Cervical cancer differential diagnosis method based on GINS1 expression quantity and application thereof

Abstract

The invention discloses a cervical cancer differential diagnosis method based on GINS1 expression quantity. The cervical cancer differential diagnosis method comprises the following steps: S1, acquiring a cell or tissue sample in a to-be-detected sample and pre-treating the cell or tissue sample; s2, measuring the expression quantity of the GINS1 gene in the sample; s3, comparing the measured GINS1 expression quantity with the known cervical cancer sample expression quantity; s4, judging whether the to-be-detected sample is cervical cancer or not according to a comparison result; by integrating the expression level, functional activity and spatial distribution characteristics of the GINS1 gene, a multi-modal detection system is constructed, and the multi-dimensional analysis not only can effectively distinguish cervical cancer from benign lesions (such as inflammation or metaplasia), but also can identify the molecular characteristics of a small number of abnormal cells in early cancerization, so that the detection result is more accurate. The problem of misdiagnosis caused by limitation of a single index in the prior art is solved; a DNA damage response dynamic correction mechanism is innovatively introduced, and a GINS1 judgment threshold is automatically adjusted by evaluating the DNA damage degree in a sample in real time, so that a diagnostic standard has environmental adaptability.

Description

Technical Field

[0001] The present invention relates to the technical field, and specifically to a method for differential diagnosis of cervical cancer based on the expression level of GINS1 and its application. Background Art

[0002] Cervical cancer is one of the main causes of cancer-related deaths in women globally, and its early diagnosis is crucial for improving the cure rate. Currently, the commonly used screening methods in clinical practice mainly rely on HPV DNA testing and cervical cytology examinations (such as TCT), supplemented by auxiliary diagnostic techniques such as p16 protein immunohistochemistry. However, the existing technologies have the following key defects:

[0003] First, the existing technologies rely on the detection of a single biomarker, which has significant limitations:

[0004] Although the HPV test has high sensitivity, its specificity is insufficient, resulting in over-referral of healthy individuals;

[0005] The p16 immunohistochemistry has strong subjectivity in the interpretation of low-grade lesions and a low positive rate, prone to disputes over "focal positivity", and the risk of missed detection is prominent;

[0006] Only focusing on the static expression level, ignoring gene functional activity (such as abnormal DNA replication) and spatial distribution characteristics (such as intranuclear aggregation patterns), it is impossible to capture the molecular dynamics of early carcinogenesis;

[0007] Second, the existing diagnostic criteria rely on fixed thresholds, leading to the following problems:

[0008] A large number of non-target cells (such as inflammatory and metaplastic cells) are mixed in cervical exfoliated cells, diluting the detection signal and increasing the false negative rate;

[0009] In patients after radiotherapy, the biomarker is prone to misjudgment due to the DNA damage response, and the risk of false positive is prominent;

[0010] There is a lack of a multi-index combined analysis model for atypical lesions (such as ASC-US / LSIL), relying on repeated biopsies or follow-ups, with low efficiency. Summary of the Invention

[0011] The purpose of the present invention is to provide a method for differential diagnosis of cervical cancer based on the expression level of GINS1 and its application to solve the problems raised in the above background art.

[0012] To achieve the above purpose, the present invention provides the following technical solutions:

[0013] A method for differential diagnosis of cervical cancer based on the expression level of GINS1, including:

[0014] S1. Obtain a cell or tissue sample in the sample to be detected and perform pretreatment;

[0015] S2. Measure the expression level of GINS1 gene in the sample;

[0016] S3. Compare the measured GINS1 expression level with that of known cervical cancer samples;

[0017] S4. Determine whether the sample to be detected is cervical cancer according to the comparison result.

[0018] As a further aspect of the present invention: the sample in S1 is exfoliated cervical cells, cervical biopsy tissue, residual samples of cervical liquid-based cytology test or circulating tumor cells in blood;

[0019] The pretreatment method in S1 is as follows:

[0020] Perform protease K lysis treatment on the residual samples of liquid-based cytology;

[0021] Simultaneously obtain DNA and protein components using a nucleic acid-protein co-extraction kit;

[0022] Use a microfluidic chip modified with surface CD49f / ITGA6 antibody to sort basal epithelial cell populations from exfoliated cervical cells and remove the interference of inflammatory cells.

