WO2021213306A1 - Test kit for measuring pd-l1 gene mutations in circulating tumor cells in peripheral blood in non-small cell lung cancer patient, and measurement method - Google Patents

Abstract

Provided in the present invention are a test kit for measuring PD-L1 gene mutations in circulating tumor cells in peripheral blood in a non-small cell lung cancer patient, and a method for same, relating to the technical field of molecular biology. The test kit comprises a diluent, a decoloring solution, a dyeing solution A, 1 mL of a dyeing solution B, a (human) PD-L1 primary antibody, goat anti-human IgG/HRP L, 0.1% Triton X-100, 0.3% H2O2, a reagent A, a reagent B, and 6xPBS buffer solution. In the measurement method provided in the present invention, expression of PD-L1 in a late-stage or relapsed non-small cell lung cancer patient can be measured without the use of puncture biopsy to collect a tissue sample. The present technology is minimally invasive, and allows for real-time measurement. The invention is able to avoid false positive results caused by edge effects possibly produced during a dyeing process, and features good stability, reduction of cell loss, and improved measurement accuracy.

Description

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

The invention provides a kit and a detection method for detecting PD-L1 gene mutations in peripheral blood circulating tumor cells of patients with non-small cell lung cancer, and belongs to the technical field of molecular biology.

Background technique

Lung cancer is one of the main malignant tumors leading to the death of cancer patients. In China, the incidence and mortality of lung cancer rank first. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and more than 70% of NSCLC patients are already at an advanced stage at the time of diagnosis. Despite the continuous improvement of treatment techniques such as surgery, radiotherapy and chemotherapy, the 5-year survival rate of NSCLC patients is still less than 20%. The main causes of death include local recurrence and distant metastasis.

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.

With the development of molecular biology, immunotherapy with PD-1/PD-L1 as an immune target has brought a new dawn to the treatment of non-small cell lung 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 non-small cell lung cancer tissue is positively correlated with tumor aggressiveness. A literature search found that there are a few reports on the detection of circulating tumor cells (CTC) PD-L1 expression using different detection methods for breast cancer, prostate cancer, colorectal cancer, and liver cancer. There are no reports of testing at home and abroad. Therefore, detecting the expression of circulating tumor cells (CTC) PD-L1 is of great value for the prognosis of non-small cell lung 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, 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 non-small cell lung 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 PD-L1 gene mutation in circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer Detection methods for non-diagnostic purposes: Use membrane filtration devices to separate CTCs in peripheral blood of patients with advanced or recurrent non-small cell lung cancer who cannot obtain tissue specimens, and further use immunohistochemistry to detect the expression of CTC PD-L1.

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

The present invention provides a kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with non-small cell lung cancer. ) 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, reagent A 0.5 mL, reagent B 1 mL, 6×PBS buffer 60 mL; the PBS buffer The pH is 7.4.

Further, the diluent is composed of 1 mmol/L EDTA + 0.1% BSA + 0.1% trehalose + 0.2% polyoxyethylene polyoxypropylene ether block copolymer, and the base liquid is a Tris-HCl buffer.

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.

Further, the reagent A is a 0.6% aqueous solution of hydroxypropyl methylcellulose; the reagent B is composed of ethanol and 1,2-propanediol in a volume ratio of 3:1.

The present invention also provides a method for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with non-small cell lung cancer for non-diagnostic purposes by using the above kit, which includes the following steps:

(1) Use membrane filtration device to separate and obtain CTC in peripheral blood of patients with advanced or recurrent non-small cell lung cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or relapsed non-small cell lung cancer who cannot obtain tissue samples: Peripheral blood of median cubital vein 5ml;

(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 overnight at 4°C, wash with PBS for 2 min× 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 mouth, a filter membrane, a filter membrane platform and a filter lower mouth. The filter membrane is placed on the filter membrane platform; the upper mouth of the filter is connected to the blood sample container, and the lower mouth of the filter is connected to the waste liquid tank through the infusion set.

