Markers and kits for predicting TA-TMA after allogeneic hematopoietic stem cell transplantation

Abstract

The present invention provides markers and kits for predicting TA-TMA after allogeneic hematopoietic stem cell transplantation. The markers include sTNFR1 and / or Ba, and the kits include reagents for detecting the contents of sTNFR1 and / or Ba in a biological sample of a subject. Through research, the present invention finds that, compared with patients who do not develop TA-TMA after transplantation, the contents of sTNFR1 and Ba in the serum of patients who develop TA-TMA after transplantation are significantly increased before transplantation pretreatment, at -1 day, +7 days, +14 days, and +28 days. The results of ROC curve analysis confirm that sTNFR1 and Ba have diagnostic value in predicting the risk of TA-TMA after allogeneic hematopoietic stem cell transplantation. The risk of TA-TMA in patients with high contents of sTNFR1 and Ba in the serum is significantly higher than that in patients with low contents. The present invention provides a marker and a kit for conveniently and rapidly predicting the risk of TA-TMA after allogeneic hematopoietic stem cell transplantation, which can assist medical staff in timely detecting potential TA-TMA patients, so as to timely formulate treatment plans, achieve early diagnosis and early treatment, and reduce the severe disease rate and mortality rate.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and particularly relates to markers and kits for predicting TA-TMA after allogeneic hematopoietic stem cell transplantation. Background Art

[0002] Hematopoietic stem cell transplantation (HSCT) is one of the important means to cure hematological diseases, and transplantation-associated thrombotic microangiopathy (TA-TMA) is a serious complication of HSCT. The main clinical manifestations of TA-TMA include microangiopathic hemolytic anemia, thrombocytopenia, microthrombus formation, and multi-organ dysfunction. According to the diagnosis time, it is divided into early-onset TA-TMA and late-onset TA-TMA. Internationally, the incidence of TA-TMA after allogeneic hematopoietic stem cell transplantation is about 0.5% - 64%, and the incidence of TA-TMA after autologous hematopoietic stem cell transplantation is <1%. According to the statistics of Laskin et al., in recent years, large-sample retrospective studies have found that the incidence of TA-TMA is 10% - 25%, and this result may be closer to the true incidence of TA-TMA. The mortality rate of TA-TMA is as high as 50% - 90%, and the delay in early diagnosis and early treatment leads to an increase in the incidence of severe TA-TMA.

[0003] At present, although certain in-depth and systematic studies have been conducted on the pathogenesis of TA-TMA, there is still a lack of an early prediction system for the occurrence of TA-TMA, and it is not possible to effectively evaluate the risk of TA-TMA occurring after transplantation in patients before transplantation, and timely screen out high-risk patients with TA-TMA, resulting in missed best intervention opportunities and increased severe illness and death risks. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide markers and kits for predicting TA-TMA after allogeneic hematopoietic stem cell transplantation, which can effectively evaluate the risk of TA-TMA occurring after transplantation in patients before transplantation and achieve early diagnosis and early treatment.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions.

[0006] In the first aspect of the present invention, there is provided the use of sTNFR1 and / or Ba in the preparation of a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

[0007] In the second aspect of the present invention, there is provided the use of sTNFR1 and / or Ba in screening a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

[0008] A third aspect of the present invention provides the use of a reagent for detecting the content of sTNFR1 and / or Ba in the preparation of a kit for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

[0009] In some embodiments, the test sample of the reagent is a serum sample.

[0010] In some embodiments, the reagent for detecting the content of sTNFR1 and / or Ba is a specific antibody against sTNFR1 and / or Ba.

[0011] In some embodiments, the specific antibody is a monoclonal antibody.

[0012] A fourth aspect of the present invention provides a method for in vitro screening of a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia, comprising the following steps: detecting the content of sTNFR1 and / or Ba in a biological sample using a candidate reagent, and if the detection can be achieved, the candidate reagent is a candidate reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

[0013] A fifth aspect of the present invention provides a kit for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia, the kit comprising a reagent for detecting the content of sTNFR1 and / or Ba.

[0014] In some embodiments, the reagent for detecting the content of sTNFR1 and / or Ba is a specific antibody against sTNFR1 and / or Ba.

[0015] In some preferred embodiments, the specific antibody is a monoclonal antibody.

