Multiplex fluorescence immunohistochemical detection panel for rare lung tumors, detection kit and application thereof

Abstract

The invention provides a multiple fluorescence immunohistochemical detection panel for rare lung tumors, a detection kit and application of the detection panel and the detection kit, and belongs to the technical field of multiple immunohistochemistry. The invention provides a multiple fluorescence immunohistochemical detection panel for rare lung tumors, and relates to 6 tumor cell related target proteins at the same time. According to the invention, the multiplex fluorescence immunohistochemical detection method and the kit thereof are used for lung cancer, especially for detection of rare subtypes. According to the method, multiple fluorescent staining of a rare lung cancer sample with a complex structure is realized on a single slice by matching a specific staining sequence and fluorescent channels, and false positive caused by staining crosstalk of each channel is prevented. The multiple detection method can be used for detecting six drug use related targets at one time, and the detection result shows the expression quantity of the related targets in a sample and can be used as a reference basis for clinical drug use.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of multiplex immunohistochemistry, and particularly relates to a multiplex fluorescence immunohistochemistry detection panel for rare lung tumors, a detection kit and application thereof. BACKGROUND

[0002] In the past 30 years, the incidence and mortality of respiratory tract cancers have been rising globally, especially tracheal, bronchial and lung cancers, and respiratory tract cancers have the trend of becoming the first cancer. Adenoid cystic carcinoma of the lung, as one of the rare lung tumors, has an incidence of only 0.04% to 0.2% of lung tumors. It occurs in the trachea or carina, and large bronchus. It grows along the wall of the tube, can invade the surrounding tissues and organs, and block the bronchial lumen, causing chest tightness, shortness of breath, difficulty breathing and even hemoptysis, which greatly affects the quality of life. Unlike common lung cancer, adenoid cystic carcinoma has a wide age range, mostly concentrated in young adults, and children can also be affected. At present, due to its rarity, the disease is relatively difficult to diagnose, and the treatment methods are relatively single, such as surgical resection, chemotherapy and radiotherapy. However, surgical resection is prone to recurrence, and chemotherapy and radiotherapy methods cannot effectively improve the survival time of patients, so immunological checkpoints, ADC drugs and CAR-T related treatment methods have entered the field of vision and are increasingly valued.

[0003] The treatment of lung cancer has gradually moved from the era of chemotherapy to the era of targeted therapy, and then to the era of Check-point inhibitors. However, small cell lung cancer, as a rare tumor accounting for less than 10% of lung cancer, has no standard treatment options after the second line, so immunotherapy may be a promising and challenging new method in clinical practice. A large number of clinical trials are exploring the clinical value of immune inhibitors in the treatment of small cell lung cancer patients. Clinical trials have confirmed that the efficacy of immunotherapy is not inferior to that of non-standard chemotherapy regimens, but there is no definite data to confirm the clinical benefit of adding immunotherapy to standard chemotherapy regimens, while increasing the incidence of adverse events and treatment-related mortality. Under the condition of a small number of patients enrolled in the trial, a number of patients have died, prompting deep thinking about the method of immunotherapy.

[0004] The efficacy of SCLC immunotherapy is lower than that of other malignant tumors. On the one hand, it may be due to the high malignancy and strong tumor heterogeneity of SCLC, and there may be other special immune therapy targets in the tumor cells that have not been discovered yet. On the other hand, SCLC has a short doubling time and progresses rapidly, while immune therapy takes a relatively long time to take effect. By the time the immune therapy has reached its maximum benefit, SCLC may have progressed and metastasized. Therefore, it is necessary to find better biomarkers, such as MET, EGFR, etc., to guide clinical treatment, and to expect that the field of SCLC treatment will soon open a new chapter.

[0005] In addition, immune checkpoint inhibitors relieve the inhibition of T cell activity by blocking the interaction of immune checkpoint molecules with ligands, enhancing T cell-mediated anti-tumor immune response. Related therapies have achieved great success in lung cancer treatment, and are also an opportunity for more targets to enter immunotherapy. However, the existing immunohistochemical techniques for detecting lung cancer, such as those in Chinese patents CN201910032641.8 and CN202311150519.3, have defects such as single immune microenvironment or single drug-related target, and the utilization rate of multiple immunohistochemical channels is a great waste. SUMMARY

[0006] The application provides a multiple fluorescence immunohistochemical detection panel for rare lung tumors, a detection kit and an application thereof, which combines the advantages of "multiple channels + drug-related targets + rare lung cancer applicability" and provides reliable basis for lung cancer targeted therapy.

