CN111521798A - Kit and method for detecting PD-L1 gene mutation of peripheral blood circulating tumor cells of renal cancer patients - Google Patents

Abstract

The invention provides a kit and a detection method for detecting the gene mutation of peripheral blood circulating tumor cells PD-L1 of a patient with renal cancer, belonging to the technical field of molecular biology. The kit comprises a diluent, a destaining solution, a staining solution A, a staining solution B, PD-L1 (human) primary antibody, goat anti-human IgG/HRP, 0.1% Triton X-100 and 0.3% H₂O₂The detection method provided by the invention can detect the PD-L1 expression condition of a patient with advanced or recurrent renal cancer without obtaining a tissue sample by biopsy, belongs to minimally invasive technology, can detect in real time, can avoid false positive results caused by edge effect possibly generated in the dyeing process, has good stability, reduces the loss of cells, and improves the detection accuracy.

Description

Kit and method for detecting PD-L1 gene mutation of peripheral blood circulating tumor cells of renal cancer patients

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a kit and a detection method for detecting gene mutation of a peripheral blood circulation tumor cell PD-L1 of a patient with renal cancer.

Background

Renal Cell Carcinoma (RCC) is one of the most common malignant tumors of the urinary system, and the incidence and mortality rate of RCC accounts for about 2-3% of that of the systemic malignant tumors, and the incidence of RCC is on the rise in recent decades. Radical surgery is an effective means for treating kidney cancer, but distant metastasis has occurred in about 20% to 30% of patients with kidney cancer at the time of treatment. Even if the patient undergoes radical renal cancer surgery, 20-40% of patients still have relapse and metastasis. With the clinical application of targeted drugs, the life cycle of patients with advanced renal cancer is greatly prolonged, but the pure targeted therapy effect of patients with high-risk metastatic renal cancer is still not ideal, and the 5-year survival rate is less than 10%. In recent years, immunotherapy targeting PD-1/PD-L1 has brought new eosin for the treatment of renal cell carcinoma.

The research finds that the immunosuppression and the immune escape are closely related to the overexpression of the tumor cell PD-L1, and the tumor cell can be combined with PD-1 on the surface of an immune cell T cell through PD-L1 on the surface of the tumor cell to transmit inhibitory signals, so that the T cell cannot recognize and attack the tumor cell, and the tumor cell is subjected to the immune escape. Based on this theory, it is hypothesized that Circulating Tumor Cells (CTCs) shed from primary foci into the circulatory system undergo apoptosis, immune clearance or survival, and metastatic fate closely related to their PD-L1 expression levels. The previous research result proves that the curative effect of the PD-1 or PD-L1 immune preparation is mostly related to the expression level of PD-L1 in tumor tissues, which suggests that PD-L1 may be a biomarker for predicting the curative effect of immunotherapy, and in addition, the research shows that the high expression of PD-L1 in renal cell carcinoma tissues is positively related to the tumor invasiveness. Therefore, the detection of the expression condition of the Circulating Tumor Cell (CTC) PD-L1 has important value for the prognosis judgment of renal cell carcinoma and the evaluation of the curative effect of immunotherapy.

At present, units such as Shandong province first medical university, Shandong province drug research institute combined with Shandong Qixin Biotechnology limited company, Shandong well-known Biotechnology limited company, Jinan Xin Biotechnology limited company, Shandong discovery biotechnology limited company and the like, carry out industrialized popularization on the key technology of detection and identification of circulating tumor cells, the project is a Shandong province major scientific and technological innovation project, the project takes the Shandong province drug research institute in Jinan school of Shandong first medical university as the core, realizes a registration system, relies on the core diagnostic technology of detection and identification of circulating tumor cells, and further registers, identifies and diagnoses a kit, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catendin, E-Cacatenin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, progestin receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A and the like are used as tracers for expression of CTCs, and the identification and diagnosis kit is registered to be an ultrasensitive, ultrafast, high-coverage, low-cost, accurate and specific kit, and industrially popularized by cooperating with Shandong Qicheng Xin Biotech Co., Ltd, Shandong well-known Biotech Co., Ltd, Jinan En Biotech Co., Ltd, Shandong discovery Biotech Co., Ltd registered in Jinan.

