PAX-5 + IMP3 + Application of double positive cells in detection of diffuse large B-cell lymphoma with bone marrow involvement

Abstract

The present application relates to PAX-5 + IMP3 + The application of double positive cells in detecting diffuse large B-cell lymphoma with bone marrow involvement. The present application unexpectedly found that PAX-5 + IMP3 + Double positive cells can be used as a biomarker to evaluate whether diffuse large B-cell lymphoma involves bone marrow. By immunohistochemical detection on bone marrow biopsy specimens, whether it contains PAX-5 + IMP3 + Double positive cells, that is, to quickly and accurately determine whether diffuse large B-cell lymphoma involves bone marrow, the detection sensitivity is as high as 95% or more, the specificity is as high as 100%, and it is especially suitable for evaluating samples with very low proportion (less than 1%) of tumor B lymphocytes in bone marrow tissue.

Description

Technical Field

[0001] This invention belongs to the field of tumor detection technology, specifically relating to the detection of diffuse large B-cell lymphoma, and more specifically to PAX-5. + IMP3 + Application of double-positive cells in the detection of diffuse large B-cell lymphoma with bone marrow involvement. Background Technology

[0002] Diffuse large B-cell lymphoma (DLBCL) is an aggressive hematologic malignancy originating from B lymphocytes, with an annual incidence of approximately 3-5 cases per 100,000. The incidence of DLBCL in China is slightly higher than in Europe and the United States, with approximately 34,000 new cases annually. About 10%-30% of DLBCL cases involve the bone marrow, indicating advanced disease progression. This significantly increases the invasiveness of the disease and the risk of relapse after treatment, and may impair the normal hematopoietic function of the bone marrow, leading to various secondary hematologic disorders.

[0003] Currently, the diagnostic methods for DLBCL involving the bone marrow rely on bone marrow pathology and immunophenotype (including flow cytometry). Flow cytometry has several limitations in assessing whether diffuse large B-cell lymphoma involves the bone marrow: ① Focal infiltration of neoplastic B lymphocytes or "peritrabecular" infiltration makes it difficult to obtain neoplastic B cells from bone marrow aspiration fluid via flow cytometry; ② Bone marrow fibrosis occurs around neoplastic B cells, making it difficult to obtain neoplastic B cells from bone marrow aspiration fluid via flow cytometry; ③ The flow cytometry immunophenotype of DLBCL patients treated with CD20 monoclonal antibodies is difficult to distinguish from reactive B progenitor cells in the bone marrow; ④ A small number of monoclonal B-cell lymphomas are easily found in the bone marrow of elderly patients or patients with autoimmune diseases, which can easily be misdiagnosed as diffuse large B-cell lymphoma. Currently, bone marrow pathological diagnosis or minimal residual disease (MRD) detection requires a comprehensive evaluation combining bone marrow pathological morphology with extensive immunohistochemical analysis (including CD20, PAX-5, Bcl-6, CD10, MuM-1, CD5, c-myc, CD34, TdT, etc.), which is cumbersome and costly. Furthermore, MRD detection is particularly challenging in DLBCL cases treated with CD20 monoclonal antibodies because the low tumor cell count makes accurate localization difficult with existing immunohistochemical methods, and some antibodies lack specificity, often failing to differentiate from pre-existing B progenitor cells in the bone marrow. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide PAX-5. + IMP3 + Application of double-positive cells in the detection of diffuse large B-cell lymphoma with bone marrow involvement, using PAX-5+ IMP3 + Double-positive cells, as biomarkers, can be used to simply, efficiently, and accurately assess whether diffuse large B-cell lymphoma involves the bone marrow.

[0005] The first aspect of the present invention is to provide PAX-5 + IMP3 + Application of double-positive cells as biomarkers in the preparation of reagents for detecting whether diffuse large B-cell lymphoma involves the bone marrow.

[0006] In some implementations, the sample being tested is bone marrow tissue.

[0007] A second aspect of the present invention is to provide a method for detecting PAX-5. + IMP3 + Application of reagents for double-positive cells in the preparation of kits for detecting whether diffuse large B-cell lymphoma involves the bone marrow.

[0008] In some implementations, the sample being tested is bone marrow tissue.

[0009] In some embodiments, the reagent is the same reagent used for detection by immunohistochemistry and / or immunofluorescence.

[0010] In some embodiments, the reagent includes antibodies that specifically bind to the IMP3 protein and antibodies that specifically bind to the PAX-5 protein.

[0011] In some embodiments, the antibody that specifically binds to the IMP3 protein and / or the antibody that specifically binds to the PAX-5 protein is a monoclonal antibody.

[0012] A third aspect of the present invention is to provide a method for detecting whether diffuse large B-cell lymphoma involves the bone marrow for non-diagnostic purposes, comprising the following steps: detecting whether the sample to be tested contains PAX-5. + IMP3 + Double-positive cells.

