CN111596058A - Kit and method for detecting FGFR gene mutation of peripheral blood circulation tumor cells of cholangiocarcinoma patient - Google Patents

Abstract

The invention provides a kit and a detection method for detecting FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma, belonging to the technical field of molecular biology. The kit includes diluent, destaining solution, staining solution A, staining solution B, FGFR (human) primary antibody, goat anti-human IgG/HRP, 0.1% Triton X‑100, 0.3% H ₂ O ₂ , reagent A, reagent B. 6×PBS buffer. ) The detection method provided by the present invention can detect the expression of FGFR in patients with cholangiocarcinoma without obtaining tissue samples by needle biopsy. The technology is minimally invasive and can be detected in real time, which 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

A test for detecting FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma Kit and detection method

technical field

The invention provides a kit and detection method for detecting FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma, especially for patients with cholangiocarcinoma with high risk of needle biopsy or unable to obtain tissue immunohistochemical detection of FGFR from biopsy, pre-assessment of advanced cholangiocarcinoma The patients were treated with FGFR inhibitor targeted therapy.

It belongs to the field of molecular biology technology.

Background technique

The incidence of intrahepatic cholangiocarcinoma accounts for about 15-20% of primary liver malignancies, and it is on the rise. Most patients with intrahepatic cholangiocarcinoma are often accompanied by local invasion or distant metastasis at the first visit and lose the chance of radical surgery. Chemotherapy with gemcitabine combined with platinum is recommended as the first-line regimen for the treatment of advanced cholangiocarcinoma, with an objective response rate (ORR) of 15-26% and frequent drug resistance. At present, there is a lack of other effective therapeutic drugs and programs in clinical practice.

The latest study shows that according to the updated data of pemigatinib second-line treatment of advanced cholangiocarcinoma (FIGHT202), the ORR of advanced cholangiocarcinoma gene detection FGFR2 fusion/rearrangement group was 35.5%, of which 3 patients had complete remission (CR), and the CR rate was 2.8% The DCR was 82%, while the ORR of the FGFR mutation and non-mutation groups was 0%. It can be seen that the application of pemigatinib targeted therapy to patients with cholangiocarcinoma based on gene detection of FGFR2 fusion/rearrangement in the future may have better curative effect, but for patients who cannot obtain tissue bile ducts Cancer patients cannot perform genetic detection of FGFR2 fusion/rearrangement status, and bile duct tumor biopsy faces risks such as bleeding, biliary fistula, infection, etc., and cannot dynamically detect FGFR2 fusion/rearrangement and mutation in real time. The detection of FGFR2 based on circulating tumor cells has important clinical guiding significance for the targeted therapy of cholangiocarcinoma, so that some patients can avoid unnecessary surgical trauma and puncture risks.

Therefore, detecting the expression of FGFR2 in circulating tumor cells (CTCs) in cholangiocarcinoma is of great value for evaluating the efficacy of targeted therapy for cholangiocarcinoma.

SUMMARY OF THE INVENTION

In order to overcome the deficiency that patients with advanced or recurrent cholangiocarcinoma cannot obtain tissue specimens in real time or repeatedly by puncturing, and thus cannot evaluate the real-time dynamic state of FGFR in patients, the present invention provides a real-time detection method for FGFR in peripheral blood of patients with cholangiocarcinoma: separation by using a membrane filtration device Circulating tumor cells (CTCs) in peripheral blood of patients with advanced cholangiocarcinoma were obtained, and the expression of FGFR on CTCs was further detected by immunohistochemistry.

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

The invention provides a kit for detecting FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma. 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; the pH of the PBS buffer 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 solution is Tris-HCl buffer.

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

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

Further, the reagent A is a 0.6% hydroxypropyl methylcellulose aqueous solution; 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 FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma using the above-mentioned kit for non-diagnostic purposes, comprising the following steps:

(1) Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of patients with intermediate and advanced cholangiocarcinoma who cannot obtain tissue samples: collect peripheral blood from patients with intermediate and advanced cholangiocarcinoma who cannot obtain tissue samples: 5ml of peripheral blood in the median cubital vein;

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

(3) Filter peripheral blood samples using membrane filtration device for separating tumor cells, and separate and obtain peripheral blood CTCs: add the pretreated peripheral blood sample to the blood sample container of the device for membrane filtration and separate tumor cells, and let it filter naturally by gravity;

(4) After filtration, 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; after the filtrate is completely filtered, add staining solution 1ml of solution B, stained for 2min, rinsed twice with 1ml of pure water, removed the filter membrane, placed it on a glass slide, and observed under a microscope after drying to determine whether CTC exists;

(5) The expression of FGFR in CTC was detected by immunohistochemical technique.

