Application of reagent for detecting SPIB protein in preparation of product for evaluating DLBCL prognosis

Abstract

The invention discloses application of a reagent for detecting SPIB protein in preparation of a product for evaluating DLBCL prognosis. The reagent is used for detecting the SPIB protein by adopting an immunohistochemical staining method. The new protein marker SPIB is screened out, the prognosis of DLBCL patients is classified and judged through simple immunohistochemical detection (IHC), and compared with gene detection, the cost is lower, the IHC technology is mature, the result is stable, paraffin sections can be used, retrospective performance is good, repeatability is high, and the application range is wider.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to application of a reagent for detecting SPIB protein in preparing a product for evaluating the prognosis of DLBCL. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common lymphatic system tumor in adults, accounting for 30%-40% of adult non-Hodgkin's lymphoma. Its clinical prognosis has improved in the rituximab era, but the prognosis of refractory subtypes (such as double-hit lymphoma and EBV-positive lymphoma) is still unsatisfactory. Therefore, it is necessary to accurately stratify patients before initial treatment to facilitate the selection of the best treatment method, and it is also necessary to find new targets to provide new basis for clinical treatment.

[0003] Existing studies have shown that MYC gene rearrangement is associated with a poor prognosis in DLBCL. In order to determine the prognosis of DLBCL patients, in principle, all DLBCL patients should undergo fluorescence in situ hybridization (FISH) examinations for MYC rearrangement. However, due to the high cost of FISH testing, for the purpose of resource conservation, currently only patients with C-MYC expression ≥ 40% are tested for FISH testing in clinical practice. Existing studies have shown that about 20% of patients with MYC gene rearrangement do not have high expression of MYC protein, and only about 10% of patients with high expression of MYC protein can detect MYC gene rearrangement. In DLBCL cases without MYC gene rearrangement, the prognosis of most cases is still poor. Therefore, those skilled in the art are in urgent need of screening out new targets. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies in the prior art and provide a reagent for detecting SPIB protein for use in preparing a product for evaluating the prognosis of DLBCL.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provided is a method for preparing a product for evaluating the prognosis of DLBCL using a reagent for detecting SPIB protein.

[0007] Furthermore, the reagent is a reagent for detecting SPIB protein using immunohistochemical staining.

[0008] Furthermore, SPIB positivity is judged as a poor prognosis for DLBCL.

[0009] Furthermore, the interpretation criteria for SPIB positivity are: SPIB protein positive reaction is brown-yellow staining localized in the cell nucleus.

[0010] Furthermore, SPIB positivity was judged as the ABC subtype with poor prognosis of DLBCL.

[0011] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0012] The present invention screens out a new protein marker SPIB, and uses a simple immunohistochemistry test (IHC) to classify and judge the prognosis of DLBCL patients. Compared with genetic testing, it has lower cost, mature IHC technology, stable results, and can use paraffin sections, with good retrospective and high repeatability, and a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Immunohistochemical staining of SPIB and MYC proteins; A is SPIB negative, B is SPIB positive; C is MYC negative; D is MYC positive.

[0014] Figure 2 Kaplan-Meier survival curves were drawn for 229 DLBCL patients grouped according to SPIB positivity. DETAILED DESCRIPTION

[0015] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0016] SPIB is a member of the Ets transcription factor family. Its expression is generally limited to lymphocytes. Its expression level is high in B cells, but low in T lymphocytes. In the process of B lymphocyte maturation, SPIB expression increases significantly, indicating that SPIB plays an important role in B cell differentiation. We performed open chromatin group sequencing on cases with positive MYC protein but negative and positive MYC gene rearrangement and found that SPIB was a core transcription factor in the two groups and was at the center of the regulatory network. According to the results of open chromatin sequencing, it was found that up to 40% of the potential transcription factor binding sites within the range of the transcription start site upstream of the MYC promoter were SPIB binding sites. And 63% of the potential binding sites of SPIB are upstream of the MYC promoter. However, MYC does not bind to the SPIB promoter region. This suggests that SPIB may be a direct upstream regulatory factor of MYC expression.

[0017] The present invention retrospectively analyzed 229 patients with newly diagnosed DLBCL who were admitted to the Affiliated Hospital of Xuzhou Medical University from 2015 to 2018 to understand the clinicopathological characteristics of SPIB-positive DLBCL patients and their relationship with prognosis.

[0018] Immunohistochemistry (IHC) staining was performed in 229 cases, and the EnVision method was used to detect the expression of SPIB, CD10, MUM-1, MYC, BCL2 and BCL6 in pathological sections.

[0019] The specific steps of immunohistochemical staining (EnVision method) are as follows: take the paraffin sections out of the 37°C oven and place them in a 65°C oven for 2 hours to further fix the tissue. Then the sections are dewaxed and hydrated: soak the sections in the following solutions in sequence: xylene-20 minutes, xylene-2 ​​for 20 minutes, 100% alcohol-15 minutes, 100% alcohol-2 for 15 minutes, 80% alcohol for 10 minutes, and 60% alcohol for 10 minutes. After soaking, rinse with PBS for 3 minutes to remove excess alcohol. Then place the slides in tris-EDTA solution and boil them in boiling water for 20 minutes for antigen retrieval. After boiling, cool them naturally at room temperature. After cooling to room temperature, wash the slides with 1×PBS for 3 times, 5 minutes each time. Then spin dry the slides, drop SPIB and c-MYC antibodies on the corresponding samples, and incubate them at 4°C overnight. The next day, take the slides out of the 4°C refrigerator, place them at room temperature for 60 minutes, and then wash them 3 times with 1×PBS to remove excess antibodies. Then drop HRP-labeled secondary antibody on the sample and incubate at room temperature for 60 minutes. After incubation, wash again with 1×PBS 3 times. Subsequently, prepare DAB developer and be careful to avoid light. Mix the developer, drop it on the glass slide, and start timing. Observe the color reaction under a microscope. After the positive point appears, immediately terminate the reaction with tap water. When the timing ends, record the reaction time. Rinse the glass slide with running water for 5 minutes to remove the residual developer. Then stain the slices with hematoxylin for 30 seconds, and rinse with running water for 5 minutes after staining. Then put the slices into gradient alcohol for dehydration: 60% alcohol, 80% alcohol, 100% alcohol one, 100 alcohol two, soak for 2 minutes each, then soak in xylene one and xylene two for 2 minutes, and then use neutral resin glue to seal the slide.

