CN112877438A - High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection - Google Patents
High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection Download PDFInfo
- Publication number
- CN112877438A CN112877438A CN202110330660.6A CN202110330660A CN112877438A CN 112877438 A CN112877438 A CN 112877438A CN 202110330660 A CN202110330660 A CN 202110330660A CN 112877438 A CN112877438 A CN 112877438A
- Authority
- CN
- China
- Prior art keywords
- risk
- pdl1
- score
- detecting
- endometrial cancer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57442—Specifically defined cancers of the uterus and endometrial
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4748—Details p53
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70532—B7 molecules, e.g. CD80, CD86
Abstract
The invention discloses a high-risk endometrial cancer (high-grade intimal carcinoma and non-intimal carcinoma) prognosis evaluation system with molecular typing and PDL1 detection, which is used for judging the stage of FIGO and the existence of Lymphatic Vascular Space Infiltration (LVSI) by HE staining on a tumor specimen of a patient with high-risk endometrial cancer receiving an operation stage. And (3) detecting and determining molecular typing through a sequencing and immunohistochemical method. Expression of PDL1 in tumor cells was determined using a multiplex fluorescence immunohistochemistry method. And respectively endowing risk scores to the expression of FIGO stages, molecular typing, LVSI and PDL1, adding the scores to obtain a prognosis score, and further layering the high-risk intima cancer patients into high-medium-risk, high-risk and ultrahigh-risk according to the scoring system. Compared with the existing FIGO staging and molecular typing, the prognosis model has higher efficiency of predicting and distinguishing prognosis, and is beneficial to avoiding over-treatment and under-treatment.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to high-risk endometrial cancer prognosis.
Background
The endometrial cancer is a common gynecological malignant tumor, the morbidity of the endometrial cancer is about 60/10 ten thousands, the mortality of the endometrial cancer is about 20/10 thousands, and the morbidity of the endometrial cancer is in an ascending trend. In 2015, 6 ten thousand new cases of Chinese and 2 ten thousand death cases. Although most endometrial cancers are early at diagnosis, prognosis is relatively good. About 15-20% of patients are diagnosed with high-risk intimal cancer, i.e., high-grade intimal cancer with deep muscle infiltration (stage IB G3 intimal cancer), no residual intimal cancer after stage II-III surgery, and stage I-III non-intimal cancer (serous carcinoma, clear cell carcinoma, carcinosarcoma, etc.). This high risk patient has a high risk of relapse and distant metastasis, and has a poor prognosis[1]. However, this high-risk intimal cancer patient has significant heterogeneity, and the pathological types include intimal and non-intimal cancers, and the FIGO stage includes stages IB to III. Without differentiating the tumor type, the 5-year survival rate of intimal cancer patients in stage I is 85% -90%, 75% -85% in stage II, 50% -65% in stage III, and 20% -25% in stage IV. While the 5-year survival rate of stage IB G3 intimal cancer is only about 58%, and the 5-year survival rate of serous cancer is only 20% -25%. Thus, for high-risk intimal cancer patients, current treatment guidelines are developed hierarchically and histologically by FIGO staging and histology, i.e., by planning treatment for stage IB G3 intimal carcinoma, stage II intimal carcinoma, stage III intimal carcinoma without lesion residual, and non-intimal carcinoma, respectively[2]. Molecular typing was added to the latest therapeutic guidelines[3]. External radiation radiotherapy is recommended for the stage IB G3 intimal cancer, and brachytherapy can also be carried out; for stage II intimal cancer, patients with G1-2 patients without LVSI recommend brachytherapy, and patients with G3 or LVSI positive recommend external irradiation treatment; for intimal-III cancer without lesion residues, external radiation therapy in combination with chemotherapy is recommended; for theNon-intimal cancers, recommended chemotherapy, and external radiation therapy is also contemplated[2]。
The treatment mode for high-risk endometrial cancer is mainly based on the operation stage and the histopathological type. However, classification is difficult due to the use of different pathological criteria, different interpretations of the same pathological diagnostic criteria, ambiguous tumor morphology; in high-grade tumors, the histomorphologic classification of the high-grade tumors is difficult to distinguish in the proportion of 26% -37%, and the pathological diagnosis is inconsistent[4,5]. In one study, 3 pathologists with gynecological tumors performed histological identification of 56 high-grade endometrial cancer cases, and the inconsistency rate of the diagnosis results was as high as 62.5%[4]. This risk stratification based on morphological pathological diagnosis affects the choice of treatment strategies for endometrial cancer patients, resulting in over-or under-treatment for some patients with intimal cancer.
