CN111521795A - Kit and method for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation - Google Patents
Kit and method for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation Download PDFInfo
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Abstract
The invention provides a kit and a detection method for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation, and belongs to the technical field of molecular biology. The kit comprises diluent, destaining solution, staining solution A, staining solution B1mL, PD-L1 (human) primary antibody, goat anti-human IgG/HRP L, 0.1% Triton X-100, 0.3% H2O2The detection method provided by the invention can detect the PD-L1 expression condition of a patient with late or recurrent non-small cell lung cancer without obtaining a tissue sample by puncture biopsy.
Description
Technical Field
The invention provides a kit and a detection method for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation, and belongs to the technical field of molecular biology.
Background
Lung cancer is one of the main malignant tumors causing death of cancer patients, and both the morbidity and mortality of lung cancer are the first in China. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers, and more than 70% of these NSCLC patients have been advanced at the time of diagnosis. Despite the increasing treatment technologies such as surgery and chemoradiotherapy, the 5-year survival rate of NSCLC patients is still less than 20%, and the main causes of death include local recurrence and distant metastasis.
Circulating Tumor Cells (CTC) are tumor cells that have been shed from solid tumors into the peripheral blood circulation, and since their discovery in 1989, various methods have been used to detect Circulating tumor cells in the peripheral blood. Recent studies have shown that their detection is of great clinical significance for assessing the prognosis of patients with tumours, especially patients with advanced tumours, and for selecting appropriate individualized treatments. CTC detection is called liquid biopsy of tumor because of its characteristics of minimal invasion, real-time detection, etc.
With the development of molecular biology, immunotherapy with PD-1/PD-L1 as an immune target brings new eosin for the treatment of non-small cell lung cancer. The research finds that the immunosuppression is closely related to immune escape and the over-expression of tumor cells PD-L1, and the tumor cells can be combined with PD-1 on the surface of T cells of immune cells through PD-L1 on the surface of the tumor cells to transmit inhibitory signals, so that the T cells cannot recognize the tumor cells and send attack signals to the tumor cells, and the immune escape of the tumor cells is caused. Based on this theory, it was hypothesized that the appearance of apoptosis, immune clearance or survival, metastatic outcome of Circulating Tumor Cells (CTCs) shed from primary foci into the circulatory system is closely related to PD-L1 expression. The curative effect of PD-1 or PD-L1 immune preparations is related to the immunohistochemical expression level of PD-L1 in tumor tissues, and the expression level of PD-L1 is possibly a biomarker for predicting the curative effect of PD-1 immunotherapy; further studies showed that high expression of PD-L1 in non-small cell lung cancer tissues was positively correlated with tumor invasiveness. Through the research of documents, the current detection methods of different applications of breast cancer, prostate cancer, colorectal cancer and liver cancer have few reports on the expression detection of Circulating Tumor Cells (CTC) PD-L1, but the detection of non-small cell lung cancer circulating tumor cells PD-L1 is not reported at home and abroad. Therefore, the detection of the expression condition of the Circulating Tumor Cell (CTC) PD-L1 has important value for the prognosis of the non-small cell lung cancer 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 patients with advanced or recurrent non-small cell lung cancer cannot obtain tissue specimens by real-time or repeated puncture and further cannot evaluate the state of PD-L1 of the patients, 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 patients with non-small cell lung cancer, which comprises the following steps: separating and obtaining CTC in peripheral blood of patients with advanced or recurrent non-small cell lung cancer, wherein the patients cannot obtain tissue specimens, 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:
the invention provides a kit for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation, which comprises 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-100100 muL and 0.3% H2O2100 mu L, 0.5mL of reagent A, 60mL of reagent B1mL and 6 × PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.4.
Further, the diluent is composed of 1mmol/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 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.
Further, the reagent A is 0.6% hydroxypropyl methyl cellulose aqueous solution; the reagent B is prepared from ethanol and 1, 2-propylene glycol according to a volume ratio of 3: 1.
The invention also provides a method for detecting the PD-L1 gene mutation of the peripheral blood circulating tumor cells of a patient with non-small cell lung cancer by using the kit for non-diagnosis purposes, which comprises the following steps:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent non-small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent non-small cell lung cancer who can not 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 for 2min × 3 times with PBS, (4) dripping 100 mu lPD-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.
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 expression condition of PD-L1 of a patient with advanced or recurrent non-small cell lung 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 lung 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 8 cases of non-small cell lung cancer patients (and simultaneously detecting 8 cases of normal human samples as negative controls) peripheral blood circulation tumor cells.
Example 1
Firstly, separating and acquiring CTCs in peripheral blood of a patient with advanced or recurrent non-small cell lung cancer, wherein a tissue specimen cannot be obtained, by using a membrane filtering 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 of diluent (component: 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylene polyoxypropylene ether block copolymer), and fixing the diluted blood sample with 3ml of 4% paraformaldehyde 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 9 healthy volunteers; except that no CTC was detected in 1 patient with recurrent non-small cell lung cancer, CTC was detected in all the patients (Table 2), the positive rate of the detection was 87.5%, and it is noted that when 0.1% trehalose or 0.2% polyoxyethylene polyoxypropylene ether block copolymer was not added to the diluent, 0.3% trehalose or 0.3% polyoxyethylene polyoxypropylene ether block copolymer was singly used to prepare a blood sample with poor stability, some blood samples were also stratified, blood cells were easily aggregated and adhered, and the final detection effect was affected.
