WO2009082856A1

WO2009082856A1 - Molecule marker used for prognosticating diffuse large b cell lymphoma

Info

Publication number: WO2009082856A1
Application number: PCT/CN2007/071367
Authority: WO
Inventors: Junmin Li; Qinghua Zhang; Zizhen Xu; Weili Zhao; Zhixiang Shen
Original assignee: Ruijin Hospital Affiliated To The Shanghai Jiao Tong University Medical School; Shanghai Biochip Co., Ltd.
Priority date: 2007-12-28
Filing date: 2007-12-28
Publication date: 2009-07-09

Abstract

The present invention claims molecule marker p-Akt and/or YB-1 used for prognosticating diffuse large B cell lymphoma and their uses. The molecule marker p-Akt and/or YB-1 can be used to prognosticate diffuse large B cell lymphoma correctly and conveniently.

Description

Molecular markers for prognosis of diffuse large B-cell lymphoma

Technical field

The invention relates to the field of hematology and medicine. More specifically, it relates to molecular targets for diffuse large B-cell lymphoma treatment regimen selection and/or prognostic evaluation and kits for detecting targets. Background technique

Diffuse large B cell lymphoma (DLBCL) is the most common non-Hodgkin's lymphoma, accounting for approximately 30-40% of adult non-Hodgkin's lymphoma. The current incidence of non-Hodgkin's lymphoma in China is about 3-4/100,000, and it is growing at a rate of 2-3% per year. Among them, diffuse large B-cell lymphoma (DLBCL) is the most common, accounting for about all lymphocytes every year. 50% of the tumors, therefore, the annual new cases in the country are about 2-3 million.

DLBCL has significant biological heterogeneity and can respond completely differently to treatment. The patient's response to treatment was significantly different, and the conventional CHOP combination chemotherapy regimen (cyclophosphamide, doxorubicin, vincristine, and peat moss) only resulted in long-term remission in approximately 40% of patients.

In recent years, the application of human-mouse chimeric anti-CD20 monoclonal antibody rituximab (IDEC-C2B8, Rituximab, Rituxan) has further improved the clinical efficacy of DLBCL. However, the cost of rituximab for each course of treatment is 10-15 million RMB, which is a heavy economic burden for most patients.

Therefore, choosing a reasonable treatment plan will help patients who need rituximab to get timely and symptomatic treatment, and also help to save the medical expenses of patients who can get good treatment and prognosis with CHOP.

The International Prognostic Index (IPI) is based on clinical indicators and analyzes the prognosis of patients based on age, physical status, clinical Ann Arbor stage, number of extranodal lesions, and lactate dehydrogenase levels. Although IPI provides convenience for patient prognosis analysis and treatment planning, this evaluation is only a combination of clinical parameters, which is out of line with the biological characteristics of DLBCL and does not accurately assess the prognosis of patients. Some patients who are classified in the low-risk, low-risk group still have a 5-year survival rate of only 32%.

The occurrence of lymphoma stems from acquired genetic abnormalities, but in addition to the occurrence of tumor genetic events, the formation, proliferation and survival of B lymphoma lymphoma requires various biological signals from the microenvironment. Activation of the signaling pathway is primarily mediated by phosphorylation of various protein kinases. Abnormal activation of various signaling pathways plays an important role in cell growth, proliferation, metabolism, and apoptosis, and leads to the formation of lymphoma. A number of recent studies have shown that abnormal activation of the PI3K/Akt/mTOR signaling pathway plays an important role in the formation and development of various lymphomas, including diffuse large B-cell lymphoma. Phosphatidylinositol-3 -kinase (PI3K) causes a threonine position at position 308 of Akt (protein kinase B, PKB) via phosphoinositide-dependent kinase (PDK) Phosphorylation at the point (Thr308) and the serine site at position 473 (Ser473) activates Akt.