[0013] In some implementations, the detection method is immunohistochemistry or immunofluorescence.

[0014] In some implementations, the sample to be tested is bone marrow tissue.

[0015] PAX-5 was unexpectedly discovered during the research and testing process of this invention. + IMP3 +Double-positive cells can serve as a biomarker for assessing whether diffuse large B-cell lymphoma involves the bone marrow. IMP3 protein is located in the cell membrane or cytoplasm, while PAX-5 protein is located in the cell nucleus. Immunohistochemical staining of bone marrow biopsy specimens can be used to observe the presence of PAX-5. + IMP3 + Double-positive cells can quickly and accurately determine whether diffuse large B-cell lymphoma has involved the bone marrow. The detection sensitivity is over 95% and the specificity is 100%. It is especially suitable for evaluating samples with a very low proportion of neoplastic B lymphocytes (less than 1%) in bone marrow tissue. Attached Figure Description

[0016] Figure 1 Immunohistochemical staining image of a bone marrow biopsy specimen with typical DLBCL involvement. Detailed Implementation

[0017] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0019] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0020] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0021] IMP3: Insulin-like growth factor II mRNA-binding protein 3 (IMP3).

[0022] PAX-5: Paired box gene 5 (PAX-5) encodes a B-cell line-specific activating protein.

[0023] PAX-5 + IMP3 + Double-positive cells: Cells that simultaneously express both PAX-5 and IMP3 proteins.

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] Example 1

[0026] This study included 140 patients with DLBCL, of whom 40 were diagnosed with bone marrow involvement and 100 were diagnosed without bone marrow involvement. Bone marrow biopsy specimens were collected from all patients, and immunohistochemical staining was used to detect the presence of PAX-5 in the bone marrow biopsy specimens. + IMP3 + Double-positive cells.

[0027] Staining Method: Bone marrow biopsy specimens were fixed with 10% neutral formaldehyde fixative, decalcified with bone marrow-specific decalcifying solution, routinely dehydrated, paraffin-embedded, sectioned to 3µm thickness, and stained with hematoxylin and eosin (HE) for light microscopic observation. Immunohistochemical staining was performed using the EnVision two-step method. IMP3 antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (catalog number ZA-0639; clone number EP286), and PAX-5 antibody was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (catalog number CPM-0244; clone number C12A5). Staining was performed using the CNT-360 fully automated immunohistochemical staining system from Sinotek, including dewaxing, antigen retrieval, and antigen-antibody (IMP3 and PAX-5 antibody) reaction. Afterward, slides were removed from the instrument, cleaned, rinsed in anhydrous ethanol, dried, and cleared in xylene, followed by mounting with neutral resin.

[0028] IMP3 protein is located in the cell membrane / cytoplasm; PAX-5 protein is located in the cell nucleus.

[0029] The criteria for judging the results are as follows:

[0030] (1) If the bone marrow biopsy specimen contains PAX-5 + IMP3 + Double-positive cells indicate DLBCL affecting the bone marrow;

[0031] (2) If the bone marrow biopsy specimen does not contain PAX-5 + IMP3 + If two cells are positive, it is determined that DLBCL has not affected the bone marrow.

[0032] The test results are shown in Table 1:

[0033] Table 1

[0034]

[0035] The results showed that PAX-5 was present in bone marrow biopsy specimens from all 40 cases of DLBCL involving the bone marrow. + IMP3 + Double-positive cells were found in bone marrow biopsy specimens from 100 cases without DLBCL involvement, and none of these specimens contained PAX-5. + IMP3 + Double-positive cells were detected, and the test results were completely consistent with the clinical diagnosis. This was based on PAX-5 bone marrow biopsy specimens. + IMP3 + The sensitivity and specificity of double-positive cells in assessing bone marrow involvement in DLBCL were 100%.

[0036] Figure 1 This is a representative staining image of a bone marrow biopsy specimen with typical DLBCL involvement. Green arrows indicate only positive nuclei (PAX-5 protein alone), representing normal B lymphocytes in the bone marrow; blue arrows indicate only positive cytoplasm (IMP3 protein alone), representing trace amounts of normal cells in the bone marrow; red arrows indicate cells with both positive nuclei and cytoplasm (PAX-5 protein alone). + IMP3 + (Double positive) diffuse large B-cell lymphoma tumor cells.

[0037] Example 2

[0038] This embodiment uses a different clinical sample than that in Example 1 to further validate the PAX-5 bone marrow biopsy specimen. + IMP3 + The detection of double-positive cells is a diagnostic efficacy for assessing whether bone marrow involvement is present in DLBCL.