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 diagnosing and treating endometrial cancer[6]. Foreign researchers have proposed a simple, economical prospective molecular classification tool to replace high-throughput sequencing, which determines the variant of POLE by the Exonucleolytic Domain Mutation (EDM) sequencing method of POLE, then determines the type of mismatch repair deficiency (dMMR) by detecting the expression of mismatch repair protein by immunohistochemistry method, and finally classifies other cases into p53 variant and p53 wild type according to the p53 immunohistochemistry result[7]. The method is finally divided into a POLE mutant type, a dMMR type, a p53 mutant type and a non-specific molecular profile (NSMP). The detection scheme for alternative molecular typing is shown in FIG. 1. Such a model has been validated by multiple teamsHas important significance for guiding endometrial cancer molecular typing, risk classification and treatment[8]。
The immunotherapy marker PDL1 and molecular typing have not been incorporated in the current stratified treatment for high-risk endometrial cancer. Therefore, there is a need to establish an objective, highly repeatable classification method that is more accurate for prognosis determination and treatment guidance.
[1].Randall M.Management of high-risk endometrial cancer:are we there yetLancet Oncol,2019,20(9):1192-1193.
[2].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.
[3].Concin N,Matias-Guiu X,Vergote I et al.ESGO/ESTRO/ESP guidelines for the management of patients with endometrial carcinoma.International Journal of Gynecologic Cancer,2021,31(1):12-39.
[4].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.
[5].Han G,Sidhu D,Duggan MA et al.Reproducibility of histological cell type in high-grade endometrial carcinoma.Mod Pathol,2013,26(12):1594-1604.
[6].Cancer Genome Atlas Research N,Kandoth C,Schultz N et al.Integrated genomic characterization of endometrial carcinoma.Nature,2013,497(7447):67-73.
[7].Vermij L,Smit V,Nout R et al.Incorporation of molecular characteristics into endometrial cancer management.Histopathology,2020,76(1):52-63.
[8] Zollia religiosa, sunward, progress in immunotherapy of refractory endometrial cancer. J.Utilis & obstetrics 2020,36(06): 415-.
Disclosure of Invention
The invention provides a high-risk endometrial cancer (IB stage G3 intimal cancer, II-III stage postoperative non-residual intimal cancer and I-III stage non-intimal cancer) prognosis evaluation system incorporating molecular typing and PDL1 detection, which has the characteristics of relative objectivity, high repeatability and clinical feasibility of the existing molecular typing, has higher prediction and differentiation prognosis efficiency, and is beneficial to avoiding over-treatment and insufficient treatment.
In order to achieve the purpose, the invention provides an application of a reagent for detecting PDL1 in preparation of a high-risk endometrial cancer prognosis evaluation preparation.
Further, the formulation also includes reagents for detecting other markers: a reagent for detecting a mutant of POLE, a reagent for detecting a mismatch repair protein and/or a p53 protein.
The invention also provides a kit for the prognosis evaluation of high-risk endometrial cancer, which comprises a reagent for detecting PDL 1.
Further, the reagent for detecting PDL1 may be a reagent for detecting PDL1 gene, PDL1 protein, and/or other biological markers.
Further, the kit also comprises reagents for detecting other markers: a reagent for detecting a mutant of POLE, a reagent for detecting a mismatch repair protein and/or a p53 protein.
Further, the mismatch repair protein comprises MLH1, MSH2, MSH6 and/or PMS 2.
Still further, the detection method is sequencing, DNA sequencing, RNA sequencing, immunohistochemistry, pathological section and/or other biological detection methods.