Activated Akt is an important mediator in this signaling pathway that regulates multiple gene families involved in cell proliferation and apoptosis. Mammalian target of rapamycin (mTOR) is the most important downstream molecule of Akt. After activation, it phosphorylates two important molecules that affect protein synthesis: ribosomal S6 kinase (ribosomal S6 kinase, p70S6K) and eukaryotic initiation factor 4E binding protein 1, 4E-BP1.

Another study found that Y-Box binding protein-1 (YB-1) is a new downstream molecule of Akt. YB-1 is a multifunctional protein involved in transcriptional regulation, translational regulation, cell proliferation, and DNA repair. The YB-1 protein plays an important role in the PI3K/Akt/mTOR signal transduction pathway and specifically inhibits translation by binding to its RNA binding domain and C-terminal binding domain. The inhibitory activity of YB-1 is decreased after phosphorylation by Akt, thereby increasing the translation and transcription of oncogenes.

The regulation of lymphocyte proliferation and the blockade of normal apoptotic processes lead to the formation of lymphoma. Studies have shown that phosphorylation regulated by Akt affects the activity of the Bcl-2 family, NF-kB, and other transcription factors that control the cell apoptosis pathway. The granulocyte leukemia-1 protein (; Mcl-1) is a member of the Bcl-2 family. Apoptosis induced by PI3K/Akt/mTOR inhibitors is closely related to the down-regulation of anti-apoptotic proteins Mcl-1 and Bcl-2. PI3K/Akt upregulates Mcl-1 at the transcriptional level, and its overexpression inhibits the exogenous and drug-induced apoptosis of various lymphoma cell lines.

However, there is no prior art that provides molecular markers that are relevant to the histological heterogeneity and prognosis of DLBCL. Therefore, DLBCL clinical treatment still urgently requires molecular markers that can be used simply and effectively as indicators of DLBCL treatment options and/or prognosis. Summary of the invention

It is an object of the present invention to provide a diagnostic kit capable of detecting p-Akt and/or YB-1, a target molecule of the AKT signaling pathway in a diffuse large B-cell lymphoma, in clinical research and diagnosis.

Another object of the present invention is to provide a method for detecting diffuse large B-cell lymphoma AKT signaling pathway target molecules p-Akt and/or YB-1, and as a detection index.

Another object of the present invention is to provide a prognostic evaluation mode for diffuse large B-cell lymphoma. Another object of the invention is to provide guidance for the selection of clinical treatment options for diffuse large B-cell lymphoma

In a first aspect of the invention there is provided a kit for use in the treatment of diffuse large B-cell lymphoma for treatment selection and/or prognosis evaluation, the kit comprising:

i) one or more reagents for detecting p-Akt expression in a biological sample; and/or

Ii) one or more reagents for detecting YB-1 expression in a biological sample.

In one embodiment of the invention, the reagents i), ii) in the kit are reagents for immunohistochemical detection of the expression of p-Akt and/or YB-1, respectively, in a biological sample.

In another embodiment of the invention, the biological sample is obtained from fresh tissue, formalin-fixed or paraffin-embedded tissue of a diffuse large B-cell lymphoma patient with or without clinical treatment.

In a preferred embodiment, the patient is not clinically treated.

In another embodiment of the invention, the clinical treatment is selected from the group consisting of: combination chemotherapy, or a combination therapy with rituximab.

In a preferred embodiment, the combination chemotherapy is CHOP combined with chemotherapy, i.e., cyclophosphamide-doxorubicin-longchunxin-prednisone combined with chemotherapy.

In another embodiment of the invention, the kit further comprises one or more selected from the group consisting of: instructions for use, a positive control, a negative control, a buffer, or an immunological adjuvant.

In another embodiment of the present invention, the instructions for use indicate that when the test result is positive for p-Akt and/or YB-1 expression, the prognosis of the combined chemotherapy is not good, but Rituximab will improve the prognosis, and combination therapy with rituximab should be used for the treatment. When the test results are negative for both p-Akt and YB-1 expression, it is only necessary for the patient. Good combination of chemotherapy can achieve good therapeutic effect and prognosis.

