Application of SP70 detection reagent in preparation of acute myelogenous leukemia patient preliminary diagnosis risk grading reagent

Abstract

The invention discloses application of a reagent for detecting SP70 in preparation of a reagent for grading the primary diagnosis risk degree of an acute myelogenous leukemia patient. The invention reports the application of serum SP70 in an AML risk grading reagent. The specific molecular marker SP70 is selected as a detection target, and compared with bone marrow cell morphology detection, detection of SP70 in peripheral blood of a patient can be noninvasive, short in detection time, simple, convenient and feasible, and can be used for dynamic monitoring. In a word, the serum SP70 is a potential biomarker for prognosis layering of AML, and a new thought and a new method are provided for clinical diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to the field of medical diagnosis and relates to the application of a reagent for detecting SP70 in preparing a reagent for grading the risk of initial diagnosis of acute myeloid leukemia patients. Background Art

[0002] Acute myeloid leukemia (AML) is a blood system malignancy characterized by malignant clonal expansion of hematopoietic stem cells accompanied by differentiation disorders and apoptosis blockage. [1] Epidemiological surveys show that AML is one of the most common hematological malignancies in adults, accounting for about 32% of all adult leukemias and is the blood disease with the highest mortality rate. [2] The 5-year survival rate is low. The 5-year survival rate of patients under 60 years old is about 40%, while the 5-year survival rate of patients over 60 years old is only about 10%. [3] .

[0003] In recent years, the incidence of acute myeloid leukemia has generally shown a slow upward trend, with short survival and high mortality as its main characteristics. [2] Chromosomal abnormalities include RUNX1-RUNX1T1 translocation and CBFB-MYH11 translocation, and gene mutations such as FLT3-ITD, NPM1, KIT, and CEBPA have been recognized as molecular markers for the diagnosis and prognostic stratification of AML patients. [4] "Guidelines for the diagnosis and treatment of adult acute myeloid leukemia (non-acute promyelocytic leukemia) in China (2023 edition)" [5] In the first part, the admission examination and diagnosis of newly diagnosed patients stipulates the genetic prognostic grouping of AML based on the changes in AML cytogenetics and molecular genetics at the time of initial diagnosis. The specific groupings are shown in Figure 6 . By stratifying the risk of AML patients, clinicians can develop more targeted treatment plans. However, this genetic prognostic grouping is based on bone marrow chromosome karyotype and molecular sequencing technology, which is relatively invasive and traumatic to patients, complex and expensive, especially in some remote areas where access to these technologies is poor. Therefore, there is a greater need for some simple, specific and sensitive new prognostic stratification tools in clinical practice. The present invention aims to provide a new tumor marker SP70 for the prognostic stratification of AML and to evaluate its efficacy.

[0004] The inventor team successfully prepared the monoclonal antibody NJ001 using the active antigen of human lung adenocarcinoma cell line SPC-A1 as an immunogen. [6] The target antigen was named tumor specific protein 70 (SP70). Previous studies have shown that SP70 plays an important role in lung cancer. [7] , breast cancer [8] , Liver cancer[9] SP70 is highly expressed in the serum and tissues of patients with various malignancies, and can be used as a new biomarker for early diagnosis of tumors. However, the expression and clinical value of SP70 in hematological malignancies have not been studied, and whether SP70 can be used as a marker for the initial risk stratification of acute myeloid leukemia patients has not been reported and is unpredictable. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a reagent for detecting SP70 for use in preparing a reagent for grading the risk of initial diagnosis of acute myeloid leukemia patients.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] Application of reagents for detecting SP70 in the preparation of reagents for grading the prognosis risk of newly diagnosed patients with acute myeloid leukemia.

[0008] As a preferred embodiment of the present invention, the reagent for detecting SP70 is the monoclonal antibody NJ001-1 of SP70, and the monoclonal antibody NJ001-1 is secreted by the hybridoma cell line NM001-1 with the deposit number of CCTCC NO: C201172. NM001-1 is disclosed in CN102391992A.

