Scoring method for the likelihood of tumor immune response based on dual-panel multiplex biomarker analysis

Abstract

This application relates to the field of biological tissue engineering technology and discloses a scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis, comprising: staining multiple cells in a tumor tissue section with multiple affinity histochemical stains; obtaining a first and / or second eigenvalue group of cells in a target analysis area defined in a digital image; determining the immunophenotyping of the tumor tissue sample based on at least part of the second eigenvalues ​​in the inner and outer regions of the tumor boundary; obtaining multiple first eigenvalues ​​of the first eigenvalue group within the inner region of the tumor boundary of a tumor tissue sample determined by immunophenotyping as either immune infiltration or immune rejection, scoring each of the multiple eigenvalues, and summing the multiple scores to obtain a score. This scoring method uses 11 cell types as biomarkers to score immune responses to predict the likelihood of tumor patients responding to immunotherapy, and has wide adaptability and strong feasibility.

Description

Technical Field

[0001] The present application relates to the field of biological tissue engineering technology, for example, to a scoring method for the possibility of tumor immune response based on dual-panel multiple biomarker analysis. Background Art

[0002] Immune checkpoint inhibitors are a class of drugs that work by blocking the binding of immune checkpoints to their ligands, relieving checkpoint-induced immune suppression and thereby reactivating immune cells to exert anti-tumor effects. Several immune checkpoint inhibitors have achieved remarkable results in clinical anti-tumor applications, representing a breakthrough in cancer treatment. However, only 10%-15% of patients are sensitive to immune checkpoint inhibitors, and drug resistance and side effects are common. Therefore, predicting cancer patients' sensitivity to immune checkpoint inhibitors is a crucial tool for improving treatment efficacy and reducing treatment risks. Furthermore, predicting cancer patients' sensitivity to immune checkpoint inhibitors has significant implications. First, it can help physicians select the most appropriate treatment plan for their patients, avoid ineffective or harmful treatments, and improve treatment efficiency and safety. Second, it can help patients understand their condition and prognosis, enhance their confidence and willingness to cooperate, and improve their quality of life. Finally, it can promote the research and development and innovation of immune checkpoint inhibitors, providing more options and possibilities for cancer immunotherapy.

[0003] During the implementation of the embodiments of the present disclosure, it was found that at least the following problems exist in the related art:

[0004] Since predicting the sensitivity of tumor patients to immune checkpoint inhibitors requires comprehensive consideration of multiple factors, although there are some predictive models and markers, there are still certain limitations and uncertainties. Summary of the Invention

[0005] In order to provide a basic understanding of some aspects of the disclosed embodiments, a brief summary is given below. The summary is not an extensive review, nor is it intended to identify key / critical elements or delineate the scope of protection of these embodiments, but rather serves as a prelude to the detailed description that follows.

[0006] The present disclosure provides a scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis, which predicts the likelihood of tumor patients responding to immunotherapy through immune response scoring.

[0007] In some embodiments, the scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis comprises:

[0008] Tumor tissue sections were obtained from the tumor tissue samples and stained with multiplex affinity histochemical staining for each of CD4, CD8, CD15, CD19, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3, and PanCK;

[0009] Delineating regions in a digital image of a multi-stained section; wherein the delineated regions include: a tumor region, a region inside the tumor region that is a first preset distance inward from the tumor region boundary, and a region outside the tumor region that is a second preset distance outward from the tumor region boundary;

[0010] Obtain the first feature value group and / or the second feature value group in the demarcated area; the first feature value group includes: CD8 + Cell ratio, CD15 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell percentage, CD4 + +FoxP3 + Cell ratio, PD-L1 + Cell percentage, Granzyme B + Cell ratio, where the ratio is the ratio of the number of corresponding cells to the number of all cells in the corresponding area; the second characteristic value group includes: CD4 + Cell density, CD8 + Cell density, CD4 + +FoxP3 + Cell density, CD8 + +Granzyme B + Cell density, CD8 + +PD-L1 + Cell density, CD15 + Cell density, CD20 + Cell density, CD56 + Cell density, CD68 + Cell density, CD86 + Cell density, where density is the ratio of the number of cells to the area of ​​the corresponding region;

[0011] Determining the immunophenotyping of the tumor tissue sample based on at least part of the second eigenvalues ​​in the second eigenvalue group of the region inside the tumor boundary and the region outside the tumor boundary; wherein the immunophenotyping includes immune infiltration type, immune rejection type, and immune desert type;

[0012] In the inner region of the tumor boundary of the tumor tissue sample determined by immunotyping as immune infiltration type or immune rejection type, multiple first eigenvalues ​​of the first eigenvalue group are obtained, and the multiple first eigenvalues ​​are scored respectively, and the multiple scores are summed to obtain a score.

[0013] The scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis provided in the embodiments of the present disclosure can achieve the following technical effects:

[0014] In the disclosed embodiments, 11 cell types—CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3, and PanCK—are used as biomarkers to perform immune response scoring to predict a patient's likelihood of responding to immunotherapy, such as immunotherapy with immune checkpoint inhibitors. The inner region of the tumor boundary reflects whether immune cells can enter the core of the tumor, while the T cell density in the outer region reflects the concentration of immune cells at the tumor edge, reflecting the immune system's initial response to the tumor and whether immune cells can access the tumor tissue. Therefore, based on at least some of the eigenvalues ​​in the second eigenvalue (cell density) group within the inner and outer regions of the tumor boundary, the immunophenotyping of the tumor tissue sample is determined, and a score is assigned to tumor tissue samples classified as immune infiltration or immune rejection. When performing the specific scoring, multiple eigenvalues ​​in the first eigenvalue (cell percentage) group are selected from a portion of the demarcated area for scoring, and the multiple scores are summed to obtain a score, thereby predicting the likelihood of an immune response. The combination of T cell density in the inner and outer areas of the tumor boundary can comprehensively reflect the distribution and potential dynamic changes of immune cells in the tumor microenvironment.