In a preferred embodiment, the combination chemotherapy is CHOP combined with chemotherapy.

A second aspect of the invention provides the use of a p-Akt expression and/or YB-1 expression detecting reagent or kit of reagents in the manufacture of a kit for the treatment of a diffuse large B-cell lymphoma for treatment selection and/or prognosis evaluation.

A third aspect of the invention provides the use of p-Akt and/or YB-1 as markers for therapeutic regimen selection and/or prognostic evaluation of diffuse large B-cell lymphoma.

A fourth aspect of the invention provides a method of assessing the prognostic effect of diffuse large B-cell lymphoma, the method comprising:

a) detecting the expression of p-Akt and/or YB-1 in biological samples obtained from patients with diffuse large B-cell lymphoma; b) analyzing the test results obtained by step a);

Among them, when the expression of p-Akt and YB-1 are negative, it suggests that the prognosis of the combination chemotherapy will be good; when the expression of p-Akt and / or YB-1 is positive, it suggests the prognosis of combination chemotherapy alone. The effect is poor, but the combination of rituximab will improve the prognosis.

In a preferred embodiment, the method further comprises assessing an international prognostic index.

In another preferred embodiment, the combination chemotherapy is CHOP combined with chemotherapy.

In another preferred embodiment, the patient is not clinically treated.

In a fifth aspect of the invention, a method of selecting a treatment regime for diffuse large B-cell lymphoma is provided, the method comprising:

a) detecting the expression of p-Akt and/or YB-1 in biological samples obtained from patients with diffuse large B-cell lymphoma;

b) analyzing the test results obtained by step a);

Among them, when the expression of p-Akt and YB-1 are negative, it suggests that combined chemotherapy should be used for this patient; when the expression of p-Akt and/or YB-1 is positive, it is suggested that the combination chemotherapy should be used for the patient. Combination therapy with toximab.

In another preferred embodiment, a method of screening for a drug or treatment regimen for ameliorating the prognostic effect of diffuse large B-cell lymphoma is provided, the method comprising:

(X) administering a drug or treatment regimen to be screened to a patient, model animal or cell of a diffuse large B-cell lymphoma;

(; b) detecting the expression of p-Akt and YB-1 in the patient, model animal or animal,

Among them, both p-Akt and YB-1 were negative, suggesting that the prognosis of the drug or treatment to be screened is good. In another preferred embodiment, the drug is a chemotherapeutic drug, a monoclonal antibody specific for diffuse large B-cell lymphoma, or a combination thereof. DRAWINGS

Figure 1: Photograph of paraffin sections (HE staining) of DLBCL samples.

Figure 2: Photograph of paraffin sections (HE staining;) of positive samples of p-Akt expression in the AKT signaling pathway target. Figure 3: Photograph of paraffin sections (HE staining;) of positive samples of AKT signaling pathway target YB-1 expression. Figure 4: Relationship between p-Akt expression and OS in 60 follow-up patients. The survival curves of patients with p-Akf and p-Akt ⁺ were shown.

Figure 5: Relationship between p-Akt expression and OS in patients treated with the CHOP regimen, respectively Survival curves of p-Akt_ patients and p-Akt ⁺ patients.

Figure 6: Relationship between p-Akt expression and OS in patients treated with the R-CHOP regimen, showing survival curves for p-Akt_ patients and p-Akt ⁺ patients, respectively. Detailed ways

The present inventors have involved various molecules involved in the PI3K/Akt/mTOR signal transduction pathway in diffuse large B-cell lymphoma tissues (for example, p-Akt, YB-1, Bcl-2, p-p70S6K, p-4E-BP1, etc.) Long-term and in-depth study of the treatment and prognosis of the disease, a better understanding of the histological heterogeneity of diffuse large B-cell lymphoma from a molecular biology perspective, and the molecular pathological features of patients will be examined Linkage with clinical treatment response and prognosis to find molecular markers on this signaling pathway that can serve as targets for biological therapy and clinical prognostic indicators.

