Biomarker for evaluating drug resistance of LGLL-combined PRCA patient to cyclosporin A and application of biomarker

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker for evaluating the drug resistance of LGLL combined PRCA patients to cyclosporin A and application of the biomarker. Wherein the TCR comprises a TCR beta chain variable region; the TCR [beta] chain variable region comprises CDR3; and the CDR3 comprises a TRBV06 gene. The sensitivity of the LGLL and PRCA combined patient to CsA treatment is evaluated in advance through detection of the biomarker, selection of treatment schemes of the LGLL and PRCA combined patient is facilitated, and the treatment effect of the LGLL and PRCA combined patient and the prognosis of the LGLL and PRCA combined patient are improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a biomarker for evaluating cyclosporine A resistance in patients with LGLL combined with PRCA and an application thereof. Background Art

[0002] T cells are the main component of the acquired immune system. T cells express antigen receptors on the cell surface, namely T cell receptors (TCRs), which recognize polypeptides bound to major histocompatibility complex (MHC) molecules presented by antigen-presenting cells. Clonal TCRs are heterodimers composed of α chains and β chains. The α chain and β chain are composed of constant region (C region), variable region (V region), transmembrane region and cytoplasmic region; the V region, also known as complementarity-determining region (CDR), is the key site for specific recognition of antigen peptide / histocompatibility antigen complex. CDR3 represents the most diverse and complex part of the variable region. Therefore, the diversity of TCR can be measured by the diversity of CDR3 sequences, which can provide potential biomarkers for the diagnosis and treatment of diseases.

[0003] Large granular lymphocytic leukemia (LGLL) is a chronic lymphocytic proliferative disease. T-LGLL accounts for 85% of LGLL, and its incidence is 0.2-0.72 / 1 million. The median age of onset is 66 years old, and the median survival period is about 10 years. Its clinical course is inert and progresses slowly. About 1 / 3 of patients are asymptomatic at the time of disease diagnosis, and most of them seek medical treatment due to abnormal blood test results for other reasons. The blood picture shows a continuous increase in LGL cells, which may be accompanied by anemia, neutropenia and thrombocytopenia. It is often accompanied by autoimmune phenomena. In Western countries, LGLL patients often show rheumatoid arthritis and neutropenia-related infections. In Asian populations, pure red cell aplasia (PRCA) is more common (47% vs. 4%, P<0.001). PRCA is a syndrome characterized by normocytic normochromic anemia, reticulocytopenia, and a significant decrease or absence of erythroid cells in the bone marrow. As a rare disease, LGLL combined with PRCA currently has no unified standard treatment guidelines, which are mainly based on small sample size retrospective studies or case reports. The commonly used treatments for LGLL combined with PRCA are cyclosporine A (CsA) and cytotoxic drugs cyclophosphamide (CTX) or methotrexate. Although CsA is the first-line drug for the treatment of LGLL combined with PRCA, a considerable number of patients still fail to respond to treatment and have CsA resistance. Therefore, the study of the mechanism of CsA resistance is an important research area at present. Summary of the invention

[0004] The purpose of the present invention is to provide a biomarker for evaluating the resistance of LGLL combined with PRCA patients to cyclosporine A and its application, and to pre-evaluate the sensitivity of LGLL combined with PRCA patients to CsA treatment through the detection of the biomarker, which is beneficial to the selection of treatment plans for LGLL combined with PRCA patients, and improves the treatment effect of LGLL combined with PRCA and the prognosis of LGLL combined with PRCA patients.

[0005] In order to achieve the above objectives, in the first aspect, the present invention provides a biomarker for evaluating CsA resistance in patients with LGLL and PRCA, wherein the biomarker is TCR; wherein the TCR includes a TCR β chain variable region; the TCR β chain variable region includes CDR3; and the CDR3 includes the TRBV06 gene.

[0006] In a second aspect, the present invention provides an application of the biomarker as described in the first aspect in evaluating CsA resistance in patients with LGLL and PRCA, wherein the application is to detect whether there is TRBV06 gene rearrangement in the sample to evaluate whether the patients with LGLL and PRCA are sensitive to CsA. Patients with TRBV06 gene rearrangement are insensitive to CsA drugs.

[0007] Preferably, the sample is selected from peripheral blood and / or bone marrow blood of patients with LGLL and PRCA.

[0008] Preferably, the detection method is TCR gene rearrangement NGS sequencing detection.