[0015] The scoring method of the disclosed embodiment has wide adaptability and strong feasibility. It comprehensively considers factors such as tumor microenvironment characteristics such as the proportion of immune cells and the spatial position relationship of immune cells, and can provide certain reference data for the possibility of tumor patients responding to immunotherapy.

[0016] The above general description and the following description are exemplary and explanatory only and are not intended to limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] One or more embodiments are exemplarily described by corresponding drawings. These exemplary descriptions and drawings do not limit the embodiments. Elements with the same reference numerals in the drawings are shown as similar elements. The drawings do not constitute a scale limitation. In addition,

[0018] Figure 1 This is a flowchart of an immune response scoring method provided by an embodiment of the present disclosure;

[0019] Figure 2 This is a digital image of a tumor tissue section after multiple immunofluorescence staining provided in Example 1 of the present disclosure (showing only PanCK / CD8 / DAPI);

[0020] Figure 3 This embodiment of the present disclosure is based on Figure 2 Schematic diagram of regional division of tumor tissue samples;

[0021] Figure 4a This is a digital image of a tumor tissue section stained with Panel-1 and a schematic diagram of tumor area division thereof, provided in Example 1 of the present disclosure;

[0022] Figure 4b This is a digital image of a tumor tissue section after Panel-2 staining and a schematic diagram of tumor area division provided in Example 1 of the present disclosure;

[0023] Figure 5 This is a digital image of a tumor tissue section after multiple immunofluorescence staining provided in Example 2 of the present disclosure (showing only PanCK / CD8 / DAPI);

[0024] Figure 6 This embodiment 2 of the present disclosure is based on Figure 5 Schematic diagram of regional division of tumor tissue samples;

[0025] Figure 7a This is a digital image of a tumor tissue section stained with Panel-1 and a schematic diagram of tumor area division thereof, provided in Example 2 of the present disclosure;

[0026] Figure 7b This is a digital image of a tumor tissue section after Panel-2 staining and a schematic diagram of tumor area division provided in Example 2 of the present disclosure;

[0027] Figure 8 This is a digital image of a tumor tissue section after multiple immunofluorescence staining provided in Example 3 of the present disclosure (showing only PanCK / CD8 / DAPI);

[0028] Figure 9 This embodiment 3 of the present disclosure is based on Figure 8 Schematic diagram of the regional division of tumor tissue samples. DETAILED DESCRIPTION

[0029] In order to be able to understand the features and technical content of the embodiments of the present disclosure in more detail, the implementation of the embodiments of the present disclosure is described in detail below in conjunction with the accompanying drawings. The accompanying drawings are for reference only and are not used to limit the embodiments of the present disclosure. In the following technical description, for the sake of convenience of explanation, a full understanding of the disclosed embodiments is provided through multiple details. However, one or more embodiments can still be implemented without these details. In other cases, to simplify the drawings, well-known structures and devices can be simplified for display.

[0030] In the description and claims of the embodiments of the present disclosure, as well as in the accompanying drawings, the terms "first," "second," and the like are used to distinguish similar items and are not necessarily used to describe a particular order or precedence. It should be understood that the terms used in this manner are interchangeable where appropriate to describe the embodiments of the present disclosure herein. In addition, the terms "including," "having," and any variations thereof are intended to cover non-exclusive inclusions.

[0031] Unless otherwise stated, the term "plurality" means two or more.

[0032] It should be noted that, unless there is any conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0033] Combine Figure 1 As shown, the present disclosure provides a method for scoring the likelihood of tumor immune response based on dual-panel multiple biomarker analysis, comprising the following steps:

[0034] S10. Obtain tumor tissue sections from the tumor tissue sample and stain the tumor tissue sections with multiple immunofluorescence staining for each biomarker of CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3 and PanCK.

[0035] In the step S10 , in the multiple immunofluorescence staining, the staining order is not limited as long as each of the above can be distinguished.

[0036] Optionally, step S10 specifically includes: obtaining continuous tumor tissue sections from the tumor tissue sample, performing H&E staining on an intermediate tumor tissue section located in the middle to obtain an H&E stained section, and when it is determined that the number of tumor cells in the tumor area of ​​the intermediate tumor tissue section meets the requirements, selecting a tumor tissue section adjacent to the intermediate tumor tissue section to perform multiple immunofluorescence staining to obtain a multiple stained section.

[0037] Determining that the tumor cell count in the intermediate tumor tissue section meets the requirements specifically includes: the number of tumor cells within the delineated tumor region is greater than or equal to a preset value. The tumor region is delineated by a senior pathologist, who then determines the number of tumor cells within the tumor region. The preset value is determined based on actual requirements, for example, 100.

[0038] S20. Delineate regions in the digital image of the multi-stained slice; the delineated regions include: a tumor area (TA), a tumor boundary inner region (TIA) at a first preset distance inward from the tumor boundary, and a tumor boundary outer region (TOA) at a second preset distance outward from the tumor boundary.

[0039] In step S20 , the digital image of the multi-stained slice can be scanned using a bright field scanner or a fluorescence scanner to obtain the digital image.

[0040] Specifically, delineating a region in a digital image of a multi-stained section includes: delineating a tumor area (TA) in the digital image of the H&E-stained section obtained in step S10; and performing region delineation on digital images of multi-stained sections of adjacent tumor tissue sections based on the H&E-stained section and the tumor area (TA) delineated thereon. The delineation of the tumor area (TA) is performed by a senior pathologist based on the H&E staining results.