[0009] The risk grading of newly diagnosed acute myeloid leukemia patients is to divide acute myeloid leukemia patients into high-risk group and low-medium risk group at the time of initial diagnosis according to the level of SP70 expression. The high-risk group corresponds to the poor prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Guidelines for the Diagnosis and Treatment of Leukemia in China (2023 edition); the low-medium risk group corresponds to the good prognosis and moderate prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Guidelines for the Diagnosis and Treatment of Leukemia in China (2023 edition).

[0010] Beneficial effects:

[0011] The preliminary results of the present invention showed that serum SP70 expression in the peripheral blood of AML patients was significantly higher than that of controls (P<0.001). In addition, there was a significant difference in serum SP70 levels between the low-risk and medium-risk groups and the high-risk groups of AML prognosis (P<0.001), indicating that serum SP70 is closely related to the occurrence and development of AML and may have a certain value in the prognostic stratification of AML. The results of further experiments with a larger sample size showed that the area under the curve (AUC) of serum SP70 for identifying patients with high-risk prognosis was 0.908 (95% CI: 0.845-0.952, sensitivity 90.5%, specificity 82.8%); the area under the curve (AUC) of HB for identifying patients with high-risk prognosis was 0.615 (95% CI: 0.526-0.699, sensitivity 92.9%, specificity 31.0%); the area under the curve (AUC) of BM blasts for identifying patients with high-risk prognosis was 0.744 (95% CI: 0.660-0.817, sensitivity 71.4%, specificity 66.7%). SP70 had the largest area under the curve, indicating the best performance. The cut-off value of serum SP70 for the best discrimination performance was 5.74 ng / mL. SP70 alone can be used for the prognostic risk grading of newly diagnosed patients with acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Comparison of serum SP70 levels between AML patients and healthy controls

[0013] Figure 2 Comparison of serum SP70 levels among healthy control group, AML patients, AML low- and medium-risk groups, and AML high-risk groups

[0014] Figure 3 Comparison of serum SP70 levels between AML patients and control group Note: Figure A is a comparison of serum SP70 levels between the control group and AML patients, and Figure B is a comparison of serum SP70 levels between the control group and AML low-, medium- and high-risk groups (the horizontal line represents the median)

[0015] Figure 4 ROC curve for predicting AML prognostic stratification

[0016] Figure 5 PFS curve of AML patients (A: SP70; B: MRD; C: bone marrow routine)

[0017] Figure 6 Screenshot of Table 1 of the Chinese Diagnosis and Treatment Guidelines for Adult Acute Myeloid Leukemia (Non-Acute Promyelocytic Leukemia) (2023 Edition) DETAILED DESCRIPTION

[0018] 1. Research subjects

[0019] This study adopts a cohort study design and includes AML patients who were first diagnosed in the Department of Hematology, the First Affiliated Hospital of Nanjing Medical University from September 2022 to January 2024. Inclusion criteria: (1) The diagnostic criteria for AML refer to the World Health Organization (WHO) (2016) Classification of Hematopoietic and Lymphoid Tumors

[10] ; (2) aged ≥18 years; (3) had not received chemotherapy before diagnosis. Exclusion criteria: (1) patients who had received chemotherapy (except hydroxyurea); (2) patients with a history of other malignant tumors; (3) patients with severe heart, lung, liver or kidney diseases; (4) patients with acute promyelocytic leukemia [11-12] ; (5) Patients with incomplete medical records and missing bone marrow routine, cytogenetics, molecular genetics and other related examinations. The control group was composed of healthy subjects and patients with benign blood diseases in the same hospital during the same period, and matched with the case group in terms of gender and age. According to the Chinese Guidelines for the Diagnosis and Treatment of Leukemia (2023 Edition) [5] According to the changes in cytogenetics and molecular genetics of AML patients at the time of initial diagnosis, their prognosis will be divided into three levels: good prognosis, moderate prognosis and poor prognosis. Those with good prognosis and moderate prognosis are classified as low-medium risk group, and those with poor prognosis are classified as high-risk group. The samples in Example 1 and Example 2 are from different individuals. This study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (approval number: 2023-SR-805), and the subjects were exempted from informed consent.

