Biomarker for molecular typing of cervical small cell carcinoma and application of biomarker

Abstract

The invention relates to the technical field of medical detection, in particular to a biomarker for molecular typing of cervical small cell carcinoma and application of the biomarker. The biomarkers are SCG3 and YAP1 (Yttrium Activated Protein 1). The molecular typing marker gene of the cervical small cell carcinoma is screened, the survival prognosis risk of a patient can be predicted through an immunohistochemical experiment, and the risk prevention of the patient after an operation and chemotherapy is better prompted.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to biomarkers for molecular subtyping of cervical small cell carcinoma and their applications. Background Technology

[0002] Small cell carcinoma of the cervix (SCCC) is a highly malignant, rapidly progressing gynecological malignancy with a low incidence rate. Due to its low incidence, large-scale biological and clinical studies are often difficult to conduct, resulting in a lack of comprehensive understanding of its biological characteristics and clinical treatment response. Current treatment for SCCC mainly references the radiotherapy and chemotherapy regimens for small cell lung cancer and the surgical procedures for cervical adenocarcinoma and squamous cell carcinoma, using FIGO staging as the indicator for surgical approach selection.

[0003] The diagnosis of small cell cervical cancer is based on pathological examination, which is considered the gold standard. Under a light microscope, tumor cells appear round, oval, or spindle-shaped, with sparse cytoplasm, dense nuclear chromatin, and inconspicuous nucleoli. Cells grow in sheets, nests, or cords, with extensive necrosis and high mitotic activity. Immunohistochemical detection is the core method for differentiating small cell cervical cancer from other poorly differentiated tumors, and various neuroendocrine markers such as Syn, CgA, CD56, and NSE are molecular markers specifically expressed in small cell cervical cancer.

[0004] However, there is currently a lack of effective methods and approaches for prognostic subtyping of cervical small cell carcinoma. In general, the lack of effective molecular subtyping and related biological research leads to limited and vague treatment options for cervical small cell carcinoma, resulting in poor patient survival. Therefore, this invention provides biomarkers for molecular subtyping of cervical small cell carcinoma and their applications. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide biomarkers for molecular subtyping of cervical small cell carcinoma and their applications. The aim is to perform molecular subtyping of cervical small cell carcinoma based on the differential expression of genes SCG3 and YAP1, and to clarify their predictive value for the survival prognosis of patients with cervical small cell carcinoma.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: Firstly, biomarkers for molecular subtyping of cervical small cell carcinoma, wherein the biomarkers are SCG3 and YAP1.

[0007] Based on the above technical solution, the present invention can be further improved as follows.

[0008] Secondly, the application of reagents and / or instruments for detecting the expression levels of the biomarkers used for molecular subtyping of cervical small cell carcinoma in the preparation of the product.

[0009] Furthermore, the product is used for prognostic assessment of survival in patients with small cell cervical cancer.

[0010] Thirdly, a product for assessing the prognostic survival of patients with small cell cervical cancer, the product comprising reagents and / or instruments for detecting the expression levels of the biomarkers used for molecular subtyping of small cell cervical cancer.

[0011] Furthermore, the product includes a reagent kit.

[0012] Fourthly, the application of the biomarkers used for molecular subtyping of cervical small cell carcinoma in constructing a prognostic assessment model for the survival of patients with cervical small cell carcinoma.

[0013] Fifthly, a model for assessing the prognostic survival of patients with small cell cervical cancer, the model being constructed based on the expression levels of the biomarkers used for molecular subtyping of small cell cervical cancer; The model is as follows: H-score = intensity grade * percentage of positive area to total tumor area; where H-score ranges from 0 to 300; intensity grade is automatically graded according to color depth; where intensity grade is: 0 - negative; 1 - weak positive, light yellow; 2 - positive, brown; 3 - strong positive, dark brown.

[0014] Furthermore, the expression level of the biomarker was determined by immunohistochemical experiments.

[0015] Furthermore, if SCG3_H_score > YAP1_H_score, then the subtype is SCG3. High / YAP1 Low This indicates a poorer prognosis and shorter overall survival for the subjects. If SCG3_H_score < YAP1_H_score, then the subtype is SCG3. Low / YAP1 High This indicates that the subjects had a better prognosis and a longer overall survival.

