CN116716404A - Device for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A2 - Google Patents

Device for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A2 Download PDF

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CN116716404A
CN116716404A CN202310697259.5A CN202310697259A CN116716404A CN 116716404 A CN116716404 A CN 116716404A CN 202310697259 A CN202310697259 A CN 202310697259A CN 116716404 A CN116716404 A CN 116716404A
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sample
expression level
clear cell
grade serous
cell carcinoma
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CN116716404B (en
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孙健
郭丹
王姝
张素梅
高艺珂
王安琪
李梅
梁智勇
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Peking Union Medical College Hospital Chinese Academy of Medical Sciences
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Abstract

The invention belongs to the field of biological medicine, and particularly relates to a device for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A 2. In particular, the present invention provides an apparatus for determining a cancer type, the apparatus comprising: a memory and a processor; the memory is used for storing program instructions; the processor is configured to invoke program instructions that, when executed, are configured to: S100A2 expression data of the sample to be detected are obtained, and the typing result of the sample to be detected is judged by comparing the S100A2 expression data with a threshold value.

Description

Device for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A2
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to equipment for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A 2.
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 more prevalent in asians than in other ethnicities, and 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 present invention provides an apparatus for determining a cancer type, the apparatus comprising: a memory and a processor;
the memory is used for storing program instructions;
the processor is configured to invoke program instructions that, when executed, are configured to: S100A2 expression data of the sample to be detected are obtained, and the typing result of the sample to be detected is judged by comparing the S100A2 expression data with a threshold value.
More specifically, the typing result is that the sample to be tested is judged to be derived from an Ovarian Clear Cell Carcinoma (OCCC) patient or a high-grade serous ovarian carcinoma (HGSC) patient, and when the S100A2 expression data is lower than a threshold value, the sample to be tested is judged to be derived from the Ovarian Clear Cell Carcinoma (OCCC) patient, and when the S100A2 expression data is higher than the threshold value, the sample to be tested is judged to be derived from the high-grade serous ovarian carcinoma (HGSC) patient.
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.
As used herein, memory includes one or more of the following: a non-transitory computer readable medium, a magnetic disk or other magnetic storage medium, an optical disk or other optical storage medium, a Random Access Memory (RAM), a Read Only Memory (ROM) or other electronic memory device or chip, or a collection of chips that are operably interconnected; an internet/intranet server from which the stored instructions may be retrieved via an internet/intranet or local area network; etc. Exemplary include various memory disks, memory sticks, memory cards, memory modules, and the like.
As used herein, a processor includes one or more of a microprocessor, microcontroller, graphics Processing Unit (GPU), application Specific Integrated Circuit (ASIC), field Programmable Gate Array (FPGA), or the like.
In another aspect, the present invention provides a system/device for determining a cancer type, the system/device comprising:
the acquisition unit is used for acquiring S100A2 expression data of the sample to be detected;
and a judging unit for comparing the S100A2 expression data with the threshold value and judging the parting result.
Preferably, the system/device may further comprise a detection unit detecting the S100A2 expression level.
Preferably, the detection unit can detect by a real-time quantitative PCR instrument, a high throughput sequencing platform, a detection chip, a chip signal reader, and the like.
Preferably, the chip includes a probe for detecting the S100A2 expression level.
Preferably, the chip comprises a protein chip and/or a gene chip.
Preferably, the system/device further includes an evaluation result transmitting unit that can transmit the typing result of the judging unit to an information communication terminal device that the patient or the medical staff can refer to.
Preferably, the system/device further comprises a user input device, a display device.
Preferably, the user input device is used for inputting personal information of the subject.
Preferably, the display device is used for displaying the conclusion drawn by the computing device.
To provide for interaction with a user, the systems and techniques described here can be implemented on a computer having: display means for displaying information to a user; and a keyboard and pointing device (e.g., a mouse) through which a user can provide input to the computer. Other kinds of devices may also be used to provide for interaction with a user; for example, feedback provided to the user may be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback); and input from the user may be received in any form, including acoustic input, speech input, or tactile input.
It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps recited in the present disclosure may be performed in parallel or sequentially or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.