[0041] In step S20, the tumor boundary inner area (TIA) at a first preset distance inward from the tumor region boundary can reflect the difference in cell proportion and distribution between the tumor region and the non-tumor region. The first preset distance is determined based on actual conditions.

[0042] Optionally, the first preset distance is less than or equal to 300 μm. The maximum value of the first preset distance is limited to 300 μm to ensure that the area of ​​the demarcated region is reasonable and adapts to the size of the tumor tissue slice, and better reflects the differences in cell proportion and distribution.

[0043] Optionally, the first preset distance is greater than or equal to 200 μm and less than or equal to 300 μm. The minimum value of the first preset distance is limited to 200 μm to ensure that the area of ​​the demarcated region is reasonable and better reflect the differences in cell proportion and distribution.

[0044] It is understandable that when the size of the delineated tumor area (TA) is small, it is meaningless to divide the TIA area or the TIA area occupies the vast majority of the TA area, then the TIA area can be omitted and the TA area can be directly used instead of the TIA area.

[0045] Optionally, when the size of the tumor area (TA) is less than or equal to a preset size, the tumor area (TA) is used instead of the tumor boundary inner area (TIA); wherein the preset size is less than or equal to a first preset distance, or the preset size is greater than the first preset distance and the difference between the preset size and the first preset distance is less than or equal to 200 μm. Optionally, the preset size is greater than the first preset distance and the difference between the preset size and the first preset distance is less than or equal to 100 μm.

[0046] Alternatively, when PanCK-positive staining areas (P+ / TA) and PanCK-negative staining areas (P- / TA) can be distinguished within the tumor area (TA), the PanCK-positive staining area (P+ / TA) will replace the TIA area. Figure 2 and Figure 3 The figure shows the clearly distinguishable P+ / TA area and P- / TA area, and the PanCK-positive and PanCK-negative areas, which can reflect the tumor parenchyma and stromal areas, respectively.

[0047] In step S20, the area outside the tumor boundary (TOA) at a second preset distance outward from the tumor boundary can reflect the difference in cell proportion and distribution between the non-tumor region and the tumor region. The second preset distance is determined based on actual conditions.

[0048] Optionally, the second preset distance is less than or equal to 500 μm. An appropriate second preset distance can be determined based on the size of the tumor tissue slice and the location of the tumor region boundary to better reflect the differences in cell proportion and distribution between the non-tumor region and the tumor region.

[0049] Optionally, the second preset distance is greater than 300 μm and less than or equal to 500 μm, which is suitable for larger tumor tissue slices.

[0050] Optionally, the second preset distance is greater than 400 μm and less than or equal to 500 μm, which is suitable for larger tumor tissue slices.

[0051] Optionally, the second preset distance is less than or equal to 300 μm, which is suitable for smaller tumor tissue slices.

[0052] Optionally, the second preset distance is greater than or equal to 200 μm and less than or equal to 300 μm.

[0053] Optionally, when the second predetermined distance of the region outside the tumor boundary is greater than or equal to 300 μm (or 400 μm) and less than or equal to 500 μm, the region outside the tumor boundary is divided into a first region outside the tumor boundary (TOA) proximal to the tumor region boundary and a second region outside the tumor boundary (TPA) distal to the tumor region boundary. It will be appreciated that the TOA represents the (first) region outside the tumor boundary proximal to the tumor region boundary.

[0054] Optionally, the area a third preset distance outward from the tumor region boundary is a first tumor outer boundary area (TOA), and the area between the third preset distance and the second preset distance is a second tumor outer boundary area (TPA); wherein the third preset distance is between one-third and two-thirds of the second preset distance. Optionally, the third preset distance is one-half of the second preset distance.

[0055] S30: Acquire a first eigenvalue group and / or a second eigenvalue group in a defined area.

[0056] Among them, the first characteristic value group includes: CD8 + Cell ratio, CD15 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + + CD86 + Cell percentage, CD4 + +FoxP3 + Cell ratio, PD-L1 + Cell percentage, Granzyme B + Cell ratio, where the ratio is the ratio of the number of corresponding cells to the number of all cells in the corresponding area; that is, the first feature value group includes multiple first feature values, and the first feature value is the cell ratio of the biomarker. Wherein, the number of all cells in the corresponding area refers to the number of all nucleated cells in the corresponding area. The second feature value group includes: CD4 + Cell density, CD8 + Cell density, CD4 + +FoxP3 + Cell density, CD8 + +Granzyme B + Cell density, CD8 + and PD-L1 + Cell density, CD15 + Cell density, CD20 + Cell density, CD56 + Cell density, CD68 + Cell density, CD86 +Cell density, where density is the ratio of the number of cells to the area of ​​the corresponding region; that is, the second eigenvalue group includes multiple second eigenvalues, and the second eigenvalue is the cell density of the biomarker.

[0057] In this step S30, it can be understood that CD4 + cells, CD8 + cells, CD15 + cells, CD20 + cells, CD56 + cells, CD68 + cells, CD86 + cells, FoxP3 + cells, PD-L1 + Cells and Granzyme B + Cell refers to the corresponding nucleated cell.

[0058] In this step S30, the first eigenvalue group and / or the second eigenvalue group are obtained in the demarcated area, including: obtaining the first eigenvalue group in the inner area of ​​the tumor boundary (tumor area); obtaining the second eigenvalue group in the inner area of ​​the tumor and the outer area of ​​the tumor boundary.

[0059] S40: Determine the immunophenotyping of the tumor tissue sample based on at least some of the second eigenvalues ​​in the second eigenvalue groups of the region inside the tumor boundary and the region outside the tumor boundary, wherein the immunophenotyping includes immune infiltration type, immune rejection type, and immune desert type.

