-risk and high-risk groups according to high-risk factors [1 ]. The specific risks are stratified as follows:

the standard was developed based on pathological diagnosis, however, in 2014 edition "WHO female reproductive organ tumor histology Classification" the intimal cancer was classified into nine major categories, i.e., endometrioid cancer, serous cancer, mucinous cancer, clear cell cancer, neuroendocrine cancer, mixed cancer, undifferentiated/dedifferentiated cancer, carcinosarcoma, and the like. This morphology-based diagnosis is poorly reproducible, especially in diagnosing high-grade endometrial cancer, where it is difficult to differentiate the type of pathology histologically. This risk stratification of ESMO based on morphological pathological diagnosis leads to over-or under-treatment of some intimal cancer patients. In one study, 3 gynecologic tumor pathologists performed histological identification of 56 cases of high-grade endometrioid cancer, respectively, and the inconsistency rate of the diagnosis results was as high as 62.5% [2 ]. Therefore, there is a need to establish an objective and highly repeatable classification method that is more accurate for prognosis determination and therapy guidance.

In 2013, the american cancer genome map program (TCGA) proposed molecular typing of endometrial cancer based on genome-wide analysis, which classified endometrial cancer into 4 types: DNA polymerase E (POLE) mutants, microsatellite instability (MSI), low/microsatellite stability and high copy number. The prognosis varies among patients of different molecular types, with the POLE hypermutant type having the best prognosis and the high copy number type having the worst prognosis. The molecular typing has important significance for diagnosis and treatment of endometrial cancer [3 ]. However, the molecular typing is proposed based on high-throughput deep sequencing, which is economically costly and time-consuming and may limit the application of molecular typing in clinics. Foreign researchers have proposed a simple, economical prospective molecular classification tool to replace high-throughput sequencing, which determines the variant of POLE by its Exonuclease Domain Mutation (EDM) sequencing method, then determines the type of mismatch repair deficiency (dMMR) by detecting the expression of mismatch repair protein by immunohistochemistry, and finally classifies other cases into p53 mutant and p53 wild-type according to the p53 immunohistochemistry result [4 ]. The method is finally divided into a POLE mutant type, a dMMR type, a p53 mutant type and a non-specific molecular profile (NSMP). This model has been validated by several groups and is of great importance for directing endometrial cancer molecular typing, risk stratification and treatment [5].

With the approval and application of tumor immunotherapy, in particular immune checkpoint inhibitors, in the clinic, the tumor immune microenvironment is receiving increasing attention. T cell subsets, macrophages, cellular immune factors and immune checkpoints in the tumor microenvironment are not only involved in the development of tumors, but also in the prognosis of tumor patients and the response rate to immunotherapy, and the immune scores of tumors are classified as prognostic factors to be independent of the tumor stage. In colorectal cancer, an immune score is performed based on the density of tumor infiltrating lymphocytes (CD3+ T cells and CD8+ T cells), which in combination with existing TNM staging, can better judge the prognosis of colorectal patients [6 ]. The international organization has suggested an immune score as a routine reporting item for the pathological diagnosis of colorectal cancer. Similarly, CD8 was also found in lung cancer as an immune score to determine the prognosis of lung cancer patients [7 ]. At present, the research on the immune microenvironment of the intimal cancer discovers that the POLE mutant type and the dMMR type tumors have more tumor infiltrating immune cells (TILs), higher tumor mutation load and stronger anti-tumor immune response. In a variety of tumors, TIL is associated with a good prognosis. The authors speculate that the better prognosis for patients with stage mutant and mmr-type intimal cancer may be associated with a stronger anti-tumor immune response.

The inventor finds that a novel immune checkpoint VISTA (V-domain immunoglobulin supply of T cell activation) is expressed in trophoblastic tumors, cervical cancer, ovarian cancer, colorectal cancer and breast cancer, the high expression of the VISTA is related to good prognosis of the ovarian cancer, the breast cancer and the colon cancer, and the expression of the VISTA in immune cells is an independent prognostic factor of the breast cancer and the colorectal cancer [8-12 ]. However, the relationship between the molecular typing and prognosis of the immune checkpoint VISTA endometrial cancer is not clear, and whether VISTA combined molecular typing can be used for judging the prognosis of early endometrial cancer remains to be developed.