[0039] The traditional detection method (Method A) and the method of the present invention (Method B) were used to analyze 60 clinically diagnosed positive cases of DLBCL involving bone marrow and 110 negative cases.

[0040] Among them, the traditional detection method combines bone marrow pathological morphology with a large number of immunohistochemicals (including CD20, PAX-5, Bcl-6, CD10, MuM-1, CD5, c-myc, P53, CD34, TdT, etc.) for comprehensive evaluation. The method is as follows: (1) Slide preparation: Tissue paraffin blocks are sliced ​​into 3μm sections; the sections are placed in a 50℃ constant temperature water bath for 20s to fully unfold; the sections are laid flat in the center of the glass slide and placed in a 50℃ constant temperature slide oven for 50min; (2) Dewaxing, antigen retrieval and antigen-antibody (CD20, PAX-5, Bcl-6, CD10, MuM-1, CD5, c-myc, P53, CD34, TdT, etc.) reactions are performed in the CNT-360 fully automated immunohistochemical instrument of Sinotec. (3) The glass slides are removed from the instrument and cleaned, then soaked in anhydrous ethanol and cleared in xylene, and then mounted with neutral resin.

[0041] The criteria for judging the results are as follows:

[0042] The proliferation of mature B lymphocytes expressing B lymphocyte markers (CD20, PAX-5) but not progenitor markers (TdT, CD34) was assessed. Then, a comprehensive evaluation was conducted on mature B lymphocytes expressing Bcl-6 and / or CD10 and / or MuM-1 and / or CD5 and / or c-myc and / or P53 to identify abnormal mature B lymphocytes. Finally, if the abnormal B lymphocytes were large lymphocytes, it was determined that DLBCL involved the bone marrow. Conversely, if they did not express large lymphocytes, it was determined that DLBCL did not involve the bone marrow.

[0043] The method of this invention uses two immunohistochemical double staining methods, PAX-5 and IMP3, to detect PAX-5. + IMP3 + Double-positive cells were used for evaluation, and the specific methods and result judgment criteria were the same as in Example 1.

[0044] The detection and analysis results are shown in Table 2 below:

[0045] Table 2

[0046]

[0047] Table 2 shows that Method A has a sensitivity of 96.7% and a specificity of 100%, while Method B has a sensitivity of 95% and a specificity of 100%. Using a chi-square test, the p-values ​​were greater than 0.05%, indicating no statistically significant difference between the results of this invention and the traditional method. However, the traditional method requires more than eight antibodies, while the method of this invention only requires two, significantly reducing the use of immunohistochemistry and reducing costs by at least 75%, resulting in a reduction of over a thousand yuan per test. Furthermore, in the traditional method (Method A), the proportion of tumor-bearing B lymphocytes in the bone marrow tissue of two false-negative cases was less than 1%, leading to ineffective detection and false negatives. However, these two false-negative cases were positive when tested using the method of this invention (Method B), suggesting that the method of this invention can accurately detect samples with a very low proportion of tumor-bearing B lymphocytes. In Method B, the three false-negative cases all showed proliferating large tumor-bearing B lymphocytes in their PAX-5 protein; therefore, although these three false-negative samples did not contain PAX-5... + IMP3 + Double-positive cells, according to the result criteria, are judged as not involved, but they also indicate to the pathologist that these three samples are actually suspicious positive results. In actual testing, the pathologist will conduct further re-examination to confirm, which can also effectively avoid false negatives.

[0048] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. PAX-5 + IMP3 + Application of double-positive cells as biomarkers in the preparation of reagents for detecting whether diffuse large B-cell lymphoma involves the bone marrow.

2. The application as described in claim 1, characterized in that, The sample tested was bone marrow tissue.

3. Testing PAX-5 + IMP3 + Application of reagents for double-positive cells in the preparation of kits for detecting whether diffuse large B-cell lymphoma involves the bone marrow.

4. The application as described in claim 3, characterized in that, The sample tested was bone marrow tissue.

5. The application as described in claim 1 or 3, characterized in that, The reagents mentioned are those used for detection by immunohistochemistry and / or immunofluorescence.

6. The application as described in claim 5, characterized in that, The reagents include antibodies that specifically bind to the IMP3 protein and antibodies that specifically bind to the PAX-5 protein.

7. The application as described in claim 6, characterized in that, The antibodies that specifically bind to the IMP3 protein and / or specifically bind to the PAX-5 protein are monoclonal antibodies.

8. A method for detecting whether diffuse large B-cell lymphoma involves the bone marrow for non-diagnostic purposes, characterized in that, Includes the following steps: Detecting whether the sample contains PAX-5 + IMP3 + Double-positive cells.

9. The method as described in claim 8, characterized in that, The detection methods are immunohistochemistry or immunofluorescence.

10. The method as described in claim 8 or 9, characterized in that, The sample to be tested was bone marrow tissue.

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