The present inventors have unexpectedly discovered that the molecules p-Akt and YB-1 in the PI3K/Akt/mTOR signal transduction pathway are indicative of prognosis of diffuse large B-cell lymphoma and have good sensitivity and can be used as good Molecular markers of clinical treatment options and prognostic effects. On the basis of this, the inventors completed the present invention. Treatment options and prognosis assessment

At present, the combination of DLBCL treatment at home and abroad is combined with chemotherapy (mainly CHOP combined with chemotherapy, ie cyclophosphamide, doxorubicin, vincristine and spongy pine;), or combined with human-mouse chimeric anti-CD20 single The cloned antibody rituximab and CHOP were combined with chemotherapy (R-CHOP;).

CHOP combined with chemotherapy can only achieve long-term remission in about 40% of patients, but its treatment costs are relatively low. The combination of rituximab and CHOP can further improve the clinical efficacy of DLBCL and improve the prognosis of patients. However, the cost of treatment for each course is 10-15 million RMB, which is a heavy economic burden for most patients. .

Therefore, patients with different treatment regimens, especially those who have a good prognosis with CHOP combined with chemotherapy, and those who have to be treated with rituximab, have improved the efficacy of clinical treatment of DLBCL and improved prognosis. It is important to reduce the burden on doctors and patients.

The inventors have unexpectedly discovered that p-Akt and/or YB-1 can serve as a molecular marker for a good clinical treatment option and prognostic effect in diffuse large B-cell lymphoma.

In the present invention, p-Akt and/or YB-1 may be employed, and only the treatment regimen may be selected or only the prognosis may be evaluated, or both may be combined to consider the overall treatment of the patient. The treatment option selection and prognosis evaluation of the present invention can also be combined with other physiological and pathological indicators such as the International Prognostic Index (IPI) to further improve the accuracy. Molecular marker

In the present invention, "molecular markers", "DLBCL treatment protocol selection and/or molecular markers of prognostic indicators" are used interchangeably, and both indicate that the present invention can be used to indicate the prognostic effect of DLBCL and have a clinical treatment option for its selection. A molecule that guides meaning. The molecular markers of the present invention are specifically p-Akt and YB-1 molecules in the PI3K/Akt/mTOR signal transduction pathway.

In the present invention, the term "p-Akt" refers to an important mediator of the PI3K/Akt/mTOR signaling pathway, which phosphorylates into its active form and regulates a number of gene families involved in cell proliferation and apoptosis. The term "YB-1" refers to Y-Box binding protein-1, which is a newly discovered downstream molecule of Akt in the PI3K/Akt/mTOR signal transduction pathway.

p-Akt is located upstream of the signal transduction pathway, while YB-1 is located downstream of the pathway. p-Akt regulates many signaling molecules in multiple downstream pathways, further regulating tumor cell proliferation and survival, and affecting cell migration, adhesion, and extracellular matrix degradation.

Downstream signaling molecules of p-Akt are involved in DNA damage repair and cell cycle regulation (eg, MDM2), protein synthesis and cell growth (eg, mTOR;), glucose metabolism (eg, GSK3P;), apoptosis (eg, BAD, FKHR) and many other important physiological and biochemical processes.

The inventors' practice has proved that not all p-Akt downstream signaling molecules have guiding significance for the selection of DLBCL prognosis and treatment options, for example, p-p70S6K, p-4E-, which are also downstream signaling molecules of p-Akt. BPl has no guiding significance. The present inventors have screened the sensitive molecules p-Akt and YB-1 molecules which have a guiding significance for the prognosis of DLBCL and the selection of clinical treatment options from numerous signal molecules.

