CN116855605A - Application of AOC1 as marker for distinguishing ovarian clear cell carcinoma and high-grade serous carcinoma - Google Patents
Application of AOC1 as marker for distinguishing ovarian clear cell carcinoma and high-grade serous carcinoma Download PDFInfo
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Abstract
The invention belongs to the field of biological medicine, and particularly relates to application of AOC1 as a marker for distinguishing ovarian clear cell carcinoma and high-grade serous carcinoma. In particular, the invention provides the use of an agent for detecting the expression level of AOC1 in the manufacture of a product for determining the type of cancer, whether it is clear cell ovarian cancer or high grade serous ovarian cancer.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to application of AOC1 as a marker for distinguishing ovarian clear cell carcinoma and high-grade serous carcinoma.
Background
Ovarian epithelial cancer (epithelial ovarian carcinoma, EOC) is one of the most common gynaecological malignancies in women, with the incidence being the seventh among female malignancies. Of these, high-grade serous ovarian cancer (High-grade serous ovarian cancer, HGSC) is the most common, accounting for about 60% of ovarian epithelial cancers. Ovarian clear cell carcinoma (Ovarian clear cell carcinoma, OCCC) is the second leading stage, accounting for about 5-11% of ovarian epithelial cancers.
There are differences in the demographic, morphological and molecular characteristics of Ovarian Clear Cell Carcinoma (OCCC) and high grade serous ovarian carcinoma (HGSC). Ovarian Clear Cell Carcinoma (OCCC) is closely associated with endometriosis. Ovarian Clear Cell Carcinoma (OCCC) exists as tubular cystic structures and clear cells, and high grade serous ovarian carcinoma (HGSC) exists as solid and papillary structures. Ovarian Clear Cell Carcinoma (OCCC) is generally WT-1 and ER negative, napsin A and HNF1B positive. However, high grade serous ovarian cancer (HGSC) is generally WT-1 and ER positive, lacking expression of either Napsin A or HNF 1B.
Surgery in combination with subsequent chemotherapy is a conventional treatment for Ovarian Clear Cell Carcinoma (OCCC) and high grade serous ovarian carcinoma (HGSC). However, ovarian Clear Cell Carcinoma (OCCC) is resistant to conventional platinum-based chemotherapy and has poor prognosis compared to high grade serous ovarian carcinoma (HGSC). Thus, exploring the cellular and molecular differences in Ovarian Clear Cell Carcinoma (OCCC) and high grade serous ovarian carcinoma (HGSC) would help to find alternative therapies.
Disclosure of Invention
The invention provides a basis for screening differential genes of Ovarian Clear Cell Carcinoma (OCCC) and high-grade serous ovarian carcinoma (HGSC), collecting biopsy samples of patients with the Ovarian Clear Cell Carcinoma (OCCC) and the high-grade serous ovarian carcinoma (HGSC), performing scRNA-seq, and verifying the application value of the biomarker in distinguishing the Ovarian Clear Cell Carcinoma (OCCC) and the high-grade serous ovarian carcinoma (HGSC) through a database and clinical samples after screening the differential genes, and provides a basis for clinically distinguishing the patients with the Ovarian Clear Cell Carcinoma (OCCC) and the high-grade serous ovarian carcinoma (HGSC).
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides the use of an agent for detecting the expression level of AOC1 in the manufacture of a product for determining the type of cancer, whether it be Ovarian Clear Cell Carcinoma (OCCC) or high grade serous ovarian carcinoma (HGSC).
Alternatively, the determining of the cancer type refers to determining the type of ovarian epithelial cancer (epithelial ovarian carcinoma, EOC).
Preferably, the expression level includes an mRNA expression level or a protein expression level.
Further, the reagent comprises a reagent for detecting the expression level of the AOC1mRNA and a reagent for detecting the expression level of the AOC1 protein.
In another specific embodiment, the agent comprises an agent that detects the number of AOC1 positive expressing cells.