[0016] Sixthly, a system for assessing the prognostic survival of patients with small cell cervical cancer, the system comprising: (1) Analysis unit, wherein the analysis unit comprises reagents and / or instruments for detecting the expression level of the biomarkers used for molecular subtyping of cervical small cell carcinoma; (2) Assessment unit, the assessment unit comprising: assessing the survival prognosis of the subject based on the expression level of the biomarker for molecular subtyping of cervical small cell carcinoma determined in step (1) and the model for prognostic assessment of survival of patients with cervical small cell carcinoma.

[0017] The beneficial effects of this invention are: (1) This invention discovered and confirmed that the expression of SCG3 protein and YAP1 protein in SCECC tissue is negatively correlated, and used this pair of combined markers to distinguish cervical small cell carcinoma subtypes with distinct molecular characteristics and clinical prognosis; overcoming the deficiency of existing technologies in lacking effective molecular subtyping markers for cervical small cell carcinoma, and providing the first simple and effective dual-marker subtyping scheme that can be used to guide clinical practice of cervical small cell carcinoma.

[0018] (2) This invention establishes a comparative interpretation rule based on the relative abundance of SCG3 and YAP1 protein expression, that is, to determine whether the H-score of SCG3 is greater than the H-score of YAP1, rather than relying on a single, absolute expression threshold; it simplifies the complex transcriptome discovery into a low-cost immunohistochemical (IHC) detection method that can be implemented in a general pathology department, which greatly enhances the clinical translation and application value of the technology.

[0019] (3) This invention clearly associates the “SCG3-High / YAP1-Low” subtype with enhanced neuroendocrine phenotype, activation of cell cycle pathways and extremely poor clinical prognosis; and associates the “SCG3-Low / YAP1-High” subtype with cell adhesion, activation of immune pathways and better clinical prognosis; it not only provides prognostic information, but also reveals potential therapeutic targets (such as cell cycle inhibitors that can be recommended for high-risk subtypes), providing a biological basis for personalized treatment. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the process of the present invention; Figure 2 This is a graph showing the regression analysis results of this invention; Figure 3 This is a diagram of immune cell infiltration analysis obtained by immunohistochemical staining according to the present invention; Figure 4 This is a diagram showing the results of the immune cell infiltration analysis of this invention; Figure 5 Immunohistochemical staining images of the candidate typing markers of this invention; Figure 6 This is a comparison of the color scores of SCG3 and YAP1 in the thermal imaging samples of this invention. Figure 7 This is a graph showing the survival analysis results of the present invention. Detailed Implementation

[0021] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0022] This embodiment relates to biomarkers for molecular subtyping of cervical small cell carcinoma, wherein the biomarkers are SCG3 and YAP1.

[0023] This embodiment also relates to the application of reagents and / or instruments for detecting the expression levels of the biomarkers used for molecular subtyping of cervical small cell carcinoma in the preparation of the product.

[0024] Preferably, the product described in this embodiment is used for prognostic assessment of survival in patients with small cell cervical cancer.

[0025] This embodiment also relates to a product for assessing the prognostic survival of patients with small cell cervical cancer, the product comprising reagents and / or instruments for detecting the expression levels of the biomarkers used for molecular subtyping of small cell cervical cancer.

[0026] Preferably, the product described in this embodiment includes a reagent kit.

[0027] This embodiment also relates to the application of the biomarkers described above for molecular subtyping of cervical small cell carcinoma in constructing a survival prognostic assessment model for patients with cervical small cell carcinoma.

[0028] This embodiment also relates to a model for assessing the prognostic survival of patients with small cell cervical cancer, the model being constructed based on the expression levels of the biomarkers used for molecular subtyping of small cell cervical cancer; The model is as follows: H-score = intensity grade * percentage of positive area to total tumor area; where H-score ranges from 0 to 300; intensity grade is automatically graded according to color depth; where intensity grade is: 0 - negative; 1 - weak positive, light yellow; 2 - positive, brown; 3 - strong positive, dark brown.

[0029] Preferably, in this embodiment, the expression level of the biomarker is determined by immunohistochemical experiments.

[0030] Preferably, in this embodiment, if SCG3_H_score > YAP1_H_score, then the subtype is SCG3. High / YAP1 Low This indicates a poorer prognosis and shorter overall survival for the subjects. If SCG3_H_score < YAP1_H_score, then the subtype is SCG3. Low / YAP1 High This indicates that the subjects had a better prognosis and a longer overall survival.