The invention further provides a high-risk endometrial cancer prognosis evaluation system and/or model, which comprises a tissue morphology judgment part, a molecular typing part and a PDL1 scoring part, wherein the tissue morphology judgment part preferably comprises a FIGO stage judgment part and a pathological parameter LVSI scoring part.
Further, each part contains a detection reagent, the tissue morphology judging part comprises FIGO stage judgment and pathological parameter LVSI judgment, and the molecular typing part comprises detection POLE, mismatch repair protein and/or p 53; the PDL1 scoring component includes detecting PDL 1.
Further, the detection method is sequencing, DNA sequencing, RNA sequencing, immunohistochemistry and/or other biological detection methods.
Further, the FIGO staging judgment standard is as follows:
and (3) stage I: the tumor is confined in the uterus and divided into IA and IB phases, wherein the IA tumor infiltrates into a muscle layer with the depth less than 1/2, and the IB is the muscle layer with the tumor infiltrates into the muscle layer with the depth more than or equal to 1/2.
And (2) in a stage II: the tumor invades the cervical interstitium, but does not spread outside the uterus.
Stage III: the local and regional spread of tumor is divided into stages IIIA, IIIB and IIIC, the stage IIIA refers to the serosal layer and adnexa of uterus involved in tumor, the stage IIIB refers to the tissues around vagina and uterus involved in tumor, and the stage IIIC refers to the metastasis of pelvic lymph and/or abdominal aorta.
Stage IV: tumors invade the mucosa and distant metastases of the bladder and rectum (exclusion).
Further, the pathology parameter LVSI performs the following decision:
at least one cluster of tumor cells is seen in the gap surrounded by the flattened endothelial cells, which is judged to be positive for LSVI or LVSI, otherwise, judged to be negative for LVSI.
Further, the molecular typing section performs the following determination:
1) firstly, judging the mutation state of the POLE gene: judging the presence of pathogenic mutation as a POLE mutant, and continuing to judge the MMR protein without POLE mutation; 2) if the expression of any protein in the 4 mismatch repair proteins is lost, judging the protein to be a dMMR type, and if the protein is not the dMMR type, continuing to judge the p 53; 3) the P53 was judged to be P53 mutant or P53 wild type based on the staining pattern of P53, and P53 wild type was judged to be non-specific molecular profile (NSMP) type.
Furthermore, the mutation state of the POLE gene is judged by extracting DNA from a paraffin tissue sample, carrying out Sanger sequencing, detecting the mutation of the No. 9 to No. 14 exons of POLE, and carrying out reverse verification on the mutated sample by using a PCR method to judge whether the POLE pathogenic mutation exists.
Further, the mismatch repair protein and/or the p53 protein are detected by an immunohistochemical method to detect the expression of the mismatch repair proteins (MLH1, MSH2, MSH6, PMS2) and the p53 protein.
Furthermore, the expression of the four mismatch repair proteins is localized in the cell nucleus, and takes the non-tumor intima gland, intima stroma and lymph cell nucleus as the positive internal control, and the positive control shows brown yellow positive coloration. The MMR protein deficiency (dMMR) is judged by complete loss of nuclear expression in the tumor area under the positive condition of the internal control.
Further, the expression of the P53 protein was determined as follows: 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, and the wild type is judged if the cell nuclei are differentially colored with different intensities.
Further, the PDL1 score section detects PDL1 expression in endometrial cancer using a multiplex fluorescence immunohistochemistry method based on Tyramine Signal Amplification (TSA) technique.
Still further, the PDL1 score moiety used AE1/AE3 antibody to label CK, positive CK labeling being tumor epithelial cells and negative CK being non-tumor cells.
Further, evaluation of the PDL 1: PDL1 positive in tumor cells (CK marker) at a rate of 1% or more was defined as PDL1 positive; PDL1 was positive in tumor cells at a rate < 1% defined as PDL1 negative.
