WO2021213323A1 - Non-diagnostic method for detecting pd-l1 gene mutation of patient with colorectal cancer by means of circulating tumor cells in peripheral blood - Google Patents

Abstract

Provided in the present invention is a non-diagnostic method for detecting a PD-L1 gene mutation of a patient with colorectal cancer by means of circulating tumor cells in peripheral blood, which belongs to the field of molecular biology. The detection method mainly comprises collecting peripheral blood, treating the peripheral blood, filtering and enriching circulating tumor cells, and using an immunohistochemical method to detect the PD-L1 gene expression of the circulating tumor cells. The detection method provided by the present invention can be used to detect the PD-L1 expression of a patient with colorectal cancer without obtaining a tissue specimen by means of needle biopsy. The technique is minimally invasive and can realize real-time detection. Moreover, the method can avoid a false positive result caused by an edge effect which may be generated in a dyeing process, has good stability, reduces cell loss, and improves detection accuracy.

Description

A non-diagnostic method for detecting PD-L1 gene mutations in patients with colorectal cancer through peripheral blood circulating tumor cells Technical field

The present invention relates to a new method for detecting PD-L1 gene mutation, in particular to a method for PDL-1 gene mutation in patients with advanced or recurrent colorectal cancer whose tissue samples cannot be obtained through peripheral blood detection.

Background technique

Colorectal cancer is one of the most common malignant tumors in the world, and the mortality rate in 2018 ranked fourth among malignant tumors. In the United States, colorectal cancer is the third most common malignant tumor. Domestically, China's annual epidemiological data in 2018: the incidence and mortality of colorectal cancer are both the fifth, with 376,300 new cases per year, and 25% of them have metastasized when they are diagnosed. Colorectal cancer is mostly asymptomatic in the early stage, and most of the symptoms appear in the middle and late stages with metastasis. Despite continuous innovations in surgical procedures and chemotherapy, the survival rate of patients with advanced colorectal cancer is still very poor. Due to the lack of better prevention methods for colorectal cancer, the most effective way to improve the prognosis of colorectal cancer and increase the survival rate is to diagnose and treat early colorectal cancer and precancerous lesions.

Immunotherapy with PD-1/PD-L1 as the immune target has brought a new dawn for the treatment of colorectal 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 predicting the efficacy of PD-1 immunotherapy; current colorectal The detection of PD-L1 in cancer circulating tumor cells has not been reported at home and abroad. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 has important value for the prognosis of colorectal cancer and the evaluation of immunotherapy efficacy.

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, 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 view of the problems in the prior art, the present invention provides a method for detecting PD-L1 gene mutations in patients with colorectal cancer through peripheral blood circulating tumor cells. A detection method for non-diagnostic purposes: using a membrane filtration device to separate and obtain unobtainable tissue specimens. The CTC in the peripheral blood of patients with posterior colorectal cancer was further used to detect the expression of PD-L1 of CTC by immunohistochemistry.

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

A kit for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with colorectal cancer, including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, PD-LI (human) primary antibody, goat Anti-human IgG/HRP 100 μL, 0.1% Triton X-100 100 μL, 0.3% H ₂ O ₂ 100 μL, Reagent A, Reagent B, 6×PBS buffer 60 mL.

Wherein, the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.

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

Wherein, the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

Wherein, the reagent A is composed of ethanol and formaldehyde in a volume ratio of 3:1; the reagent B is xylene.

A method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of colorectal cancer by using the above kit for non-diagnostic purposes, including the following steps:

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

(2) Peripheral blood sample pretreatment: Dilute the collected peripheral blood sample 10 times with diluent, add paraformaldehyde to fix the peripheral blood sample 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.

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, 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, the filter 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 colorectal 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 isolated from peripheral blood of patients with colorectal cancer; the arrow is CTM.

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	100μL
Goat anti-human IgG/HRP	100μL
0.1% Triton X-100	100μL
0.3% H 2 O 2	100μL
Reagent A	0.5mL
Reagent B	1mL

An example of using this technique to separate, obtain and identify 6 cases of colorectal cancer patients (7 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 colorectal cancer patients who cannot obtain tissue samples to determine whether CTCs exist:

Collect 5ml of fasting blood for 8-12 hours from the median cubital vein, dilute the peripheral blood with 45ml of diluent, 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. What needs to be explained here is that the diluent plays the role of de-adhesion and dispersion. The laurate and poloxamer are used together to ensure that blood cells and CTC do not adhere and are fully dispersed in the diluent to effectively pass through the filter membrane. 7 was detained.

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; 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 a glass slide; dry the filter under the microscope Observe to determine whether there is a CTC.

Through observation, no CTC was found in 7 healthy volunteers; except for 2 patients with colorectal cancer, which was not detected (1 patient with advanced colorectal cancer + 1 patient with recurrent colorectal cancer), the remaining 4 cases were all detected CTC (Table 1), the positive rate of this test was 66%.

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 staining 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 in running water for 5 minutes, stain with hematoxylin for 5 minutes; differentiate with hydrochloric acid and alcohol for 8 seconds, and tap water to turn blue for 5 minutes; 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol dehydration, then add 0.5ml reagent A, After shaking evenly, add 1ml of Reagent B, shake and mix evenly, centrifuge to precipitate, air-dry the precipitate, and seal with neutral resin; microscopic examination under an optical microscope, cytopathology experts read the film, according to the degree of coloration of the cell membrane and cytoplasm Determine the expression of PD-L1.

More, here, xylene + neutral resin is used for sealing, which needs to be fixed and dewatered and maintain neutrality. Therefore, reagent A (ethanol + formaldehyde) and reagent B (xylene) are used together.

Figure 4 is an image of circulating tumor cells isolated from the peripheral blood of a colorectal cancer patient. The nucleus is large 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 of colorectal cancer specimens to observe the differences, mainly for patients with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells , To guide the targeted therapy of colorectal cancer and provide new ideas for targeted therapy of colorectal cancer.

Claims (7)

1. A kit for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells in patients with colorectal cancer, which is characterized in that it includes 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-LI human primary antibody , Goat anti-human IgG/HRP 100μL, 0.1% Triton X-100 100μL, 0.3% H ₂ O ₂ 100μL, Reagent A, Reagent B, 6×PBS buffer 60mL.
2. The kit according to claim 1, wherein the diluent is composed of 1 mmol/L EDTA + 1 mmol moon silicate + 0.1% BSA + 0.2% poloxamer.
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 composed of ethanol and formaldehyde in a volume ratio of 3:1; and the reagent B is xylene.
6. A method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of colorectal 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 a membrane filtration device to separate and obtain CTCs in peripheral blood of patients with recurrent colorectal cancer who cannot obtain tissue samples: Collect peripheral blood of patients with colorectal 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, 95% ethanol, 100% ethanol gradient ethanol to dehydrate the CTC after returning to blue, then add 0.5mL reagent A, after shaking evenly, add 1mL reagent B, shake to mix evenly, centrifuge to precipitate, The sediment is sealed with neutral resin;

   (10) Microscopic examination under an optical microscope.

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