[0016] A sixth aspect of the present invention provides a method for non-diagnostic purposes, characterized in that the biomarker comprises sTNFR1 and / or Ba; the method comprises the following steps: collecting a biological sample of a subject to be tested, detecting the content of the biomarker in the biological sample, and predicting the risk of TA-TMA occurring after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

[0017] Through research, the present invention has obtained biomarkers sTNFR1 and / or Ba that can effectively predict the risk of TA-TMA after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia. Compared with patients who did not develop TA-TMA after transplantation, the levels of sTNFR1 and Ba in the serum of patients who developed TA-TMA after transplantation were significantly increased at pre-transplant conditioning, -1 day, +7 days, +14 days, and +28 days. The results of receiver operating characteristic curve (ROC curve) analysis showed that sTNFR1 and Ba had the value of predicting the occurrence of TA-TMA after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia before transplantation. At the same time, the results of KM curve analysis indicated that the risk of developing TA-TMA after transplantation in patients with high levels of sTNFR1 and Ba in the serum was significantly higher than that in patients with low levels. The present invention provides a marker and kit for conveniently and rapidly predicting the risk of TA-TMA after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia, which can assist medical staff in timely detecting potential TA-TMA patients before transplantation, so as to timely formulate treatment plans, achieve early diagnosis and early treatment, and reduce the severe disease rate and mortality rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Detection results of the levels of the marker in the serum of patients at different detection time points.

[0019] Figure 2 Detection results of the levels of the marker in the serum at different detection time points after excluding patients with grade III-IVa GVHD.

[0020] Figure 3 ROC curve results for sTNFR1 predicting the risk of TA-TMA in patients.

[0021] Figure 4 ROC curve results for Ba predicting the risk of TA-TMA in patients.

[0022] Figure 5 KM curve analysis results for sTNFR1 before pre-transplant conditioning.

[0023] Figure 6 KM curve analysis results for sTNFR1 at +14 days.

[0024] Figure 7 KM curve analysis results for Ba at +14 days. DETAILED DESCRIPTION OF THE INVENTION

[0025] In the following examples of the present invention, the experimental methods without specific conditions noted are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. All kinds of common chemical reagents used in the examples are commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0027] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that includes a series of steps is not limited to the listed steps or modules, but may optionally further include steps not listed, or may optionally further include other steps inherent to these processes, methods, products or devices.

[0028] The "and / or" mentioned in the present invention describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0029] The following is described in conjunction with detailed embodiments.

[0030] Example 1

[0031] I. Research method

[0032] A prospective-nested case-control study was adopted. From August 2022 to January 2024, 96 patients with severe aplastic anemia undergoing transplantation were prospectively enrolled, and the contents of candidate markers in the sera of the patients at different time points (before pretreatment: the situation before transplantation; -1 day before transplantation: pretreatment + CNI injury; +7 days after transplantation: pretreatment + CNI + immune injury; +14 days after transplantation: pretreatment + CNI + immune injury; +28 days after transplantation: CNI + immune injury) were detected. The candidate markers are markers that the inventors obtained through research and may be used to predict the risk of TA-TMA occurrence, including Ba, interferon-γ, CXCL-9, ds-DNA, REG3a, sC5b-9, sST2, sTNFR1, IL-6, and IL-8.

[0033] During the research process, the enrolled patients were evaluated for whether they developed TA-TMA according to the TA-TMA diagnostic criteria. The patients who developed TA-TMA were grouped together as the case group, and a group was selected as the control group by matching the case group according to (gender, donor type, whether drug-resistant aGVHD occurred). Control group: The number of patients in the case group was in a ratio of 2:1.

[0034] The diagnostic criteria for TA-TMA are as follows: ① Lactate dehydrogenase (LDH) exceeds the upper limit of the normal value; ② Proteinuria (random urine protein exceeds the upper limit of the normal value or random urine protein / creatinine ≥ 2 mg / mg); ③ Hypertension (for those under 18 years old: blood pressure is higher than the upper limit of the normal reference value of healthy people of the same age, gender, and height; for those 18 years old and above: blood pressure ≥ 140 / 90 mmHg); ④ New-onset thrombocytopenia (platelet count < 50×10 9 / L or platelet count decreases by ≥ 50% compared to the baseline level); ⑤ New-onset anemia (hemoglobin value is lower than the lower limit of the normal reference value or the need for blood transfusion increases); ⑥ Evidence of microvascular lesions (fragmented red blood cells are present in peripheral blood or the pathological examination results of tissue specimens suggest microangiopathy); ⑦ Terminal complement activation (plasma sC5b-9 value is higher than the upper limit of the normal value of healthy people).

[0035] According to the above criteria, 17 patients developed TA-TMA and 79 patients did not develop TA-TMA (non-TA-TMA) among the enrolled patients, with an incidence rate of 17.7% (17 / 96). According to the propensity score at a ratio of 1:2, 34 patients who did not develop TA-TMA were selected as controls. The comparison results of patient characteristics when selecting and not selecting control patients are shown in Table 1:

[0036] Table 1

[0037]

[0038]

[0039] As can be seen from Table 1, the selection of enrolled control patients was reasonable.