TABLE 2 results of CTC assay in examples
Secondly, detecting the EGFR expression condition of the 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 at room temperature for 10min, washing with PBS for 2min × 3 times, dripping 100 μ L PD-L1 (human) primary antibody, incubating at room temperature for 2h (or overnight at 4 ℃), washing with PBS for 2min × 3 times, dripping 100 μ L goat anti-human IgG/HRP, incubating at room temperature (18-26 ℃) for 20min, washing with PBS for 2min × 3 times, dripping 100 μ L 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, dyeing with hematoxylin for 5min, differentiating with hydrochloric acid alcohol for 8 seconds, returning the running water to blue for 5min, 75% ethanol (1 min), 95% ethanol (1 min), dehydrating with 100% ethanol (1 min), and adding 0.6% hydroxypropyl methylUniformly oscillating the cellulose aqueous solution, adding a mixed solvent of ethanol and 1, 2-propylene glycol (V: V =3: 1), uniformly mixing by shaking, centrifugally precipitating, drying the precipitate in the air, and sealing by neutral resin; and (4) performing microscopic examination under an optical microscope, and judging the expression condition of PD-L1 by a cytopathologist according to the staining degree of cell membranes and cytoplasm.
When the reagent B adopts single ethanol or 1, 2-propylene glycol and is sealed by neutral resin, the detection accuracy of the mixed solvent of the ethanol and the 1, 2-propylene glycol can reach 100 percent after sealing, the accuracy of the single ethanol is 85 percent, and the accuracy of the single 1, 2-propylene glycol is only 70 percent, so that false positive results caused by edge effect possibly generated in the dyeing process can be avoided, the stability is good, the loss of cells is reduced, and the detection accuracy is improved.
FIG. 4 is a diagram of a circulating tumor cell image obtained by separating peripheral blood of a patient with non-small cell lung cancer, wherein the image is 1 CTC cell, the cell nucleus is abnormal, the karyoplasmic ratio is more than 0.8, the cell diameter (long end) is more than 15 μm, the cell nucleus is larger due to deep staining (due to the increase of cancer cell chromatin, coarsening of particles and deep staining), and the shape of the cell nucleus is irregular; nuclear chromatin border shift, abnormal nuclear division.
The detected circulating tumor cells are applied to immunohistochemistry to confirm that the expression of PD-L1 is compared with the result of a non-small cell lung cancer general specimen PD-L1, the difference is observed, the targeted therapy of the non-small cell lung cancer is guided mainly aiming at patients with negative expression of the general specimen PD-L1 and positive expression of the circulating tumor cells, and a new thought is provided for the targeted therapy of the non-small cell lung cancer.
Claims (7)
1. A kit for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation 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-100100 muL and 0.3% H2O2100 mu L, 0.5mL of reagent A, 60mL of reagent B1mL and 6 × PBS buffer solution, wherein the pH value of the PBS buffer solution is 7.4.
2. The kit of claim 1, wherein the diluent is composed of 1mmol/L EDTA +0.1% BSA +0.1% trehalose +0.2% polyoxyethylenepolyoxypropylene ether block copolymer and the base is Tris-HCl buffer.
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. The kit of claim 1, wherein the reagent a is a 0.6% hydroxypropyl methylcellulose aqueous solution; the reagent B is prepared from ethanol and 1, 2-propylene glycol according to a volume ratio of 3: 1.
6. A method for detecting the PD-L1 gene mutation in the peripheral blood circulating tumor cells of a patient with non-small cell lung cancer using the kit of any one of claims 1-5 for non-diagnostic purposes, comprising the steps of:
(1) separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent non-small cell lung cancer, wherein the patients cannot obtain tissue specimens, by using a membrane filtration device: collecting peripheral blood of patients with advanced or recurrent non-small cell lung cancer who can not 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.
7. The method of detecting according to claim 6, 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 mu lPD-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.
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| PCT/CN2021/088053 WO2021213306A1 (en) | 2020-04-21 | 2021-04-19 | 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 |
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| WO2021213306A1 (en) * | 2020-04-21 | 2021-10-28 | 山东第一医科大学(山东省医学科学院) | 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 |
| WO2022001824A1 (en) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | Kit and method for detecting pd-l1 gene mutations in circulating tumor cells in peripheral blood of patient with small cell lung cancer |
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| CN105588943B (en) * | 2016-01-28 | 2018-03-02 | 山东省肿瘤防治研究院 | A kind of detection method of the genes of peripheral blood from patients with gastric cancer circulating tumor cell Her 2 |
| CN108020666A (en) * | 2018-02-07 | 2018-05-11 | 福州大学 | It is a kind of can simultaneous quantitative detection blood in CEA and CA19-9 magnetic immuno-chromatographic test paper strip and preparation method |
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| CN111521795A (en) * | 2020-04-21 | 2020-08-11 | 山东第一医科大学(山东省医学科学院) | Kit and method for detecting non-small cell lung cancer patient peripheral blood circulating tumor cell PD-L1 gene mutation |
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| WO2022001824A1 (en) * | 2020-07-01 | 2022-01-06 | 山东第一医科大学第二附属医院 | Kit and method for detecting pd-l1 gene mutations in circulating tumor cells in peripheral blood of patient with small cell lung cancer |
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