WO2021213261A1 - Kit and detection method for detecting pd-l1 gene mutations in peripheral blood circulating tumor cells of patient with gastric cancer - Google Patents

Abstract

Disclosed are a kit and detection method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of a patient with gastric cancer. A membrane filter device is used to separate and obtain CTCs in peripheral blood of a patient with advanced or recurrent gastric cancer from whom a tissue sample cannot be obtained, and immunohistochemistry technology is further applied to detect the PD-L1 expression of the CTCs. The detection method provided by the present invention may detect the PD-L1 expression of patients with advanced or recurrent gastric cancer without a needle biopsy to obtain tissue samples. The technology is minimally invasive and may be used for real-time detection. The method provided by the present invention may avoid prevent positive results caused by an edge effect that may be generated during a staining process, has good stability, reduces the loss of cells, 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 gastric cancer Technical field

The invention provides a kit and a detection method for detecting the PD-L1 gene mutation of a patient through peripheral blood circulating tumor cells and gastric cancer, and belongs to the technical field of molecular biology.

Background technique

Gastric cancer (GC) is a highly fatal malignant tumor with the fourth highest incidence in the world (950,000 new cases per year) and the third highest among cancer-related deaths in 2012. Although the diagnosis and treatment of gastric cancer have improved in recent years, the 5-year survival rate of gastric cancer patients is less than 30%, and about 50% of gastric cancer patients suffer tumor recurrence or metastasis after curative resection. Tumor recurrence and metastasis are the main causes of death in patients with gastric cancer.

Circulating tumor cells (CTC) are tumor cells that fall off from solid tumors and enter the peripheral blood circulation. Since they were discovered in 1989, there have been a variety of methods for detecting circulating tumor cells in the peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of cancer patients, especially advanced cancer patients, and selecting appropriate individualized treatments. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.

The immunotherapy with PD-1/PD-L1 as an immune target has brought a new light to the treatment of gastric cancer. Studies have found that immune suppression is closely related to immune escape and the overexpression of PD-L1 in 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 conduct inhibitory signals to make T cells The inability to recognize tumor cells and send out attack signals to tumor cells leads to immune escape of tumor cells. Based on this theory, a hypothesis is proposed that circulating tumor cells (CTC) that fall off from the primary tumor and enter the circulatory system appear apoptosis, immune clearance or survival, and the outcome of metastasis is closely related to the expression of PD-L1. The efficacy of PD-1 or PD-L1 immune preparations is mostly related to the immunohistochemical expression level of PD-L1 in tumor tissues, suggesting that the expression level of PD-L1 may be a biomarker for predicting the efficacy of PD-1 immunotherapy; there are also studies It shows that the high expression of PD-L1 in gastric cancer tissue is positively correlated with tumor aggressiveness. According to literature search, there are a few reports on the detection of circulating tumor cell (CTC) PD-L1 expression using different detection methods for breast cancer, prostate cancer, colorectal cancer, and liver cancer. However, the detection of circulating tumor cell PD-L1 in gastric cancer is reported at home and abroad There is no report. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 is of great value for the prognosis of gastric cancer 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 industrialization 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 Provincial 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, CA125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc. are used as tracers for the expression of CTCs, registered as ultra-sensitive, ultra-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 gastric cancer to obtain tissue specimens in real time or repeatedly, and thus unable to assess the patient's PD-L1 status, the present invention provides a method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of gastric cancer patients for non-diagnostic purposes : Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with advanced or recurrent gastric cancer who cannot obtain tissue specimens, 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 gastric cancer, including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, PD-L1 (human) primary antibody 100μL, goat antibody 100 μL of human IgG/HRP, 100 μL of 0.1% Triton X-100, 100 μL of 0.3% H ₂ O ₂ , 0.5 mL of reagent A, 60 mL of 6×PBS buffer.

Preferably, the composition of the diluent is: 1 mmol/L EDTA+0.1% BSA+0.1% trehalose+0.2% tyrosine+PBS buffer 150 mmol/L, wherein the percentage is a volume ratio.

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

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

Preferably, the reagent A is xylene.

The method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of patients with gastric cancer for non-diagnostic purposes of the kit described above includes the following steps:

(1) Use membrane filtration device to separate and obtain CTC in peripheral blood of patients with advanced or recurrent gastric cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or recurrent gastric cancer who cannot obtain tissue samples: 5ml of peripheral blood in 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.