[1].Colombo N,Creutzberg C,Amant F et al.ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer:diagnosis,treatment and follow-up.Ann Oncol,2016,27(1):16-41.

[2].Gilks CB,Oliva E,Soslow RA.Poor interobserver reproducibility in the diagnosis of high-grade endometrial carcinoma.Am J Surg Pathol,2013,37(6):874-881.

[3].Cancer Genome Atlas Research N,Kandoth C,Schultz N et al.Integrated genomic characterization of endometrial carcinoma.Nature,2013,497(7447):67-73.

[4].Vermij L,Smit V,Nout R et al.Incorporation of molecular characteristics into endometrial cancer management.Histopathology,2020,76(1):52-63.

[5] Zollia religiosa, sunward, progress in immunotherapy of refractory endometrial cancer. J.Utilis & obstetrics 2020,36(06): 415-.

[6].Pagès F,Mlecnik B,Marliot F et al.International validation of the consensus Immunoscore for the classification of colon cancer:a prognostic and accuracy study.Lancet,2018,391(10135):2128-2139.

[7].Donnem T,Hald SM,Paulsen EE et al.Stromal CD8+T-cell Density-A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.Clin Cancer Res,2015,21(11):2635-2643.

[8].Zong L,Mo S,Yu S et al.Expression of the immune checkpoint VISTA in breast cancer.Cancer Immunol Immunother,2020,69(8):1437-1446.

[9].Zong L,Yu S,Mo S et al.High VISTA Expression Correlates With a Favorable Prognosis in Patients With Colorectal Cancer.Journal of Immunotherapy,2021,44(1):22-28.

[10].Zong L,Zhang M,Wang W et al.PD-L1,B7-H3 and VISTA are highly expressed in gestational trophoblastic neoplasia.Histopathology,2019,75(3):421-430.

[11].Zong L,Zhang Q,Zhou Y et al.Expression and significance of immune checkpoints in clear cell carcinoma of the uterine cervix.J Immunol Res,2020,2020:1283632.

[12].Zong L,Zhou Y,Zhang M et al.VISTA expression is associated with a favorable prognosis in patients with high-grade serous ovarian cancer.Cancer Immunol Immunother,2020,69(1):33-42

Disclosure of Invention

The invention aims to establish an objective, clinically feasible and high-repeatability comprehensive risk model based on molecular typing, VISTA immune scoring and pathological parameter comprehensive scoring, which is used for predicting prognosis of early endometrial cancer (FIGO stages I and II) and guiding layered treatment of early endometrial cancer. Another objective of the study is to compare the predictive power of the new model with that of the currently applied ESMO model, expecting that the new model can predict the prognosis of the patient more objectively and accurately, thereby avoiding over-treatment and under-treatment and achieving the goal of accurate treatment and individualized treatment.

The invention provides a risk model for judging postoperative recurrence and survival time of early endometrial cancer, and the prognosis model is a comprehensive risk scoring system based on intimal cancer molecular typing, VISTA scoring and tumor grade and can predict postoperative recurrence and survival of early endometrial cancer.

The invention provides application of a reagent for detecting VISTA in preparation of a preparation for judging the risk of endometrial cancer prognosis. The formulation also includes reagents for detecting other markers. The endometrial cancer is early stage endometrial cancer. Preferably, the early stage endometrial cancer is stage I and stage II of the fix.

The invention also provides a kit for judging the risk of early endometrial cancer prognosis, which contains a reagent for detecting VISTA; the kit also comprises reagents for detecting other markers.

The invention also provides a kit for judging the risk of early endometrial cancer prognosis, which comprises a reagent for detecting VISTA and other markers.

