TWI454577B - Method and kit for predicting therapeutic effect of interferon therapy - Google Patents

Description

Method and kit for predicting the efficacy of interferon

The present invention relates to a method for predicting the response of an individual infected with hepatitis C to an interferon in vitro. The invention also relates to a kit for predicting the efficacy response of interferon in the treatment of hepatitis C.

Hepatitis C virus (HCV) is one of the major causes of chronic hepatitis, cirrhosis, and hepatocellular carcinoma in the world and in Taiwan. Hepatitis C virus currently has six major genotypes and many minor subtypes. In Western countries, such as Europe and the United States, genotype 1 or 4 is most prevalent, while genotype 2 or 3 is in the eastern state. There are more. At present, the distribution of HCV genotypes in Taiwan is similar to that of Japan, with subtype 1b as the main type, accounting for 45% to 71% of all HCV in Taiwan.

Treatment of chronic hepatitis C in clinical efficacy is prescribed for the treatment of hybrid interferon -α (Interferon- α, IFN- α) and ribavirin (ribavirin) preferred; clinically judged whether the eradication of HCV infection, This was determined by the achievement of a sustained virologic response (SVR, defined as RNA in which no HCV was detected in the serum 24 weeks after discontinuation of the drug). In addition, the early virologic response (EVR, whether the RNA of HCV disappears after 12 weeks of treatment or the logarithm of the virus is reduced by 100 times) is used to predict whether the positive response can be obtained after the end of treatment. In patients with HCV genotype type 1. In addition, factors such as age, sex, ethnicity, insulin resistance, host immune response or mononucleotide polymorphism may affect the efficacy of interferon therapy.

Micro-deoxyribonucleic acid (microRNA, miRNA), a small fragment of RNA, about 20 to 22 nucleotides in length, transcribed from DNA, but cannot be further transcribed into protein RNA; its function is to regulate genes. Characterizes or inhibits genes, thus regulating many cell growth processes such as cell growth, differentiation, proliferation, suicide, and metabolism. In virology, miRNAs can be used as important biomarkers for predicting the course, diagnosis and treatment of viral infections. Studies have shown that some specific miRNAs are associated with liver diseases such as cirrhosis, B and C virus infection, liver cancer cell metastasis or hyperplasia (Jiang J, Gusev Y et al, Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection). , cirrhosis, and patient survival. Clin Cancer Res , 2008, 14: 419-27; Yao J, Liang L et al, MicroRNA-30d promotes tumor invasion and metastasis by targeting Galphai2 in hepatocellular carcinoma. Hepatology , 2010, 51: 846- 56); There are also reports that the expression of miRNA 122 in liver cells infected with HCV is reduced, and the therapeutic effect of interferon is also reduced (Sarasin-Filipowicz M et al, Decreased levels of microRNA miR-122 in individuals with Hepatitis C responding poorly to interferon therapy. Nat Med , 2009, 15: 31-3).

Before treatment, the therapeutic effect of drug treatment for chronic hepatitis C was evaluated. The current HCV genotype of the patient, the RNA concentration of HCV in the serum before treatment, and the genotype variation of the host interleukin-28B (IL28B) were performed. Evaluation. Liver biopsy before treatment is often necessary. In addition to determining the diagnosis, the urgency of the treatment and the response to the estimated treatment can be assessed. However, if the liver sample is used for analysis, it is an invasive method and may have problems such as sample contamination; therefore, it is necessary to use other reliable and non-invasive methods. Therefore, in addition to liver cells, it has been pointed out that peripheral blood mononuclear cells (PBMC) can detect the RNA expression of HCV, indicating that HCV also infects mononuclear cells for replication and reproduction (Tacke RS et Al, Extracellular hepatitis C virus core protein activates STAT3 in human monocytes/macrophages/dendritic cells via an IL-6 autocrine pathway. J Biol Chem , 2011, 286: 10847-55). Therefore, PBMC can be an important non-invasive biomarker for assessing HCV infection.

The miRNA expression of PBMC is also associated with some diseases. However, there is no relevant study to explore the relationship between the efficacy of interferon in miRNAs of PBMC and HCV patients.