[0060] In step S40 , the immunophenotyping of the tumor tissue sample is determined by comparing the differences in some second characteristic values ​​between the region inside the tumor and the region outside the tumor.

[0061] S50. In the inner region of the tumor boundary of the tumor tissue sample determined by immunotyping to be immune infiltration type or immune rejection type, obtain multiple eigenvalues ​​of the first eigenvalue group, score the multiple first eigenvalues ​​respectively, and sum the multiple scores to obtain a score.

[0062] In the scoring method for the possibility of tumor immune response of the embodiment of the present disclosure, 11 cells including CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3 and PanCK are used as biomarkers to perform immune response scoring to predict the possibility of tumor patients responding to immunotherapy, such as predicting the possibility of responding to immunotherapy using immune checkpoint inhibitors. Among them, the immunotyping of the tumor tissue sample is determined based on at least part of the eigenvalues ​​in the second eigenvalue group of the inner area and the outer area of ​​the tumor, and the tumor tissue sample with the immunotyping of immune infiltration type or immune rejection type is scored. Specifically, multiple eigenvalues ​​in the first eigenvalue group of some areas in the demarcated area are selected for scoring, and the multiple scores are summed to obtain a score, thereby predicting the possibility of immune response. This prediction method has wide adaptability and strong feasibility, and can provide certain reference data for the possibility of tumor patients responding to immunotherapy. It can be understood that the higher the score result, the greater the possibility of immune response.

[0063] In some embodiments, step S10 includes: obtaining consecutive tumor tissue sections from a tumor tissue sample, performing H&E staining on an intermediate tumor tissue section to obtain an H&E-stained section, and when it is determined that the number of tumor cells in the intermediate tumor tissue section meets the requirement, selecting two tumor tissue sections adjacent to the intermediate tumor tissue section for multiple immunofluorescence staining, and performing the following two panel stainings on the two tumor tissue sections in a one-to-one correspondence:

[0064] Panel-1: CD4 / CD8 / FoxP3 / Granzyme B / PD-L1 / PanCK / DAPI;

[0065] Panel-2: CD15 / CD20 / CD56 / CD68 / CD86 / PanCK / DAPI.

[0066] That is, CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3 and PanCK are divided into two groups, and one of the two tumor tissue sections adjacent to the middle tumor tissue section is stained with Panel-1, and the other is stained with Panel-2, thereby obtaining two stained multiple staining sections accordingly, so as to better obtain the first characteristic value and / or second characteristic value of each biomarker in different delineated areas.

[0067] It is understood that the number of consecutive tumor tissue sections is at least three to meet the needs of one H&E staining and two multiple immunofluorescence stainings. The H&E staining and multiple immunofluorescence staining can be performed using conventional procedures.

[0068] In this embodiment, determining that the number of tumor cells in the intermediate tumor tissue slice meets the requirement specifically includes: the number of tumor cells within the delineated tumor region is greater than or equal to a preset value. The tumor region is delineated by a senior pathologist. The preset value is determined based on actual requirements, for example, 100.

[0069] Optionally, in step S10, during the staining process of Panel-1, the antibodies and dyes used for each biomarker and the corresponding fluorescence test channels are shown in Table 1 below:

[0070] Table 1

[0071]

[0072] In the fluorescence channel, each value is the fluorescence wavelength, and its unit is the fluorescence wavelength unit, nm.

[0073] Optionally, in the staining process of Panel-2, the antibodies and dyes used for each biomarker and the corresponding fluorescence test channels are shown in Table 2 below:

[0074] Table 2

[0075]

[0076] In the fluorescence channel, each value is the fluorescence wavelength, and its unit is the fluorescence wavelength unit, nm.

[0077] In this example, two panels were stained for 11 biomarkers, and corresponding antibodies and dyes were specifically screened to precisely target these biomarkers, improving signal detection accuracy and signal-to-noise ratio, enabling simultaneous detection of multiple markers, and enhancing multiple staining compatibility. Furthermore, this method clearly visualizes the spatial distribution of cells and their tissue structure, providing reliable data for quantitative analysis.

[0078] In some embodiments, in step S40, part of the second feature values ​​in "at least part of the second feature values ​​in the second feature value group according to the inner region of the tumor boundary and the outer region of the tumor boundary" include CD4 + Cell density, CD8 + Cell density, CD20 + Cell density, CD56 + Cell density and CD68 +By comparing the difference and change trend of at least part of the second characteristic values ​​in the tumor inner region and the tumor outer region, the immunophenotyping of the tumor tissue sample can be determined.

[0079] Optionally, determining the immunophenotyping of a tumor tissue sample based on at least some of the second eigenvalues ​​in the second eigenvalue groups for the inner and outer regions of the tumor boundary includes the following steps: S401: obtaining a second eigenvalue (ρ) for the inner region of the tumor boundary and a corresponding second eigenvalue (ρ') for the outer region of the tumor boundary, and summing the two second eigenvalues ​​(ρ + ρ') to obtain a second sum α. That is, α = ρ + ρ'. S402: determining the tumor tissue sample as an immune desert type if the second sum α is less than or equal to a second preset value. S403: determining the tumor tissue sample as an immune rejection type if the second sum α is greater than the second preset value and the ratio of the second eigenvalues ​​in the outer region of the tumor boundary to the second eigenvalues ​​in the inner region of the tumor boundary is greater than a third preset value. S404: determining the tumor tissue sample as an immune infiltration type if the second sum α is greater than the second preset value and the ratio of the second eigenvalues ​​in the outer region of the tumor boundary to the second eigenvalues ​​in the inner region of the tumor boundary is less than or equal to a third preset value.