Further, the reagent for detecting VISTA can be a reagent for detecting VISTA gene, a reagent for detecting VISTA protein and/or other biological markers.

Further, the reagent for detecting other markers may be a reagent for detecting POLE, mismatch repair protein and/or p 53.

Further, the reagent for detecting other markers may be a reagent for detecting genes, a reagent for detecting proteins, and/or a reagent for detecting other biological markers.

Further, the mismatch repair protein comprises MLH1, MSH2, MSH6 and/or PMS 2.

Further, the detection method is sequencing, DNA sequencing, RNA sequencing, immunohistochemistry and/or other biological detection methods.

The invention provides a VMG prognosis model (VMG model), which comprises three factors of VISTA, Molecular typing (Molecular type) and tumor Grade (Grade).

The invention also provides a device, system and/or model for determining the prognosis of early stage endometrial cancer comprising a VISTA assessment moiety.

Further, the VISTA typing moiety includes a reagent for detecting VISTA.

Further, the reagent for detecting VISTA can be a reagent for detecting VISTA gene, a VISTA protein and/or other biological markers.

Further, the detection may be sequencing, DNA sequencing, RNA sequencing, immunohistochemistry, and/or other biological detection methods.

Furthermore, the VISAT typing part defines that the VISTA is more than or equal to 1% positive in tumor interstitial immune cells as being positive, and less than 1% as being negative.

Further, the system also includes a molecular typing component and a tumor grade component.

Further, the molecular typing moiety includes a reagent for detecting POLE, mismatch repair protein and/or p 53.

Further, the molecular typing moiety detects the expression of POLE, mismatch repair protein (MLH1, MSH2, MSH6, PMS2) and/or p53 protein by DNA sequencing, immunohistochemistry and/or other biological methods.

Further, the molecular typing performs judgment as follows,

1) the mutation state of the POLE gene is judged firstly: judging the pathogenic mutation as a POLE mutation type, and judging the MMR protein if the POLE mutation is not existed;

2) if the expression of any protein in the 4 mismatch repair proteins is deleted, judging the protein to be a dMMR type, and if the protein is not the dMMR type, judging the p 53;

3) the staining of p53 was judged as p53 mutant or p53 wild type, and p53 wild type was judged as non-specific molecular profile (NSMP) type.

Further, the tumor grade moiety comprises an agent that grades a tumor.

Further, the tumor grade is classified into a high grade and a low grade.

Further, the high level is a FIGO3 level, and the low level is a FIGO1 level or a 2 level.

In the VISTA assessment portion, a score of 0 is determined as being positive for VISTA, and a score of 1 is determined as being negative for VISTA.

Further, in the molecular typing part, the POLE mutant type was judged as score 0, the dMMR/NSMP type was judged as score 1, and the P53 mutant type was judged as score 2.

Further, among the tumor grade portions, the low grade was judged as 0, and the high grade was judged as 1.

Further, the low-grade, POLE-type and VISTA-positive scores were judged as 0, the high-grade, dMMR/NSMP-type and VISTA-negative scores were judged as 1, and the p 53-mutant score was judged as 2.

Further, the scores of the three factors are added to obtain a total score, the total score range is 0-4, and the risk stratification is as follows: 0-1 is low risk, 2 is medium risk, and 3-4 is high risk.

The invention provides a method for constructing a risk model for judging early endometrial cancer prognosis, which comprises the following steps:

1. determining histological grade of tumor

Formalin-fixed paraffin-embedded intimal cancer tumor specimens were stained with Hematoxylin and Eosin (HE), and the histological grade of the tumor was judged under a microscope: grade 1 (G1), i.e. well differentiated one is called high differentiation, tumor cells are close to corresponding normal tissue of origin, and the degree of malignancy is low; grade 2 (G2), with a tissue abnormality between grade I and grade III, with intermediate malignancy; grade 3 (G3), the less differentiated cells are called poorly differentiated, and the tumor cells are highly malignant, with large differences from the corresponding normal tissue of origin. For endometrioid carcinoma, the FIGO1 grade tumor is composed of well differentiated glands, and part of the glands are fused; gland structures are partially visible in FIGO 2-grade tumors, and part of the gland structures are unclear, are in solid sheets and are moderately heterotypic cells; the FIGO3 grade tumor has unobvious glandular cavity structure and obvious cellular heterogeneity, and most of nuclear division and focal necrosis can be seen.