In addition, the current clinical treatment is mainly based on a combination of long-acting interferon (IFN- α ) and ribavirin. After treatment with interferon in patients with HCV genotype 1 infection, only about 10-20% of patients in the virus can be completely eliminated; if combined with interferon and ribavirin, 40% to 50% of patients can be cured, The second and third genotypes are likely to be cured by about 50% or 70 to 80% respectively. However, after the treatment is over, that is, after 24 weeks of withdrawal, there is still a risk of recurrence. If such patients are treated again with the same course of treatment, the chance of recurrence is high, but if a more effective treatment is adopted at the beginning, the virus will continue to respond. The ratio of (SVR) may be 40% to 50%. Therefore, in order to provide appropriate and effective treatment for patients with HCV infection, it is necessary to evaluate the therapeutic effect before treatment, which can improve the treatment effect and avoid unnecessary medical treatment waste.

The present invention explores the clinical relevance of interferon and ribavirin in the treatment of C-type hepatitis patients with microRNA (miRNA), and the results show that the response of the treatment is related to the peripheral blood mononuclear cells of the host ( PBMC) is involved in microRNA-125b (miR-125b) and microRNA-550 (miR-550), and it is found that these two miRNAs are significant in patients with viral persistent response (SVR) and non-SVR patients. Differences existed; further analysis was performed in patients with IL28 rs8099917 TT genotype (there was a high chance of achieving SVR response after interferon treatment), and patients with SVR response were found to have low performance of miR-125b and miR-550. the amount. All results show that miR-125b and miR-550 play an important role in interferon therapy, so the performance of miR-125b and miR-550 on PBMC can be used to evaluate and predict interferon for HCV-infected patients. Therapeutic response.

Therefore, the present invention can be used to detect the expression of miR-125b and/or miR-550 in an HCV-infected individual, and to evaluate and predict the effect of the individual on interferon therapy, and the application level thereof comprises: producing a test for miR-125b and/or miR. -550 test reagents or biochips; prepare probes or primers for detection of miR-125b and miR-550; develop miR-125b and/or miR-550 as biomarkers for evaluation with other projects or factors such as IL28B gene The efficacy of interferon, etc.; or the development of miR-125b and/or miR-550 as the main or auxiliary therapeutic targets, by gene therapy or other biotechnology, reduce the performance of miR-125b and / or miR-550 in individuals Amount to treat hepatitis C or Improve the efficacy of treatment of hepatitis C.

The "interferon" referred to in the present invention includes, but is not limited to, any derivative which is effective for hepatitis C, such as interferon alpha or beta, PEG (polyethylene glycol) interferon. The term "interferon therapeutic response" as used herein means a therapeutic response including, but not limited to, treatment with interferon (IFN) alone or interferon in combination with ribavirin. In addition, the term "effect response" means including, but not limited to, whether the amount of RNA expression of HCV is reduced or disappeared before, during, and after treatment. Reference herein to "miR-125b" means a sequence comprising, but not limited to, miR-125b (SEQ ID NO: 1), a complete or partial sequence of its reverse transcribed cDNA, or a precursor thereof, such as SEQ ID NO: 3 or SEQ ID NO: 4. "miR-550" means a sequence comprising, but not limited to, miR-550 RNA (SEQ ID NO: 2), a complete or partial sequence of its reverse transcribed cDNA, or a precursor thereof, such as SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

The present invention provides a method for predicting the response of an individual infected with hepatitis C to an interferon in vitro, comprising: (a) providing a sample of the individual; and (b) detecting the microRNA-125b of the sample (miR) -125b) and/or the amount of microRNA-550 (miR-550). In a preferred embodiment, wherein step (b) further comprises detecting a miR-125b expression in a control group of a hepatitis C infected individual exhibiting a viral persistent response (SVR), wherein if the sample has a low miR-125b expression The amount of miR-125b expression in a control group of hepatitis C infected individuals presenting a viral persistent response (SVR) indicates that the individual is suitable for treatment with interferon. In another embodiment, wherein step (b) further comprises detecting a hepatitis C infection presenting a viral persistent response (SVR) The miR-550 expression level of the control group, wherein if the miR-550 expression of the sample is lower than the miR-550 expression of the control group of the hepatitis C infected individual exhibiting the viral persistent response (SVR), it indicates that the individual is suitable Treated with interferon. In a preferred embodiment, the system is a mammal or a human. In still other preferred embodiments, the test system is blood, plasma, serum or body fluid. In a more preferred embodiment, the test system peripheral blood mononuclear cells. In the individual mentioned herein, the individual may be infected with hepatitis C or have symptoms of hepatitis C or are susceptible to hepatitis C. The "control group for hepatitis C-infected individuals presenting a viral sustained response (SVR)" as used herein refers to hepatitis C including, but not limited to, a viral persistent response (SVR), a rapid viral response (RVR), or a therapeutic response. Infected individual control group.