[0040] The following methods were used to detect the contents of Ba, interferon-γ, CXCL-9, ds-DNA, REG3a, sC5b-9, sST2, sTNFR1, IL-6, and IL-8 in the serum of patients respectively:

[0041] (1) sC5b-9 and Ba: sC5b-9 (MicroVue sC5b-9PlusEIA, A020, range 110 - 252 ng / ml) was measured using a commercial kit (Quidel, San Diego, California, USA) according to the manufacturer's instructions for EDTA plasma samples; Ba (MicroVue Ba EIA, A033 / A034 XUS) was measured using a commercial kit (Quidel, San Diego, California, USA) according to the manufacturer's instructions for EDTA plasma samples.

[0042] (2) Detection of ds-DNA concentration by colorimetry (Invitrogen Qubit 4 fluorometer): ds-DNA dye (Quant-iT PicoGreen dsDNA Assay Kit, P7589), purchased as a commercial kit [Life Technologies, Molecular Probes Division (Eugene, OR, PN-P7589)], and detected according to the manufacturer's instructions.

[0043] (3) Detection of interferon-γ, CXCL-9, REG3a, sTNFR1, sST2, IL-6, and IL-8 concentrations using the Luminex Assays-200 platform: Customized reagents (Bio-Techne, R&D Systems) were used and detected according to the kit instructions.

[0044] II. Research Results

[0045] The detection results of the contents of Ba, CXCL-9, ds-DNA, REG3a, sC5b-9, sST2, and sTNFR1 in the sera of patients at different detection time points are as Figure 1 shown. The values of IL-6, IL-8, and interferon-γ in most patients were below the minimum threshold of the kit and were excluded from the analysis. As Figure 1 can be seen, compared with the non-TA-TMA group, the concentrations of sTNFR1, Ba, and REG3a in the sera of patients in the TA-TMA group were significantly increased at each detection time point.

[0046] Current studies have shown that sST2, REG3a, and sTNFR1 are related to grade III-IVa GVHD. Nine patients in our study developed grade III-IVa GVHD (7 with TA-TMA and 2 with non-TA-TMA). Therefore, after further excluding these 9 patients, the relationship between the detection indicators and TA-TMA was explored. The results are as Figure 2 shown. After excluding the interference of grade III-IVa GVHD, sTNFR1, Ba, and REG3a also showed the role of predicting the occurrence of TA-TMA.

[0047] Furthermore, the predictive value of the preliminary screening indicators was analyzed by ROC curve. The results showed that sTNFR1 could well predict the risk of TA-TMA in patients from before pretreatment to +14 days after transplantation ( Figure 3 , Table 2); Ba could predict the risk of TA-TMA in patients from before pretreatment to +14 days after transplantation ( Figure 4, Table 3). It shows that sTNFR1 and Ba have the value in predicting the occurrence of TA-TMA after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia before transplantation, and the risk of TA-TMA after transplantation can be predicted before the patients undergo transplantation. Since REG3a has low specificity, its diagnostic value is not high.

[0048] Table 2

[0049]

[0050] Table 3

[0051]

[0052]

[0053] The above results indicate that both sTNFR1 and Ba are significantly elevated from before pretreatment to before the occurrence of TA-TMA, with good sensitivity and specificity. They may be good early predictors of TA-TMA after transplantation for severe aplastic anemia and even potential early treatment target sites.

[0054] Next, the ability of Ba and sTNFR1 to predict TA-TMA before pretreatment and 14 days after transplantation was analyzed in detail.

[0055] 1. Data analysis before pretreatment

[0056] Since there was a significant difference in age between the TA-TMA group and the non-TA-TMA group in the baseline information, age was included in the multivariate COX analysis of Ba and sTNFR1 before pretreatment.

[0057] (1) Multivariate COX analysis of sTNFR1

[0058] As shown in Table 4, after adjusting for covariates (patient age), sTNFR1 was an independent risk factor for patients to develop TA-TMA (p = 0.0481). For every 1 ng / ml increase in the sTNFR1 level of patients before pretreatment, the risk of TA-TMA occurrence increased by 34%.

[0059] Table 4

[0060]

[0061] Before pretreatment, sTNFR1 was divided into two groups, high (≥1.93) and low (<1.93), according to the optimal cut-off value of 1.93 ng / ml. The results of the KM curve analysis showed that the hazard ratio (HR) of TA-TMA in patients with a pre-treatment sTNFR1 concentration ≥1.93 ng / ml was 6.14 times that in patients with a sTNFR1 concentration <1.93 ng / ml (95% CI, 2.21 - 17.1, p<0.0001), as specifically shown in Figure 5 as follows.