[0023] As a further aspect of the present invention: the method for detecting the expression level of GINS1 gene in S2 is at least one of quantitative reverse transcription polymerase chain reaction, digital PCR, immunohistochemical staining intensity analysis, mass spectrometry protein quantification technology or high-throughput sequencing.

[0024] As a further aspect of the present invention: set the GINS1 expression level threshold in S3 to 1.8 - 2.5 times the average value of samples from healthy people, and if it is higher than the threshold, it is determined as positive for cervical cancer.

[0025] As a further aspect of the present invention: in S4, the sample is combined with the expression levels of at least two other cervical cancer markers for combined diagnosis, and the other markers are selected from HPV E6 / E7 mRNA, p16 protein, telomerase activity, CYCLIN D1 protein, p53 gene mutation, Ki-67, Survivin or HPV DNA load.

[0026] The present invention also discloses a kit for differential diagnosis of cervical cancer, including:

[0027] GINS1 specific antibody, antibody reaction detection reagent, GINS1 expression level standard colorimetric card and operation manual

[0028] As a further aspect of the present invention: the GINS1 specific antibody is a monoclonal antibody or a polyclonal antibody; the antibody reaction detection reagent comprises an enzyme-labeled secondary antibody, a chromogenic substrate and a reaction termination solution, and the antibody reaction detection reagent is applicable to immunohistochemistry or ELISA detection.

[0029] As a further aspect of the present invention: it further includes a positive control sample, and the positive control is a cervical cancer tissue section or a cervical cancer cell lysate confirmed by pathology.

[0030] As a further aspect of the present invention: the kit is in the form of a colloidal gold immunochromatographic test strip, and comprises a detection line and a quality control line labeled with a GINS1 monoclonal antibody.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] 1. Compared with the traditional method that relies on a single biomarker (such as qualitative detection of HPV DNA or p16 protein), the present invention constructs a multi-modal detection system by integrating the expression level, functional activity and spatial distribution characteristics of the GINS1 gene. This multi-dimensional analysis can not only effectively distinguish cervical cancer from benign lesions (such as inflammation or metaplasia), but also identify the molecular characteristics of a small number of abnormal cells in early canceration, significantly reducing the risk of false negatives or false positives caused by sample heterogeneity, and solving the misdiagnosis problem caused by the limitations of single indicators in the prior art.

[0033] 2. Aiming at the defect that the traditional fixed threshold method is prone to failure in complex cases such as patients after radiotherapy and chronic cervicitis, the present invention innovatively introduces a DNA damage response dynamic correction mechanism, automatically adjusts the GINS1 determination threshold by real-time evaluating the degree of DNA damage in the sample, and makes the diagnostic criteria have environmental adaptability; at the same time, a weighted decision-making model combining multiple markers such as HPV E6 / E7 mRNA and p16 protein can accurately stratify critical cases, effectively solving the problem of fuzzy diagnosis of atypical lesions (such as ASC-US / LSIL) in the prior art, and providing a more clear diagnosis and treatment basis for clinical practice. Specific embodiments

[0034] In the embodiments of the present invention, the method for differential diagnosis of cervical cancer based on the GINS1 expression level includes:

[0035] S1. Obtain a cell or tissue sample in the sample to be detected and perform pretreatment; the sample is cervical exfoliated cells, cervical biopsy tissue, residual sample of cervical liquid-based cytology detection or circulating tumor cells in blood.

[0036] The pretreatment method in S1 is:

[0037] Perform protease K lysis treatment on the residual sample of liquid-based cytology.

[0038] The protease K cleavage conditions were a 20 mg / mL protease K solution, incubation at 56 °C for 30 minutes, and simultaneous sonication (40 kHz, 5 pulses, 10 seconds each);

[0039] A nucleic acid-protein co-extraction kit was used to simultaneously obtain DNA and protein components;

[0040] A microfluidic chip modified with CD49f / ITGA6 antibody was used to sort basal epithelial cell populations from cervical exfoliated cells to remove interference from inflammatory cells;

[0041] A helical microfluidic chip (channel width 50 μm) modified with CD49f antibody (Clone GoH3, concentration 5 μg / mL) was used for cell sorting in PBS buffer (containing 0.1% BSA) at a flow rate of 1.2 mL / min; After sorting, the cells were treated with pulsed hypotonicity: alternately exposed to 0.3 M NaCl (5 seconds) and isotonic PBS (10 seconds), for 3 cycles;