In a preferred embodiment of the invention, when a positive expression of p-Akt and/or YB-1 is detected in a sample obtained from a patient with DLBCL, the prognosis of the combined chemotherapy is indicated to be poor, but the combination is beneficial. Tetuximab will improve the prognosis, and combination therapy with rituximab should be used for the treatment. When the test results are negative for both p-Akt and YB-1 expression, it is suggested that the patient only needs to use the combination. Chemotherapy can achieve good therapeutic effects and prognosis.

In the present invention, the term "negative" means that the test result indicates that the marker molecule is not expressed in the sample or has no statistical or histological difference compared to the negative control. Conversely, the term "positive" means that the test results indicate that the marker molecule is expressed in the sample or that the expression is increased or statistically or histologically compared to the negative control. The obvious difference.

In a specific embodiment of the present invention, an immunohistochemical detection method is adopted, wherein a tumor cell with a positive definition of p-Akt and YB-1 is >10%, and a granular brown or brownish color is clearly located, and a negative is Defined as <10% cell coloration in the field of view.

The expression of p-Akt or YB-1 can be tested separately and used for prognostic evaluation and treatment options. The results of the expression of these two signaling molecules can also be combined to more accurately perform prognostic evaluation and treatment options. Kit

Also provided in the invention is a kit for the treatment of diffuse large B-cell lymphoma treatment options and/or prognosis, the kit comprising: i) one or more reagents for detecting p-Akt expression in a biological sample; And/or ii) one or more reagents for detecting YB-1 expression in a biological sample.

The biological sample may be fresh tissue obtained from a patient with diffuse large B-cell lymphoma, formalin-fixed or paraffin-embedded tissue, body fluid, blood, or cells, etc., preferably fresh tissue, formalin-fixed or paraffin-embedded organization. These samples may be in various forms suitable for detection such as slicing, smears, suspensions, solutions, etc., for example, in the detection of immunohistochemistry, paraffin section specimens are preferably employed. Preferably, the patient is not clinically treated prior to collecting the sample.

The detection method used in the present invention is preferably an immunohistochemical assay which is extremely simple and effective for detecting and determining the expression of p-Akt and/or YB-1 in a sample, and is very suitable for clinical application. Of course, the present invention does not exclude other detection methods that can be employed by those skilled in the art, such as Western blotting, ELISA, flow cytometry, biochip assay, etc., but the procedure may be preferred to immunohistochemistry in the present invention. The method is cumbersome and can be selected by a person skilled in the art as needed.

A reagent or reagent set for detecting p-Akt and/or YB-1 expression can be provided in the kit as needed according to various detection principles and methods. In the present invention, "reagent set" refers to a combination of reagents containing a plurality of reagents required for detection.

In a specific embodiment of the present invention, the detection method used is an immunohistochemical assay, and thus the reagent for detecting p-Akt contained in the kit includes: primary antibody against secondary p-Akt, secondary antibody, and immune antibody. Other reagents (such as diluents) required for histification.

In addition, the kit of the present invention may further comprise, as needed, a container, a control (including a positive or negative control;), instructions for use, a buffer, an immunological adjuvant, etc., which can be selected by a person skilled in the art according to the specific circumstances. Advantages of the invention

1. This study analyzed the expression of p-Akt and YB-1 in the diffuse large B-cell lymphoma tissue of the AKT signaling pathway.

2. This study confirmed that the AKT signaling pathway target p-Akt, YB-1 positive expression patients have poor response to treatment, no progression survival and short overall survival, poor clinical prognosis, suggesting continuous activation of this pathway It is a poor molecular biology prognostic factor for DLBCL.

3. Rituximab combined with chemotherapy (R-CHOP) abolished the negative impact of the expression of this pathway on prognosis. Akt, YB-1 molecules can be used as new DLBCL biological therapeutic targets and clinical prognostic indicators. Example

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with the conditions described in the conventional conditions, or in accordance with the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the present invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1. Specimen collection

The patients enrolled in the study were 73 patients with newly diagnosed DLBCL, including 42 males and 31 females, aged 22-80 years, with a median age of 53 years. The following clinical data were collected from patients: gender, age, behavioral status, Ann Arbor clinical stage, LDH level and extranodic lesions, tumor size, and scored according to the International Prognostic Index (IPI). The patient signed informed consent as required by the institution. Patients were treated with 6 cycles of standard doses of CHOP or R-CHOP, depending on the condition with or without radiation therapy.