Furthermore, the FIGO stage judgment part is endowed with 0 score in the stage I, and 1 score is endowed in the stages II to III; the negative score of the pathological parameter LVSI score part is 0, and the positive score is 1; the molecular typing part, the POLE mutant type, the dMMR/NSMP type and the p53 mutant type are respectively assigned with a score of 0, a score of 1 and a score of 2; PDL1 positive gave a score of 0 and PDL1 negative gave a score of 1 in tumor cells.
Further, the scores of the parts are added to obtain a total score, the total score ranges from 0 to 5, and the risk is layered as follows: 0-2 is high-medium-risk, 3 is high-risk, and 4-5 is ultra-high-risk.
Advantageous effects
(1) A prognostic scoring system according to the present invention is established based on 289 cases of high-risk endometrial cancer. The high-risk endometrial cancer is further subjected to risk stratification, which is divided into high-medium-risk, high-risk and ultrahigh-risk: 67 high-intermediate-risk patients, wherein 1 patient has relapse, and the 5-year relapse-free survival Rate (RFS) is 98%; 96 high-risk patients, 13 of which recur, and 5-year RFS is 83%; 126 patients with ultra-high risk had recurrence in 34 of them, with 5-year RFS of 77.5%. While 111 patients in stage I, according to the fido staging, had 11 relapses with a 5-year RFS of 91.2%; 32 patients in stage II, with 6 relapses and 77.5% RFS in 5 years; stage III patients had 146 cases with 31 relapses and a 5-year RFS of 84.6%. The survival curves for progression-free survival and disease-specific survival of patients based on FIGO stages, molecular typing and the prognostic models of the present invention are shown in FIG. 3.
(2) For high-risk intimal cancer patients, the prognosis scoring system of the invention has higher prediction capability than the existing FIGO stages and molecular typing. 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 of patients, the C-index of the model of the invention is 0.84 (95% confidence interval 0.75-0.93), while the C-index of the FIGO stage is 0.71 (95% confidence interval 0.59-0.92), the C-index of the molecular typing is 0.66 (95% confidence interval 0.53-0.79), and the C-index of the model of the invention is significantly higher than the C-index of the FIGO stage and the molecular typing (p < 0.01). For the disease specific survival of patients, the C index of the model of the invention is 0.86 (95% confidence interval 0.76-0.95), while the C index of the FIGO stage is 0.72 (95% confidence interval 0.58-0.86), the C index of the molecular typing is 0.63 (95% confidence interval 0.48-0.79), and the C index of the model of the invention is significantly higher than the C index of the FIGO stage and the molecular typing (p < 0.01). A comparison of the C indices is shown in FIG. 4.
(3) The system/model IDI of the invention has high value and better prediction capability. 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, 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. For the prediction capability of 2-year relapse-free survival period, compared with the FIGO, the IDI of the model is 8.4%, the 95% credible interval is 3.7% -14.5%, and p is less than 0.001; for 2-year disease specific survival period, IDI is 3.3%, 95% confidence interval is 1.1% -6.3%, and p is less than 0.001; compared with the FIGO, the model in the invention has the advantages that the IDI is 11.7%, the 95% confidence interval is 3.8% -19.6%, and p is 0.002 for the prediction capability of the 5-year relapse-free survival period; for 5-year disease specific survival, the IDI is 12.2%, the 95% confidence interval is 1.9% -24.7%, and p is less than 0.001. For the prediction capability of 2-year relapse-free survival period, compared with molecular typing, the model disclosed by the invention has the advantages that IDI is 9.4%, the 95% confidence interval is 2.4% -17.7%, and p is 0.02; for 2-year disease specific survival, the IDI is 3.5%, the 95% confidence interval is 0.9% -9.2%, and p is less than 0.001. It can be seen that the model of the present invention has better predictive power than the existing FIGO stages and molecular typing, and has stronger ability to predict patient relapse and death.