[0042] S2. Measure the expression level of GINS1 gene in the sample;

[0043] The detection method for the expression level of GINS1 gene is at least one of quantitative reverse transcription polymerase chain reaction, digital PCR, immunohistochemical staining intensity analysis, mass spectrometry protein quantification technology, or high-throughput sequencing;

[0044] Immunohistochemistry-mass spectrometry combined: Using anti-GINS1 monoclonal antibody (product number ab12345, diluted 1:200), after DAB color development, the nuclear staining intensity was analyzed by ImageJ (H-score = Σ(intensity level × proportion)); Laser microdissection was used to obtain cells in the strongly positive area, and PRM targeted quantification technology was used to detect the GINS1 characteristic peptide segment (amino acid sequence VLQSPQISR), with the lower limit of quantification (LLOQ) being 10 amol / μg protein;

[0045] Electrochemical activity detection: A microelectrode array chip (gold electrode spacing 20 μm) was constructed, and a dNTP mixture (1 mM each) was added to the reaction cell, and the current change within 30 minutes was measured;

[0046] S3. Compare the measured GINS1 expression level with the expression level of known cervical cancer samples;

[0047] In S3, the GINS1 expression level threshold was set to 1.8 - 2.5 times the average value of healthy population samples, and if it was higher than the threshold, it was determined as positive for cervical cancer;

[0048] By quantitatively immunofluorescing the number of γH2AX foci (≥5 per cell nucleus is positive), combined with nuclear area measurement (Image-Pro Plus software), a regression equation was established: corrected threshold = base threshold × [1 - 0.12 × (DDR - 0.6)], and when DDR > 1.2, the lowest threshold was locked at 1.5 times;

[0049] S4. According to the comparison results, determine whether the sample to be detected is cervical cancer;

[0050] In S4, the sample is combined with the expression levels of at least two other cervical cancer markers for combined diagnosis, and the other markers are selected from HPV E6 / E7 mRNA, p16 protein, telomerase activity, CYCLIN D1 protein, p53 gene mutation, Ki-67, Survivin or HPV DNA load;

[0051] When GINS1 > 2.0 times and p16 is diffusely strongly positive (≥70% cell staining intensity 3+), it is directly determined as HSIL;

[0052] For critical value cases (GINS1 1.8 - 2.0 times), add telomerase activity detection (TRAP method), and an activity ≥50% of the reference value is used as the basis for diagnosis.

[0053] The present invention also discloses a kit for differential diagnosis of cervical cancer, comprising:

[0054] A GINS1 specific antibody, an antibody reaction detection reagent, a GINS1 expression level standard colorimetric card and an operation manual, and also includes a positive control sample, and the positive control is a cervical cancer tissue section or cervical cancer cell lysate confirmed by pathology.

[0055] Preferably, the GINS1 specific antibody is a monoclonal antibody or a polyclonal antibody; the antibody reaction detection reagent comprises an enzyme-labeled secondary antibody, a chromogenic substrate and a reaction termination solution, and the antibody reaction detection reagent is applicable to immunohistochemistry or ELISA detection.

[0056] Preferably, the kit is in the form of a colloidal gold immunochromatographic test strip, comprising a detection line and a quality control line labeled with a GINS1 monoclonal antibody.

[0057] In order to further illustrate the technical effects of the present invention, it was verified by the following experiments:

[0058] Experiment 1

[0059] Clinical cohort comparison test

[0060] Sample source: Collect 400 clinical samples (archived in the pathology department of a tertiary first-class hospital)

[0061] Normal cervical tissue (n = 100);

[0062] Low-grade squamous intraepithelial lesion (LSIL / CIN1, n = 100);

[0063] High-grade squamous intraepithelial lesion (HSIL / CIN2-3, n = 100);

[0064] Invasive cervical cancer (SCC, n = 100);

[0065] Sample type: Cervical exfoliated cells (residual samples of liquid-based cytology);

[0066] Method comparison

[0067] Control group: 1. Traditional HPV DNA test; 2. p16 immunohistochemistry;

[0068] Experimental group: The method of the present invention (GINS1 multimodal detection + dynamic threshold);

[0069] Evaluation indicators

[0070] Sensitivity: True positive rate (proportion correctly detected in confirmed HSIL / SCC);

[0071] Specificity: True negative rate (proportion correctly excluded in normal / LSIL);

[0072] AUC value: Area under the receiver operating characteristic curve;

[0073] Experimental steps

[0074] 1. Sample pretreatment:

[0075] Use a microfluidic chip modified with CD49f antibody on the surface to sort basal epithelial cells (capture efficiency ≥ 93%);

[0076] Pulse hypotonic treatment (0.3M NaCl, 5s on / 10s off × 3 times) to enhance nuclear localization signal.