[0020] 2. The research methods are as follows:

[0021] 1. Data collection:

[0022] The electronic medical record system was consulted to collect patients’ general information (sex, age, admission time, diagnosis), clinical characteristics (first typical symptoms such as fever, bleeding, anemia, bone and joint pain, and concomitant diseases), bone marrow routine reports, bone marrow pathology reports, cytogenetics, molecular genetics and other related examination reports.

[0023] 2. Serum SP70 test:

[0024] All blood samples were centrifuged at room temperature for 10 min at 1600 × g within 2 h after collection to separate serum. The serum SP70 concentration was determined using the “Tumor Specific Protein 70 Detection Kit (Enzyme-Linked Immunoassay)” (Jiangsu Kede Biopharmaceutical Technology Co., Ltd., batch number: MY01GIYT010). [6] .

[0025] 3. Laboratory routine blood index testing:

[0026] The routine blood test of newly diagnosed AML patients before chemotherapy was tested using the Japanese Sysmex XN series fully automatic blood analyzer. The routine bone marrow test was performed by two experienced inspectors in the hematology laboratory for smear, staining, microscopic examination and review.

[0027] 4. Genetic indicator detection:

[0028] Cytogenetics uses R banding for chromosome karyotype analysis according to the International System of Nomenclature for Human Cytogenetics (ISCN2020).

[13] Describe karyotype abnormalities. Molecular genetics used targeted sequencing of 20 genes, including ASXL1 (exon 12), CBL (exon 8, 9), FLT3 (exon 14, 15, 20), IDH1 (exon 4), IDH2 (exon 4), KIT (exon 2, 8-11, 13, 17), NPM1 (exon 12), NRAS (exon 2-4), SETBP1 (exon 4), SF3B1 (exon 12-15), SRSF2 (exon 1), U2AF1 (exon 2, 6), and CEBPA, DNMT3A, ETV6, EZH2, PHF6, TET2, TP53, and ZRSR2 coding regions.

[14] At present, the genetic prognostic grouping of AML is mainly based on the cytogenetics and molecular genetic changes of AML at the time of initial diagnosis.

[16] .

[0029] 5. Grouping criteria

[0030] The risk grading of newly diagnosed acute myeloid leukemia patients is to divide acute myeloid leukemia patients into low-risk and medium-risk groups and high-risk groups at the time of initial diagnosis according to the level of SP70 expression. The low-risk and medium-risk groups correspond to the good prognosis and moderate prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Chinese Leukemia Diagnosis and Treatment Guidelines (2023 edition); the high-risk group corresponds to the poor prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Chinese Leukemia Diagnosis and Treatment Guidelines (2023 edition).

[0031] 6. Statistical methods

[0032] SPSS24.0 statistical software and R 4.0.2 software were used for statistical analysis. Continuous variable data were analyzed by Kolmogorov-Smirnov test. If the data followed a skewed distribution, the median (quartile) [M(Q1,Q3)] was used to represent them. Categorical variables were compared using the chi-square test or Fisher's exact test, and continuous variables between two different groups were compared using the Mann-Whitney U test. Univariate and multivariate logistic regression were used to analyze the risk factors for the risk level of patients with newly diagnosed acute myeloid leukemia, and ROC curves were drawn.