FIGO grades

1 and 2 are low grade tumors and FIGO grade 3 is high grade tumors.

2. Molecular typing

And (3) extracting DNA from paraffin tissue specimens to carry out Sanger sequencing to detect mutation of the POLE No. 9-14 exons, and carrying out reverse verification on the mutated specimens by using a PCR (polymerase chain reaction) method. The expression of mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) and p53 protein is detected by an immunohistochemical method. The expression of 4 mismatch repair proteins is localized in the nucleus, and non-tumor intima gland, intima stroma and lymph cell nucleus are used as positive internal control, and the nucleus is brownish yellow positive coloration. The complete loss of nuclear expression in the tumor region can be judged as MMR protein loss (dMMR) only under the positive condition of the internal control. Non-tumorous intimal glands, intimal stroma and lymphocytes were used as wild-type internal controls for p53 staining, with differential staining of the nuclei with varying intensity. More than 70% of tumor cell nuclei are diffuse strong positive and judged as missense mutant, all tumor cell nuclei are not colored and judged as nonsense mutant, and the cell nuclei are differentially colored in different strengths and judged as wild type. Judging process of molecular typing: 1) the mutation state of the POLE gene is judged firstly: judging the pathogenic mutation as a POLE mutation type, and judging the MMR protein if the POLE mutation is not existed; 2) if the expression of any protein in the 4 mismatch repair proteins is deleted, judging the protein to be a dMMR type, and if the protein is not the dMMR type, judging the p 53; 3) the protein is judged to be p53 mutant type or p53 wild type according to the staining condition of p53, and the p53 wild type is a non-specific molecular profile (NSMP) type.

3. Evaluation of VISTA

VISTA expression in endometrial cancer was examined using immunohistochemical methods. Evaluation of VISTA: the positive proportion of the VISTA in tumor interstitial immune cells is more than or equal to 1 percent and is defined as VISTA positive, and the positive proportion of the VISTA in tumor interstitial immune cells is less than 1 percent and is defined as VISTA negative.

4. Prognostic risk assigning systems are as follows:

prognostic factor	0 point (min)	1 minute (1)	2 is divided into
				Tumor grade	Low level	High grade
Molecular typing	POLE mutant	dMMR/NSMP type	Mutant p53
				VISTA score	VISTA positive	VISTA negative

And adding the scores of the three prognostic factors to obtain a total score, wherein the total score range is 0-4, and the risk is layered as follows: 0-1 is low risk, 2 is medium risk, and 3-4 is high risk. The prognosis model was incorporated into the VISTA, Molecular typing (Molecular type) and tumor Grade (Grade) definitions as the VMG prognosis model for early stage intimal cancer.

Has the advantages that:

the risk scoring system for judging the prognosis of early endometrial cancer is constructed by analyzing 351 cases of early endometrial cancer and based on molecular typing, VISTA immune scoring and the histological grade of tumors, and patients with early endometrial cancer are classified into low-risk, medium-risk and high-risk according to the risk scoring system.

(1) The VMG model of the present invention has a higher predictive power than existing ESMO models. In the VMG model, survival curves of low-risk and medium-risk and high-risk patients are completely separated, and survival curves of different risk patients in the existing ESMO model are partially crossed.

(2) Compared with the existing ESMO model, the VMG model in the invention is more objective and simpler. The ESMO model was interpreted based on histomorphology only, incorporating the histological type of the tumor, the depth of tumor infiltration, the histological grade of the tumor, and the lymphatic vascular space infiltration. The histological types of the tumors are 9 at present, the diagnosis consistency of pathologists is low, particularly in high-grade tumors, the histological types are difficult to identify, and the diagnosis consistency among pathologists is low. The VMG model is a scoring system established based on a molecular pathology, immunohistochemistry and tumor grade classification method, is not included in a histology type, and can judge the recurrence risk of the intimal cancer according to a prognosis score. Therefore, the VMG model in the invention is more objective and concise.