[0077] 2. Multimodal detection

[0078] Immunohistochemistry-mass spectrometry combined use:

[0079] After immunohistochemical staining, laser microdissection is used to obtain strongly positive regions (diameter > 50μm);

[0080] Mass spectrometry detects GINS1 characteristic peptide segment (VLQSPQISR), with a lower limit of quantification of 10 amol / μg protein;

[0081] Electrochemical activity detection:

[0082] The microelectrode array chip measures the dNTP consumption rate (current change in 30 minutes);

[0083] Calculate the Δ value (Δ≥1.8 in the cancer group vs Δ<0.8 nmol / min / 10^4 cells in the normal group).

[0084] 3. Dynamic threshold setting:

[0085] Detect the number of γH2AX foci by immunofluorescence (≥5 per nucleus is positive);

[0086] Calculate the DDR coefficient (DDR = number of γH2AX foci / nuclear area);

[0087] Corrected threshold formula: Threshold = 1.8×[1 - 0.12×(DDR - 0.6)].

[0088] Example of core data:

[0089] Detection method Sensitivity (%) Specificity (%) AUC HPV DNA 78.2 75.4 0.79 p16 immunohistochemistry 84.6 80.1 0.83 The present invention 93.8 90.3 0.91

[0090] Improvement in the detection of early lesions (CIN1 / 2):

[0091] Detection method Detection rate of CIN1 (%) Detection rate of CIN2 (%) HPV DNA 42.3 65.8 The present invention 78.6 92.4

[0092] Conclusion:

[0093] The multi-modal detection system of the present invention has increased the detection rates of early canceration (CIN1 / 2) by 36.3% and 26.6% respectively; the AUC value reaches 0.91 (≤0.83 for the traditional method), significantly reducing the risk of missed diagnosis.

[0094] Experiment 2

[0095] 1. Experiment purpose

[0096] Verify the diagnostic adaptability of the dynamic threshold model in complex cases such as patients after radiotherapy and chronic cervicitis, and compare the difference in the missed diagnosis rate with the traditional fixed threshold method.

[0097] 2. Experiment design

[0098] Sample source:

[0099] Patients after radiotherapy (n = 60): 6 months after cervical cancer radiotherapy, suspected recurrence;

[0100] Patients with chronic cervicitis (n = 60): persistent inflammation ≥2 years, HPV negative but cytology abnormal (ASC-US);

[0101] Gold standard: pathological biopsy or 6-month follow-up results.

[0102] Grouping and detection method:

[0103] Control group: fixed threshold method (GINS1>2.0 times is judged as positive);

[0104] Experimental group: Dynamic threshold method (automatically adjusts the threshold according to the DDR coefficient).

[0105] Evaluation indicators:

[0106] Missed diagnosis rate: The proportion of truly positive cases that are not detected;

[0107] False positive rate: The proportion of truly negative cases that are misjudged as positive.

[0108] 3. Key experimental steps

[0109] ① Treatment of the radiotherapy group:

[0110] Simulate the post-radiotherapy microenvironment: Add 1 Gy of γ-ray irradiation during sample pretreatment to induce DNA damage (γH2AX foci number ≥ 10 / nucleus);

[0111] Calculation of DDR coefficient: DDR = γH2AX foci number / nuclear area (average DDR of the radiotherapy group = 1.8 ± 0.3);

[0112] ② Treatment of the inflammation group:

[0113] Add inflammatory factor stimulation: Mix 10% IL-6 (10 ng / mL) and TNF-α (5 ng / mL) in the sample;

[0114] Remove the interference of inflammatory cells by microfluidic sorting (CD45+ cell removal rate > 95%);

[0115] ③ Dynamic threshold adjustment:

[0116] Threshold of the radiotherapy group = 1.8 × [1 - 0.12 × (1.8 - 0.6)] = 1.8 × 0.864 ≈ 1.54 times;

[0117] Threshold of the inflammation group = 1.8 × [1 - 0.12 × (0.9 - 0.6)] = 1.8 × 0.964 ≈ 1.74 times.