[0033] Example 1

[0034] 1. Clinical characteristics and serum SP70 levels in AML patients

[0035] There were 80 AML patients, including 27 in the high-risk group and 53 in the low- to medium-risk group. The age of the high-risk group [61 (49, 69) years] was significantly higher than that of the low- to medium-risk group [51 (35, 64) years, Z = -2.291, P = 0.022], and the proportion of males (20 cases, 74.1%) was significantly higher than that of the low- to medium-risk group (25 cases, 47.2%, χ 2 =5.261, P=0.022). There was no significant difference in the incidence of typical clinical symptoms including fever, bleeding, anemia and bone and joint pain between the two groups (all P>0.05). There was no significant difference in gender composition and age distribution between the 80 AML patients and the 80 controls (all P>0.05). The serum SP70 level of AML patients was 5.69 (3.99, 7.17) ng / ml, which was significantly higher than that of the control group [0.60 (0.60, 1.07) ng / ml, P<0.001, Figure 1 In addition, the serum SP70 level of patients in the high-risk group was 7.75 (6.30, 9.59) ng / ml, which was significantly higher than that of patients in the low- and medium-risk group [4.45 (3.33, 5.74) ng / ml], and the difference was statistically significant (P<0.001, Figure 2 ).

[0036] Example 2

[0037] 1. Clinical characteristics and laboratory indicators of AML patients

[0038] There were 129 AML patients, including 42 in the high-risk group and 87 in the low- to medium-risk group. The age of the high-risk group [62 (48, 67) years] was significantly higher than that of the low- to medium-risk group [50 (35, 63) years, Z = -2.381, P = 0.017], and the proportion of males (30 cases, 71.4%) was significantly higher than that of the low- to medium-risk group (41 cases, 47.1%, χ 2=6.760, P=0.009). There was no significant difference in the incidence of typical clinical symptoms including fever, bleeding, anemia and bone and joint pain between the two groups (all P>0.05). The neutrophil count (N), peripheral blood blasts (PM blasts) and bone marrow blasts (BM blasts) of the high-risk AML group were significantly higher than those of the low- and medium-risk AML groups (all P<0.05), while the hemoglobin concentration (HB) and platelet count (PLT) were significantly lower than those of the low- and medium-risk AML groups (all P<0.05).

[0039] 2. Serum SP70 levels in AML patients

[0040] There was no significant difference in gender and age distribution between the 129 AML patients and the 127 controls (all P>0.05). The serum SP70 level in AML patients was 5.29 (3.86, 6.89) ng / ml, which was significantly higher than that in the control group [0.60 (0.60, 1.05) ng / ml, P<0.001, Figure 3 In addition, the serum SP70 level of patients in the high-risk group was 7.19 (6.17, 9.10) ng / ml, which was significantly higher than that of patients in the low- and medium-risk group [4.37 (3.44, 5.39) ng / ml], and the difference was statistically significant (P<0.001, Figure 3 B).

[0041] 3. Logistic regression analysis of prognostic stratification of AML patients

[0042] The results of univariate logistic regression analysis showed that gender, age, serum SP70, N, HB, PLT, PMblasts and BM blasts were significantly associated with the prognostic stratification of patients (all P<0.05). Multivariate logistic regression analysis showed that SP70 (OR=2.54, 95%CI: 1.68-3.84, P<0.001), HB (OR=0.96, 95%CI: 0.93-0.99, P<0.05) and BM blasts (OR=1.07, 95%CI: 1.02-1.13, P<0.05) were independent risk factors for high-risk prognosis (Table 1).

[0043] Table 1 Univariate and multivariate logistic regression analysis of high-risk AML patients

[0044]

[0045]

[0046] 4. ROC analysis of prognostic stratification of AML patients

[0047] ROC curve analysis showed that the area under the curve (AUC) of serum SP70 for identifying patients with high-risk prognosis was 0.908 (95% CI: 0.845-0.952, sensitivity 90.5%, specificity 82.8%); HB was 0.615 (95% CI: 0.526-0.699, sensitivity 92.9%, specificity 31.0%); BM blasts was 0.744 (95% CI: 0.660-0.817, sensitivity 71.4%, specificity 66.7%). The cut-off value of serum SP70 for optimal identification efficiency was 5.74 ng / mL. The area under the curve of SP70 was the largest, indicating optimal efficacy. The cut-off value of serum SP70 for optimal identification efficiency was 5.74 ng / mL. SP70 alone can be used for prognostic risk grading in newly diagnosed patients with acute myeloid leukemia ( Figure 4 ).

