WO2021213318A1

WO2021213318A1 - Non-diagnostic method for measuring braf gene v600e mutations in colorectal cancer patient by means of circulating tumor cells in peripheral blood

Info

Publication number: WO2021213318A1
Application number: PCT/CN2021/088101
Authority: WO
Inventors: 李胜; 黄宁; 刘智鸿; 李�浩
Original assignee: 山东第一医科大学(山东省医学科学院)
Priority date: 2020-04-21
Filing date: 2021-04-19
Publication date: 2021-10-28
Also published as: CN111521797A

Abstract

A non-diagnostic method for measuring BRAF gene V600E mutations in a colorectal cancer patient by means of circulating tumor cells in peripheral blood, the method primarily comprising collecting peripheral blood, processing the peripheral blood, filtering and enriching circulating tumor cells, and measuring BRAF gene V600E mutation expression in the circulating tumor cells using immunohistochemistry. In the measurement method provided in the present invention, expression of BRAF gene V600E mutations in a colorectal cancer patient can be measured without the use of puncture biopsy to collect a tissue sample. The method is minimally invasive and allows for real-time measurement, and 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 non-diagnostic method for detecting BRAF gene V600E mutations in colorectal cancer patients by peripheral blood circulating tumor cells

Technical field

The present invention relates to a new method for detecting BRAF gene V600E mutation, in particular to a method for BRAF gene V600E mutation in patients with advanced or recurrent colorectal cancer whose tissue samples cannot be obtained through peripheral blood detection.

Background technique

Colorectal cancer (CRC) is one of the common malignant tumors of the digestive tract in China. According to statistics, the incidence and mortality of CRC are among the forefront of all malignant tumors and are on the rise. However, its pathogenic factors and pathogenesis are still not fully understood. It is currently considered to be a complex process involving multiple genes in the regulation, involving abnormal activation of oncogenes (such as RAF, etc.) and inactivation of tumor suppressor genes (such as APC, P53, etc.). Studies have shown that BRAF gene plays an important role in the pathogenesis and treatment of colorectal cancer. However, the relationship between BRAF gene mutation characteristics in colorectal cancer and the relationship between the two and the clinical pathology has not been reported in many studies.

The BRAF gene is located in the proto-oncogene of human chromosome 7q34, which encodes a serine/threonine specific kinase. It is an activator of the RAF gene and MAPK signal transduction cascade that regulates the serine/threonine kinase. After activating mutations, it can develop in cancer. Plays a carcinogenic effect in. Studies have found that BRAF mutations can inhibit colonic epithelial cell apoptosis and promote cell proliferation, and promote the immortal proliferation, survival and migration of cancer cells. Even in the absence of cytokines and other stimuli, the V600E mutation in BRAF can activate this signaling pathway, leading to immortal cell proliferation and eventually cancer. Based on the mutation of the tumor BRAF gene, the current clinical specimens are mainly tumor tissues, which are derived from surgery or needle biopsy, and it is difficult to achieve multiple or real-time detection.

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 BRAF gene V600E mutations in colorectal cancer patients by using peripheral blood circulating tumor cells. A detection method for non-diagnostic purposes: the use of a membrane filter device to separate and obtain tissue specimens that cannot be obtained after surgery The CTC in the peripheral blood of patients with colorectal cancer was further used to detect the expression of the BRAF gene V600E of the CTC by immunohistochemistry.

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

A kit for detecting BRAF gene V600E mutation in peripheral blood circulating tumor cells in patients with colorectal cancer, including diluent 45mL, decolorizing solution 1mL, staining solution A 0.5mL, staining solution B 1mL, mouse anti-human BRAFV600E mutant protein monoclonal antibody (one Anti), goat anti-mouse 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 BRAF gene V600E mutations in peripheral blood circulating tumor cells of colorectal cancer for non-diagnostic purposes using the above-mentioned kit includes the following steps:

(1) Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of cancer patients who cannot obtain tissue samples: Collect peripheral blood of colorectal cancer patients who cannot obtain tissue samples: 5ml of peripheral blood of 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 pre-treated 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 immunohistochemical technology to detect the expression of BRAF gene V600E of CTC.

Wherein, the specific method for detecting the expression of the BRAF gene V600E 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 mouse anti-human BRAFV600E mutant protein monoclonal antibody (primary antibody) dropwise, incubate at room temperature for 2h or 4℃ overnight, wash with PBS for 2min×3 times;

(5) Add 100μl goat anti-mouse 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 to mix evenly, 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 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 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 BRAF gene V600E expression of patients with colorectal cancer without puncture biopsy to obtain tissue samples. The 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 the image of circulating tumor cells obtained from the peripheral blood of patients with colorectal cancer; the cutting head is CTM; in the figure: 1 iron stand, 2 blood sample container, 3 filter, 4 infusion set, 5 waste liquid tank, 6 filter on top Port, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 stands, and 13 supports.

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

Using this technology method to separate, obtain and identify 10 cases of colorectal cancer patients (10 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 10 healthy volunteers. Except for 3 cases of colorectal cancer, which were not detected (1 case of advanced colorectal cancer + 2 cases of recurrent colorectal cancer), the remaining 7 cases were all detected CTC (Table 1), the positive rate of this test was 70%.

Table 2 Example CTC detection results

2. Using immunohistochemical technology to detect the expression of BRAF gene V600E of 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; dropwise add 100 μl mouse anti-human BRAF gene V600E monoclonal antibody, incubate at room temperature for 2h (or overnight at 4℃), wash with PBS for 2min×3 times; add 100μl goat anti-mouse 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 reagent B, shake and mix well, centrifuge to precipitate, air-dry the precipitate, and seal with neutral resin; check under an optical microscope, read by cytopathologists, and color according to cell membrane and cytoplasm To determine the degree of BRAF gene V600E expression.

More, here, xylene + neutral resin is used for sealing, which needs to be fixed to remove water 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 patient with colorectal cancer. 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 to confirm the expression of BRAF gene V600E and compared with the results of BRAF gene V600E in gross specimens of colorectal cancer, to observe the differences, mainly for patients with negative expression of BRAF gene V600E 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

A kit for detecting BRAF gene V600E mutations in peripheral blood circulating tumor cells of 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 mouse anti-human BRAFV600E mutant protein single Cloned antibody, goat anti-mouse 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 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.
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.
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.
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.
A method for detecting BRAF gene V600E mutation in peripheral blood circulating tumor cells of colorectal cancer for non-diagnostic purposes using the kit according to any one of claims 1-5, which is characterized in that it comprises the following steps:

(1) Use a membrane filtration device to separate and obtain CTCs in the peripheral blood of cancer patients who cannot obtain tissue samples: Collect peripheral blood of colorectal cancer patients who cannot obtain tissue samples: 5ml of peripheral blood of 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 immunohistochemical technology to detect the expression of BRAF gene V600E of CTC.
The detection method according to claim 6, wherein the specific method for detecting the expression of BRAF gene V600E 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 mouse anti-human BRAFV600E mutant protein monoclonal antibody dropwise, incubate at room temperature for 2h or 4℃ overnight, wash with PBS for 2min×3 times;

(5) Add 100μl goat anti-mouse 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.