Disclosure of Invention

In order to overcome the defects that a patient with advanced or recurrent renal cancer cannot obtain a tissue sample by real-time or repeated puncture and further cannot evaluate the state of PD-L1 of the patient, the invention provides a detection method for the non-diagnosis purpose of the mutation of peripheral blood circulating tumor cells PD-L1 gene of the patient with renal cancer: separating and obtaining CTC in peripheral blood of patients with advanced or recurrent non-renal cancer who can not obtain tissue specimens by using a membrane filtration device, and further detecting the PD-L1 expression condition of the CTC by using an immunohistochemical technology.

The technical scheme adopted by the invention is as follows:

a kit for detecting gene mutation of peripheral blood circulating tumor cells PD-L1 of renal cancer patients comprises 50mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of PD-L1 (human) primary antibody, 100 muL of goat anti-human IgG/HRP, 0.1% Triton X-100100 muL and 0.3% H₂O₂100. mu.L of 6 × PBS buffer 60 mL.

Further, the diluent consists of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene lauryl ether.

Further, the decoloring solution is composed of 95% alcohol and 100% xylene according to a volume ratio of 1: 1.

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

The method for detecting the PD-L1 gene mutation of the peripheral blood circulation tumor cells of the renal cancer patient by using the kit for non-diagnostic purposes comprises the following steps:

(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent renal cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of late stage or recurrent renal cancer patients who cannot obtain tissue samples: 5ml of peripheral blood of the median cubital vein;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(5) detecting the expression of PD-L1 of CTC by using immunohistochemical technology.

The specific method for detecting the PD-L1 expression of CTC comprises the following steps:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;

(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;

(3) 100 μ l of 0.3% H was added dropwise₂O₂Incubating for 10min at room temperature, washing with PBS for 2min × 3 times, (4) dropping 100 μ L PD-L1 (human) antibody, incubating at room temperatureIncubating for 2h or overnight at 4 ℃, washing with PBS for 2min × 3 times;

(5) dropping 100 mul goat anti-human IgG/HRP, incubating for 20min at 18-26 ℃, washing for 2min x 3 times with PBS;

(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;

(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;

(9) dehydrating the rewound CTC by using 75% ethanol (1 min), 95% ethanol (1 min) and 100% ethanol (1 min) in a gradient manner, then adding 0.5mL of reagent A, oscillating uniformly, adding 1mL of reagent B, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;

(10) and (5) performing microscopic examination by using an optical microscope.

The device for separating tumor cells by membrane filtration comprises a filter, a blood sample container, a waste liquid tank and an iron stand, wherein the iron stand is provided with a base, a vertical frame and a support, the blood sample container is arranged at the upper part of the iron stand through the support, the filter is arranged below the blood sample container, the filter is communicated to the waste liquid tank through an infusion apparatus, and the waste liquid tank is arranged on the base.

The filter comprises a filter upper opening, a filter membrane carrying platform and a filter lower opening, and the filter membrane is arranged on the filter membrane carrying platform; the upper port of the filter is connected with a blood sample container, and the lower port of the filter is connected with a waste liquid cylinder through an infusion apparatus.

The filter membrane is made of hydrophobic materials, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane.

The invention has the beneficial effects that:

(1) the detection method provided by the invention can detect the PD-L1 expression condition of a patient with advanced or recurrent renal cancer without taking a tissue sample by a puncture biopsy. The technology belongs to minimally invasive and can detect in real time.

(2) The method provided by the invention can avoid false positive results caused by edge effect possibly generated in the dyeing process, has good stability, reduces the loss of cells and improves the detection accuracy.