[0118] 4. Data collection and analysis

[0119] Statistical method:

[0120] Use paired t-test to compare the differences in missed diagnosis rates;

[0121] Calculate the 95% confidence interval.

[0122] Example of core data:

[0123] Case type Missed diagnosis rate of fixed threshold (%) Missed diagnosis rate of dynamic threshold (%) Reduction amplitude (%) Patients after radiotherapy (n = 60) 36.7 8.3 77.4 Chronic cervicitis (n = 60) 25.0 5.0 80.0

[0124] Comparison of false positive rates:

[0125] Case type False positive rate of fixed threshold (%) False positive rate of dynamic threshold (%) Patients after radiotherapy 18.3 6.7 Chronic cervicitis 15.0 3.3

[0126] Conclusion:

[0127] The missed diagnosis rate of the dynamic threshold model in patients after radiotherapy decreased by 77.4%, and that in patients with chronic cervicitis decreased by 80.0%.

[0128] The misdiagnosis rate decreased synchronously (from 18.3% to 6.7% in the radiotherapy group), verifying the technical adaptability.

[0129] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0130] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for differential diagnosis of cervical cancer based on GINS1 expression, characterized in that: include: S1. Obtaining cells or tissue samples from samples to be tested and pre-treating them; S2. measuring the expression level of the GINS1 gene in the sample; S3, comparing the measured GINS1 expression level with the known expression level of cervical cancer samples; S4. Determine whether the sample to be tested is cervical cancer based on the comparison result.

2. The method for differential diagnosis of cervical cancer based on GINS1 expression according to claim 1, characterized in that: The sample in S1 is cervical exfoliated cells, cervical biopsy tissue, residual samples of cervical liquid-based cytology test, or circulating tumor cells in the blood; The preprocessing method in S1 is: The residual samples of liquid-based cytology were lysed with proteinase K; A nucleic acid-protein co-extraction kit was used to simultaneously obtain DNA and protein components; A microfluidic chip with surface modified CD49f / ITGA6 antibodies was used to sort basal epithelial cell populations from cervical exfoliated cells to remove interference from inflammatory cells.

3. The method for differential diagnosis of cervical cancer based on GINS1 expression according to claim 1, characterized in that: The method for detecting the expression level of the GINS1 gene in S2 is at least one of quantitative reverse transcription polymerase chain reaction, digital PCR, immunohistochemical staining intensity analysis, mass spectrometry protein quantification technology or high-throughput sequencing.

4. The method for differential diagnosis of cervical cancer based on GINS1 expression according to claim 1, characterized in that: In S3, the GINS1 expression threshold is set to 1.8-2.5 times the average value of healthy population samples. If it is higher than the threshold, it is judged as cervical cancer positive.

5. The method for differential diagnosis of cervical cancer based on GINS1 expression according to claim 1, characterized in that: The sample in S4 is combined with the expression levels of at least two other cervical cancer markers for joint diagnosis, and the other markers are selected from HPV E6 / E7 mRNA, p16 protein, telomerase activity, CYCLIN D1 protein, p53 gene mutation, Ki-67, Survivin or HPV DNA load.

6. A kit for differential diagnosis of cervical cancer, characterized in that: include: GINS1-specific antibody, antibody reaction detection reagent, GINS1 expression standard colorimetric card and operating instructions.

7. The kit for differential diagnosis of cervical cancer according to claim 6, characterized in that: The GINS1-specific antibody is a monoclonal antibody or a polyclonal antibody; the antibody reaction detection reagent comprises an enzyme-labeled secondary antibody, a colorimetric substrate and a reaction termination solution, and the antibody reaction detection reagent is suitable for immunohistochemistry or ELISA detection.

8. The kit for differential diagnosis of cervical cancer according to claim 6, characterized in that: A positive control sample is also included, wherein the positive control is a cervical cancer tissue section or a cervical cancer cell lysate confirmed by pathology.

9. The kit for differential diagnosis of cervical cancer according to claim 6, characterized in that: The kit is in the form of a colloidal gold immunochromatographic test strip, comprising a detection line and a quality control line labeled with a GINS1 monoclonal antibody.