[0031] This embodiment also relates to a system for assessing the prognostic survival of patients with small cell cervical cancer, the system comprising: (1) Analysis unit, wherein the analysis unit comprises reagents and / or instruments for detecting the expression level of the biomarkers used for molecular subtyping of cervical small cell carcinoma; (2) Assessment unit, the assessment unit comprising: assessing the survival prognosis of the subject based on the expression level of the biomarker for molecular subtyping of cervical small cell carcinoma determined in step (1) and the model for prognostic assessment of survival of patients with cervical small cell carcinoma.

[0032] In summary, this invention clarifies that there are two subtypes of cervical small cell carcinoma that differ significantly in molecular biological expression and clinical prognosis; it screens and validates a method for distinguishing cervical small cell carcinoma subtypes using transcriptome sequencing abundance and immunohistochemical staining intensity as data indicators for the genes SCG3 and YAP1; and it analyzes the indicative significance of SCG3 and YAP1 as molecular subtypes for the survival prognosis of cervical small cell carcinoma patients. Specific examples are further illustrated below.

[0033] Example 1. Screening and validation of molecular markers ( Figure 1 ).

[0034] 1.1 Research Sample and Data Collection: In this study, patients diagnosed with cervical small cell carcinoma from 2003 to 2015 from 12 hospitals in China were searched in 2016. Clinicopathological information of 265 cervical small cell carcinoma patients was transcribed, and paraffin-embedded tumor samples from 214 cervical small cell carcinoma cases and 19 fresh tumor samples were collected. Transcriptome sequencing (Illumina HiSeq 2500 sequencing platform) and expression analysis were performed on the 19 fresh cervical small cell carcinoma tumor samples in the same year. All transcriptome sequencing data passed raw data quality checks, data cleaning and filtering, alignment quality assessment, and gene expression level assessment, and the raw data were uploaded to GSA (GSA-Human: HRA002655). In this study, all paraffin-embedded samples and fresh tumor tissues were examined and confirmed by at least two pathologists. The inclusion criteria for samples included in the survival analysis were: 1. Pathological diagnosis of small cell cervical cancer and FIGO stage (FIGO 2009) of IA-IIB; 2. Age between 18 and 79 years; 3. Patients who have undergone treatment in accordance with the NCCN cervical cancer guidelines.

[0035] 1.2 Subtyping of cervical small cell carcinoma using nonnegative matrix classification: Transcriptome sequencing data from 19 cases of cervical small cell carcinoma were analyzed, and two subtypes of cervical small cell carcinoma were distinguished by nonnegative matrix fractionation (NMF). The two subtypes were cervical small cell carcinoma subtype A and cervical small cell carcinoma subtype B.

[0036] 1.3 Screening and Validation Methods: Subtypes were grouped, and differential expression analysis and linear regression models were used to screen out SCG3 and YAP1 as molecular markers to distinguish cervical small cell carcinoma subtypes. In the two subtypes of cervical small cell carcinoma samples, the expression of SCG3 and YAP1 was mutually exclusive, that is, when SCG3 was highly expressed in cervical small cell carcinoma subtype A, YAP1 expression was low; when SCG3 was low in cervical small cell carcinoma subtype B, YAP1 expression was high.

[0037] The expression patterns of these two genes were identified in the transcriptome data of 19 SCCC samples, and further immunohistochemical staining of paraffin sections verified the consistency between protein expression and messenger RNA expression. Based on this, the two subtypes were named cervical small cell carcinoma SCG3. High / YAP1 Low and cervical small cell carcinoma SCG3 Low / YAP1 High .

[0038] The molecular biological characteristics of these two subtypes have been clarified. (SCG3 in cervical small cell carcinoma) High / YAP1 Low Compared to another subtype, this sample was characterized by high neuroendocrine expression, suppressed immune pathway expression, and significantly active cell cycle and nucleic acid replication-related pathways. Cervical small cell carcinoma SCG3... Low / YAP1 High The opposite is true; cell adhesion-related pathways are upregulated, and immune-related pathways are actively expressed.

[0039] The gene screening and validation methods include the following specific steps: (1) Using the overall data of transcriptome expression in 19 cases of cervical small cell carcinoma, we screened genes with the highest TPM expression level and the highest TPM standard deviation. The intersection of the two sets of genes with high expression and large expression differences among samples was obtained. (2) Through differential expression analysis (R package: limma), we screened genes with statistical significance between the two subtypes obtained by non-negative matrix factorization, and obtained gene set 2 by taking the intersection with gene set 1 above. This gene set is the gene with significant expression difference between the two subtypes, relatively high expression level in cervical small cell carcinoma samples, and large expression fluctuation between samples. (3) Based on the subtypes obtained by nonnegative matrix factorization, the gene expression values ​​of the screened genes were subjected to linear regression model. The genes that were significant in distinguishing subtypes in the model analysis included AP3B2, CHI3L1, GADD45B, IER3, NCAM1, PRR15L, RUNDC3A, SCG3, SULT4A1, and UNC13A.

