WO2021213316A1 - Kit for detecting peripheral blood circulating tumor cell pd-l1 gene mutation of patient with kidney cancer, and detection method - Google Patents

Abstract

Provided in the present invention are a kit for detecting a peripheral blood circulating tumor cell PD-L1 gene mutation of a patient with kidney cancer, and a detection method, which fall within the technical field of molecular biology. The kit comprises a diluent, a decolorizing solution, a staining solution A, a staining solution B, a PD-L1 (human) primary antibody, goat anti-human IgG/HRP antibody, 0.1% Triton X-100, 0.3% H₂O₂ and a 6×PBS buffer solution. By means of the detection method provided by the present invention, the PD-L1 expression of a patient with advanced or recurrent kidney cancer can be detected without acquiring a tissue specimen by means of needle biopsy. The technology is a minimally invasive technology, can realize real-time detection, can avoid false positive results caused by edge effects possibly generated in the staining process, has good stability, reduces cell loss and improves the accuracy of detection.

Description

A kit and detection method for detecting PD-L1 gene mutation in peripheral blood circulating tumor cells of patients with renal cell carcinoma Technical field

The invention belongs to the technical field of molecular biology, and particularly relates to a kit and a detection method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of renal cancer patients.

Background technique

Renal cell carcinoma (RCC) is one of the most common malignant tumors in the urinary system. The morbidity and mortality rate account for about 2-3% of systemic malignancies, and its incidence has been on the rise in recent decades. Radical surgery is an effective means to treat kidney cancer, but about 20% to 30% of kidney cancer patients have seen distant metastases when they visit a doctor. Even after radical renal cancer surgery, there are still 20%-40% of patients will have recurrence and metastasis. With the clinical application of targeted drugs, the survival period of patients with advanced renal cell carcinoma has been greatly prolonged, but for patients with high-risk metastatic renal cell carcinoma, the effect of targeted therapy alone is still not satisfactory, and the 5-year survival rate is less than 10%. In recent years, immunotherapy targeting PD-1/PD-L1 has brought a new dawn to the treatment of renal cell carcinoma.

Studies have found that immunosuppression and immune escape are closely related to the overexpression of PD-L1 on tumor cells. Tumor cells can bind to PD-1 on the surface of immune cells and T cells through PD-L1 on their surface and transmit inhibitory signals to make T cells Failure to recognize and attack tumor cells, leading to immune escape of tumor cells. Based on this theory, the hypothesis is proposed that circulating tumor cells (CTC) that fall off from the primary tumor and enter the circulatory system undergo apoptosis, immune clearance or survival, and the outcome of metastasis is closely related to their PD-L1 expression level. Previous studies have confirmed that the efficacy of PD-1 or PD-L1 immune preparations is mostly related to the expression level of PD-L1 in tumor tissues, suggesting that PD-L1 may be a biomarker for predicting the efficacy of immunotherapy. In addition, studies have shown that The high expression of PD-L1 in renal cell carcinoma tissue is positively correlated with tumor aggressiveness. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 is of great value for the prognosis of renal cell carcinoma and the evaluation of the efficacy of immunotherapy.

At present, Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have Research on the industrial promotion of key technologies for tumor cell detection and identification. This project is a major scientific and technological innovation project in Shandong Province. This project will take the Shandong Pharmaceutical Research Institute on the Jinan Campus of Shandong First Medical University as the core and implement the registrant system. Circulating tumor cell detection and identification of core diagnostic technology, and further registration and identification of diagnostic kits to include PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, pregnancy Hormone receptors, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc. are used as tracers for CTCs expression. Registration is super sensitive, super fast, high coverage, low cost, accurate and specific The identification and diagnosis kits are industrialized and promoted through cooperation with Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., and Shandong Discovery Biotechnology Co., Ltd., which are registered in Jinan.

Summary of the invention

In order to overcome the inability of patients with advanced or recurrent renal cancer to obtain tissue specimens in real time or repeatedly through puncture, thereby failing to assess the patient's PD-L1 status, the present invention provides a peripheral blood circulating tumor cell PD-L1 gene mutation in renal cancer patients for non-diagnostic purposes Detection method: Separate CTC in the peripheral blood of patients with advanced or recurrent non-kidney cancer who cannot obtain tissue samples by using a membrane filtration device, and further use immunohistochemistry to detect the expression of CTC's PD-L1.