Further, the reagent for detecting the mRNA expression level of the AOC1 comprises a reagent for detecting the mRNA expression level of the AOC1 by adopting the following method: PCR-based detection methods, southern hybridization methods, northern hybridization methods, dot hybridization methods, fluorescent in situ hybridization methods, DNA microarray methods, ASO methods, high throughput sequencing platform methods, chip methods.
Further, the reagent for detecting the expression level of the AOC1 protein comprises a reagent for detecting the expression level of the AOC1 protein by adopting the following method: western Blot (Western Blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescent resolution technique, and protein chip method.
Preferably, the reagent comprises: a primer pair, probe or antisense nucleotide that specifically binds to a gene of AOC 1; or, an antibody, ligand, nanoparticle or aptamer that specifically binds to an AOC1 protein or peptide fragment.
Preferably, the assay is an assay performed on a sample from a subject.
Further, the sample refers to a composition obtained or derived from a subject comprising cellular entities and/or other molecular entities to be characterized and/or identified, e.g. based on physical, biochemical, chemical and/or physiological characteristics. The sample may be obtained from blood and other fluid samples of biological origin and tissue samples of the subject, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue, such as tissue from fresh, frozen and/or preserved organs or tissue samples, biopsy tissue or aspirates; blood or any blood component; body fluid; cells from any time of gestation or development of an individual; or plasma. The term sample includes biological samples that have been treated in any way after they have been obtained, such as by treatment with reagents, stabilization, or enrichment for certain components (such as proteins or polynucleotides), or embedding in a semi-solid or solid matrix for sectioning purposes.
Preferably, the sample comprises: tissue, cell culture, blood, urine, saliva, mucosal samples, stool, intestinal lavage, joint fluid, cerebrospinal fluid, bile samples, respiratory secretions, bronchoalveolar lavage samples.
More preferably, the sample comprises a tissue or cell culture.
Preferably, the product comprises a kit, a chip and a test strip.
Preferably, the product may further include an mRNA expression level auxiliary detection reagent, including but not limited to: reagents for visualizing the amplicon corresponding to the primer, for example, reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, or the like; an RNA extraction reagent; a reverse transcription reagent; cDNA amplification reagents; preparing a standard substance used for a standard curve; positive control.
Preferably, the kit may further comprise a protein expression level-aiding detection reagent including, but not limited to: a sealing liquid; an antibody diluent; washing buffer; a color development stop solution; standard for standard curve was prepared.
Specifically, the AOC1 is expressed in an amount higher in Ovarian Clear Cell Carcinoma (OCCC) than in high grade serous ovarian carcinoma (HGSC), and the AOC1 is classified as Ovarian Clear Cell Carcinoma (OCCC) when the detected AOC1 expression is higher than a threshold value, and classified as high grade serous ovarian carcinoma (HGSC) when the detected AOC1 expression is lower than the threshold value.
The threshold value is well known in the art and depends on the particular measurement technique, the determination of the threshold value is within the skill of the person skilled in the art.
The terms "expression level", "expression" and "expression" as used herein refer to the amount of a polynucleotide or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into a structure that is present and operational in a cell. Thus, "expression" of a biomarker as used herein refers to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Transcribed polynucleotides, translated proteins, or fragments of post-translationally modified proteins are also considered expressed, whether they originate from transcripts produced by alternative splicing or degraded transcripts, or from post-translational processing of proteins (e.g., by proteolysis).
The term "subject" as used herein means any animal, and also refers to human and non-human animals (e.g., bovine, equine, ovine, porcine, canine, feline, rodent, primate).
More preferably, the subject of the invention is a patient with ovarian epithelial cancer (epithelial ovarian carcinoma, EOC), or the subject is a patient with suspected Ovarian Clear Cell Carcinoma (OCCC), high grade serous ovarian cancer (HGSC).
In another aspect, the present invention provides an apparatus/system for determining a cancer type, the apparatus/system comprising a judgment means which runs the following judgment criteria (judgment program): the test samples were judged to be from patients with Ovarian Clear Cell Carcinoma (OCCC) when the AOC1 expression level was above the threshold, and from patients with high grade serous ovarian carcinoma (HGSC).