[0080] In this embodiment, the immune desert phenotype is determined based on the sum of the second characteristic values ​​(e.g., the cell density of a particular biomarker) within the tumor boundary and the area outside the tumor boundary relative to a second preset value. If the tumor is not immune desert, a ratio is introduced to distinguish between immune rejection and immune infiltration. This method is reliable and effective.

[0081] In step S401 of this embodiment, a second characteristic value is the cell density of one of the biomarkers, for example, selected from CD4 + Cell density, CD8 + Cell density, CD20 + Cell density, CD56 + Cell density and CD68 + For example, the second characteristic value is CD8 + Cell density.

[0082] In steps S403 and S404 of this embodiment, when the tumor boundary outer region is divided into a first tumor boundary outer region (TOA) and a second tumor boundary outer region (TPA), the ratio of the second characteristic value within the tumor boundary outer region to the second characteristic value within the tumor boundary inner region includes a first ratio and a second ratio, wherein the first ratio is the ratio of the second characteristic value of the first tumor boundary outer region to the tumor boundary inner region, and the second ratio is the ratio of the second characteristic value of the second tumor boundary outer region to the tumor boundary inner region. Then, in step S403, if the first ratio and / or the second ratio is greater than a third preset value, the tumor tissue sample is determined to be of the immune rejection type; and in step S404, if the first ratio and / or the second ratio is less than or equal to the third preset value, the tumor tissue sample is determined to be of the immune infiltration type.

[0083] In step S402, step S403 and step S404 of this embodiment, the second preset value can be determined according to relevant parameters such as tumor type. For example, the second preset value is 80 to 150 cells / mm 2 For example, the second preset value is 100 cells / mm 2 .

[0084] In step S403 and step S404 of this embodiment, the third preset value can be determined based on relevant parameters such as tumor type. For example, the third preset value is 2 to 4. For example, the third preset value is 3.

[0085] In some embodiments, in step S50, a plurality of first feature values ​​in the first feature value group are obtained, and the plurality of first feature values ​​are scored respectively, including: S501, obtaining CD8 in the first feature value group; + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage, Granzyme B + Cell percentage, CD4 + +FoxP3 + Cell proportion and CD15 + Cell ratio. S502, when CD8 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage and Granzyme B +When the cell ratio is greater than or equal to the threshold, each corresponding score is calculated. + +FoxP3 + Cell proportion and CD15 + When the cell proportions are less than or equal to the threshold, each cell is scored. S504: Sum the scores corresponding to the first feature values, and the sum is the score.

[0086] In this embodiment, it is understood that when multiple first eigenvalues ​​all meet their respective conditions and can be scored, the highest score is obtained. This highest score can be a number of tens, such as 10, 20, 50, or 100, for convenient scoring. Of course, it can also be a number other than tens, which can be determined according to actual circumstances.

[0087] In this embodiment, a higher score indicates a more sensitive immune response; a lower score indicates a less sensitive immune response.

[0088] In this embodiment, it is understood that in step S502, when the proportion of each cell is less than the threshold, no score is calculated, that is, 0. Similarly, in step S503, when the proportion of each cell is greater than the threshold, no score is calculated, that is, 0.

[0089] In this embodiment, the threshold value is determined according to the actual situation. Optionally, the threshold value includes 5%. It is understandable that if CD8 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage and Granzyme B + If the cell ratio is less than the threshold, no score will be given. + +FoxP3 + Cell proportion and CD15 + If the cell ratio is greater than the threshold, no score will be given.

[0090] Alternatively, when CD8 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage and Granzyme B + When the cell ratio is greater than or equal to the threshold, each is scored accordingly; when CD4 + +FoxP3 +Cell proportion and CD15 + When the cell ratio is less than or equal to the threshold, each corresponding score is scored; including: when CD8 + Cell proportion and PD-L1 + When the cell ratio is greater than or equal to the first threshold, the first score and the second score are recorded respectively; when Granzyme B + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell percentage and CD20 + When the cell ratio is greater than or equal to the second threshold, the corresponding scores are the third, fourth, fifth and sixth scores; when the CD4 + +FoxP3 + Cell proportion and CD15 + When the cell proportion is less than or equal to the third threshold, the seventh and eighth scores are assigned to each, respectively; wherein the first threshold is greater than the second threshold, and the second threshold is greater than the third threshold; and the values ​​of the first to eighth scores show a decreasing trend. In this embodiment, different weights are assigned to different biomarkers or biomarker combinations by different scoring values ​​to improve the accuracy of the scoring.

[0091] Optionally, the first threshold includes 5%, and / or the second threshold includes 3%, and / or the third threshold includes 2%.

[0092] Optionally, the sum of the first to eighth scores is 10. That is, the highest score is 10 points.

[0093] Optionally, the first score is 3, the second score is 2, the third score is 1.5, the fourth score and the fifth score are both 1, and the sixth score, the seventh score and the eighth score are both 0.5.

[0094] In the disclosed embodiment, in conjunction with steps S40 and S50, it can be seen that the scoring step for the first characteristic value in step S50 is only performed for tumor tissue samples determined to be immune-infiltrating and immune-rejecting in step S40. That is, in step S30, the first characteristic value group and / or the second characteristic value group are obtained in the delineated area, including two situations: the first situation, the second characteristic value group is obtained in the delineated area; the second situation, the first characteristic value group and the second characteristic value group are obtained in the delineated area. It is understandable that steps S30 to S50 can be performed using the following steps S31, S41, and S51.