The concrete method that the present invention detects the FGFR expression of CTC is as follows:

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

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

(3) Add 100 μl 0.3% H ₂ O ₂ dropwise, incubate at room temperature for 10 min, wash with PBS for 2 min×3 times; (4) Add 100 μl FGFR (human) primary antibody dropwise, incubate at room temperature for 2 h or 4°C overnight, wash with PBS for 2 min×3 Second-rate;

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

(6) Add 100 μl of DAB color developing solution dropwise, incubate at 18-26°C 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 developing solution, rinse with running water for 5 minutes, and stain with hematoxylin for 5 minutes;

(8) Hydrochloric acid alcohol differentiation for 8 seconds, tap water returns to blue for 5 minutes;

(9) Dehydrate the blue-returned CTC with gradient ethanol of 75% ethanol (1 min), 95% ethanol (1 min), and 100% ethanol (1 min), then add 0.5 mL of reagent A, and after shaking evenly, add 1 mL of reagent B, After shaking and mixing evenly, centrifuge for precipitation, 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 tank and an iron stand, the iron stand is provided with a base, a stand and a bracket, and the blood sample container is arranged on the upper part of the iron stand through the stand, Below the blood sample container is a filter, and the filter is connected to a waste liquid tank through an infusion set, and the waste liquid tank is arranged on the base.

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

The filter membrane is made of hydrophobic material, and is evenly covered with filter holes with a diameter of 8 microns.

The beneficial effects of the present invention are:

(1) The detection method provided by the present invention can detect the expression of FGFR in patients with middle-advanced cholangiocarcinoma without obtaining tissue samples by needle biopsy. The technique is minimally invasive and enables real-time detection.

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

Description of drawings

1 is a schematic structural diagram of a 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;

Fig. 3 is the structural representation 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 cholangiocarcinoma.

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

Detailed ways

The present invention is described below with reference to the accompanying drawings and embodiments.

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

Table 1

An example of using this technique to isolate and identify peripheral blood circulating tumor cells from 8 patients with advanced cholangiocarcinoma (8 normal human samples were also detected as negative controls).

Example 1

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

Collect 5ml of fasting blood for 8-12 hours from the median cubital vein, and use 45ml of diluent (component: 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2% polyoxyethylene polyoxypropylene ether block copolymer) Dilute 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 Figure 1, Figure 2, Figure 3, the filter 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, then add the fixed peripheral blood sample to the blood sample container 2 of the membrane filtration device, so that it is naturally filtered by gravity, and 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, when the peripheral blood containing CTCs is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, while the diameter of CTCs is larger than The filter pores 10 are trapped on the filter membrane 7 .

After 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 min, and rinse with PBS buffer; the filtrate is completely filtered Then add B solution, 1ml, stain for 2min, pure water 1ml, PBS buffer to rinse filter 3, remove filter 7 with ophthalmic tweezers, place the cell side up on a glass slide;

The filter was dried and observed under a microscope to determine the presence of CTCs.

Through observation, no CTCs were detected in 8 healthy volunteers; CTCs were detected in 3 patients except 5 patients with cholangiocarcinoma (Table 2). The positive rate of this test was 37.5%. It is worth noting Yes, when the diluent does not add 0.1% trehalose or does not add 0.2% polyoxyethylene polyoxypropylene ether block copolymer, single use 0.3% trehalose or 0.3% polyoxyethylene polyoxypropylene ether block copolymer , the stability of the prepared blood sample is poor, and some blood samples will form layers, and blood cells are prone to aggregation and adhesion, which affects the final detection effect.

Table 2 Embodiment CTC detection results

2. Using immunohistochemical techniques to detect the expression of FGFR in CTCs:

Remove the CTC-loaded filter 7 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. Add 100 μl 0.1% Triton X-100 dropwise, incubate at room temperature for 15 min, wash with DI water for 2 min x 3 times; add dropwise 100 μl 0.3% H ₂ O ₂ , incubate at room temperature for 10 min, wash with PBS for 2 min x 3 times; add dropwise 100 μl FGFR (human ) primary antibody, incubate at room temperature for 2h (or overnight at 4°C), wash with PBS for 2min×3 times; add 100 μl goat anti-human IgG/HRP dropwise, incubate at room temperature (18~26°C) for 20min, wash with PBS for 2min×3 times; add dropwise 100μl DAB color developing solution, incubate at room temperature (18~26℃) and observe the color development under the microscope at any time (usually 3~10min, the time should not exceed 10min); after the color development is completed, discard the DAB color developing solution and rinse with running water 5min, hematoxylin staining for 5min; hydrochloric acid alcohol differentiation for 8 seconds, tap water to turn blue for 5min; 75% ethanol (1min), 95% ethanol (1min), 100% ethanol (1min) gradient ethanol dehydration, and then add 0.6% hydroxypropyl methyl alcohol cellulose-based aqueous solution, after shaking evenly, add ethanol and 1,2-propanediol mixed solvent (V:V=3:1), shake and mix evenly, centrifuge for precipitation, air dry the precipitate, and mount with neutral resin; optical Microscopic examination, cytopathology experts read the film, and determine the expression of FGFR according to the staining degree of cell membrane and cytoplasm.