(3) The VMG model of the invention may be used to guide adjuvant therapy after early stage endometrial cancer. For low-risk patients, the recurrence risk is extremely low, the 5-year survival rate after operation is 99.2 percent, and the patients can be subjected to follow-up visit after operation and do not receive auxiliary treatment; for the middle-risk patients, the patients have certain recurrence risk, the 5-year survival rate after operation is 85.2 percent, and the patients are recommended to receive brachytherapy; for high-risk patients, the recurrence risk is high, the 5-year survival rate after operation is only 57.2%, and external radiotherapy or combined chemotherapy is recommended and closely followed.

Drawings

FIG. 1 detection procedure for endometrial cancer molecular typing

FIG. 2 molecular typing, VISTA expression and tumor grade correlate with survival time in patients with early stage endometrial cancer.

Vista expression is associated with relapse free survival in patients with early stage endometrial cancer;

B. the histological grade of the tumor is associated with relapse-free survival of patients with early stage endometrial cancer;

C. molecular typing is associated with relapse-free survival of patients with early stage endometrial cancer;

vista expression is associated with disease-specific survival in patients with early stage endometrial cancer;

E. the histological grade of the tumor is associated with disease-specific survival of early stage endometrial cancer patients;

F. molecular typing is associated with disease-specific survival in patients with early stage endometrial cancer.

FIG. 3 survival curves for patients with different risk stratification in the VMG model of the present invention and the existing ESMO model.

A. A relapse-free survival curve for patients risk stratification according to the VMG model of the invention;

B. stratifying the risk of the patient according to the existing ESMO model without recurrence survival curve;

C. stratifying the patient's disease-specific survival curve according to the VMG model risk in the present invention; D. disease-specific survival curves for patients were risk-stratified according to the existing ESMO model.

FIG. 4 comparison of the C indices of the VMG model of the present invention and the existing ESMO model shows that the C index of the MG model is significantly higher than that of the existing ESMO model in predicting recurrence and survival.

Detailed Description

Examples

First, research object

The study object of this embodiment is a tumor tissue specimen after operation of patients with stage I-II endometrial cancer of FIGO. Inclusion criteria were: a definitive pathological diagnosis of endometrial cancer; FIGO stage I-II; the pathological section and the paraffin specimen are complete; the follow-up time is at least 3 months; the clinical data is complete.

Exclusion criteria: FIGO stages III-IV; before operation, receiving new auxiliary chemotherapy; receiving radiation therapy before operation; combined with gynecological malignant tumors such as ovarian cancer and cervical cancer; no tumor tissue exists in the operation specimen after uterine curettage; pathological section or paraffin specimen loss; the follow-up time is less than 3 months.

Second, research method

1. Collection of clinical data

The patient's age, operation mode, ascites cytology result, whether to receive chemotherapy before operation, whether to receive adjuvant therapy after operation, and the mode of adjuvant therapy are recorded by referring to the patient's medical record of hospitalization and the medical record of outpatient service, and whether the patient has relapsed, the relapse time, the relapse part, the treatment mode after relapse, whether the patient dies, the death time, and the reason for death are recorded by telephone or follow-up of the medical record of outpatient service.

2. Interpretation of pathological parameters

Pathological HE sections are observed under a microscope, and the following pathological parameters including histological type, differentiation degree, tumor grading, tumor infiltration depth, LVSI and cervical interstitial infiltration condition are judged. Determining FIGO stages according to pathological parameters and imaging examination.

3. POLE sequencing

Extracting tumor tissue DNA of the intimal cancer by using a DNA extraction reagent set QIAamp of Qiagen, and amplifying exons 9 to 14 of POLE by using a PCR method, wherein the sequences of PCR primers are as follows:

product utilization of PCR

The Terminator v3.1 cycle sequencing kit was tested on-line in an ABI3730 sequencer and the mutated POLE was verified in both directions using Sanger sequencing.