[0009] In the above technical scheme, the inventors found that LGLL patients with PRCA and TRBV06 gene rearrangement had poor sensitivity to CsA. Detecting whether there is TRBV06 gene rearrangement can be used as a biomarker to evaluate whether LGLL patients with PRCA are sensitive to CsA, thereby screening out patients who are insensitive to CsA and adopting other treatment options to improve the treatment effect.

[0010] The present invention utilizes TCR sequencing and data analysis of clinical samples and finds that TRBV06 gene rearrangement is associated with sensitivity to CsA, and TRBV06 gene rearrangement can enhance the drug resistance of patients with LGLL combined with PRCA to CsA, thereby indicating that TRBV06 gene rearrangement can be used as a marker for evaluating the drug resistance of patients with LGLL combined with PRCA to CsA, and the gene can be detected through clinical samples (peripheral blood, bone marrow blood, etc.), providing a new detection target for CsA resistance, which is helpful to pre-evaluate the effect of CsA drug treatment on patients with LGLL combined with PRCA.

[0011] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0013] Figure 1 is a schematic diagram of the distribution of different responses of patients with TRBV06 gene rearrangement and patients without TRBV06 gene rearrangement when treated with CsA in Example 1;

[0014] Figure 2 Schematic diagram of the distribution of different responses in CsA-resistant patients treated with cyclophosphamide combined with prednisone (CP) regimen. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] In a first aspect, the present invention provides a biomarker for evaluating CsA resistance in patients with LGLL and PRCA, wherein the biomarker is TCR; wherein the TCR includes a TCR β chain variable region; the TCR β chain variable region includes CDR3; and the CDR3 includes the TRBV06 gene.

[0018] The inventors found that LGLL patients with PRCA and TRBV06 gene rearrangement had poor sensitivity to CsA. Detecting whether TRBV06 gene rearrangement exists can be used as a biomarker to evaluate whether LGLL patients with PRCA are sensitive to CsA, thereby screening out patients who are insensitive to CsA and adopting other treatment options to improve the treatment effect.

[0019] In a second aspect, the present invention provides an application of the biomarker as described in the first aspect in evaluating CsA resistance in patients with LGLL and PRCA, wherein the application is to detect whether there is TRBV06 gene rearrangement in the sample to evaluate whether the patients with LGLL and PRCA are sensitive to CsA. Patients with TRBV06 gene rearrangement are insensitive to CsA drugs.

[0020] In a preferred embodiment of the present invention, the sample is selected from peripheral blood and / or bone marrow blood of patients with LGLL and PRCA.

[0021] In a preferred embodiment of the present invention, the detection method is TCR gene rearrangement NGS sequencing detection.

[0022] The present invention will be described in detail below through examples, but the protection scope of the present invention is not limited thereto.

[0023] Example 1

[0024] TRBV06 gene rearrangement is present in patients with LGLL and PRCA, and the efficacy of CsA is poor.

[0025] (1) A total of 61 patients with LGLL and PRCA were selected, and all of them underwent NGS sequencing of TCR gene rearrangement. CsA was the first choice for treatment, with a starting dose of 3-5 mg / (kg·d). The serum drug trough concentration was adjusted to 150-200 ng / mL according to adverse reactions. The effective dose was maintained for 12 months and then slowly reduced;

[0026] Patients with CsA resistance were treated with cyclophosphamide combined with prednisone (CP): the initial dose of cyclophosphamide (CTX) was 100 mg / d and continued until effective. CTX was effective when the lymphocyte ratio was lower than normal or the granulocyte count was less than 1.0×10 9 / L, the dose is reduced to 50 mg / d; when the lymphocyte ratio is lower than 50% of the normal level, or the granulocyte count is lower than 0.5×10 9 The initial dose of prednisone is 0.5-1 mg / (kg·d), which should be gradually reduced to discontinuation after 4 weeks.

[0027] The response criteria are as follows: complete response (CR) is defined as hemoglobin >100 g / L, maintained for at least 2 months; partial response (PR) is defined as improvement of anemia symptoms, independence from blood transfusion, hemoglobin level increased by more than 30 g / L compared with baseline, maintained for 3 months without decline; no response (NR) is failure to meet the above criteria and presence of transfusion dependence; relapse is defined as a significant decrease in hemoglobin, requiring re-transfusion or resumption of adequate drug treatment; overall response rate (ORR) is the ratio of CR and PR to the total number of treated patients; complete remission rate (CRR) is the ratio of CR to the total number of treated patients.