The specific composition of the CHOP regimen is as follows: cyclophosphamide 750 mg/m ² , static push, day 1; doxorubicin 50 mg/m ² , static push, day 1; vincristine 1.4 mg/m ² (maximum dose, 2.0 mg), static push, day 1; and prednisone 60 mg, orally, day 1-5.

The specific composition of the R-CHOP protocol is as follows: rituximab 375 mg/m ² , slow intravenous infusion, day 1; cyclophosphamide 750 mg/m ² , static push, day 2; doxorubicin 50 mg/m ² , static push, day 2; vincristine 1.4 mg/m ² (maximum dose, 2.0 mg), static push, day 2; and prednisone 60 mg, oral, 2-6 day.

Efficacy evaluation was performed 1 month after 6 courses of treatment. All patients must have a CT scan of the neck, chest, and abdomen, regardless of whether these sites are involved at the time of onset. Treatment response was divided into complete response (CR;), clinical complete response (Cm;), partial response (PR;), disease stabilization (SD), and disease progression (PD;) according to International Workshop criteria. . The total efficiency is defined as the sum of CR, Cm and PR.

Of the 73 cases, 60 were followable, and the follow-up deadline was August 30, 2007. The primary end point of this study was progression-free survival (PFS) and overall survival (OS;). PFS is defined as: Treatment effective from the onset of lymphoma progression, recurrence, death, or last follow-up. The OS is defined as the initial diagnosis to death or the last follow-up time. Example 2. Immunohistochemistry detection and result determination

Tissue sample processing

73 cases of DLBCL samples were fixed by formaldehyde. According to histological and immunohistochemical analysis, two high-grade pathologists determined the tissue type according to the 2001 WHO lymphoma classification criteria.

Cut 3 micron sections, routine dewaxing, dehydration, microwave cooling, natural cooling for 30 minutes, 1.5% hydrogen peroxide to remove endogenous peroxidase, 1% bovine serum albumin (BSA) for 20 minutes at room temperature, add the following Primary antibody: anti-p-Akt, anti-YB-1 (Cell Signaling Technology, Beverly, MA) or anti-p-p70S6K, anti-p-4E-BPl (Cell Signaling Technology, Beverly, MA), overnight incubation at 4°C, Dako Envision HRP (Dako Cytomation) labeled secondary antibody, DAB color development, gradient alcohol dehydration, hematoxylin counterstained nuclei after xylene translucent.

Ten representative fields were selected under high power microscope, and the number of positive cells in 1000 tumor cells was calculated as a percentage. P-Akt, YB-1 positively defined as >10% of tumor cells showed clear, granular brown or brownish yellow coloration. The positive control was breast cancer or colon cancer tissue, and TBS replaced the primary antibody as a negative control. All stained sections were judged by two blood pathologists on the same day, and if there was a dispute, re-evaluation was reached until consensus was reached.

2. Results determination and analysis

The immunohistochemistry results of paraffin sections and cells were observed using a Leica CTR MIC microscope observation system (Leica Microsystem, Wetzlar, Germany), and a 3CCD camera system (HV-C20AMP, Hitachi Kokusai Electric Inc., Tokyo, Japan) was used for photographing, image capture. Software for Matrox Intellicam Version 2.06 (Matrox Electronic Systems Ltd., Dorval, Quebec, Canada), late image The processing software is Adobe Photoshop CS2 9.0 (Adobe Systems, San Jose, CA). Statistical analysis of all data was performed using SPSS 13.0 software. The frequency comparison data was analyzed using the χ ² test and the Fisher exact test. The calculations of PFS and OS are based on the Kaplan-Meier method and the survival curves are depicted. The analysis of the impact of single factor on survival (comparison of survival data;) was performed by Log rank t test. Example 3. Detection results and therapeutic effects of p-Akt and YB-1

1. Immunohistochemical test results

The positive rates of p-Akt and YB-1 in DLBCL paraffin tissues were 54.8% (40/73) and 65.8% (48/73), respectively. Both p-Akt and YB-1 were positively located in the cytoplasm and stained in a brownish-yellow uniform sheet. YB-1 strong positive can be expressed as membrane, perinuclear and nucleus tan granule staining. As shown in Figure 1 to Figure 3.