[0095] Specifically, S31, a second feature value group is obtained within the demarcated region; S41, the immunophenotyping of the tumor tissue sample is determined based on at least some of the second feature values ​​in the second feature value group in the region inside and outside the tumor boundary; wherein immunophenotyping includes immune infiltration type, immune rejection type, and immune desert type; S51, a first feature value group is obtained within the tumor region of the tumor tissue sample determined by immunophenotyping to be immune infiltration type or immune rejection type; and multiple first feature values ​​in the first feature value group are scored to predict the likelihood of immune response to treatment. In this embodiment, if the tumor tissue sample is determined to be immune desert type, the acquisition of the first feature value is not required, thereby reducing unnecessary steps and workload.

[0096] The following is a specific example of a scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis to further illustrate the present disclosure, but does not limit the scope of the present disclosure.

[0097] Example 1

[0098] A scoring method for the likelihood of tumor immune response based on dual-panel multiplex biomarker analysis comprises the following steps:

[0099] S101. Obtain serial tumor tissue sections from colorectal cancer tumor tissue sample I. Perform H&E staining (conventional staining process) on an intermediate tumor tissue section to obtain an H&E-stained section. After confirming that the number of tumor cells in the intermediate tumor tissue section meets the requirement, select two tumor tissue sections adjacent to the intermediate tumor tissue section for multiple immunofluorescence staining. The two tumor tissue sections are stained one by one using the following two panels:

[0100] Panel-1: CD4 / CD8 / FoxP3 / Granzyme B / PD-L1 / PanCK / DAPI;

[0101] Panel-2: CD15 / CD20 / CD56 / CD68 / CD86 / PanCK / DAPI.

[0102] The multiple affinity histochemical staining process is described in detail using Panel-1 staining as an example:

[0103] (a) One of the tumor tissue sections was dewaxed in xylene, rehydrated with graded alcohol, and antigen retrieved before blocking with blocking solution.

[0104] (b) CD4 antibody (clone 4B12) was used for staining and incubated at room temperature.

[0105] (c) After the sections were incubated with the primary antibody, they were washed with the cleaning solution and incubated with the secondary antibody at room temperature.

[0106] (d) After incubation with secondary antibodies, the sections were washed with cleaning solution and then incubated with fluorescent dye (ATTO425-Tyramide) diluted with a specific signal amplification solution (TSA signal amplification reaction solution) at room temperature.

[0107] (e) The stained sections were then repaired with antigen retrieval solution (citrate buffer), the bound CD4 antibody was stripped, and the sections were blocked with blocking solution.

[0108] (f) Repeat steps (b) to (e) to sequentially complete the staining process for CD8, FoxP3, Granzyme B, PD-L1, and PanCK. The antibodies and dyes used are shown in Table 1.

[0109] (g) After the sections were incubated with fluorescent dyes, they were washed with cleaning solution, stained for nuclei, and sealed; Panel-1 stained sections (e.g. Figure 4a shown).

[0110] Same as above steps, refer to the corresponding antibodies and dyes shown in Table 2 above, obtain Panel-2 stained sections (such as Figure 4b shown).

[0111] S102. Use a fluorescent scanner to scan and obtain digital images of Panel-1 stained sections and Panel-2 stained sections respectively; delineate the tumor area (TA) based on the digital image of the H&E stained section, and delineate the area on the digital image of the multiple stained sections of the adjacent tumor tissue sections based on the H&E stained section and the tumor area (TA) delineated thereon (the delineation of the tumor area (TA) is performed by a senior pathologist based on the H&E staining results); the delineated areas include: the tumor area (TA), the tumor boundary inner area (TIA) at a first preset distance inward from the tumor boundary (for example, a first preset distance of 200 to 300 μm), the first tumor boundary outer area (TOA) close to the tumor boundary at a third preset distance outward from the tumor boundary (for example, the third preset distance is 150 to 300 μm), and the second tumor boundary outer area (TPA) at a third preset distance outward from the tumor boundary to a second preset distance (for example, the second preset distance is 300 to 500 μm, and is greater than the third preset distance). Figure 2 and Figure 3As shown, in this embodiment 1, the PanCK-positive staining area (P+ / TA) and the PanCK-negative staining area (P- / TA) can be distinguished within the tumor area (TA). Therefore, the PanCK-positive staining area (P+ / TA) replaces the inner area of ​​the tumor boundary (TIA), that is, the P+ / TA area is defined as the TIA area.

[0112] S103. Obtaining the second characteristic measurement of the area inside the tumor boundary and the area outside the tumor boundary - biomarker CD8 + As shown in Table 3 below, the cell density of CD8 + The cell density is 435.6 cells / mm 2 (greater than 100 cells / mm 2 ), and CD8 + The cell density and CD8 + The ratios of the cell densities of the tumor tissue samples were 41.9 and 57.2 (greater than 3), respectively, confirming that the immunophenotyping of the tumor tissue sample was immune-exclusion type.

[0113] Table 3

[0114]

[0115] S104. Obtain a group of first eigenvalues ​​within the tumor area (TA); score each of the multiple first eigenvalues ​​(see Table 4). As shown in Table 4, the sum of the scores corresponding to the multiple first eigenvalues ​​is used to form the score, which is 6.5. This indicates mild sensitivity, meaning the immune response is mildly sensitive and potentially responsive to immunotherapy.

[0116] Table 4

[0117]

[0118] Example 2

[0119] A scoring method for the likelihood of tumor immune response based on dual-panel multiplex biomarker analysis comprises the following steps:

[0120] S201, using colorectal cancer tumor tissue sample II, complete the staining operation according to step S101 of Example 1; the obtained Panel-1 staining section is as shown Figure 7a As shown, the Panel-2 stained sections obtained were as follows Figure 7b shown.