Drawings

FIG. 1 is a schematic structural view of a membrane filtration apparatus according to the present invention;

FIG. 2 is a schematic sectional view showing the structure of a filter of the membrane filtration apparatus of the present invention;

FIG. 3 is a schematic view showing the structure of a filter membrane of the membrane filtration apparatus of the present invention;

FIG. 4 is a diagram of a circulating tumor cell image obtained by separating peripheral blood of a patient with renal cancer;

in the figure: 1 iron stand, 2 blood sample containers, 3 filters, 4 transfusion devices, 5 waste liquid jars, 6 filter upper ports, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 vertical frames and 13 supports.

Detailed Description

The invention is elucidated below with reference to the figures and embodiments.

The specific specification of the kit used in the invention is shown in table 1:

TABLE 1

The technical method is applied to the embodiment of separating, obtaining and identifying 10 patients with kidney cancer (detecting 10 normal human samples as negative controls at the same time) peripheral blood circulation tumor cells.

Example 1

Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent renal cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device, and determining whether the CTCs exist:

collecting 5ml of fasting 8-12 hr fasting blood from the median cubital vein, diluting peripheral blood with 45ml diluent (component: 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene lauryl ether), and adding 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 min;

at fixed intervals, a membrane filtration device was assembled: as shown in fig. 1, 2 and 3, the filter device comprises a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5 and an iron stand 1;

wetting the filter 3 with 10ml of PBS, then adding the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, allowing it to naturally filter by gravity, and the CTC being trapped on the filter membrane 7;

the tumor cells are typically larger than 15 microns in diameter, while the blood cells (including red blood cells, white blood cells) are typically smaller than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells can be filtered by being smaller than filter pores 10, while the CTCs are retained on filter membrane 7 by being larger than filter pores 10.

After the filtration is finished, taking the filter 3 from the filter device, opening and removing the upper opening 6 of the filter, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; filtering the filtrate completely, adding solution B, 1ml, staining for 2min, and pure water 1ml, washing filter 3 with PBS buffer solution, taking down filter membrane 7 with ophthalmic forceps with cell surface facing upwards, and placing on glass slide;

the filters were dried and observed under a microscope to determine the presence of CTCs.

By observation, no CTCs were detected in 10 healthy volunteers; except that CTCs are not detected in 1 patient with advanced kidney cancer and 1 patient with recurrent kidney cancer, CTCs are detected in the other 8 patients (table 1), the positive rate of the detection is 80%, and it is noted that when 0.1% trehalose or 0.2% polyoxyethylene lauryl ether is not added to the diluent, 0.3% trehalose or 0.3% polyoxyethylene lauryl ether is singly used, the prepared blood sample has poor stability, a part of the blood sample can form stratification, blood cells are easy to aggregate and adhere, and the final detection effect is affected.

TABLE 1 results of CTC assay in examples

Secondly, detecting the expression condition of PD-L1 of CTC by using an immunohistochemical technology:

taking down the filter membrane 7 carrying CTC on the glass slide from the glass slide, soaking in a destaining solution of 95% alcohol and 100% xylene uniformly mixed according to a volume ratio of 1:1 for 4-6 hours, removing the CTC staining solution, dripping 100 mul of 0.1% Triton X-100, incubating at room temperature for 15min, washing with DI water for 2min × 3 times, dripping 100 mul of 0.3% H₂O₂Incubating for 10min at room temperature, washing for 2min × 3 times by PBS, dripping 100 mul PD-L1 (human) primary antibody, incubating for 2h (or overnight at 4 ℃), washing for 2min × 3 times by PBS, dripping 100 mul goat anti-human IgG/HRP, incubating for 20min at room temperature (18-26 ℃), washing for 2min × 3 times by PBS, dripping 100 mul DAB developing solution, incubating at room temperature (18-26 ℃) and observing the developing condition under a microscope at any time (generally 3-10 min, the time can not exceed 10 min), discarding the DAB developing solution after the developing is finished, flushing with running water for 5min, staining with hematoxylin for 5min, differentiating for 8 seconds by hydrochloric acid alcohol, returning blue by running water for 5min, 75% ethanol (1 min), 95% ethanol (1 min), dehydrating by 100% ethanol (1 min), drying the precipitate, sealing with neutral resin, examining under an optical microscope, and reading by cytopathology experts, and judging the PD-L1 expression condition according to the coloring degree of cell membranes and cytoplasm.