[0007] The application provides a multiple fluorescence immunohistochemical detection panel for rare lung tumors, which comprises a monoclonal antibody group, fluorescent dyes specifically combined with the monoclonal antibody group, and secondary antibodies.

[0008] The monoclonal antibody group comprises the following antibodies: ROS1 monoclonal antibody, HER2 monoclonal antibody, HER3 monoclonal antibody, MET monoclonal antibody, ALK monoclonal antibody and EGFR monoclonal antibody.

[0009] In a preferred mode of the application, the secondary antibodies comprise HPR enzyme-labeled anti-mouse / rabbit IgG polymer secondary antibodies.

[0010] In a preferred mode of the application, the fluorescent dyes comprise 480 fluorescent dyes, 520 fluorescent dyes, 570 fluorescent dyes, 620 fluorescent dyes, 670 fluorescent dyes and 780 fluorescent dyes.

[0011] The application further provides a multiple immunohistochemical kit comprising the above-mentioned multiple fluorescence immunohistochemical detection panel.

[0012] In a preferred mode of the application, the kit further comprises buffer solution, eluent, blocking solution, biotin, DAPI nuclear staining reagent, antigen repair solution and signal amplification solution.

[0013] In a preferred mode of the application, the buffer solution is a mixed solution composed of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20 and ProClin300.

[0014] The blocking solution is a mixed solution composed of BSA, TBS and ProClin300;

[0015] The signal amplification solution is a mixed solution composed of magnesium sulfate, 4-bromophenylboronic acid and Tris-HCl;

[0016] The antigen repair solution is a solution composed of disodium ethylenediaminetetraacetate and tris-hydroxymethyl aminomethane;

[0017] The elution solution is a solution composed of disodium ethylenediaminetetraacetate and tris-hydroxymethyl aminomethane, which has the same components as the antigen repair solution and is used for elution of the primary and secondary antibody complexes after completing a round of fluorescent staining.

[0018] The application also provides a use method of the multiplex immunohistochemical kit, which comprises the following steps:

[0019] (1) placing the detection sample on the X30 full-automatic staining instrument, and obtaining a pretreated section through baking, dewaxing, antigen repair, buffer cleaning and blocking with the blocking solution;

[0020] (2) incubating the pretreated section with ROS1 monoclonal antibody, marking 480 fluorescent dye after binding with secondary antibody, and obtaining a first fluorescent staining section through elution and blocking;

[0021] (3) incubating the first fluorescent staining section with HER2 monoclonal antibody, marking 570 fluorescent dye after binding with secondary antibody, and obtaining a second fluorescent staining section through elution and blocking;

[0022] (4) incubating the second fluorescent staining section with HER3 monoclonal antibody, marking 670 fluorescent dye after binding with secondary antibody, and obtaining a third fluorescent staining section through elution and blocking;

[0023] (5) incubating the third fluorescent staining section with MET monoclonal antibody, marking 520 fluorescent dye after binding with secondary antibody, and obtaining a fourth fluorescent staining section through elution and blocking;

[0024] (6) incubating the fourth fluorescent staining section with ALK monoclonal antibody, marking 620 fluorescent dye after binding with secondary antibody, and obtaining a fifth fluorescent staining section through elution and blocking;

[0025] (7) incubating the fifth fluorescent staining section with EGFR monoclonal antibody, marking biotin after binding with secondary antibody, and marking 680 fluorescent dye in sequence through elution again; ​​​​​​780 fluorescent dyes and DAPI nuclear stain.

[0026] In a preferred mode of the present application, in steps (2) to (7), the temperature of the incubation is 25-40℃, and the time is 15-90min.

[0027] In a preferred mode of the present application, in steps (2) to (7), the temperature of the incubation is 25-40℃, and the time is 15-90min.

[0028] The present application also provides the use of the above-mentioned multiplex fluorescent immunohistochemical detection panel or the above-mentioned multiplex immunohistochemical kit in the preparation of the following products, and the use includes at least one of the following: (1) a product for predicting the effectiveness of an immune checkpoint inhibitor for lung cancer treatment;

[0029] (2) a product for evaluating the screening of targeted drugs for rare tumors in the lung;

[0030] (3) a product for predicting the expression amount of marker proteins in lung cancer samples.