Drawings
FIG. 1. detection scheme for endometrial cancer replacement molecule typing;
FIG. 2 is a method and system for detecting further risk stratification of high-risk endometrial cancer according to the present invention; wherein, HE: haematoxylin eosin; LVSI: lymphatic vascular space infiltration; and (3) IHC: immunohistochemical staining; MMR: a mismatch repair protein; NSMP: no specific molecular characteristics;
FIG. 3 is a high risk endometrial cancer patient survival curve plotted according to the prognostic model, FIGO stages and molecular typing of the present invention. A: a recurrence-free survival curve drawn according to the prognostic model of the present invention; b: a disease specific survival curve drawn according to the prognosis model of the invention; c: a recurrence-free survival curve is drawn according to FIGO stages; d: disease specific survival curves drawn according to FIGO stages; e: a recurrence-free survival curve is drawn according to molecular typing; f: a disease specific survival curve drawn according to molecular typing;
FIG. 4. prognostic models, FIGO stages and molecular typing in the present invention predict the concordance index for tumor recurrence and tumor-specific death in patients with high risk endometrial cancer.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
1. Study object
The study object of this embodiment is a tumor tissue specimen after surgery of a patient with high-risk endometrial cancer. Inclusion criteria were: stage IB high-grade intimal carcinoma (i.e., stage I G3 intimal carcinoma with deep muscle layer infiltration), stage II and stage III intimal carcinoma without lesion residues, stage I-III non-intimal carcinoma (including serous carcinoma, clear cell carcinoma, small cell carcinoma, carcinosarcoma, mucinous adenocarcinoma and mixed carcinoma), pathological section and paraffin specimen are intact; the follow-up time is at least 3 months; the clinical data is complete.
Exclusion criteria: FIGO stage 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.
2. Research method
2.1 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, the medical record of outpatient service and telephone follow-up visit, and whether the patient has relapse, relapse time, relapse position, treatment mode after relapse, whether to die, death time, and death reason are recorded by telephone or outpatient service medical record follow-up visit.
2.2 interpretation of pathological parameters
Pathological HE sections are observed under a microscope, and the following pathological parameters including histological type, tumor grading, tumor infiltration depth, LVSI and cervical interstitial infiltration condition are judged. Determining FIGO stages according to pathological parameters and imaging examination.
2.3POLE 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 ABI 3730 sequencer and the mutation-containing POLE was verified in both directions by Sanger sequencing.
2.4 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: putting the slices into xylene I, xylene II, 100% alcohol, 95% alcohol, 90% alcohol, 85% alcohol, 75% alcohol, 50% alcohol, 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 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 MSH2, MSH6, MLH1, PMS2 (antibodies to four mismatch repair proteins from VENTANA MMR IHC kit) and p53 antibody (ZA-0408, dilution ratio 1: 200, gold bridge of Cunninghamia lanceolata) were added dropwise to the tissue sections and placed in a wet box 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.
2.5 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.
2.6 step of molecular typing
As shown in fig. 1: 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; 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 non-specific molecular profile (NSMP) type.
2.7 multiple fluorescent immunohistochemistry
Reagent preparation
Sterilized water, dimethylbenzene, ethanol, AR6 repairing liquid, primary anti-diluent/blocking liquid anti-diluent, secondary anti-working liquid Polymer HRP, TSA dye, TBST, DAPI, anti-quenching sealing tablet, colorless nail polish, AE1/AE3 antibody and PDL1 antibody
Dyeing step
1) Baking slices: at 65 ℃ for 1 hour, placing the mixture obliquely
2) Dewaxing and hydrating: placing in xylene 1 for 10 min; placing in xylene 2 for 10 min; placing in xylene 3 for 10 min; standing in anhydrous ethanol for 5 min; standing in anhydrous ethanol for 5 min; standing in 95% ethanol for 5 min; standing in 75% ethanol for 2 min; washing with sterile water for 3 times, shaking gently for 1min, and pouring off.