Alternatively, the means/system for determining cancer typing may also be referred to as differentiating means/system for determining ovarian epithelial cancer (epithelial ovarian carcinoma, EOC) typing, differentiating between Ovarian Clear Cell Carcinoma (OCCC) and high grade serous ovarian carcinoma (HGSC).
Optionally, one or more of a detection means, an input means, an output means, a communication means may also be included in the device/system.
Further, the detection means is for detecting an expression amount of AOC1 in a subject sample.
Further, the input module is configured to input an expression amount of AOC1, where the expression amount of AOC1 may be detected by the detecting member.
Further, the output module is used for outputting the judging result of the judging component.
Illustratively, the input module includes one or more of a mouse, a keyboard, a touch screen display, one or more buttons, one or more switches, one or more triggers, and the like; the output module includes one or more of a Liquid Crystal Display (LCD), a Light Emitting Diode (LED) display, a plasma display, a projection display, a touch screen display, and the like.
As will be appreciated by one of skill in the art, the present invention may be embodied as an apparatus, method or computer program product. Accordingly, the present disclosure may be embodied in either entirely hardware, entirely software (including firmware, resident software, micro-code, etc.), or in a combination of hardware and software.
The components described as separate components may or may not be physically separate, and components shown as units may or may not be physical units, may be located in one place, or may be distributed over multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In another aspect, the invention also provides a method of distinguishing between an Ovarian Clear Cell Carcinoma (OCCC) patient and a high grade serous ovarian carcinoma (HGSC) patient, the method comprising determining that the AOC1 is from an Ovarian Clear Cell Carcinoma (OCCC) patient or a high grade serous ovarian carcinoma (HGSC) patient based on the amount of expression of the AOC1 in a sample from the subject.
In another aspect, the invention also provides a method of differentiating between Ovarian Clear Cell Carcinoma (OCCC) cells and high grade serous ovarian carcinoma (HGSC) cells, the method comprising determining whether an AOC1 is an Ovarian Clear Cell Carcinoma (OCCC) cell or a high grade serous ovarian carcinoma (HGSC) cell based on the amount of expression of the AOC1 in the cells.
Preferably, the method of distinguishing cells is of non-diagnostic interest.
Implementation of the methods of the present invention may include performing or completing selected steps manually, automatically, or a combination thereof.
Drawings
FIG. 1 is a graph showing the results of HE staining and immunohistochemical staining sections of OCCC and HGSC.
FIG. 2 is a Umap diagram of cell visualizations performed by the RunUMAP function.
FIG. 3 is a comparison of AOC1 expression levels in epithelial cell populations (epithelial subpopulations) of 5 OCCC patients and 5 HGSC patients.
FIG. 4 shows the results of RT-PCR and immunoblotting.
FIG. 5 shows the results of differential expression gene validation performed on GSE189553 database.
Fig. 6 is a representative image in a tissue chip.
Detailed Description
The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.
Example 1 screening for novel signature molecular markers in OCCC and HGSC
All patients received surgical resection in Beijing co-ordination and all patients had no treatment prior to surgery. Patients with mixed cancer, ovarian and uterine double primary cancer were excluded. All patients were diagnosed by 3 experienced pathologists, each according to the 2020 world health organization classification, and were staged (typed) using the NCCN ovarian cancer guidelines (Version 3.2022). The study was approved by the ethical committee of Beijing co-ordinates, hospital (JS-3553). All patients had informed consent.
The present invention collects tumor biopsies from the above 5 OCCC patients and 5 HGSC patients, detailed clinical information is shown in Table 1, and representative H & E and IHC staining images are shown in FIG. 1.
TABLE 1 clinical pathological characteristics of patients in single cell sequencing
Note that: tumor size is a measure of the longest axis of the primary tumor. The size of bilateral tumors is listed as the size of left tumor/the size of right tumor.
Tumor biopsies of the above patients were subjected to 3'sc RNA-seq, library construction using Chromium Next GEM Single Cell 3'GEM,Library&Gel Bead Kit v3.1 (10 x Genomics, USA) and single cell sequencing using Illumina Nova Seq 6000 (norstanding, beijing, china). After filtering out low quality cells and diploids we integrated all 10 samples into one gene expression matrix for a total of 101,672 cells, with an average of 2000 genes per cell.