4. Immunohistochemical staining

1) Screening pathological specimens: selecting a paraffin tissue specimen containing tumor tissue, and preparing a tissue chip of a paraffin embedded specimen under certain conditions.

2) Slicing, spreading, pasting and baking: the slice thickness is 4 μm, then the unfolded slice is taken out with an anti-drop adhesive glass slide, put on a slice rack for air drying, and baked on a baking machine for 30min at 70 ℃.

3) Dewaxing: placing the slices into xylene I, xylene II, 100% ethanol, 95% ethanol, 90% ethanol, 85% ethanol, 75% ethanol, 50% ethanol, and distilled water for 5min, respectively, and dewaxing.

4) Antigen retrieval: adding a certain amount of sodium citrate antigen retrieval solution (pH is 6.0) into a beaker, putting the beaker into a pressure cooker, heating to boil, boiling at high pressure for 2-3 min, cooling the antigen retrieval solution to return to room temperature, and washing with PBS for 2 times.

5) Inactivating peroxidase: 3% hydrogen peroxide solution was added dropwise to the tissue sections, incubated at room temperature for 15min, and washed 3 times with PBS to block endogenous peroxidase.

6) And (3) sealing: the tissue specimen part was outlined with a waterproof marker pen, and sealing serum was added dropwise, placed in a wet box and sealed at room temperature for 30min, and washed with PBS 3 times.

7) Incubating the primary antibody: antibodies against VISTA (cat # D1L2G, dilution ratio 1: 200; manufacturer Cell Signaling Technology), MSH2, MSH6, MLH1, PMS2 (antibodies to four mismatch repair proteins from VENTANA MMR IHC kit) and p53 antibody (ZA-0408, dilution ratio 1:200, King-bridge, Beijing) were added dropwise to the tissue sections in a wet box and left overnight at 4 ℃. Isotype IgG served as negative control.

8) Incubation of secondary antibody: after the primary antibody is incubated at 4 ℃ overnight, taking out, rewarming for 1h at 37 ℃, washing with PBS for three times, dripping the secondary antibody, and incubating for 1h at room temperature; PBS was then washed three times.

9) DAB color development: and after the incubation is finished, dropwise adding a freshly prepared DAB developing solution, developing for about 5min, observing the dyeing condition, washing for 10min by flowing water according to the dyeing degree, and stopping developing in time.

10) Hematoxylin counterstain, blue return: staining the slices in hematoxylin staining solution at room temperature for about 2min, washing with flowing water, adding into 0.1% hydrochloric acid ethanol for 5-10s, washing with tap water, and adding into ammonia water for 5-10 s.

11) And (3) dehydrating: sequentially dehydrating for 5min at the concentration of 50% alcohol → 70% alcohol → 80% alcohol → 90% alcohol → 95% alcohol → 100% alcohol (twice), the xylene is transparent: and the xylene I and the xylene II are transparent for 10 min.

12) Sealing: sealing the neutral gum, covering with a cover glass, paying attention to avoid bubbles, and observing in a fume hood after drying.

5. Evaluation of immunohistochemical results

Interpretation of mismatch repair proteins: the expression of 4 mismatch repair proteins is localized in the nucleus, and non-tumor intima gland, intima stroma and lymph cell nucleus are used as positive internal control, and the nucleus is brownish yellow positive coloration. Only under the positive precondition of the internal control, the complete loss of any MMR expression in the cell nucleus of the tumor area can be judged as MMR protein loss (dMMR).

Evaluation of P53: non-tumorous intimal glands, intimal stroma and lymphocytes were used as wild-type internal controls for p53 staining, with differential staining of the nuclei with varying intensity. The missense mutation type is judged if more than 70 percent of tumor cell nuclei are diffuse strong positive, the nonsense mutation type is judged if all tumor cell nuclei are not colored, the missense mutation type and the nonsense mutation type are judged as the p53 mutation type, and the wild type p53 is judged if the cell nuclei are differentially colored in different strengths.