3) Reinforcing the sample and attaching: soaking in 10% neutral formalin for 10min, sterilizing with water for 3 times, and gently shaking for 1min
4) Microwave repair: placing the slide in a repair cup, and immersing the AR Buffer; boiling with high fire in a microwave oven, and maintaining with low fire for 15 min; taking out and naturally cooling to room temperature (at least 20min)
5) And (3) sealing: sterilizing and washing the film for 1 time; soaking the slices in TBST for 2 min; taking out the slide, removing water, drawing a tissue by a group drawing pen, and dripping the antisense diluent; incubating at room temperature for 10min
6) CK (AE1/AE3) primary antibody incubation: discarding residual confining liquid, dropwise adding diluted AE1/AE3 primary antibody, and incubating at room temperature for 1 h; TBST washing, 2min × 3 times.
7) And (3) secondary antibody incubation: removing residual washing liquid, dripping Polymer HRP antigen solution, and incubating at room temperature for 10 min; TBST washing, 2min X3 times
8) TSA incubation staining: discarding the residual washing solution, adding TSA dye (opal 520) dropwise, and incubating at room temperature for 10 min; TBST washing, 2min X3 times
9) Microwave repairing (removing primary antibody), and repeating the steps 4-5
10) PDL1 primary antibody incubation: discarding the residual confining liquid, dropwise adding diluted PDL1 primary antibody, and incubating at room temperature for 1 h; TBST washing, 2min × 3 times.
11) Repeat step 7
12) TSA incubation staining: discarding the residual washing solution, adding TSA dye (opal 570) dropwise, and incubating at room temperature for 10 min; TBST washing, 2min X3 times
13) Dyeing of DAPI: adding DAPI dropwise and incubating for 5 min; TBST washing for 2 min; washing with sterilized water for 2min
14) Sealing: anti-quenching sealing tablet, colorless nail polish sealing tablet
Interpretation of PDL 1: scanning and imaging in a multispectral imager, judging according to a TSA dye, capturing blue cell nuclei, green CK (tumor cells) and red PDL1, analyzing the positive proportion of PDL1 in the tumor cells (CK positive) by using inform software, wherein the positive proportion of PDL1 is more than or equal to 1 percent, namely PDL1 is positive in the tumor cells, and the negative proportion of PDL1 is less than 1 percent, namely PDL1 in the tumor cells.
2.8 determining the recurrence risk of high-risk endometrial cancer according to a prognostic scoring system
Prognostic risk assignment was performed according to the following table:
and adding the scores of the three prognostic factors to obtain a total score, wherein the total score range is 0-5, and the risk is layered as follows: 0-2 is high-medium-risk, 3 is high-risk, and 4-5 is ultra-high-risk.
2.9 statistical analysis
The method comprises the steps of drawing a kaplan-meier survival curve by utilizing graphpad prism software, comparing the C index of a new model with the C index of the existing FIGO stage by utilizing a subvalur packet and a subvcomp packet in the R language, and calculating the comprehensive Discrimination Improvement index (IDI) of the two models by utilizing a subvIDINR packet in the R language. 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. For the prediction capability of 2-year relapse-free survival period, compared with the FIGO, the IDI of the model is 8.4%, the 95% credible interval is 3.7% -14.5%, and p is less than 0.001; for 2-year disease specific survival period, IDI is 3.3%, 95% confidence interval is 1.1% -6.3%, and p is less than 0.001; compared with the FIGO, the model in the invention has the advantages that the IDI is 11.7%, the 95% confidence interval is 3.8% -19.6%, and p is 0.002 for the prediction capability of the 5-year relapse-free survival period; for 5-year disease specific survival, the IDI is 12.2%, the 95% confidence interval is 1.9% -24.7%, and p is less than 0.001. For the prediction capability of 2-year relapse-free survival period, compared with molecular typing, the model disclosed by the invention has the advantages that IDI is 9.4%, the 95% confidence interval is 2.4% -17.7%, and p is 0.02; for 2-year disease specific survival, the IDI is 3.5%, the 95% confidence interval is 0.9% -9.2%, and p is less than 0.001. It can be seen that the model of the present invention has better predictive power than the existing FIGO stages and molecular typing, and has stronger ability to predict patient relapse and death.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
SEQUENCE LISTING
<110> Beijing coordination hospital of Chinese academy of medical sciences
<120> high-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection
<130> P210044
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 1
tcttttaaca accagaggga ggt 23
<210> 2
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 2
ttgctcccat tcctggacta a 21
<210> 3
<211> 22
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 3
cacattgctg tggacttctt tg 22
<210> 4
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 4
ctcatggagc tgcaattctg a 21
<210> 5
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 5
atgaggctgc tgcttctgaa c 21
<210> 6
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 6
ccaggagcca cctcctaagt c 21
<210> 7
<211> 20
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 7
<210> 8
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 8
tcccatgaga tgtggtgaca g 21
<210> 9
<211> 19
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 9
gcccagtttt gccagttct 19
<210> 10
<211> 18
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 10
gagcgggctg gcatacat 18
<210> 11
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 11
ctgtgccggt ctccttactg t 21
<210> 12
<211> 21
<212> DNA
<213> Artificial sequences (synthetic sequences)
<400> 12
gggacatcca cctccattca g 21
Claims (10)
1. Application of reagent for detecting PDL1 in preparation of high-risk endometrial cancer prognosis evaluation preparation.