2. The relationship between the expression of p-Akt and YB-1 and the prognosis of different treatment regimens

Among the 73 patients enrolled, the single factor χ2 test showed that the overall effective rate of treatment for p-Akt_ patients was 91.0%, and the overall response rate for p-Akt ⁺ patients was 60.0%. There was a statistical difference between the two.

Similarly, the positive response rate of YB-1 positive patients to chemotherapy was low, and the P value was 0.002. The overall response rate of p-p70S6K- was 82.1%, and the overall response rate of p-p70S6K+ was 64.7%. There was no significant difference between the two (P = 0.113). The overall response rate of P-4E-BP1-77.5%, ρ-4Ε- The overall response rate of ΒΡ1+ was 69.7, and there was no statistical difference between the two (P=0.593).

According to the treatment response group, univariate analysis showed that the overall effective rate of p-Akf patients in the CHOP group was 85.7%, and the overall effective rate of p-Akt ⁺ patients was 37.5%. There was a statistical difference between the two (P=0.011). o The overall effective rate of p-Akt_ patients in the R-CHOP group was 94.7%, and the overall effective rate of p-Akt ⁺ patients was 75.0%. There was no significant difference between the two (P=0.112). Similar to p-Akt, the overall efficiency of YB-1 expression in the CHOP group was low, with a P value of 0.018, and no statistical difference in the R-CHOP group.

Sixty patients with diffuse large B in this study were followed up for a median follow-up of 14 months (3-61 months;), 2-year estimated progression-free survival (PFS) and overall survival (OS) were 75 ± 7 % and 80 ± 5%. Univariate analysis of the relationship between each molecular marker and PFS and OS. P- and P-Aff were better than P-Akt ⁺ (; PFS, 89% vs 62%, P = 0.008; OS, 97% vs 65%, P = 0.034) (survival curve is shown in Figure 4) . Similarly, positive expression of YB-1 was associated with poor prognosis (PFS, P=0.011; OS, P=0.030). In contrast, there was no significant difference in p-p70S6K-PFS, OS and p-p70S6K+ (P = 0.158; 0.306); ρ-4Ε-ΒΡ1-PFS, OS and p-4E-BPl+ were not statistically different (P =0.903; 0.972).

According to the treatment plan, the PFS and OS of the p-Akf in the CHOP group were better than those of the p-Akt ⁺ PFS. 100% vs 56%, P = 0.017; OS, 100% vs 58%, P = 0.018) (survival curve is shown in Figure 5). In the R-CHOP group, there was no significant difference in PFS (P=0.156) and OS (P=0.270) between p-Akt_ patients and p-Akt ⁺ patients (the survival curve is shown in Figure 6). Also in the CHOP group, YB-1 and Bcl-2 positive expression had a poor prognosis, and there was no significant difference in the R-CHOP group. The IPI index has prognostic significance in both the CHOP group and the R-CHOP group.