[0020] Interpretation method and standard of immunohistochemical staining results: Two pathologists read and judged the films respectively. SPIB protein positive reaction was brown-yellow staining located in the cell nucleus. Tumor cells were defined as SPIB positive expression group, and vice versa as SPIB protein negative expression group. MYC protein positive reaction was brown-yellow staining located in the cell nucleus. The percentage of stained tumor cells in all tumor cells was counted under high-power microscope (400×). More than 40% of tumor cells were positive and defined as MYC protein high expression group, and vice versa as MYC protein low expression group. Figure 1 .

[0021] 1. Basic pathological characteristics of 229 DLBCL patients

[0022] The age distribution of 229 DLBCL patients ranged from 25 to 91 years old, with an average age of 61.4 years and a median age of 63 years. Elderly patients over 60 years old were slightly more common, and there was no significant difference between men and women. The first site was more common outside the lymph nodes, and it could occur in various organs, with a total of 168 cases. The most common nasopharyngeal and oral cavity was 44 cases, and other common extra-lymphatic organs included the gastrointestinal tract in 35 cases, the brain in 33 cases, and the tonsils in 10 cases. The chi-square test analysis found that the first site of the SPIB-positive group was more common outside the lymph nodes, and the difference was statistically significant (P<0.05). The Ann Arbor staging system divides DLBCL patients into four stages: stage I, stage II, stage III, and stage IV. The chi-square test analysis found that the SPIB-positive group stage III and stage IV were more common than stage I and stage II, and the difference was statistically significant (P<0.05). In the laboratory examination of SPIB-positive patients, lymphocyte abnormalities were more common, and the chi-square test analysis found that the difference was statistically significant (P<0.05). See Table 1:

[0023] Table 1

[0024]

[0025]

[0026] *Fisher's exact test

[0027] 2. SPIB positive / negative and DLBCL immunophenotype

[0028] The results of immunohistochemistry of SPIB positive / negative patients were further analyzed. The χ2 test analysis showed that the SPIB positive group was often accompanied by BCL2 expression, and the difference was statistically significant (P<0.05). In addition, the SPIB positive group was often accompanied by high expression of MYC, and the χ2 test analysis showed that the difference was statistically significant (P<0.05), as shown in Table 2.

[0029] Table 2

[0030]

[0031] 3. Survival analysis of SPIB-positive / -negative DLBCL patients

[0032] The results of univariate Cox regression analysis showed that SPIB positivity was an influencing factor for the prognosis of DLBCL patients (P<0.05), see Table 3 (univariate and multivariate survival analysis of OS in 229 DLBCL patients).

[0033] Table 3

[0034]

[0035]

[0036] 229 DLBCL patients were grouped according to SPIB positivity, and Kaplan-Meier survival curves were drawn. Log-rank test was used to analyze whether there was a statistical difference. The results showed that patients with SPIB positivity had a worse prognosis. Figure 2 .

[0037] 4. Analysis of differences in COO typing between SPIB-positive and -negative DLBCL

[0038] Of the 229 primary DLBCL pathologies studied, 47 underwent molecular testing. DNA targeted sequencing and COO assay DNA was extracted from paraffin-embedded sections of 47 patients and commissioned to Nanjing Shihe Gene Co., Ltd. for targeted DNA sequencing. The library probes covered all exon sequences of 475 lymphoma-related genes.

[0039] Among the 47 cases of DLBCL, 9 cases expressed SPIB protein (SPIB positive), and the staining was localized in the nuclei of tumor cells, showing uniform and diffuse positive. 38 cases did not express SPIB protein (SPIB negative). Based on this result, the 47 cases of DLBCL were divided into SPIB positive group (n = 9) and SPIB negative group (n = 38). Lymph2Cx detection method based on Nanostring platform was used to determine the cell-of-origin (COO) of 47 cases of DLBCL, and the COO typing was analyzed. The results showed that all the SPIB positive group were ABC subtypes, and most of the SPIB negative group were GCB subtypes. The differences were statistically significant after χ2 and Fisher's exact test analysis (P < 0.05, Table 4).

[0040] Table 4

[0041]

[0042] The above clinical studies have demonstrated that PIB positivity is associated with the clinical characteristics and poor prognosis of DLBCL. Therefore, SPIB positivity is a subtype of DLBCL with a poor prognosis, and SPIB positivity can be used as a marker for prognostic stratification of DLBCL.

[0043] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of a reagent for detecting SPIB protein in the preparation of a product for evaluating the prognosis of DLBCL.

2. The use according to claim 1, characterized in that: The reagent is a reagent for detecting SPIB protein by immunohistochemical staining.

3. The use according to claim 1, characterized in that: SPIB positivity indicates a poor prognosis for DLBCL.

4. The use according to claim 3, characterized in that: The interpretation criteria for SPIB positivity are: SPIB protein positive reaction is brown-yellow staining localized in the cell nucleus.

5. The use according to claim 3, characterized in that: SPIB positivity was judged as ABC subtype of DLBCL with poor prognosis.

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