More preferably, 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) Add 100μl PD-L1 (human) primary antibody dropwise, incubate at room temperature for 2h or 4℃ overnight, wash with PBS for 2min×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) Drop 100μl DAB color developing solution, 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, then add 0.5mL reagent A, shake and mix well, centrifuge to precipitate, The sediment is sealed 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 PD-L1 expression in patients with advanced or recurrent gastric 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 the peripheral blood of a patient with gastric cancer;

Figure 5 is a stained image of circulating tumor cells in the peripheral blood of a patient with advanced gastric cancer.

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	0.5mL
Staining Solution B	1mL
PD-L1 (human) primary antibody (commercially available)	100μL
Goat anti-human IgG/HRP (commercially available)	100μL
0.1% Triton X-100	100μL
0.3% H 2 O 2	100μL
Reagent A	0.5mL

Examples of using this technique to separate, obtain and identify 8 cases of gastric cancer patients (8 cases of normal human samples were tested at the same time as 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 gastric cancer who cannot obtain tissue samples, and determine whether CTCs exist:

Collect 5ml of fasting blood for 8-12 hours from the median cubital vein, and use 45ml of diluent (composition: 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2%tyrosine+PBS buffer 150mmol/L, in which The percentage is the volume ratio) Dilute the peripheral blood, 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 port 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; after the filtrate is filtered completely Add solution B, 1ml, stain for 2min, pure water 1ml, PBS buffer solution to rinse filter 3, remove filter 7 with ophthalmic tweezers, cell side up, 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 8 healthy volunteers; except for 1 patient with recurrent gastric cancer and 1 patient with advanced gastric cancer, CTC was not detected, the remaining 6 cases were all detected with CTC (Table 2), the positive rate of this test was 75%. It is worth noting that when the diluent is not added with 0.1% trehalose or 0.2% tyrosine, 0.3% trehalose or 0.3% tyrosine is used alone, the stability of the prepared blood sample is poor, and part of the blood sample is still It will form stratification, blood cells are prone to aggregation and adhesion, which will affect the final detection effect.

Table 2 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 can not exceed 10min); after the color development is completed, discard the DAB chromogenic solution, Rinse in running water for 5 minutes, stain with hematoxylin for 5 minutes; differentiate with hydrochloric acid and alcohol for 8 seconds, and turn to blue for 5 minutes in tap water; 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol dehydration, then add xylene, shake to mix After uniformity, centrifugal precipitation, air-dry the precipitate, and seal with neutral resin; microscopic examination under an optical microscope, cytopathologists read the film, and determine 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 gastric cancer patient. The nucleus is large and the nucleus is irregular in shape; it has a high nucleus to cytoplasmic ratio. As can be seen in the figure: nuclear atypia, nuclear-to-plasma ratio greater than 0.8, cell diameter (long end) greater than 15um, and hyperchromatic nuclei (due to the increased chromatin of cancer cells, the particles become thicker and the nuclei are hyperchromatic).

Figure 5 is a stained image of circulating tumor cells in the peripheral blood of a patient with gastric cancer.

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 gastric cancer to observe the differences. It is mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells. Targeted therapy of gastric cancer provides new ideas for targeted therapy of gastric cancer.

Claims (7)

1. A kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with gastric cancer, which is characterized by comprising 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) primary antibody 100 μL, goat anti-human IgG/HRP 100 μL, 0.1% Triton X-100 100 μL, 0.3% H ₂ O ₂ 100 μL, 0.5 mL reagent A, and 6×PBS buffer 60 mL.
2. The kit according to claim 1, wherein the composition of the diluent is: 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2%tyrosine+PBS buffer 150mmol/L, wherein The percentages mentioned are volume ratios.
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. The kit according to claim 1, wherein the reagent A is xylene.
6. A method for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with gastric cancer for non-diagnostic purposes using the kit according to any one of claims 1 to 5, characterized in that it comprises the following steps:

   (1) Use membrane filtration device to separate and obtain CTC in peripheral blood of patients with advanced or recurrent gastric cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or recurrent gastric cancer who cannot obtain tissue samples: 5ml of peripheral blood in 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.
7. The detection method according to claim 6, 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) Add 100μl PD-L1 (human) primary antibody dropwise, incubate at room temperature for 2h or 4℃ overnight, wash with PBS for 2min×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, then add 0.5mL reagent A, shake and mix well, centrifuge to precipitate, The sediment is sealed with neutral resin;

   (10) Microscopic examination under an optical microscope.

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