All of the above results indicate that p-Akt and YB-1 can be used as molecular markers to select a preferred treatment regimen for patients with different DLBCL, and the prognosis can be evaluated. When the expression of p-Akt and / or YB-1 is positive, it suggests that the prognosis of the combination chemotherapy will be poor, but the combination of rituximab will improve the prognosis, and the combination chemotherapy and rituximab should be used for the treatment. Combination therapy for resistance. When the test results were negative for both p-Akt and YB-1 expression, it was suggested that the patient should be treated with combination chemotherapy to obtain good therapeutic effect and prognosis. Combination chemotherapy should be used. Example 4. Preparation of test kit

The kit of the present embodiment was prepared as follows (the following temperature is a suitable storage temperature)

Reagent A: Unmasking Buffer 5. lg, 2-8 °C _;

Reagent B: Blocking Buffer 5ml, 2-8 °C _;

Reagent CI: anti-p-Akt monoclonal antibody 50μ1, -20 °C _;

C2: anti-YB-1 monoclonal antibody 50μ1, -20. C

Reagent D: - anti-dilution buffer 18ml, 2-8 °C;

Reagent E: secondary antibody (HRP labeling) ready-to-use type 5ml, 2-8 °C _;

Reagent F: DAB working solution F1 - concentrated buffer 100μ1 (20Χ), 2-8°C (protected from light);

F2-DAB solution 100 μ 1 (20 X) 2-8 °C (protected from light);

F3—concentrated H ₂ O ₂ 100 μ 1 (20 X), 2-8 ° C (protected from light);

Reagent G: 3% H ₂ O ₂ - methanol solution ready-to-use type 1.5ml, 2-8 ° C (protected from light);

Reagent H: Hematoxylin ready-to-use 1.5ml, 2-8°C.

Tissue samples isolated from patients with DLBCL were tested using this kit. P-Akt was positively localized in the cytoplasm and stained in a brownish-white uniform sheet. YB-1 strong positive can be expressed as cell membrane, perinuclear and nucleus tan granule staining. Tumor cells with a positive definition of >10% showed a clear, granular brown or brownish coloration. The prognosis of the patient is evaluated based on the results obtained by the method of the present invention, and the evaluation results are consistent with clinical practice. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

Rights request

A kit for the treatment of a diffuse large B-cell lymphoma for treatment and/or prognosis evaluation, the kit comprising:

Ii) one or more reagents for detecting YB-1 expression in a biological sample.

2. The kit according to claim 1, wherein the reagents i), ii) in the kit are used for immunohistochemistry of expression of p-Akt and/or YB-1, respectively, in a biological sample. Chemical detection reagents.

3. The kit according to claim 1, wherein the biological sample is obtained from fresh tissue, formalin-fixed or paraffin-embedded tissue of a diffuse large B-cell lymphoma patient with or without clinical treatment. .

The kit according to claim 3, wherein the clinical treatment is selected from the group consisting of: combined chemotherapy, or a combination therapy with rituximab.

5. The kit according to claim 1, wherein the kit further comprises one or more selected from the group consisting of: instructions for use, a positive control, a negative control, a buffer, or an immune aid. Agent.

6. The kit according to claim 5, wherein the instructions for use indicate that when the test result is positive for p-Akt and/or YB-1 expression, the patient is prognosed for combination chemotherapy. It will be poor, but the combination of rituximab will improve the prognosis. Combination therapy with rituximab should be used for the treatment. When the test results are negative for both p-Akt and YB-1 expression, It is suggested that the patient can obtain good therapeutic effect and prognosis by using combined chemotherapy.

7. Use of a p-Akt expression and/or YB-1 expression detection reagent or kit in the kit for the preparation of a therapeutic regimen and/or prognosis for diffuse large B cell lymphoma.

8. Use of p-Akt and/or YB-1 as markers of treatment options and/or prognostic assessment for diffuse large B-cell lymphoma.

9. A method of assessing the prognostic effect of diffuse large B-cell lymphoma, the method comprising: a) detecting the expression of p-Akt and/or YB-1 in a biological sample obtained from a patient with diffuse large B-cell lymphoma;

b) analyzing the test results obtained by step a);

10. A method of selecting a treatment regime for diffuse large B-cell lymphoma, the method comprising: a) detecting the expression of p-Akt and/or YB-1 in biological samples obtained from patients with diffuse large B-cell lymphoma;

b) analyzing the test results obtained by step a);