2. The use according to claim 1, wherein the formulation further comprises reagents for detecting other markers: a reagent for detecting a mutant of POLE, a reagent for detecting a mismatch repair protein and/or a p53 protein.
3. A kit for the prognosis evaluation of high-risk endometrial cancer, which is characterized by comprising a reagent for detecting PDL 1.
4. The kit of claim 3, further comprising reagents for detecting additional markers: a reagent for detecting a mutant of POLE, a reagent for detecting a mismatch repair protein and/or a p53 protein.
5. The kit according to claim 4, wherein the mismatch repair protein comprises MLH1, MSH2, MSH6 and/or PMS 2.
6. The kit of claim 3, wherein the detection method is sequencing, DNA sequencing, RNA sequencing, immunohistochemistry, pathological section and/or other biological detection method.
7. A high-risk endometrial cancer prognosis evaluation system and/or model is characterized by comprising a tissue morphology judging part, a molecular typing part and a PDL1 scoring part, wherein the tissue morphology judging part preferably comprises a FIGO stage judging part and a pathological parameter LVSI scoring part.
8. The assessment system and/or model according to claim 7, wherein said fractions contain detection reagents, said tissue morphology decision fraction comprises FIGO staging decision and LVSI decision as pathological parameters, said molecular typing fraction comprises detection POLE, mismatch repair protein and/or p 53; the PDL1 scoring component includes detecting PDL 1.
9. The evaluation system and/or model of claim 7, wherein the FIGO stage determination is given a score of 0 in stage I and 1 in stages II-III; the negative score of the pathological parameter LVSI score part is 0, and the positive score is 1; the molecular typing part, the POLE mutant type, the dMMR/NSMP type and the p53 mutant type are respectively assigned with a score of 0, a score of 1 and a score of 2; PDL1 positive gave a score of 0 and PDL1 negative gave a score of 1 in tumor cells.