The technical scheme adopted by the present invention is as follows:

A kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with renal cell carcinoma, including dilution solution 50mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, PD-L1 (human) primary antibody 100μL, goat Anti-human IgG/HRP 100 μL, 0.1% Triton X-100 100 μL, 0.3% H ₂ O ₂ 100 μL, 6×PBS buffer 60 mL.

Further, the diluent is composed of 1 mmol/L EDTA+0.1%BSA+0.1% trehalose+0.2% laureth.

Further, the decolorizing liquid is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.

Further, the staining solution A is a DAB staining solution; the staining solution B is a hematoxylin staining solution.

A method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of patients with renal cell carcinoma for non-diagnostic purposes of the above kit includes the following steps:

(1) Use a membrane filter device to separate and obtain CTCs in peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples: 5ml of peripheral blood from the median cubital vein;

(2) Pretreatment of peripheral blood samples: Dilute the collected peripheral blood samples 10 times with diluent, add paraformaldehyde to fix the peripheral blood samples for 10 minutes after dilution, and fix the final concentration to 0.25%;

(3) Use membrane filtration device to separate tumor cells to filter peripheral blood samples to separate and obtain peripheral blood CTC: add the pretreated peripheral blood sample to the blood sample container of the membrane filtration device to separate tumor cells so that it can be filtered naturally by gravity;

(4) After filtering, remove the filter from the device for separating tumor cells by membrane filtration, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 minutes, and rinse with PBS buffer; add staining solution after filtering the filtrate 1ml of B solution, staining for 2min, and washing with 1ml of pure water twice, remove the filter membrane, place it on a glass slide, and observe under a microscope after drying to determine whether there is CTC;

(5) Use immunohistochemistry technology to detect the expression of CTC PD-L1.

The specific method of the present invention for detecting the expression of PD-L1 of CTC is as follows:

(1) Decolorization: Remove the filter membrane with CTC from the glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution;

(2) Add 100μl 0.1% Triton X-100 dropwise, incubate at room temperature for 15min, wash with DI water for 2min×3 times;

(3) Add 100μl 0.3% H ₂ O ₂ dropwise, incubate at room temperature for 10 min, wash with PBS for 2 min×3 times; (4) drop 100 μl PD-L1 (human) primary antibody, incubate at room temperature for 2 hours or 4°C overnight, wash with PBS for 2 minutes ×3 times;

(5) Add 100μl goat anti-human IgG/HRP dropwise, incubate at 18～26℃ for 20min, wash with PBS for 2min×3 times;

(6) Add 100μl DAB color developing solution dropwise, incubate at 18～26℃ and observe the color development under the microscope at any time, the observation time is 3～10min;

(7) After the color development is completed, discard the DAB color development solution, rinse with running water for 5 minutes, and stain with hematoxylin for 5 minutes;

(8) The hydrochloric acid and alcohol are differentiated for 8 seconds, and the tap water returns to blue for 5 minutes;

(9) Use 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol to dehydrate the CTC after returning to blue, and then add 0.5mL reagent A, after shaking evenly, add 1mL reagent B, After shaking and mixing uniformly, centrifuge to settle, and seal the precipitate with neutral resin;

(10) Microscopic examination under an optical microscope.

The membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid cylinder, and an iron stand. The iron stand is provided with a base, a stand and a bracket. The blood sample container is set on the upper part of the iron stand through the bracket. Below the blood sample container is a filter, which is connected to the waste liquid tank through the infusion set, and the waste liquid tank is arranged on the base.

The filter includes a filter upper port, a filter membrane, a filter membrane platform and a filter lower port. The filter membrane is placed on the filter membrane platform; the upper port of the filter is connected to the blood sample container, and the lower port of the filter is connected to the waste liquid tank through the infusion device.

The filter membrane is made of a hydrophobic material, and the filter holes with a diameter of 8 micrometers are evenly spread on it.

The beneficial effects of the present invention are:

(1) The detection method provided by the present invention can detect the expression of PD-L1 in patients with advanced or recurrent renal cancer without puncture biopsy to obtain tissue samples. This technology is minimally invasive and can be detected in real time.

(2) The method provided by the present invention can avoid false positive results caused by edge effects that may occur during the staining process, has good stability, reduces cell loss, and improves detection accuracy.