Raw sequencing data from the above 10 patients were processed according to the GRCh38 human reference genome, according to chromanum's Cell Ranger pipeline (version 4.0.0). The data was filtered from individual cell counts in all samples using the setup analysis package in R (v4.1.0) and individual setup objects were created. We further pooled all samples and used a typical correlation analysis (CCA) for dimension reduction (dimension) and batch effect removal (batch effect). Standard unit clustering was then performed using the Scale Data function, principal Component Analysis (PCA) dimensions were calculated using the RunPCA function, and unsupervised clustering was performed on the Data using the Find Neighbors and Find Cluster functions (unsupervised clustering analysis).
Cell visualization by RunUMAP function visible cell aggregation into 3 main populations: epithelial (epihelial), immune (Immune) and stroma (Stromal).
Analysis of the Differential Expression Genes (DEGs) of the epithelial cell populations (epithelial subpopulations) of the above 5 OCCC patients and 5 HGSC patients showed that AOC1 was significantly higher in OCCC than HGSC (fig. 3).
Example 2, RT-PCR and Western blot to verify differential expression of AOC1
RT-PCR detection: total RNA was extracted using TRIZOL reagent (Thermo Fisher Scientific) and reverse transcribed into cDNA using Prime ScriptTM RT Master Mix (TAKARA Co., japan). Real-Time fluorescent quantitative PCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific) and Quant studio 7Flex Real-Time PCR System (Thermo Fisher Scientific). The gene expression uses GAPDH as an internal reference, and the primers of the AOC1 are as follows:
AOC1-F：5′-CCTAAGCAACCAAGAGCTGAA-3′，
AOC1-R：5′-CGGTGACATTGGGATGCTCC-3′
western blot detection: cells were lysed with RIPA lysate (bejing aoqing biotechnology limited) containing Prot Lystic Protease Inhibitor Cocktail (New cell & Molecular Biotech), BCA Assay Kit (Pierce Biotechnology, USA) to determine protein concentration. Cell lysates were separated by Nu PAGETM 4-12% gel (Thermo Fisher Scientific) and transferred to polyvinylidene difluoride (PVDF) (Millipore, USA) membranes. After blocking the membrane, it is incubated with primary antibody and then with secondary antibody. The bands were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Peirce).
The detection result is consistent with the sc RNA-seq sequencing analysis result, the expression of AOC1 in OCCC is obviously higher than that of HGSC, and the result is shown in figure 4.
Example 3 verification of the Distinguishing function of AOC1 in GEO database
Gene expression profiles of primary ovarian cancer specimens (OCCC, n=11, HGSC, n=8) in GSE189553 were obtained in the GEO database (http:// www.ncbi.nlm.nih.gov/GEO /), and the limma package (v3.50.3) was used to identify Differentially Expressed Genes (DEGs) between OCCC samples and HGSC samples. The analysis showed that AOC1 was expressed higher in OCCC than HGSC, consistent with the foregoing (FIG. 5).
Example 4 verification of the Distinguishing function of AOC1 in clinical samples
Collection of 128 OCCCs and 81 HGSCs patients constructed 2 tissue chips (TMAs). A representative image is shown in fig. 6. The above patients included patients who underwent surgical excision in Beijing co-ordination hospitals from 1 month 2019 to 5 months 2022 and had sufficient archived tissue for immunohistochemical detection. Finally, 128 OCCC patients and 81 HGSC patients were included. Clinical information is shown in Table 2.
Table 2 clinical features of inclusion queue in validation
Note that: the relapse time is defined as the number of months between the first surgery and the time of relapse, considering only patients who relapse.
Two tissue chips were constructed from formalin-fixed, paraffin-embedded tissue. IHC staining procedure was performed as per standard with DAKO Autostainer Link and AOC1 as primary antibody ab278497 (Abcam).
AOC1 expression in cytoplasm and cell membrane (positive control with cells at kidney and negative control with matrix cells) was detected in immunohistochemical staining, and (0-12) was scored comprehensively according to the intensity and percentage of positive cells. Cases with a composite score of 4 or more were considered positive, and other cases were considered negative.