[0040] (4) Based on the comprehensive differential expression analysis results, combined with pathway analysis between the two groups, gene functions were linked and queried to further screen YAP1, SCG3, and CHI3L1 as candidate subtyping marker genes. Immunohistochemical staining was performed to verify the consistency between gene messenger ribonucleotide expression and protein expression, clarifying SCG3 and YAP1 as molecular markers for subtyping cervical small cell carcinoma. The distribution of immunohistochemical staining between the two groups was mutually exclusive, which could significantly distinguish different subtypes of tumor cells in tumor tissue. Figure 2 ).

[0041] To clarify the differences in the tumor immune microenvironment between the two subtypes, deconvolution analysis was first used to analyze the immune cell infiltration of transcriptome data from 19 cases of cervical small cell carcinoma. The results showed that SCG3 in cervical small cell carcinoma... Low / YAP1 High The number of CD8+ T cells in the sample was significantly higher than that in cervical small cell carcinoma SCG3. High / YAP1 Low Samples. Immunohistochemical staining for SCG3, YAP1, and CD8A was used to identify SCG3 in cervical small cell carcinoma. Low / YAP1 High In the sample, CD8+ T cells infiltrated the tumor margins and center, while SCG3 was present in small cell cervical cancer. High / YAP1 Low CD8+ T cell infiltration was almost absent in the tumor area of ​​the sample. This confirms the biological significance of SCG3 and YAP1 as molecular markers in differentiating the immune invasive status of cervical small cell carcinoma. Figures 3 to 5 ).

[0042] 2. The prognostic value of SCG3 and YAP1 as molecular subtyping for patients with small cell cervical cancer.

[0043] An immunohistochemical antibody kit and its application procedure for using SCG3 and YAP1 as molecular subtyping markers were confirmed. Furthermore, by expanding the sample size and applying the kit to paraffin section staining, the prognostic significance of SCG3 and YAP1 as molecular subtyping markers for patients with small cell cervical cancer was clarified.

[0044] 2.1 Molecular subtyping of small cell cervical cancer based on differential expression of SCG3 and YAP1 genes: Ninety-two cervical small cell carcinoma samples with well-preserved paraffin sections were selected for immunohistochemical staining with SCG3 and YAP1. Molecular subtyping was performed based on the immunohistochemical staining results. The immunohistochemical staining and scoring process indicators are as follows: (1) Immunohistochemical staining: Baking (65°C, 0.5 h); dewaxing (three times, 5 minutes each); hydration (100%, 95%, 75% ethanol, 5 minutes each); antigen retrieval (1 mM EDTA: Cat#G1203, Servicebio, 50°C, 10 minutes, then 30°C, 10 minutes); blocking endogenous peroxidase activity (3% H2O2, 25 minutes), followed by phosphate buffer washing; blocking (3% bovine serum albumin Cat: G5001, Servicebio, room temperature, 0.5 h); primary antibody incubation (room temperature, 1 h; YAP1: CST, Cat#14074, dilution 1:200; SCG3 10954-1-AP, dilution 1:200); thorough cleaning followed by secondary antibody incubation (Cat: G1211, Servicebio; room temperature, 1 h); TBST buffer washing three times; hematoxylin staining; air drying and mounting.

[0045] (2) Immunohistochemical score: ① Data Acquisition (Digital Image Acquisition): Tissue sections stained with SCG3 and YAP1 antibodies are placed under a digital pathology scanner. The scanner captures a full-field digital image of the entire tissue section and stores it as an image file at high resolution (e.g., 40x). ② Data Processing (Image Analysis and Quantification, Protein Expression Quantification: Within the identified tumor regions, SCG3 and YAP1 staining were analyzed separately. For each gene: Positive Judgment: Based on the RGB or optical density values ​​of the tannin-absorbed (DAB) area, a threshold was set to determine whether each cell nucleus or cytoplasm was positive. Intensity Grading: Positive areas were automatically graded according to color depth (typically 0 – negative, 1 – weakly positive, light yellow, 2 – positive, brown, 3 – strongly positive, dark brown). H-score Calculation: The algorithm automatically calculated H-score = intensity grade * percentage of positive area to total tumor area, ranging from 0-300; ③ Subtyping: Read the SCG3 H-score and YAP1 H-score of the current sample. If SCG3_H_score > YAP1_H_score, the subtype is SCG3-High / YAP1-Low, indicating a higher risk and shorter survival for the patient; otherwise, SCG3... Low / YAP1 High They have a better survival prognosis.