Description of the drawings

Figure 1 is a schematic diagram of the structure of the membrane filtration device of the present invention;

2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention;

3 is a schematic diagram of the structure of the filter membrane of the membrane filtration device of the present invention;

Figure 4 is an image of circulating tumor cells obtained from peripheral blood of a renal cancer patient;

In the picture: 1 iron stand, 2 blood sample container, 3 filter, 4 infusion set, 5 waste liquid tank, 6 filter upper mouth, 7 filter membrane, 8 filter membrane platform, 9 filter lower mouth, 10 filter holes, 11 base, 12 stand, 13 support.

Detailed ways

The present invention will be described below in conjunction with the drawings and embodiments.

The specific specifications of the kit used in the present invention are shown in Table 1:

Table 1

Component		content
6×PBS buffer	60mL
Diluent	45mL
Decolorizing liquid	1mL
Staining solution A	0.3mL
Staining Solution B	1mL
PD-L1 (human)-anti	100μL
Goat anti-human IgG/HRP	100μL
0.1%Trition X-100	100μL
0.3% H 2 O 2	100μL

This technique was used to separate, obtain and identify 10 cases of renal cancer patients (10 normal samples were tested at the same time as a negative control) examples of circulating tumor cells in the peripheral blood.

Example 1

1. Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue specimens to determine whether CTCs exist:

Collect 5ml of fasting blood for 8-12 hours from the median cubital vein, and dilute the peripheral blood with 45ml of diluent (composition: 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2% laureth), and then Add 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;

In a fixed interval, assemble the membrane filtration device: as shown in Figures 1, 2, and 3, the filtration device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;

Wet the filter 3 with 10ml PBS, and then add the fixed peripheral blood sample to the blood sample container 2 of the membrane filtration device, so that it can be filtered naturally by gravity, and the CTC is trapped on the filter membrane 7;

The diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, after the peripheral blood containing CTC is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, and the CTC is larger than the diameter. The filter hole 10 is trapped on the filter membrane 7.

After the filtration, remove the filter 3 from the filter device, open and remove the upper mouth 6 of the filter, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 minutes, and rinse with PBS buffer; the filtrate is completely filtered Then add B solution, 1ml, stain for 2min, pure water 1ml, PBS buffer solution to rinse the filter 3 clean, remove the filter membrane 7 with ophthalmic tweezers, place the cell side up, and place it on the glass slide;

After drying the filter membrane, observe under a microscope to determine whether there is CTC.

Through observation, no CTC was found in 10 healthy volunteers; except for 1 patient with advanced renal cancer and 1 patient with recurrent renal cancer, no CTC was detected, the remaining 8 cases were all detected with CTC (Table 1), this test was positive The rate is 80%. It is worth noting that when the diluent does not add 0.1% trehalose or 0.2% laureth, only 0.3% trehalose or 0.3% laureth is used to prepare The stability of blood samples is poor, and some blood samples will also form stratification, and blood cells are prone to aggregation and adhesion, which affects the final detection effect.

Table 1 Example CTC detection results

2. Use immunohistochemistry technology to detect the expression of CTC PD-L1:

Remove the filter membrane 7 with CTC on the glass slide from the glass slide, and soak it in a decolorizing solution of 95% alcohol and 100% xylene in a volume ratio of 1:1 for 4-6 hours to remove the CTC stain Dropwise add 100μl 0.1% Triton X-100, incubate at room temperature for 15min, wash with DI water for 2min×3 times; add dropwise 100μl 0.3% H ₂ O ₂ , incubate at room temperature for 10 min, wash with PBS for 2min×3 times; add 100μl PD-L1 dropwise (Human) primary antibody, incubate at room temperature for 2h (or overnight at 4℃), wash with PBS for 2min×3 times; add 100μl goat anti-human IgG/HRP dropwise, incubate at room temperature (18～26℃) for 20min, wash with PBS for 2min×3 times; Add 100μl DAB chromogenic solution dropwise, incubate at room temperature (18～26℃) and observe the color development under the microscope at any time (generally 3-10min, the time cannot exceed 10min); after the color development is completed, discard the DAB chromogenic solution, Rinse with running water for 5 minutes, stain with hematoxylin for 5 minutes; differentiate with hydrochloric acid and alcohol for 8 seconds, and then turn the tap water to blue for 5 minutes; 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol dehydration, dry the precipitate, medium Sealing with sex resin; microscopic examination under an optical microscope, cytopathologists reading the pictures, and determining the expression of PD-L1 according to the degree of staining of the cell membrane and cytoplasm.