Interpretation of VISTA: only VISTA expression in stromal immune cells was assessed, and VISTA expression in tumor cells was not assessed. VISTA positive in tumor interstitial immune cells with the positive proportion of more than or equal to 1% is defined as VISTA positive, and VISTA negative with the positive proportion of less than 1%.

6. Step of molecular typing

As shown in fig. 1: 1) judging the mutation state of the POLE gene: judging whether the mutation is a POLE mutation type or not, and judging whether the MMR protein is absent or not; 2) if the expression of any protein in the 4 mismatch repair proteins is deleted, judging the protein to be a dMMR type, and if the protein is not the dMMR type, judging the p 53; 3) the staining of p53 was judged as p53 mutant or p53 wild type, and p53 wild type was judged as non-specific molecular profile (NSMP) type.

7. Determining recurrence risk according to VMG model

VISTA, Molecular typing (Molecular type) and tumor Grade (Grade) were included and we named this model as a VMG prognostic model of early intimal carcinoma. The VMG model prognosis risk assigning system is as follows:

and adding the scores of the three prognostic factors to obtain a total score, wherein the total score range is 0-4, and the risk is layered as follows: 0-1 is low risk, 2 is medium risk, and 3-4 is high risk.

8. Determining a Risk of relapse level according to the ESMO method

The grade of the risk of relapse was judged according to the following table, according to the pathological parameters and the ESMO criteria:

9. statistical analysis

The survival package and the ggplot package in the R language are used for drawing patient survival curves of the two models, the survival package and the survivcomp package in the R language are used for comparing the C index of the new model with the C index of the existing ESMO model, and the survivIDINR package in the R language is used for calculating the comprehensive discriminant Improvement index (IDI) of the two models. Overall the larger the IDI, the better the predictive power of the new model is suggested. If IDI >0, positive improvement indicates that the prediction ability of the new model is improved compared to the old model, if IDI <0, negative improvement indicates that the prediction ability of the new model is decreased, and if IDI is 0, it is considered that the new model is not improved.

Third, data analysis

The VMG model of the present invention has a higher predictive power than existing ESMO models. In the invention, the results of the multi-factor regression analysis show that: molecular typing, expression of VISTA and tumor grade are independent prognostic factors of early endometrial cancer, p53 mutant patients, VISTA negative and high grade tumor patients have poorer prognosis in molecular typing, and the survival curve is shown in FIG. 2. In the VMG model of the invention, the survival curves of low-risk and medium-risk and high-risk patients are completely separated, while the survival curves of different risk patients in the existing ESMO model are partially crossed, as shown in FIG. 3.

The C-index is a consistency index (C-index) used to evaluate the predictive ability of the model. The C index is the proportion of pairs in all patient pairs for which the predicted outcome is consistent with the actual outcome, and estimates the probability that the predicted outcome is consistent with the actual observed outcome. For Relapse Free Survival (RFS) of patients, the C-index of the VMG model was 0.90 (95% confidence interval 0.84-0.96), while the C-index of the ESMO was 0.75 (95% confidence interval 0.65-0.85), with a difference in the predictive power of the two models (p < 0.001). For disease-specific survival (DSS) in patients, the C-index for the VMG model was 0.91 (95% confidence interval 0.85-0.97), while the C-index for ESMO was 0.78 (95% confidence interval 0.67-0.89), with a difference in the predictive power of the two models (p 0.004), see figure 4.