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

Figure 4 is an image of circulating tumor cells isolated from peripheral blood of a patient with cholangiocarcinoma. The nuclei are large and irregular in shape, with a high nuclear-to-cytoplasmic ratio.

The detected circulating tumor cells were detected by immunohistochemistry to detect the expression of FGFR and compared with the FGFR expression results of cholangiocarcinoma surgical or puncture tissue specimens to observe the differences, and to verify the potential of CTCs as a non-invasive biopsy for real-time assessment of FGFR expression in cholangiocarcinoma. It provides an important reference for evaluating the prognosis of cholangiocarcinoma targeted therapy.

Claims (7)

1. a test kit for detecting FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma, it is characterized in that, comprises dilution solution 45mL, decolorization solution 1mL, staining solution A 0.5mL, staining solution B 1mL, FGFR (human) primary antibody 100μL , goat anti-human IgG/HRP 100 μL, 0.1% Triton X-100 100 μL, 0.3% H ₂ O ₂ 100 μL, Reagent A 0.5 mL, Reagent B 1 mL, 6×PBS buffer 60 mL; the pH of the PBS buffer is 7.4. 2. test kit according to claim 1, is characterized in that, described dilution is made up 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. test kit according to claim 1, is characterized in that, described decolorizing solution is made up of 95% alcohol and 100% xylene by volume ratio 1:1. 4. The kit according to claim 1, wherein the dyeing solution A is DAB dyeing solution; the dyeing solution B is hematoxylin dyeing solution. 5. test kit according to claim 1, is characterized in that, described reagent A is the hydroxypropyl methylcellulose aqueous solution of 0.6%; Described reagent B is ethanol and 1,2-propylene glycol according to volume ratio 3: 1 composition. 6. a method utilizing the non-diagnostic purpose of the test kit described in any one of claims 1-5 to detect the FGFR gene mutation in peripheral blood circulating tumor cells of patients with cholangiocarcinoma, is characterized in that, comprises the following steps: (1) Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of intermediate-advanced cholangiocarcinoma for which no tissue specimens can be obtained: collect peripheral blood from patients with intermediate-advanced cholangiocarcinoma for which no tissue specimens can be obtained: 5ml of peripheral blood from the median cubital vein; (2) Pretreatment of peripheral blood samples: The collected peripheral blood samples were diluted 10 times with diluent, and after dilution, paraformaldehyde was added to fix the peripheral blood samples for 10 minutes, and the final concentration was 0.25%; (3) Filter peripheral blood samples using membrane filtration device for separating tumor cells, and separate and obtain peripheral blood CTCs: add the pretreated peripheral blood sample to the blood sample container of the device for membrane filtration and separate tumor cells, and let it filter naturally by gravity; (4) After filtration, 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; after the filtrate is completely filtered, add staining solution 1ml of solution B, stained for 2min, rinsed twice with 1ml of pure water, removed the filter membrane, placed it on a glass slide, and observed under a microscope after drying to determine whether CTC exists; (5) The expression of FGFR in CTC was detected by immunohistochemical technique. 7. detection method according to claim 6, is characterized in that, the concrete method that the FGFR expression of described detection CTC is as follows: (1) Decolorization: Remove the filter membrane with CTC from the slide, soak it in the decolorizing solution for 4-6 hours, and remove the CTC staining solution; (2) Add 100 μl 0.1% Triton X-100 dropwise, incubate at room temperature for 15 min, and wash with DI water for 2 min × 3 times; (3) Add 100 μl 0.3% H ₂ O ₂ dropwise, incubate at room temperature for 10 min, and wash with PBS for 2 min × 3 times; (4) Add 100 μl FGFR (human) primary antibody dropwise, incubate at room temperature for 2 h or 4 °C overnight, and wash with PBS for 2 min × 3 times; (5) Add 100 μl goat anti-human IgG/HRP dropwise, incubate at 18-26 °C for 20 min, and wash with PBS for 2 min × 3 times; (6) Add 100 μl of DAB color developing solution dropwise, incubate at 18-26°C 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 developing solution, rinse with running water for 5 minutes, and stain with hematoxylin for 5 minutes; (8) Hydrochloric acid alcohol differentiation for 8 seconds, tap water returns to blue for 5 minutes; (9) Dehydrate the blue-returned CTC with gradient ethanol of 75% ethanol (1 min), 95% ethanol (1 min), and 100% ethanol (1 min), then add 0.5 mL of reagent A, and after shaking evenly, add 1 mL of reagent B, After shaking and mixing evenly, centrifuge for precipitation, and seal the precipitate with neutral resin; (10) Microscopic examination under an optical microscope.

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