[0121] S202, referring to step S102 of embodiment 1, perform the region demarcation operation; Figure 5 and 6 As shown, the delineated areas include: the tumor area (TA), a first tumor-outer-border area (TOA) located near the tumor area boundary and extending a third predetermined distance outward from the tumor area boundary (e.g., the third predetermined distance is 150-300 μm), and a second tumor-outer-border area (TPA) extending from the third predetermined distance to a second predetermined distance outward from the tumor area boundary (e.g., the second predetermined distance is 300-500 μm and is greater than the third predetermined distance). Due to the small size of the tumor area TA (less than approximately 300 μm), the tumor-inner-border area (TIA) is not delineated. In this case, the tumor area TA is the tumor-inner-border area TIA.

[0122] S203. Obtaining the second characteristic measurement of the area inside the tumor boundary and the area outside the tumor boundary - biomarker CD8 + As shown in Table 5 below, the cell density of CD8 + The cell density is 1543 cells / mm 2 (greater than 100 cells / mm 2 ), and CD8 + The cell density and CD8 + The ratios of the cell densities of the tumor tissue samples were 0.1 and 0.07 (less than 3), respectively, confirming that the immunophenotyping of the tumor tissue sample was immune infiltration type.

[0123] Table 5

[0124]

[0125] S204: Obtain a first eigenvalue group within the tumor area (TA); score each of the multiple first eigenvalues ​​(see Table 6). As shown in Table 6, the multiple scores corresponding to the multiple first eigenvalues ​​are summed to form a score of 9.5. This indicates sensitivity, indicating a sensitive immune response and the potential for response to immunotherapy.

[0126] Table 6

[0127]

[0128] Example 3

[0129] A scoring method for the likelihood of tumor immune response based on dual-panel multiplex biomarker analysis comprises the following steps:

[0130] S301. Using intestinal cancer tumor tissue sample III, perform a staining operation according to step S11 of Example 1.

[0131] S302, referring to step S102 of embodiment 1, perform the region demarcation operation; Figure 8 and Figure 9 As shown, the delineated areas include: the tumor area (TA), a first tumor-outer-border area (TOA) located near the tumor area boundary and extending a third predetermined distance outward from the tumor area boundary (e.g., the third predetermined distance is 150-300 μm), and a second tumor-outer-border area (TPA) extending from the third predetermined distance to a second predetermined distance outward from the tumor area boundary (e.g., the second predetermined distance is 300-500 μm and is greater than the third predetermined distance). Due to the small size of the tumor area TA (less than approximately 300 μm), the tumor-inner-border area (TIA) is not delineated. In this case, the tumor area TA is the tumor-inner-border area TIA.

[0132] S303. Obtaining the second characteristic measurement of the area inside the tumor boundary and the area outside the tumor boundary - biomarker CD8 + As shown in Table 7 below, the cell density of CD8 + The cell density is 25.9 cells / mm 2 (less than 100 cells / mm 2 ), the immunophenotyping of the tumor tissue sample was determined to be immune desert type. At the same time, the CD8 + The cell density and CD8 + The cell density ratio was 0.4 (less than 3) as a reference.

[0133] Tumor tissue samples identified as immune deserts are less likely to respond to immunotherapy.

[0134] Table 7

[0135]

[0136] The above description and the accompanying drawings sufficiently illustrate the embodiments of the present disclosure to enable those skilled in the art to practice them. Other embodiments may include structural and other changes. The embodiments represent only possible variations. Unless expressly required, individual components and functions are optional, and the order of operations may vary. Portions and features of some embodiments may be included in or replace portions and features of other embodiments. The embodiments of the present disclosure are not limited to the structures described above and shown in the accompanying drawings, and various modifications and changes may be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A scoring method for the likelihood of tumor immune response based on dual-panel multiple biomarker analysis, characterized in that: include: Obtaining a tumor tissue section from a tumor tissue sample, and staining the tumor tissue section with multiple immunofluorescence staining for each biomarker of CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3, and PanCK to obtain a multiplex staining section; Delineating regions in a digital image of a multi-stained section; wherein the delineated regions include: a tumor region, a region inside the tumor region that is a first preset distance inward from the tumor region boundary, and a region outside the tumor region that is a second preset distance outward from the tumor region boundary; Obtain the first feature value group and / or the second feature value group in the demarcated area; the first feature value group includes: CD8 + Cell ratio, CD15 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell percentage, CD4 + +FoxP3 + Cell ratio, PD-L1 + Cell percentage, Granzyme B + Cell ratio, where the ratio is the ratio of the number of corresponding cells to the number of all cells in the corresponding area; the second characteristic value group includes: CD4 + Cell density, CD8 + Cell density, CD4 + +FoxP3 + Cell density, CD8 + +Granzyme B + Cell density, CD8 + +PD-L1 + Cell density, CD15 + Cell density, CD20 + Cell density, CD56 + Cell density, CD68 + Cell density, CD86 + Cell density, where density is the ratio of the number of cells to the area of ​​the corresponding region; Determining the immunophenotyping of the tumor tissue sample based on at least part of the second eigenvalues ​​in the second eigenvalue group of the region inside the tumor boundary and the region outside the tumor boundary; wherein the immunophenotyping includes immune infiltration type, immune rejection type, and immune desert type; In the inner region of the tumor boundary of the tumor tissue sample determined by immunotyping as immune infiltration type or immune rejection type, multiple first eigenvalues ​​of the first eigenvalue group are obtained, and the multiple first eigenvalues ​​are scored respectively, and the multiple scores are summed to obtain a score.