[0062] 2. Data analysis on day +14 after transplantation

[0063] Since the correlation coefficient between Ba and sTNFR1 was 0.931, indicating collinearity, multivariate COX analysis was performed on Ba and sTNFR1 separately.

[0064] (1) Multivariate COX analysis of sTNFR1

[0065] After adjusting for the covariate (patient age), sTNFR1 was an independent risk factor for TA-TMA in patients (p<0.001); on day +14 after transplantation, for every 1 ng / ml increase in the patient's sTNFR1 level, the risk of TA-TMA increased by 30%, as specifically shown in Table 5:

[0066] Table 5

[0067]

[0068] On day +14 after transplantation, sTNFR1 was divided into two groups, high (≥3.74) and low (<3.74), according to the optimal cut-off value of 3.74 ng / ml. The results of the KM curve analysis ( Figure 6 ) showed that the hazard ratio (HR) of TA-TMA in patients with a sTNFR1 concentration ≥3.74 ng / ml on day +14 after transplantation was 7.77 times that in patients with a sTNFR1 concentration <3.74 ng / ml (95% CI, 2.88 - 21, p<0.0001).

[0069] (2) Multivariate COX analysis of Ba

[0070] After adjusting for the covariate (patient age), Ba was an independent risk factor for TA-TMA in patients (p<0.001), and on day +14 after transplantation, for every 100 ng / ml increase in the patient's Ba level, the risk of TA-TMA increased by 12%, as specifically shown in Table 6:

[0071] Table 6

[0072]

[0073] At +14 days after transplantation, Ba was divided into two groups, high (≥568) and low (<568), according to the optimal cut-off value of 568 ng / ml. The results of the KM curve analysis ( Figure 7 ) showed that the hazard ratio (HR) of TA-TMA in patients with a Ba concentration ≥568 ng / ml at +14 days after transplantation was 7.28 times that in patients with a Ba concentration <568 ng / ml (95% CI, 2.08 - 25.5, p = 0.00031).

[0074] The above results indicate that sTNFR1 and Ba can be used as effective biomarkers for predicting the risk of TA-TMA in patients with severe aplastic anemia after allogeneic hematopoietic stem cell transplantation.

[0075] Example 2

[0076] In this example, sTNFR1 and Ba were used as biomarkers to predict the risk of TA-TMA in patients with severe aplastic anemia after allogeneic hematopoietic stem cell transplantation.

[0077] Serum samples of 20 patients with severe aplastic anemia who needed allogeneic hematopoietic stem cell transplantation were collected at different time points (the same as in Example 1). The contents of sTNFR1 and Ba were detected to predict the risk of TA-TMA in patients with severe aplastic anemia after allogeneic hematopoietic stem cell transplantation, and the results were compared with the clinical diagnosis. After clinical diagnosis, among the 20 patients who underwent allogeneic hematopoietic stem cell transplantation for severe aplastic anemia, 3 developed TA-TMA and 17 did not. The prediction results using sTNFR1 and Ba as markers were consistent with the clinical diagnosis, accurately predicting that 3 patients would develop TA-TMA within 100 days.

[0078] In summary, sTNFR1 and / or Ba can be used as effective biomarkers for predicting the risk of TA-TMA in patients with severe aplastic anemia after allogeneic hematopoietic stem cell transplantation, conveniently and rapidly predicting the occurrence risk of TA-TMA in patients with severe aplastic anemia after allogeneic hematopoietic stem cell transplantation, assisting medical staff to timely detect potential TA-TMA patients, so as to timely formulate treatment plans, achieve early diagnosis and early treatment, and reduce the severity and mortality.

[0079] The technical features of the above embodiments can be combined arbitrarily. For the sake of brief description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0080] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims.

Claims

1. Use of sTNFR1 in the preparation of a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

2. Use of sTNFR1 in the screening of a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

3. Use of a reagent for detecting the content of sTNFR1 in the preparation of a kit for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

4. The application according to claim 3, characterized in that The test sample of the reagent is a serum sample.

5. The application according to claim 3, wherein The reagent for detecting the content of sTNFR1 is a specific antibody against sTNFR1.

6. The application according to claim 5, wherein The specific antibody is a monoclonal antibody.

7. A method for in vitro screening of a reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia, characterized in that, It includes the following steps: Detect the content of sTNFR1 in a biological sample using a candidate reagent. If the detection can be achieved, the candidate reagent is a candidate reagent for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

8. A method for predicting whether TA-TMA occurs after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia for non-diagnostic purposes, characterized in that, The biomarker includes sTNFR1; the method includes the following steps: collect a biological sample of a subject to be tested, detect the content of the biomarker in the biological sample, and predict the risk of TA-TMA occurring after allogeneic hematopoietic stem cell transplantation in patients with severe aplastic anemia.

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