10. The assessment system and/or model of claim 9, wherein said partial scores are added to provide a total score in the range of 0-5, further stratifying the risk of high risk endometrial cancer as follows: 0-2 is high-medium-risk, 3 is high-risk, and 4-5 is ultra-high-risk.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110330660.6A CN112877438A (en) | 2021-03-26 | 2021-03-26 | High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110330660.6A CN112877438A (en) | 2021-03-26 | 2021-03-26 | High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112877438A true CN112877438A (en) | 2021-06-01 |
Family
ID=76040897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110330660.6A Pending CN112877438A (en) | 2021-03-26 | 2021-03-26 | High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112877438A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116842436A (en) * | 2023-06-07 | 2023-10-03 | 中国医学科学院北京协和医院 | Multispectral combined skin basal cell carcinoma identification method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017008165A1 (en) * | 2015-07-14 | 2017-01-19 | British Columbia Cancer Agency Branch | Classification method and treatment for endometrial cancers |
| US20180045727A1 (en) * | 2015-03-03 | 2018-02-15 | Caris Mpi, Inc. | Molecular profiling for cancer |
-
2021
- 2021-03-26 CN CN202110330660.6A patent/CN112877438A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20180045727A1 (en) * | 2015-03-03 | 2018-02-15 | Caris Mpi, Inc. | Molecular profiling for cancer |
| WO2017008165A1 (en) * | 2015-07-14 | 2017-01-19 | British Columbia Cancer Agency Branch | Classification method and treatment for endometrial cancers |
Non-Patent Citations (3)
| Title |
|---|
| SHUANG ZHANG等: "PD-L1 and CD4 are independent prognostic factors for overall survival in endometrial carcinomas", 《BMC CANCER》 * |
| UMRAN KUCUKGOZ GULEC等，: "programmed death-1(PD-1) and programmed death-ligand 1 (PD-L1) expressions in type 2 endometrial cancer", 《ARCHIVES OF GYNECOLOGY AND OBSTETRICS》 * |
| 汤永峰等: "子宫内膜透明细胞癌分子分型临床应用及其与PD-L1表达的关系", 《临床与实验病理学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116842436A (en) * | 2023-06-07 | 2023-10-03 | 中国医学科学院北京协和医院 | Multispectral combined skin basal cell carcinoma identification method |
| CN116842436B (en) * | 2023-06-07 | 2024-01-16 | 中国医学科学院北京协和医院 | Multispectral combined skin basal cell carcinoma identification method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Reed et al. | Loss of expression of the p16/cyclin-dependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression | |
| Ried et al. | Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromsomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas | |
| Luk et al. | Mammary analogue secretory carcinoma: an evaluation of its clinicopathological and genetic characteristics | |
| PT1781814E (en) | Methods and kit for the prognosis of breast cancer | |
| CN108179134B (en) | EpCAM/PSMA-based double-antibody functionalized microfluidic chip and preparation method and application thereof | |
| CN113046438B (en) | Endometrial cancer prognosis evaluation system incorporating molecular typing and immune scoring | |
| CN113156120B (en) | Application of B7H4 in preparation of endometrial cancer molecular typing reagent and system | |
| CN112877438A (en) | High-risk endometrial cancer prognosis evaluation system incorporating molecular typing and PDL1 detection | |
| CN110244058B (en) | Application of ENPP1 in preparation of high-grade serous ovarian cancer diagnosis and prognosis kit | |
| CN107266567A (en) | LCRMP4 monoclonal antibodies and preparation method and application | |
| CN106706915A (en) | Application of SUSD2 in preparation of kit for diagnosis and/or prognostic judgement of high-grade serous ovarian carcinoma | |
| CN112961921B (en) | Preparation for judging prognosis of early endometrial cancer and recurrence risk model | |
| CN105785004B (en) | Purposes of the CDC GAP-associated protein GAP 2 in diagnosis of pancreatic cancer or prognosis | |
| CN112229997B (en) | Prognostic diagnosis marker Claudin23 for ovarian cancer and application thereof | |
| CN111665358B (en) | Application of NALCN protein in prognosis prediction of esophageal squamous cell carcinoma | |
| CN112229998B (en) | Prognostic diagnosis marker Claudin22 for ovarian cancer and application thereof | |
| CN111948395A (en) | Quadruple marker for diagnosing immune regulation subtype of triple negative breast cancer and application thereof | |
| JP4887505B2 (en) | Method for examining metastasis and invasiveness of cancer | |
| CN113552355B (en) | Cervical cancer prognosis scoring system and application thereof | |
| El-Mansi et al. | Validation of tissue microarray technology using cervical adenocarcinoma and its precursors as a model system | |
| CN112229999B (en) | Prognostic diagnosis marker Claudin21 for ovarian cancer and application thereof | |
| CN110244057B (en) | Application of ADORA3 in preparation of high-grade serous ovarian cancer diagnosis and prognosis kit | |
| CN116590416A (en) | Application of DHX33 protein as thyroid tumor diagnosis marker | |
| CN117230188A (en) | Application of DHX33 protein as kidney cancer diagnosis marker | |
| Chen et al. | Performance of a novel eight‐color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210601 |