[0031] Beneficial effects: the present application provides a multiplex fluorescent immunohistochemical detection panel for rare tumors in the lung, which simultaneously involves six tumor cell-related target proteins (ROS1, HER2, HER3, ALK, MET, EGFR). The present application uses the multiplex fluorescent immunohistochemical (mIHC) detection method and its kit for lung cancer, especially the simultaneous detection of multiple drug-related target proteins (ROS1, HER2, HER3, ALK, MET, EGFR) in rare subtypes of lung cancer, such as lung adenoid cystic carcinoma and small cell lung cancer, and combines the advantages of "multi-channel + drug-related target + rare lung cancer application", which provides a reliable basis for targeted treatment of lung cancer.

[0032] The ROS1, HER2, HER3, ALK, MET, EGFR protein target multiplex immunohistochemical detection is carried out on a single slice, the sample quantity is saved while the spatial position information of each protein target is increased; the multiplex immunohistochemical advantage is that arbitrary two targets can be recognized in the same cell co-localization positive, part of ALK positive cells exist in the position of EGFR positive, and the two do not completely coincide, so that the sample slice quantity is saved while the detection target flux is increased. The six protein targets are realized multiplex fluorescence staining of complex structure rare lung cancer samples in a specific staining order and fluorescence channel matching, false positives caused by staining crosstalk of each channel are prevented; the high sensitivity and strong specificity of the biotin-streptavidin system make up for the defects of the conventional 780 near-infrared spectrum channel which is weak and prone to false negatives, and realize the improvement of more than 10 times fluorescence intensity of conventional staining. The biotin-streptavidin system can complete the detection of complex structure samples such as lung adenoid cystic carcinoma and small cell lung cancer which need high signal-to-noise ratio and signal amplification, by virtue of its high sensitivity and strong specificity.

[0033] The targets detected in the detection Panel of the application have associated drugs on the market or in the clinical stage, such as EGFR overexpression corresponding drugs (cetuximab, necitumumab), ALK / ROS1 rearrangement and overexpression can use corresponding ALK / ROS1 inhibitors (such as crizotinib, lorlatinib), MET amplification / overexpression can use corresponding MET inhibitors (such as capmatinib, tepotinib), and HER2 / HER3 abnormalities may indicate that HER family targeted drugs (such as ADC drug dacetuzumab or TKI class drugs) can be used. The multiplex detection method used in the application can detect six drug-related targets at a time, and the detection results reflect the expression amount of the related targets in the sample, which can be used as a reference for clinical drug use. Unlike the prior art which detects only a single immune microenvironment or a single drug-related target, the actual utilization rate of the Panel channel is increased, which provides the possibility for the majority of patients to benefit more practically. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Schematic diagram of fluorescence channel staining without crosstalk for example 1 HER2- 570 / MET- 520 fluorescence channel without crosstalk schematic diagram;

[0035] Figure 2 Schematic diagram of fluorescence channel staining without crosstalk for example 1 HER2- 570+DAPI fluorescence channel staining schematic diagram;

[0036] Figure 3 Schematic diagram of fluorescence channel staining without crosstalk for example 1 MET- 520+DAPI fluorescence channel staining schematic diagram;

[0037] Figure 4 HER2- for the control 1 570 / HER3- 520 fluorescence channel staining crosstalk schematic diagram;

[0038] Figure 5 HER2- for the control 1 570+DAPI fluorescence channel staining schematic diagram;

[0039] Figure 6 HER3- for the control 1 520+DAPI fluorescence channel staining schematic diagram;

[0040] Figure 7 is a lung squamous carcinoma sample multiplex fluorescence immunohistochemical detection result diagram, in the drawing A: Panel combination schematic diagram-ROS1 / HER3 / HER2 / ALK / MET / EGFR / DAPI, B: ROS1( 480)+DAPI, C: MET( 520)+DAPI, D: HER2( 570)+DAPI, E: ALK( 620)+DAPI, F: HER3( 670)+DAPI, G: EGFR( 780))+DAPI;