[0048] Example 3 Serum SP70 detection for prognosis assessment of AML patients after chemotherapy

[0049] 1. Follow-up

[0050] Follow-up was conducted by hospital reexamination or telephone contact after 1-2 cycles of chemotherapy. The follow-up content included serum SP70 test, bone marrow routine report, measurable residual disease (MRD), etc. The follow-up deadline was June 30, 2024, and the median follow-up time was [11 (4, 16) months]. The follow-up endpoint was the progression, recurrence or death of the patient after treatment. The interval from treatment to disease progression or death of AML patients was progression-free survival (PFS).

[0051] 2. Statistical methods

[0052] SPSS24.0 statistical software and GraphPad Prism 9.5.1 software were used for statistical analysis and drawing. The levels of SP70 and MRD in AML patients after chemotherapy were tested by Kolmogorov-Smirnov test and showed non-normal distribution, so the median (quartile) [M(Q1,Q3)] was used to represent it. The Kaplan-Meier method was used for survival analysis and the survival curve was drawn. The Log-rank test was used to compare the survival time of each group. P<0.05 was considered statistically significant.

[0053] 3. Results

[0054] The median SP70 value of the 129 patients at the time of initial diagnosis was 5.29 ng / mL, and the patients were divided into high SP70 subgroup (SP70>5.29 ng / mL) and low SP70 subgroup (SP70≤5.29 ng / mL) based on this median value; 110 cases of MRD were tracked after 1-2 chemotherapy cycles, and 19 cases were censored. Cases without MRD detection were classified as MRD-negative group, and cases with MRD detection were classified as MRD-positive group. PFS and prognosis of each group were compared.

[0055] The survival curve showed that the PFS of the low SP70 subgroup [12.5 (7.3, 16) months] was significantly longer than that of the high SP70 subgroup [9 (2, 14.5) months], and the difference was statistically significant (χ 2 =7.079, P=0.008, Figure 5 A); PFS in the MRD-negative group [14 (10, 17) months] was longer than that in the MRD-positive group [6 (3, 10) months], and the difference was statistically significant (χ 2 =49.489, P=0.000, Figure 5 B); PFS in the bone marrow routine remission group [13 (10, 16) months] was longer than that in the bone marrow routine non-remission group [5 (2, 8.5) months], and the difference was statistically significant (χ 2 =61.108,P=0.000, Figure 5 C).

[0056] It can be seen that the progression-free survival of the high SP70 group was significantly shorter than that of the low SP70 group, which verified the accuracy of SP70 in the prognosis classification of newly diagnosed AML. Serum SP70 can be used as a new biomarker to evaluate the prognosis and efficacy of AML, and its efficacy is comparable to bone marrow MRD and bone marrow routine examination.

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Claims

1. Application of reagents for detecting SP70 in the preparation of reagents for stratifying the prognosis risk of newly diagnosed patients with acute myeloid leukemia.

2. The use according to claim 1, characterized in that: The reagent for detecting SP70 is the monoclonal antibody NJ001-1 of SP70, and the monoclonal antibody NJ001-1 has the accession number of CCTCC NO: Secretion of hybridoma cell line NM001-1 of C201172.

3. The use according to claim 1, characterized in that The risk grading of newly diagnosed acute myeloid leukemia patients is to divide acute myeloid leukemia patients into high-risk group and low-medium risk group at the time of initial diagnosis according to the level of SP70 expression. The high-risk group corresponds to the poor prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Guidelines for the Diagnosis and Treatment of Leukemia in China (2023 edition); the low-medium risk group corresponds to the good prognosis and moderate prognosis in the risk grading of cytogenetic / molecular genetic indicators in the Guidelines for the Diagnosis and Treatment of Leukemia in China (2023 edition).

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