In another aspect, the present invention provides a computer readable storage medium having stored thereon a computer program which when executed by a processor performs the following operations:
S100A2 expression data of the sample to be detected are obtained, and the typing result of the sample to be detected is judged by comparing the S100A2 expression data with a threshold value.
In the context of this document, a computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device. The computer readable storage medium may be, for example, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination thereof.
As used herein, the terms "sample," "test sample" may be any biological sample isolated from a subject. For example, the sample may include, but is not limited to, body fluids, whole blood, platelets, serum, plasma, stool, erythrocytes, leukocytes or leukocytes, endothelial cells, tissue, synovial fluid, lymph, ascites, interstitial or extracellular fluid, fluid in the intercellular space, including gingival crevicular fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine, nasal brush fluid, pap smear fluid, or any other body fluid. The body fluid may include saliva, blood or serum.
The sample to be tested according to the invention is preferably a tissue, in particular a biopsy tissue sample or a tissue culture or a cell derived therefrom. The source of tissue may be solid tissue, such as from fresh, frozen and/or preserved organs or tissue samples, biopsy tissue or aspirates.
Specifically, the sample to be tested is collected from a subject; preferably, the subject is a patient with ovarian epithelial cancer (epithelial ovarian carcinoma, EOC), or the subject is a patient with suspected Ovarian Clear Cell Cancer (OCCC), high grade serous ovarian cancer (HGSC).
The term "determining the type of cancer" according to the present invention is understood to mean determining the type of ovarian epithelial cancer (epithelial ovarian carcinoma, EOC), as a result of which the type is Ovarian Clear Cell Carcinoma (OCCC) or high grade serous ovarian carcinoma (HGSC).
The "S100A2 expression data" as used herein refers to the amount of the polynucleotide of S100A2 or the amino acid product or protein of S100A2 in a sample, and may be referred to as "S100A2 expression level" or "S100A2 expression level".
In another aspect, the invention provides the use of a reagent for detecting the amount of S100A2 expression in the preparation of a product for determining the type of cancer.
In particular, the typing is 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.
Preferably, the reagent comprises:
a reagent for detecting the mRNA expression level of S100A2 in a sample;
a reagent for detecting the protein expression level of S100A2 in a sample; or (b)
And (3) detecting the number of S100A2 positive expression cells in the sample.
Further, the product comprises a detection kit and a biochip.
Further, the detection kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent.
Further, the detection kit includes: RT-PCR detection kit, ELISA detection kit, protein chip detection kit, rapid detection kit, DNA chip detection kit, immunohistochemical detection kit, or MRM (multiple reaction monitoring) detection kit.
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 patient is from an Ovarian Clear Cell Carcinoma (OCCC) patient or a high grade serous ovarian carcinoma (HGSC) patient based on the amount of S100A2 expressed in a sample from the subject.
In another aspect, the invention provides a method for differentiating between ovarian clear cell carcinoma cells and high-grade serous ovarian carcinoma cells, the method comprising determining whether the ovarian clear cell carcinoma cells or the high-grade serous ovarian carcinoma cells are in accordance with the expression level of S100A2 in the test cells.
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 S100A2 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 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 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 Differentially Expressed Genes (DEGs) of the epithelial cell populations (epithelial subpopulations) of the above 5 OCCC patients and 5 HGSC patients showed that S100A2 was significantly lower in OCCC than HGSC (fig. 3).
Example 2, RT-PCR and Western blot to verify differential expression of S100A2
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 S100A2 are as follows:
S100A2-F：5′-TGCCAAGAGGGCGACAAGTTCA-3′，
S100A2-R：5′-AAGTCCACCTGCTGGTCACTGT-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 S100A2 in OCCC is obviously lower than that of HGSC, and the result is shown in FIG. 4.
Example 3 verification of the Distinguishing function of S100A2 in clinical samples
Collection of 128 OCCCs and 81 HGSCs patients constructed 2 tissue chips (TMAs). A representative image is shown in fig. 5. 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, the primary antibody of S100A2 being ab109494 (Abcam).
The expression of S100A2 in cytoplasm and cell membrane (with cells at esophagus as positive control and matrix cells as negative control) was detected in immunohistochemical staining, and (0-12) was comprehensively scored 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.
Table 3 comparison of immunohistochemical results for OCCC and HGSC
Note that: the number is the number (percentage) of patients positive for immunohistochemical staining, a is statistically significant
It was calculated that the Sensitivity (Sensitivity) was 33.3%, the Specificity (Specificity) was 96.9%, PPV (positive predictive value) 87.1.1% and the NPV (negative predictive value) was 69.7% in the discrimination between OCCC and HGSC by S100A 2.