[0041] Figure 8 is a adenoid cystic carcinoma sample multiplex fluorescence immunohistochemical detection result diagram, in the drawing A: Panel combination schematic diagram-ROS1 / HER3 / HER2 / ALK / MET / EGFR / DAPI, B: ROS1( 480)+DAPI, C: MET( 520)+DAPI, D: HER2( 570)+DAPI, E: ALK( 620)+DAPI, F: HER3( 670)+DAPI, G: EGFR( 780))+DAPI;

[0042] Figure 9 is a small cell lung cancer sample multiplex fluorescence immunohistochemical detection result diagram, in the drawing A: Panel combination schematic diagram-ROS1 / HER3 / HER2 / ALK / MET / EGFR / DAPI, B: ROS1( 480)+DAPI, C: MET( 520)+DAPI, D: HER2( 570) + DAPI, E: ALK( 620) + DAPI, F: HER3( 670) + DAPI, G: EGFR( 780)) + DAPI. DETAILED DESCRIPTION

[0043] The present application provides a multiplex fluorescence immunohistochemical detection panel for rare lung tumors, comprising a monoclonal antibody group, fluorescent dyes specifically combined with the monoclonal antibody group, and secondary antibodies.

[0044] The monoclonal antibody group comprises the following antibodies: ROS1 monoclonal antibody, HER2 monoclonal antibody, HER3 monoclonal antibody, MET monoclonal antibody, ALK monoclonal antibody and EGFR monoclonal antibody.

[0045] In the present application, the Chinese names and full English expressions of various abbreviations used are as shown in Table 1.

[0046] Table 1 Abbreviation explanation of the present application

[0047] Chinese expression Complete English expression English abbreviation immunohistochemistry Immunohistochemistry IHC multiplex immunohistochemistry Multiplex Immunohistochemistry mIHC small cell lung cancer Small Cell Lung Cancer SCLC tyramide signal amplification Tyramide Signal Amplification TSA proto-oncogene tyrosine-protein kinase 1 Proto-oncogenetyrosine-proteinkinase1 ROS1 receptor tyrosine-protein kinase erbB-2 Receptortyrosine-proteinkinaseerbB-2 HER2 receptor tyrosine-protein kinase erbB-3 Receptortyrosine-proteinkinaseerbB-3 HER3 ALK tyrosine kinase receptor ALKtyrosinekinasereceptor ALK hepatocyte growth factor receptor Hepatocytegrowthfactorreceptor MET epidermal growth factor receptor Epidermalgrowthfactorreceptor EGFR

[0048] Each monoclonal antibody in the panel described in the present application can be derived from a conventional commercially available product, such as the sources and catalog numbers of each monoclonal antibody used in the examples as follows: ROS1 Recombinant Rabbit Monoclonal Antibody [PD01-27] - HUABIO (Catalog # HA721420); HER2 / ErbB2 Recombinant Rabbit Monoclonal Antibody [PD00-53] - HUABIO (Catalog # HA721178); ErbB3 / HER3 Recombinant Rabbit Monoclonal Antibody [PD00-44] - HUABIO (Catalog # HA721194); ALK-1 Rabbit Polyclonal Antibody - HUABIO (Catalog # ER62643); c-Met Mouse Monoclonal Antibody [A9A4] - HUABIO (Catalog # HA601092); EGFR Recombinant Rabbit Monoclonal Antibody [SZ40-19] - HUABIO (Catalog # ET1603-37).

[0049] The secondary antibody includes HPR enzyme-labeled anti-mouse / rabbit IgG polymer secondary antibody. The fluorescent dyes include 480 fluorescent dyes, 520 fluorescent dyes, 570 fluorescent dyes, 620 fluorescent dyes, 670 fluorescent dyes, and 780 fluorescent dyes. In one embodiment, each of the fluorescent dyes is derived from Alpha 7-color fluorescent staining kit - Alpha X Bio (Catalog # AXT37100041). Of course, the panel also includes BIOTIN, DAPI nuclear staining reagent, and the like.

[0050] The application also provides a multiplex immunohistochemical kit comprising the multiplex fluorescent immunohistochemical detection panel.

[0051] In one preferred embodiment of the application, the kit further comprises a buffer solution, an eluent, a blocking solution, biotin, a DAPI nuclear staining reagent, an antigen retrieval solution, and a signal amplification solution.

[0052] In one preferred embodiment of the application, the buffer solution is a mixed solution composed of tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, and ProClin 300.