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 non-small cell lung 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 lung 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 buffer	60mL
Diluent	45mL
Decolorizing liquid	1mL
Staining solution A	0.5mL
Staining Solution B	1mL
PD-L1 (human)-anti	100μL
Goat anti-human IgG/HRP	100μL
0.1%Trition X-10	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 8 cases of non-small cell lung cancer patients (8 normal human 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 non-small cell lung 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 use 45ml of diluent (composition: 1mmol/L EDTA+0.1%BSA+0.1% trehalose+0.2% polyoxyethylene polyoxypropylene ether block copolymer) Dilute the peripheral blood, 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 9 healthy volunteers. Except for 1 patient with relapsed non-small cell lung cancer, which was not detected, CTC was detected in the rest (Table 2). The positive rate of this test was 87.5%, which is worth noting. However, when the diluent does not add 0.1% trehalose or 0.2% polyoxyethylene polyoxypropylene ether block copolymer, 0.3% trehalose or 0.3% polyoxyethylene polyoxypropylene ether block copolymer is used alone The stability of the prepared blood sample 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 2 Example CTC detection results

2. Use immunohistochemical technology to detect the expression of EGFR in CTC:

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°C), wash with PBS for 2min×3 times; add 100μl goat anti-human IgG/HRP dropwise, incubate at room temperature (18～26°C) 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 with running water for 5 minutes, stain with hematoxylin for 5 minutes; differentiate with hydrochloric acid and alcohol for 8 seconds, tap water to turn blue for 5 minutes; 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol dehydration, then add 0.6% hydroxypropyl Methyl cellulose aqueous solution, after shaking evenly, add a mixed solvent of ethanol and 1,2-propanediol (V:V=3:1), shake and mix evenly, centrifuge to precipitate, dry the precipitate, and seal with neutral resin ; Microscopic examination under an optical microscope, cytopathology experts read the pictures, and judge the expression of PD-L1 according to the degree of staining of cell membrane and cytoplasm.

When a single ethanol or 1,2-propanediol is used for reagent B, after solid sealing with a neutral resin, the detection accuracy of the ethanol and 1,2-propanediol mixed solvent of the present invention after solid sealing can reach 100%, and The accuracy of a single ethanol is 85%, while the accuracy of using a single 1,2-propanediol is only 70%, which can avoid false positive results caused by edge effects that may occur during the staining process, and has good stability and reduces cells. The loss of detection, improve the accuracy of detection.

Figure 4 is an image of circulating tumor cells isolated from peripheral blood of a patient with non-small cell lung cancer. It is a CTC cell with nuclear atypia, nuclear to cytoplasm ratio greater than 0.8, cell diameter (long end) greater than 15 μm, and nuclear deep staining ( Due to the increase in chromatin of cancer cells, the particles become thicker, and the nucleus is deeply stained. The nucleus is larger and the shape of the nucleus is irregular; the chromatin of the nucleus is shifted and abnormal nuclear division.

The detected circulating tumor cells were confirmed by immunohistochemistry for the expression of PD-L1 and compared with the results of non-small cell lung cancer specimens of PD-L1 to observe the differences, mainly for those with negative expression of PD-L1 in gross specimens and positive expression of circulating tumor cells Patients, guide the targeted therapy of non-small cell lung cancer, and provide new ideas for targeted therapy of non-small cell lung cancer.

Claims (7)

1. A kit for detecting PD-L1 gene mutations in circulating tumor cells in the peripheral blood of patients with non-small cell lung 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, reagent A 0.5 mL, reagent B 1 mL, 6×PBS buffer 60 mL; the PBS buffer The pH is 7.4.
2. The kit according to claim 1, wherein the diluent is composed of 1mmol/L EDTA+0.1%BSA+0.1% trehalose+0.2% polyoxyethylene polyoxypropylene ether block copolymer. The base solution is Tris-HCl buffer.
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 a 0.6% aqueous solution of hydroxypropyl methylcellulose; the reagent B is composed of ethanol and 1,2-propanediol in a volume ratio of 3:1 .
6. A method for detecting PD-L1 gene mutations in circulating tumor cells in peripheral blood of patients with non-small cell lung 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 non-small cell lung cancer who cannot obtain tissue samples: Collect peripheral blood of patients with advanced or relapsed non-small cell lung cancer who cannot obtain tissue samples: Peripheral blood of median cubital vein 5ml;

   (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) drop 100 μl PD-L1 (human) primary antibody, incubate at room temperature for 2 hours or overnight at 4°C, wash with PBS for 2 min× 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.

Images