[0028] (2) Collect peripheral blood or bone marrow blood samples from patients before treatment and perform NGS sequencing of TCR gene rearrangement. The method is: extract gDNA or cfDNA from gDNA or MRD tracking monitoring samples (such as peripheral blood leukocytes, peripheral blood plasma collected from free DNA storage tubes, bone marrow, enriched / sorted hematopoietic stem cells, etc.), and use this as a template to perform multiple PCR amplification of the V(D)J variable region of the TCR gene. After the PCR amplification product is purified, a second round of PCR is performed to add a double-end index tag, and then the amplified library is sequenced using the Illumina high-throughput sequencing platform; after sequencing, the V(D)J variable region sequence of the TCR gene is filtered and quality controlled, and then compared to the IMGT database (https: / / www.imgt.org / ). Combined with the abundance ratio of the V(D)J variable region sequence of the TCR gene in the test sample, the specific TCR clone of the test sample is determined;

[0029] (3) Based on the above data, the differential gene rearrangement characteristics of the two groups of samples, the CsA-insensitive group (i.e., CsA-resistant group) and the CsA-sensitive group, were compared.

[0030] Analysis of TCR rearrangement sequencing results in T-LGLL-related PRCA: Among 61 patients, the dominant clonal type in 48 patients was β-type rearrangement. In the TCRβ product rearrangement, the variable region (V region) gene with the highest usage frequency was TRBV06, with 12 cases accounting for 25% (12 / 48), indicating that the TRBV06 gene may be a T-LGLL-specific high-frequency usage gene.

[0031] (4) To analyze the treatment response of patients with TRBV06 gene rearrangement and patients without TRBV06 gene rearrangement to CsA treatment and those who failed CsA treatment and then received CP treatment:

[0032] Combination Figure 1 The results showed that among the 36 patients with non-TRBV06 gene rearrangement who received CsA treatment, 13 had CR, 8 had PR, and 15 had NR. The ORR and CRR of CsA treatment were 58% and 36% respectively. The 15 patients who failed CsA treatment were sequentially treated with CP regimen, of which 12 had CR, 2 had PR, and 1 had NR. The ORR and CRR were 93% and 80% respectively. It can be seen that in patients with non-TRBV06 gene rearrangement, the ORR (P=0.01) and CRR (P=0.003) of CP treatment were better than those of CsA treatment.

[0033] Combination Figure 2The results showed that among the 12 patients with TRBV06 gene rearrangement who received CsA treatment, 1 had CR, 1 had PR, and 10 had NR. The ORR and CRR of CsA treatment were 17% and 8% respectively. The 10 patients who failed CsA treatment were sequentially treated with CP regimen, among whom 5 had CR, 2 had PR, and 3 had NR. The ORR and CRR were 70% and 50% respectively. It can be seen that the treatment response of patients with TRBV06 gene rearrangement treated with CsA was not as good as that of patients without TRBV06 gene rearrangement treated with CsA (17% vs 58%, P=0.01), while the ORR and CRR of patients with TRBV06 gene rearrangement treated with CP were higher than those of patients with TRBV06 gene rearrangement treated with CsA (70% vs 17%, P=0.01; 50% vs 8%, P=0.03), and the difference was statistically significant.

[0034] In summary, TRBV06 gene rearrangement indicates CsA resistance, and patients with CsA resistance can benefit from the CP regimen. Whether the patient has TRBV06 gene rearrangement has a certain correlation with the prediction of CsA resistance. TRBV06 can be used as a potential biomarker, and biomarkers can be applied to personalized drug selection.

[0035] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0036] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0037] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A biomarker for evaluating cyclosporine A resistance in patients with LGLL and PRCA, characterized in that: The biomarker is TCR; wherein, The TCR comprises a TCR β chain variable region; The TCR β chain variable region includes CDR3; The CDR3 includes the TRBV06 gene.

2. A use of the biomarker according to claim 1 in evaluating cyclosporine A resistance in patients with LGLL combined with PRCA, characterized in that: The application is to detect whether there is TRBV06 gene rearrangement in the sample to evaluate whether LGLL patients with PRCA are sensitive to cyclosporine A. Patients with TRBV06 gene rearrangement are insensitive to cyclosporine A.

3. The use according to claim 2, characterized in that: The sample is selected from peripheral blood and / or bone marrow blood of patients with LGLL and PRCA.

4. The use according to claim 2 or 3, characterized in that: The detection method is TCR gene rearrangement NGS sequencing detection.

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