[0053] The blocking solution is a mixed solution composed of BSA, TBS, and ProClin 300.

[0054] The signal amplification solution is a mixed solution composed of magnesium sulfate, 4-bromophenylboronic acid, and Tris-HCl.

[0055] The antigen retrieval solution is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane.

[0056] The eluent is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl)aminomethane, which has the same components as the antigen retrieval solution and is used to elute the primary and secondary antibody complexes after completing a round of fluorescent staining.

[0057] The specifications of the multiplex immunohistochemical kit constructed by the application can be as shown in Table 2:

[0058] Table 2: One specification of the multiplex immunohistochemical kit of the application

[0059]

[0060]

[0061] The application also provides a method for using the multiplex immunohistochemical kit, comprising the following steps:

[0062] (1) placing a detection sample on an X30 full-automatic staining instrument, and performing baking, dewaxing, antigen repairing, buffer cleaning and blocking with blocking solution to obtain a pretreated section;

[0063] (2) incubating the pretreated section with a ROS1 monoclonal antibody, marking with a secondary antibody, and then marking with a 480 fluorescent dye after elution to obtain a first fluorescently stained section;

[0064] (3) incubating the first fluorescently stained section with a HER2 monoclonal antibody, marking with a secondary antibody, and then marking with a 570 fluorescent dye after elution to obtain a second fluorescently stained section;

[0065] (4) incubating the second fluorescently stained section with a HER3 monoclonal antibody, marking with a secondary antibody, and then marking with a 670 fluorescent dye after elution to obtain a third fluorescently stained section;

[0066] (5) incubating the third fluorescently stained section with a MET monoclonal antibody, marking with a secondary antibody, and then marking with a 520 fluorescent dye after elution to obtain a fourth fluorescently stained section;

[0067] (6) incubating the fourth fluorescently stained section with an ALK monoclonal antibody, marking with a secondary antibody, and then marking with a 620 fluorescent dye after elution to obtain a fifth fluorescently stained section;

[0068] (7) incubating the fifth fluorescently stained section with an EGFR monoclonal antibody, marking with a secondary antibody, and then marking with a 780 fluorescent dye and DAPI cell nucleus dye in sequence after elution.

[0069] In the application, the fluorescent dyes 480, 520, 570, 620, 670 and 780 are used for staining, and a fluorescent dye working solution is required, wherein the fluorescent dye working solution takes a signal amplification solution as a solvent, for example, a fluorescent dye stock solution and a signal amplification solution in the kit are configured according to a volume ratio of 1:150.

[0070] The detection platform of the multiplex immunohistochemical kit comprises ​​​​​​​​X30 multi-target pathological staining machine-Alpha X Bio and ZEISS AXIOSCAN 7 whole section imaging system-ZEISS; its detection process includes dewaxing, antigen repair, six antibodies and their matching fluorescent cyclical labeling (primary antibody incubation, secondary antibody binding, fluorescent labeling), cell nucleus re-staining and other steps, the whole staining process is completed on X30 automatic staining instrument, the time and temperature of each reagent incubation step are strictly controlled by the instrument. Among them, dewaxing and antigen repair can expose the antigens in the tissue sections, EGFR, HER2, HER3, MET, ROS1 and ALK antibodies specifically bind to the corresponding proteins in the tissue, the secondary antibody in the multiplex immunohistochemical staining kit binds to the primary antibody, and then the fluorescent is connected to the secondary antibody for labeling, after 6 cycles of labeling, after DAPI cell nucleus re-staining, the staining process is completed. After the stained sections are sealed with an anti-fluorescence sealing agent, the expression of EGFR, HER2, HER3, MET, ROS1 and ALK proteins in the sample can be observed by a fluorescence scanning imaging system and software.

[0071] The staining procedure of the target protein described in the application is shown in Table 3, and the room temperature referred to in the application refers to 15-30℃.

[0072] Table 3 Staining procedure of target protein

[0073]

[0074]

[0075] The staining sequence and fluorescent matching of each protein target antibody in the detection method of the application are shown in Table 4. The six protein targets are subjected to multiplex fluorescent staining of the sample in the specific staining sequence and fluorescent channel matching in the following table, and the fluorescent channel is displayed at the specific position without staining cross talk.