Figure 4 is an image of circulating tumor cells isolated from the peripheral blood of a renal cancer patient. The CTC cells have nuclear atypia, with a nuclear to cytoplasm ratio greater than 0.8, cell diameter (long end) greater than 15 μm, and hyperchromatic nuclei (due to increased chromatin in cancer cells, The granules become thicker and the nucleus is deeply stained). The nucleus is larger and the shape of the nucleus is irregular; the ratio of nucleus to cytoplasm is high.

The detected circulating tumor cells were confirmed by immunohistochemistry for the expression of PD-L1 and compared with the results of PD-L1 in gross specimens of renal cancer, to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells. Guide the targeted therapy of kidney cancer and provide new ideas for targeted therapy of kidney cancer.

Claims (6)

1. A kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with renal cell carcinoma, which is characterized in that it comprises 45 mL of dilution solution, 1 mL of decolorizing solution, 0.5 mL of staining solution A, 1 mL of staining solution B, and PD-L1 (human) one Anti-100 μL, goat anti-human IgG/HRP 100 μL, 0.1% Triton X-100 100 μL, 0.3% H ₂ O ₂ 100 μL, 6×PBS buffer 60 mL, and the pH value of the PBS buffer is 7.6.
2. The kit according to claim 1, wherein the diluent is composed of 1 mmol/L EDTA+0.1%BSA+0.1% trehalose+0.2% laureth.
3. The kit according to claim 1, wherein the decolorizing solution is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
4. The kit according to claim 1, wherein the staining solution A is a DAB staining solution; the staining solution B is a hematoxylin staining solution.
5. A method for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with renal cell carcinoma for non-diagnostic purposes using the kit according to any one of claims 1 to 4, characterized in that it comprises the following steps:

   (1) Use a membrane filter device to separate and obtain CTCs in peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or recurrent renal cancer who cannot obtain tissue samples: 5ml of peripheral blood from the median cubital vein;

   (2) Pretreatment of peripheral blood samples: Dilute the collected peripheral blood samples 10 times with diluent, add paraformaldehyde to fix the peripheral blood samples for 10 minutes after dilution, and fix the final concentration to 0.25%;

   (3) Use membrane filtration device to separate tumor cells to filter peripheral blood samples to separate and obtain peripheral blood CTC: add the pretreated peripheral blood sample to the blood sample container of the membrane filtration device to separate tumor cells so that it can be filtered naturally by gravity;

   (4) After filtering, remove the filter from the device for separating tumor cells by membrane filtration, add 0.5 ml of circulating tumor cell staining solution A to the filter, stain for 3 minutes, and rinse with PBS buffer; add staining solution after filtering the filtrate 1ml of B solution, staining for 2min, and washing with 1ml of pure water twice, remove the filter membrane, place it on a glass slide, and observe under a microscope after drying to determine whether there is CTC;

   (5) Use immunohistochemistry technology to detect the expression of CTC PD-L1.
6. The detection method according to claim 5, wherein the specific method for detecting the expression of PD-L1 of CTC is as follows:

   (1) Decolorization: Remove the filter membrane with CTC from the glass slide, soak in the decolorization solution for 4-6 hours, and remove the CTC staining solution;

   (2) Add 100μl 0.1% Triton X-100 dropwise, incubate at room temperature for 15min, wash with DI water for 2min×3 times;

   (3) Add 100μl 0.3% H ₂ O ₂ dropwise, incubate at room temperature for 10 min, wash with PBS for 2 min×3 times; (4) drop 100 μl PD-L1 (human) primary antibody, incubate at room temperature for 2 hours or 4°C overnight, wash with PBS for 2 minutes ×3 times;

   (5) Add 100μl goat anti-human IgG/HRP dropwise, incubate at 18～26℃ for 20min, wash with PBS for 2min×3 times;

   (6) Add 100μl DAB color developing solution dropwise, incubate at 18～26℃ and observe the color development under the microscope at any time, the observation time is 3～10min;

   (7) After the color development is completed, discard the DAB color development solution, rinse with running water for 5 minutes, and stain with hematoxylin for 5 minutes;

   (8) The hydrochloric acid and alcohol are differentiated for 8 seconds, and the tap water returns to blue for 5 minutes;

   (9) Use 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol to dehydrate the CTC after returning to blue, and then add 0.5mL reagent A, after shaking evenly, add 1mL reagent B, After shaking and mixing uniformly, centrifuge to settle, and seal the precipitate with neutral resin;

   (10) Microscopic examination under an optical microscope.

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