It was calculated that the Sensitivity (Sensitivity) was 62.5%, the Specificity (Specificity) was 88.9%, PPV (positive predictive value) 89.9.89.9% and the NPV (negative predictive value) was 60.0% in the discrimination between OCCC and HGSC by AOC 1.
Table 3 comparison of immunohistochemical results for OCCC and HGSC
Note that: the number is the number (percentage) of patients positive for immunohistochemical staining, and a is statistically significant.
Claims (10)
1. Use of an agent that detects the amount of AOC1 expression in the manufacture of a product for determining the type of cancer, said type being ovarian clear cell cancer or high grade serous ovarian cancer;
preferably, the expression level includes an mRNA expression level or a protein expression level.
2. The use of claim 1, wherein the reagent comprises a reagent for detecting the mRNA expression level of AOC1, a reagent for detecting the expression level of AOC1 protein, or a reagent for detecting the number of AOC1 positive expressing cells;
further, the reagent for detecting the mRNA expression level of the AOC1 comprises a reagent for detecting the mRNA expression level of the AOC1 by adopting the following method: PCR-based detection methods, southern hybridization methods, northern hybridization methods, dot hybridization methods, fluorescent in situ hybridization methods, DNA microarray methods, ASO methods, high throughput sequencing platform methods, chip methods;
further, the reagent for detecting the expression level of the AOC1 protein comprises a reagent for detecting the expression level of the AOC1 protein by adopting the following method: western blot, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescence resolution technique, and protein chip method;
preferably, the reagent comprises:
a primer pair, probe or antisense nucleotide that specifically binds to a gene of AOC 1; or alternatively, the first and second heat exchangers may be,
an antibody, ligand, nanoparticle or aptamer that specifically binds to an AOC1 protein or peptide fragment.
3. The use of claim 1, wherein the assay is an assay performed on a sample from a subject;
preferably, the sample comprises: tissue, cell culture, blood, urine, saliva, mucosal samples, stool, intestinal lavage, joint fluid, cerebrospinal fluid, bile samples, respiratory secretions, bronchoalveolar lavage samples;
more preferably, the sample comprises a tissue or cell culture;
more preferably, the subject of the invention is a patient with ovarian epithelial cancer, or the subject is a patient with suspected ovarian clear cell cancer, a patient with high grade serous ovarian cancer;
preferably, the product comprises a kit, a chip and a test strip.
4. The use according to claim 1, wherein the product further comprises an mRNA expression level-assisted detection reagent and/or a protein expression level-assisted detection reagent;
preferably, the mRNA expression level-assisting detection reagent includes: reagents for visualizing the amplicon corresponding to the primer, for example, reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, or the like; an RNA extraction reagent; a reverse transcription reagent; cDNA amplification reagents; preparing a standard substance used for a standard curve; a positive control;
preferably, the protein expression level-assisted detection reagent includes, but is not limited to: blocking solution, antibody dilution, washing buffer, chromogenic stop solution, and standard for preparing standard curve.
5. A device/system for determining a cancer type, the device/system comprising a judgment means which performs the following judgment criteria: the test sample has an AOC1 expression level higher than the threshold value, and is judged to be from an ovarian clear cell carcinoma patient, otherwise, from a high-grade serous ovarian carcinoma patient.
6. The device/system of claim 5, further comprising one or more of a detection means, an input means, an output means, and a communication means.
7. The device/system of claim 6, wherein the detection means is for detecting the amount of AOC1 expressed in a subject sample.
8. The apparatus/system according to claim 6, wherein the input module is configured to input an expression level of AOC1, and the expression level of AOC1 is detected by the detecting means.
9. The apparatus/system according to claim 6, wherein the output module is configured to output a determination result of the determination means.
10. A method of distinguishing between ovarian clear cell carcinoma cells and high-grade serous ovarian carcinoma cells, the method comprising determining whether an ovarian clear cell carcinoma cell or a high-grade serous ovarian carcinoma cell is based on the amount of AOC1 expressed in the cell;
preferably, the method is of non-diagnostic interest.
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