[0076] Table 4 Staining sequence and fluorescent matching

[0077]

[0078]

[0079] In the embodiment of the application, the target of DAPI used is the cell nucleus, and the color is blue.

[0080] The application also provides the use of the above-mentioned multiplex fluorescent immunohistochemical detection panel or the above-mentioned multiplex immunohistochemical kit in the preparation of the following products, and the use includes at least one of the following: (1) a product for predicting the effectiveness of an immune checkpoint inhibitor for lung cancer treatment;

[0081] (2) Evaluation of products for screening targeted therapies for rare lung tumors;

[0082] (3) Products that predict the expression levels of marker proteins in lung cancer samples.

[0083] The detection method of this invention is applicable to rare subtypes of lung cancer: adenoid cystic carcinoma and small cell lung cancer.

[0084] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a multiplex fluorescent immunohistochemical detection panel, detection kit, and their applications for rare lung tumors provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] 1.1 Testing Platform

[0087] X30 Multi-Target Pathological Staining System - AlphaX Bio;

[0088] ZEISS AXIOSCAN 7 Full Slice Imaging System - ZEISS.

[0089] 1.2 Testing Process

[0090] Using squamous cell carcinoma, adenoid cystic carcinoma, and small cell lung cancer as samples, this detection procedure includes dewaxing, antigen retrieval, the use of six antibodies and optimized staining conditions as shown in Table 3, fluorophore cyclic labeling (primary antibody incubation, secondary antibody binding, fluorophore labeling), and nuclear counterstaining. The entire staining process is carried out within... The staining process was performed using the X30 fully automated staining system, with strict control over the time and temperature of each reagent incubation step. Dewaxing and antigen retrieval exposed the antigens in the tissue sections. Antibodies against EGFR, HER2, HER3, MET, ROS1, and ALK were used to specifically bind to their corresponding proteins in the tissue. The secondary antibody in the multiplex immunohistochemical staining kit was bound to the primary antibody, and then fluorescein was used to conjugate the secondary antibody. After six cycles of labeling, the staining was completed by counterstaining the cell nuclei with DAPI. After mounting with anti-fluorescence attenuation mounting media, the expression of EGFR, HER2, HER3, MET, ROS1, and ALK proteins in the samples could be observed using a fluorescence scanning imaging system and software.

[0091] Six protein targets were subjected to multiplex fluorescence staining of the samples using the specific staining order and fluorescence channel combinations shown in Table 3. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, HER2-positive cells (orange) and MET-positive cells (green) are located at their respective specific locations, indicating no staining crosstalk in the fluorescence channels.

[0092] Comparative Example 1

[0093] The rest of the parameters are the same except for the order of antibody staining which is different from Example 1.

[0094] Table 5 Staining order of Comparative Example 1

[0095]

[0096] The results are shown in Figure 4 , Figure 5 and Figure 6 , HER2 positive cells (orange) and HER3 positive cells (green) are in coincidence (yellow) in most locations, and there is staining crosstalk in both fluorescence channels, resulting in false positive phenomenon in one channel.

[0097] Example 2 Lung Squamous Carcinoma

[0098] After the same staining and imaging steps as Example 1, the lung squamous carcinoma sample can obtain the 6 target and nucleus staining target combination shown in Figure 7 and the individual target and nucleus staining target combination, and the expression of each target can be evaluated. Among them, the expression of MET, HER3, and HER2 is low, about 10%, 10%, and 15% respectively, the expression of ALK and ROS1 is about 20% and 35% respectively, and the expression of EGFR is high, about 80%.

[0099] Example 3 Adenoid Cystic Carcinoma

[0100] After the same staining and imaging steps as Example 1, the lung adenoid cystic carcinoma sample can obtain the 6 target and nucleus staining target combination shown in Figure 8 and the individual target and nucleus staining target combination, and the expression of each target can be evaluated. Among them, the expression of MET, ALK, and HER2 is low, about 5%, 10%, and 10% respectively, the expression of HER3 and ROS1 is about 30% and 50% respectively, and the expression of EGFR is high, about 80%.

[0101] Example 4 Small Cell Lung Cancer

[0102] After the same staining and imaging steps as Example 1, the small cell lung sample can obtain the 6 target and nucleus staining target combination shown in Figure 9 and the individual target and nucleus staining target combination, and the expression of each target can be evaluated. Among them, the expression of MET, ALK, and HER2 is low, about 5%, 5%, and 15% respectively, the expression of HER3 and ROS1 is about 20% and 40% respectively, and the expression of EGFR is high, about 90%.