[0046] Through the above experiments, immunohistochemical staining was completed on a total of 91 samples. The SCG3 H-Score ranged from 0 to 227, and the YAP1 H-Score ranged from 0 to 190. The color scores of SCG3 and YAP1 in the samples were compared using heat maps. Figure 6 In this cohort, the expression of SCG3 and YAP1 remained mutually exclusive, validating the results of the analysis of the 19 transcriptome data mentioned above.

[0047] 2.2 Validation of the prognostic value of SCG3 and YAP1 as molecular subtyping in patients with small cell cervical cancer: KM survival analysis was performed on collected cervical small cell carcinoma samples, combined with clinical survival and recurrence information of cervical small cell carcinoma. Figure 7 ), clearly identifying SCG3 in cervical small cell carcinoma High / YAP1 Low The patient had SCG3 more cervical small cell carcinoma. Low / YAP1 High Patients have a worse survival prognosis and a shorter disease-free survival time.

[0048] With a follow-up period of 150 months, SCG3 High / YAP1 Low The patient mortality rate was 77.4% (24 / 31), SCG3 Low / YAP1 High The mortality rate was 31.8% (7 / 22). The median survival time in the high-risk group (SCG3-High / YAP1-Low) was 33.3 months, and the median disease-free survival time was 25.5 months; while the mortality rate (7 / 22) and relapse rate in the low-risk group (SCG3-Low / YAP1-High) were both less than 50% (6 / 22).

[0049] In summary, this invention, by screening for molecular subtyping marker genes of cervical small cell carcinoma, can predict the risk level of patient survival prognosis through immunohistochemical (IHC) experiments, thus providing better guidance for risk prevention after surgery and chemotherapy.

[0050] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A biomarker for molecular subtyping of cervical small cell carcinoma, characterized in that, The biomarkers are SCG3 and YAP1.

2. The use of reagents and / or instruments for detecting the expression level of biomarkers for molecular subtyping of cervical small cell carcinoma as described in claim 1 in the preparation of the product.

3. The application according to claim 2, characterized in that, The product is used for prognostic assessment of survival in patients with small cell cervical cancer.

4. A product for assessing the prognostic survival of patients with small cell cervical cancer, characterized in that, The product comprises reagents and / or instruments for detecting the expression levels of the biomarkers for molecular subtyping of cervical small cell carcinoma as described in claim 1.

5. The product for prognostic assessment of survival in patients with small cell cervical cancer as described in claim 1, characterized in that, The product includes a reagent kit.

6. The application of the biomarker for molecular subtyping of cervical small cell carcinoma as described in claim 1 in constructing a survival prognostic assessment model for patients with cervical small cell carcinoma.

7. A model for assessing the prognostic survival of patients with small cell cervical cancer, characterized in that, The model was constructed based on the expression levels of the biomarkers for molecular subtyping of cervical small cell carcinoma as described in claim 1; The model is as follows: H-score = intensity grade * percentage of positive area to total tumor area; where H-score ranges from 0 to 300; intensity grade is automatically determined based on color depth.

8. The model for prognostic assessment of survival in patients with small cell cervical cancer according to claim 7, characterized in that, The expression level of the biomarker was determined by immunohistochemical experiments.

9. The model for prognostic assessment of survival in patients with small cell cervical cancer according to claim 7, characterized in that, If SCG3_H_score > YAP1_H_score, then the subtype is SCG3. High / YAP1 Low This indicates a poorer prognosis and shorter overall survival for the subjects. If SCG3_H_score < YAP1_H_score, then the subtype is SCG3. Low / YAP1 High This indicates that the subjects had a better prognosis and a longer overall survival.

10. A system for assessing the prognostic survival of patients with small cell cervical cancer, characterized in that, The system includes: (1) An analysis unit comprising a reagent and / or instrument for detecting the expression level of the biomarker for molecular subtyping of cervical small cell carcinoma as described in claim 1; (2) An assessment unit comprising: assessing the survival prognosis of the subject based on the expression level of the biomarker for molecular subtyping of cervical small cell carcinoma determined in step (1), according to any one of claims 7 to 9.

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