2. The method for scoring the likelihood of a tumor immune response according to claim 1, wherein: Tumor tissue sections were obtained from the tumor tissue samples and stained with multiple immunofluorescence staining for each of the cell markers CD4, CD8, CD15, CD20, CD56, CD68, CD86, Granzyme B, PD-L1, FoxP3 and PanCK, including: Serial tumor tissue sections were obtained from the tumor tissue sample. An intermediate tumor tissue section was stained with H&E to obtain an H&E-stained section. After confirming that the number of tumor cells in the tumor region of the intermediate tumor tissue section met the requirements, two tumor tissue sections adjacent to the intermediate tumor tissue section were selected for multiple immunofluorescence staining. The two tumor tissue sections were stained with the following two panels in a one-to-one correspondence: Panel-1: CD4 / CD8 / FoxP3 / Granzyme B / PD-L1 / PanCK / DAPI; Panel-2: CD15 / CD20 / CD56 / CD68 / CD86 / PanCK / DAPI.

3. The method for scoring the possibility of tumor immune response according to claim 2, characterized in that: In the staining process of Panel-1, the antibodies and dyes used for each biomarker, as well as the corresponding fluorescence test channels, are as follows: and / or, In the staining process of Panel-2, the antibodies and dyes used for each biomarker, as well as the corresponding fluorescence test channels, are as follows:

4. The method for scoring the likelihood of a tumor immune response according to claim 1, wherein: The first preset distance is less than or equal to 300 μm; and / or The second preset distance is less than or equal to 500 μm.

5. The method for scoring the likelihood of tumor immune response according to claim 1, wherein: When the size of the tumor region is less than or equal to a preset size, the tumor region is used to replace the inner region of the tumor boundary; wherein the preset size is less than or equal to a first preset distance, or the preset size is greater than the first preset distance and the difference between the preset size and the first preset distance is less than or equal to 200 μm; or the preset size is greater than the first preset distance and the difference between the preset size and the first preset distance is less than or equal to 100 μm; or When PanCK-positive and PanCK-negative areas can be distinguished within the tumor region, the PanCK-positive area is used to replace the area inside the tumor boundary; or When the second preset distance of the tumor boundary outer region is greater than or equal to 400 μm and less than or equal to 500 μm, the tumor boundary outer region is divided into a first tumor boundary outer region close to the tumor region boundary and a second tumor boundary outer region far from the tumor region boundary.

6. The method for scoring the likelihood of tumor immune response according to claim 1, wherein: At least part of the second feature values ​​in the second feature value group of the tumor boundary inner region and the tumor boundary outer region include CD4 + Cell density, CD8 + Cell density, CD20 + Cell density, CD56 + Cell density and CD68 + One or more of the cell densities.

7. The method for scoring the likelihood of tumor immune response according to any one of claims 1 to 6, characterized in that: Determining the immunophenotyping of the tumor tissue sample based on at least some of the second feature values ​​in the second feature value groups of the region inside the tumor boundary and the region outside the tumor boundary includes: Obtaining a second eigenvalue of the region inside the tumor boundary and a corresponding second eigenvalue of the region outside the tumor boundary, and adding the two second eigenvalues ​​to obtain a second sum; When the second sum is less than or equal to a second preset value, determining that the tumor tissue sample is of immune desert type; When the second sum is greater than a second preset value and the ratio of the second characteristic value in the area outside the tumor boundary to the second characteristic value in the area inside the tumor boundary is greater than a third preset value, determining that the tumor tissue sample is of immune rejection type; When the second sum is greater than the second preset value and the ratio of the second characteristic value in the area outside the tumor boundary to the second characteristic value in the area inside the tumor boundary is less than or equal to a third preset value, the tumor tissue sample is determined to be an immune infiltration type.

8. The method for scoring the likelihood of tumor immune response according to claim 1, wherein: Obtaining multiple first eigenvalues ​​in the first eigenvalue group, scoring the multiple first eigenvalues ​​respectively, and summing the multiple scores to obtain a score, including: Get CD8 in the first eigenvalue group + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage, Granzyme B + Cell percentage, CD4 + +FoxP3 + Cell proportion and CD15 + Cell ratio; When CD8 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage and Granzyme B + When the cell proportions are greater than or equal to the threshold, they are scored accordingly; When CD4 + +FoxP3 + Cell proportion and CD15 + When the cell proportions are less than or equal to the threshold, they are scored accordingly; The multiple scoring scores corresponding to the multiple first eigenvalues ​​are added together, and the sum obtained is the score.

9. The method for scoring the likelihood of tumor immune response according to claim 8, wherein: When CD8 + Cell ratio, CD20 + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell ratio, PD-L1 + Cell percentage and Granzyme B + When the cell ratio is greater than or equal to the threshold, each is scored accordingly; when CD4 + +FoxP3 + Cell proportion and CD15 + When the cell ratio is less than or equal to the threshold, the corresponding score is calculated; including: When CD8 + Cell proportion and PD-L1 + When the cell proportions are respectively greater than or equal to the first threshold, the first score and the second score are recorded respectively; When Granzyme B + Cell ratio, CD56 + Cell ratio, CD68 + +CD86 + Cell percentage and CD20 + When the cell proportions are respectively greater than or equal to the second threshold, the corresponding scores are recorded as the third score, the fourth score, the fifth score and the sixth score; When CD4 + +FoxP3 + Cell proportion and CD15 + When the cell proportions are less than or equal to the third threshold, the seventh and eighth scores are recorded respectively; Wherein, the first threshold is greater than the second threshold, and the second threshold is greater than the third threshold; The values ​​from the first score to the eighth score show a decreasing trend.

10. The method for scoring the possibility of tumor immune response according to claim 8 or 9, characterized in that: Thresholds include 5%; Alternatively, the first threshold value includes 5%, and / or the second threshold value includes 3%, and / or the third threshold value includes 2%; Alternatively, the first score is 3, the second score is 2, the third score is 1.5, the fourth score and the fifth score are both 1, and the sixth score, the seventh score and the eighth score are each 0.5.

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