[0103] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A multiplexed fluorescence immunohistochemistry detection panel for rare lung tumors, characterized in that, The monoclonal antibody group comprises ROS1 monoclonal antibody, HER2 monoclonal antibody, HER3 monoclonal antibody, MET monoclonal antibody, ALK monoclonal antibody and EGFR monoclonal antibody. The secondary antibody comprises HPR enzyme-labeled anti-mouse / rabbit IgG polymer secondary antibody.

2. The multiplexed fluorescent immunohistochemical detection panel of claim 1, wherein, 4. A multiplex immunohistochemistry kit comprising the multiplex fluorescent immunohistochemistry detection panel according to any one of claims 1-3.

3. The multiplexed fluorescent immunohistochemical detection panel of claim 1, wherein, The fluorescent dyes include 480 fluorescent dyes, 520 fluorescent dyes, 570 fluorescent dyes, 620 fluorescent dyes, 670 fluorescent dyes, and 780 fluorescent dyes. The kit further comprises buffer solution, eluent, blocking solution, biotin, DAPI nuclear staining reagent, antigen repair solution and signal amplification solution.

5. The multiplex immunohistochemistry kit of claim 4, wherein, The buffer solution is a mixed solution composed of tris(hydroxymethyl) aminomethane, sodium chloride, Tween-20 and ProClin300; 6. The multiplexed immunohistochemistry kit of claim 5, wherein, The blocking solution is a mixed solution composed of BSA, TBS and ProClin300; The signal amplification solution is a mixed solution composed of magnesium sulfate, 4-bromophenylboronic acid and Tris-HCl; The antigen repair solution is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl) aminomethane; The eluent is a solution composed of disodium ethylenediaminetetraacetate and tris(hydroxymethyl) aminomethane. In steps (2) to (7), the temperature of the incubation is 25-40℃, and the time is 15-90 min.

7. A method of using the multiplexed immunohistochemistry kit according to any one of claims 4 to 6, characterized in that, The steps include: (1) placing the test sample in On the X30 fully automated staining instrument, after baking, dewaxing, antigen retrieval, buffer washing and blocking, pretreated sections are obtained; (2) Using pre-treatment sections incubated with ROS1 monoclonal antibody, labeled after binding with secondary antibody 480 Fluorescent dye, after elution, blocked, first fluorescent dye-stained sections obtained; (3) The first fluorescent dye-stained section is incubated with HER2 monoclonal antibody, and labeled after binding with secondary antibody 570fluorescent dye, and the second fluorescent dye-stained section is obtained after blocking (4) Incubate the second fluorescently stained section with HER3 monoclonal antibody, and label after binding with secondary antibody 670 Fluorescent dye, after elution, block, and obtain the third fluorescently stained section; (5) Incubate the third fluorescently stained section with MET monoclonal antibody, and label after binding with secondary antibody 520 Fluorescent dye, after elution, block to obtain the fourth fluorescently stained section; (6) The fourth fluorescent dye-stained section is incubated with ALK monoclonal antibody, and labeled after binding with secondary antibody 620 The fluorescent dye is blocked after elution, and the fifth fluorescent dye-stained section is obtained; (7) Using the fifth fluorescent staining section to incubate with EGFR monoclonal antibody, after binding with secondary antibody, labeling biotin, after elution, in turn labeling 780fluorescent dyes and DAPI nuclear dyes.

8. The method of use of claim 7, wherein, In steps (2) to (7), the temperature of the labeling is 15-30℃, and the time is 5-30 min.

9. The method of use of claim 7, wherein, The application comprises at least one of the following: (1) a product for predicting the effectiveness of an immune checkpoint inhibitor on lung cancer treatment; 10. Use of the multiplexed fluorescence immuno-histochemistry detection panel according to any one of claims 1 to 3 or the multiplexed immuno-histochemistry kit according to any one of claims 4 to 6 for the manufacture of a product, characterized in that, (2) a product for evaluating the screening of targeted drugs for rare tumors in the lung; (3) a product for predicting the expression amount of marker proteins in lung cancer samples. ​

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