FIG. 4 is a photograph of a circulating tumor cell isolated from peripheral blood of a patient with renal cancer, wherein the CTC cell has abnormal cell nucleus shape, a karyoplasmic ratio of more than 0.8, a cell diameter (long end) of more than 15 μm, a nucleus deep stain (due to chromatin increase, coarseness of particles and deep stain of cancer cells) with a large nucleus and an irregular shape of the nucleus; high nuclear-to-mass ratio.

The detected circulating tumor cells are applied to immunohistochemistry to confirm the expression of PD-L1 and are compared with the result of PD-L1 of a renal cancer general sample, the difference is observed, the target treatment of the renal cancer is guided mainly aiming at patients with negative expression of PD-L1 of the general sample and positive expression of the circulating tumor cells, and a new thought is provided for the target treatment of the renal cancer.

Claims (6)

1. A kit for detecting the gene mutation of peripheral blood circulating tumor cells PD-L1 of a renal cancer patient is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 100 muL of PD-L1 (human) primary antibody, 100 muL of goat anti-human IgG/HRP, 0.1% Triton X-100100μL、0.3% H₂O₂60mL of 6 × PBS buffer with a pH of 7.6.

2. The kit according to claim 1, characterized in that the diluent consists of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% laureth alcohol.

3. The kit of claim 1, wherein the destaining solution is comprised of 95% alcohol to 100% xylene in a volume ratio of 1: 1.

4. The kit according to claim 1, wherein the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

5. A method for detecting a mutation in the PD-L1 gene of a peripheral blood circulating tumor cell of a patient with renal cancer using the kit of any one of claims 1 to 4 for non-diagnostic purposes, which comprises the steps of:

(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent renal cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of late stage or recurrent renal cancer patients who cannot obtain tissue samples: 5ml of peripheral blood of the median cubital vein;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; adding 1ml of staining solution B after completely filtering the filtrate, staining for 2min, washing for 2 times by using 1ml of pure water, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(5) detecting the expression of PD-L1 of CTC by using immunohistochemical technology.

6. The method of detecting according to claim 5, wherein the specific method for detecting PD-L1 expression in CTCs is as follows:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, and removing the CTC staining solution;

(2) dropping 100 μ l of 0.1% Triton X-100, incubating at room temperature for 15min, and washing with DI water for 2min × 3 times;

(3) 100 μ l of 0.3% H was added dropwise₂O₂Incubating for 10min at room temperature, washing for 2min × 3 times with PBS, (4) dripping 100 μ L PD-L1 (human) primary antibody, incubating for 2h at room temperature or overnight at 4 ℃, and washing for 2min × 3 times with PBS;

(5) dropping 100 mul goat anti-human IgG/HRP, incubating for 20min at 18-26 ℃, washing for 2min x 3 times with PBS;

(6) dripping 100 mul of DAB color development solution, incubating at 18-26 ℃, and observing the color development condition under a microscope at any time, wherein the observation time is 3-10 min;

(7) after the color development is finished, discarding DAB color development liquid, flushing with running water for 5min, and dyeing with hematoxylin for 5 min;

(8) the hydrochloric acid alcohol is differentiated for 8 seconds, and tap water is rewound for 5 min;

(9) dehydrating the rewound CTC by using 75% ethanol (1 min), 95% ethanol (1 min) and 100% ethanol (1 min) in a gradient manner, then adding 0.5mL of reagent A, oscillating uniformly, adding 1mL of reagent B, shaking and mixing uniformly, centrifuging and precipitating, and sealing the precipitate by using neutral resin;

(10) and (5) performing microscopic examination by using an optical microscope.

Images