Claims (10)

1. An apparatus for determining a cancer type, the apparatus comprising: a memory and a processor;
the memory is used for storing program instructions;
the processor is configured to invoke program instructions that, when executed, are configured to: S100A2 expression data of the sample to be detected is obtained, the typing result of the sample to be detected is judged by comparing the S100A2 expression data with a threshold value,
and the typing result is that the sample to be tested is judged to be derived from an ovarian clear cell carcinoma patient or a high-grade serous ovarian carcinoma patient.
2. A system/device for determining a cancer type, the system/device comprising:
the acquisition unit is used for acquiring S100A2 expression data of the sample to be detected;
a judging unit comparing the S100A2 expression data with the threshold value, judging the parting result,
and the typing result is that the sample to be tested is judged to be derived from an ovarian clear cell carcinoma patient or a high-grade serous ovarian carcinoma patient.
3. The system/apparatus according to claim 2, further comprising a detection unit that detects an S100A2 expression amount;
preferably, the detection unit detects through a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip, chip signal reading and the like;
preferably, the chip comprises a protein chip and/or a gene chip.
4. The system/apparatus according to claim 2, further comprising an evaluation result transmitting unit that transmits the typing result of the judging unit to an information communication terminal apparatus that can be referred to by the patient or the medical staff;
preferably, the system/device further comprises a user input device, a display device;
preferably, the user input device is used for inputting personal information of a subject;
preferably, the display device is used for displaying the conclusion drawn by the computing device.
5. A computer readable storage medium having stored thereon a computer program which when executed by a processor performs the operations of:
S100A2 expression data of a sample to be detected is obtained, and the parting result of the sample to be detected is judged by comparing the S100A2 expression data with a threshold value;
and the typing result is that the sample to be tested is judged to be derived from an ovarian clear cell carcinoma patient or a high-grade serous ovarian carcinoma patient.
6. Use of a reagent for detecting the expression level of S100A2 in the preparation of a product for determining the type of cancer, the type resulting in ovarian clear cell carcinoma or high-grade serous ovarian carcinoma;
preferably, the expression level includes an mRNA expression level or a protein expression level;
preferably, the reagent comprises:
a reagent for detecting the mRNA expression level of S100A2 in a sample;
a reagent for detecting the protein expression level of S100A2 in a sample; or (b)
And (3) detecting the number of S100A2 positive expression cells in the sample.
7. The use according to claim 6, wherein the product comprises a detection kit, a biochip;
further included in the product is one or more selected from the group consisting of: a container, instructions for use, positive control, negative control, buffer, adjuvant, or solvent.
8. The use of claim 7, the detection kit comprising: RT-PCR detection kit, ELISA detection kit, protein chip detection kit, rapid detection kit, DNA chip detection kit, immunohistochemical detection kit, or MRM detection kit.
9. The use according to claim 6, wherein the S100A2 expression level is detected for a sample from a subject;
preferably, the sample is tissue;
preferably, the tissue comprises a biopsy tissue sample or a tissue culture or cell derived therefrom;
preferably, the tissue comprises fresh, frozen and/or preserved tissue.
10. A method for distinguishing ovarian clear cell carcinoma cells from high-grade serous ovarian carcinoma cells, the method comprising judging whether the ovarian clear cell carcinoma cells or the high-grade serous ovarian carcinoma cells are the ovarian clear cell carcinoma cells or the high-grade serous ovarian carcinoma cells according to the expression level of S100A2 in the cells to be detected;
preferably, the expression level includes an mRNA expression level or a protein expression level;
preferably, the reagent comprises:
a reagent for detecting the mRNA expression level of S100A2 in a sample;
a reagent for detecting the protein expression level of S100A2 in a sample; or (b)
And (3) detecting the number of S100A2 positive expression cells in the sample.
CN202310697259.5A 2023-06-13 2023-06-13 Device for distinguishing ovarian clear cell carcinoma from high-grade serous carcinoma based on S100A2 Active CN116716404B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010132958A1 (en) * 2009-05-22 2010-11-25 Garvan Institute Of Medical Research Methods for predicting responsiveness to treatment
GB201607393D0 (en) * 2016-04-28 2016-06-15 Isis Innovation Biomarkers for early diagnosis of ovarian cancer
CN116129998A (en) * 2023-01-19 2023-05-16 中国医学科学院肿瘤医院 Esophageal squamous cell carcinoma data processing method and system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010132958A1 (en) * 2009-05-22 2010-11-25 Garvan Institute Of Medical Research Methods for predicting responsiveness to treatment
GB201607393D0 (en) * 2016-04-28 2016-06-15 Isis Innovation Biomarkers for early diagnosis of ovarian cancer
CN116129998A (en) * 2023-01-19 2023-05-16 中国医学科学院肿瘤医院 Esophageal squamous cell carcinoma data processing method and system

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