The comprehensive discriminant Improvement index (IDI) indicates that the larger the IDI is, the better the prediction capability of the new model is. If IDI >0, the improvement is positive, indicating that the prediction capability of the new model is improved compared to the old model, if IDI <0, the improvement is negative, the prediction capability of the new model is decreased, and if IDI is 0, the new model is considered not to be improved. For the prediction capability of 2-year relapse-free survival, compared with the ESMO model, the VMG model has an IDI of 6%, a 95% confidence interval of 0.2% to 13.9%, and p of 0.038; for 2-year disease specific survival, the confidence interval of 95% is 0.1% -8.4% and the confidence interval of IDI is 2.2%, and p is 0.04; for the prediction capability of the 5-year relapse-free survival period, compared with the ESMO model, the VMG model has the IDI of 13.7 percent, the 95 percent credibility interval of 2.3 to 27.7 percent and the p of 0.014; for 5-year disease specific survival, IDI is 8.9%, 95% confidence interval is-1.8% -21.2%, and p is 0.078. It can be seen that the VMG model of the invention is better able to predict ESMO and more robust in predicting patient prognosis than the existing models.

Compared with the existing ESMO model, the VMG model in the invention is more objective and simpler. The ESMO model was interpreted based on histomorphology only, incorporating the histological type of the tumor, the depth of tumor infiltration, the histological grade of the tumor, and the lymphatic vascular space infiltration. The histological types of the tumors are 9 at present, the diagnosis consistency of pathologists is low, particularly in high-grade tumors, the histological types are difficult to identify, and the diagnosis consistency among pathologists is low. The VMG model is a scoring system established based on a molecular pathology, immunohistochemistry and tumor grade classification method, is not included in a histology type, and can judge the recurrence risk of the intimal cancer according to a prognosis score. Therefore, the VMG model in the invention is more objective and concise.

The VMG model of the invention may be used to guide adjuvant therapy after early stages of endometrial cancer surgery. For low-risk patients, the recurrence risk is extremely low, the 5-year survival rate after operation is 99.2 percent, and the patients can be subjected to follow-up visit after operation and do not receive auxiliary treatment; for the middle-risk patients, the patients have certain recurrence risk, the 5-year survival rate after operation is 85.2 percent, and the patients are recommended to receive brachytherapy; for high-risk patients, the recurrence risk is high, the 5-year survival rate after operation is only 57.2%, and external radiotherapy or combined chemotherapy is recommended and closely followed.

SEQUENCE LISTING

<110> Beijing coordination hospital of Chinese academy of medical sciences

<120> preparation for judging prognosis of early endometrial cancer and recurrence risk model

<130> P210028

<160> 12

<170> PatentIn version 3.5

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Claims

1. A system for prognostic stratification of early stage endometrial cancer comprising a VISTA assessment portion, a molecular typing portion and a tumor grade portion;

the VISTA assessing moiety comprises an agent that detects VISTA; the VISTA evaluation portion defines the ratio of VISTA positive in tumor stromal immune cells >1% as VISTA positive, <1% as VISTA negative; the VISTA positive judgment score is 0, and the VISTA negative judgment score is 1;

the tumor grade portion comprises an agent that grades a tumor; the tumor grade is classified as high grade, low grade; the high level is a FIGO3 level, and the low level is a FIGO1 level or a FIGO 2 level; the low level is judged as 0 point, and the high level is judged as 1 point;

the molecular typing moiety includes reagents for detecting POLE, mismatch repair protein and p53, performs judgment such as,

1) the mutation state of the POLE gene is judged firstly: judging whether the mutation is a POLE mutation type or not, and judging whether the MMR protein is absent or not;

2) if the expression of any protein in the 4 mismatch repair proteins is deleted, judging the protein to be a dMMR type, and if the protein is not the dMMR type, judging the p 53; the mismatch repair proteins comprise MSH2, MSH6, MLH1 and PMS 2;

3) judging the type of the mutant p53 or the wild p53 according to the staining condition of p53, wherein the wild p53 is the NSMP type;

the POLE mutant type is judged to be 0, the dMMR/NSMP type is judged to be 1, and the P53 mutant type is judged to be 2;

the scores of the VISTA evaluation part, the molecular typing part and the tumor grading part are added to obtain a total score, the total score range is 0-4, and the risk is graded as follows: 0-1 is low risk, 2 is medium risk, and 3-4 is high risk.