The invention also provides a kit for predicting the therapeutic response of interferon in the treatment of hepatitis C, comprising: a nucleotide primer for detecting miR-125b, which is used for detecting the expression amount of miR-125b; and/or a detection A nucleotide primer for miR-550, which is used to detect the amount of expression of miR-550. In a preferred embodiment, the nucleotide primer that detects miR-125b will hybridize to SEQ ID NO: 1. In a more preferred embodiment, the nucleotide primer that detects miR-550 will hybridize to SEQ ID NO: 2. In still other preferred embodiments, the nucleotide primer for detecting miR-125b or miR-550 can be further immobilized on a detection wafer.

The "nucleotide primer" of the present invention includes, but is not limited to, an primer, a probe, an RNA/DNA segment, an oligonucleotide or an "oligomer" (a relatively short polynucleotide), a gene, and the like. The term "hybrid" as used herein is meant to include, but is not limited to, the meaning of binding, recognizing, recognizing or further including amplification.

The following examples are presented to illustrate and not to limit the various aspects and features of the invention.

(1) Research object

Patients who were untreated and who responded positively to hepatitis C genotype 1 (ie, anti-HCV antibodies and RNA responses for more than six months) were enrolled in the trial. However, patients with the following diseases or conditions will be excluded from the experiment, such as hepatitis B, HIV, autoimmune hepatitis, primary cholestasis cirrhosis, sclerosing cholangitis, Wilson's disease (Wilson) Disease), α1-antitrypsin deficiency, compensatory cirrhosis, excessive liver failure, current or past history of alcohol abuse (20 grams per day or more), mental status, liver transplantation, liver tumors or other cancers.

All participants in the study received standard medical care for 48 weeks of long-acting interferon (IFN-α) and a mixed prescription of ribavirin based on body weight, and at least 80% of the designated treatment process And disposal plan. After the patient has received the treatment, real-time PCR is used to detect the degree of HCV RNA expression in the patient's serum. According to the difference in the degree of performance, the patients are divided into three groups: the first group includes 3 Treatment of patients who are completely non-reactive (null-responders) (ie, the amount of RNA attenuation of HCV is lower than the baseline value of 2 times in the course of treatment, which means it is difficult to treat); the second group includes 10 rapid viral responses (rapid) Patients with virological response (RVR) and viral persistent response (SVR) (representing ease of treatment), while the above two groups served as training sets; and the third group included 48 patients as validation sets (validation set) ) for analysis with candidate microRNAs (miRNAs) of the training cohort.

(two) evaluation of the efficacy of interferon

Efficacy evaluation is based on the patient's SVR response, that is, within 24 weeks after the end of treatment, HCV RNA is not detected in the serum, which means that the treatment with interferon is effective; other non-SVR (non-SVR) patients return In the fourth week of treatment, the serum of the patient is negative for HCV, ie, the RNA expression of HCV is not detected; and the treatment is completely non-reactive ((R-) is defined as the non-responders; Null-responders) is the presence of RNA of HCV in the patient's serum, regardless of the duration of the treatment or the end.

(three) miRNA extraction

The entire RNA was isolated from peripheral blood mononuclear cells (PBMC) of the patient during the first two weeks of treatment.

(4) MicroRNA analysis of the training group

The miRNA array analysis of the present invention is through a miRNA detection platform (TaqMan Array Human MicroRNA Panel v3.0), each array includes a card A and a card B, and the two cards contain 768 TaqMan miRNAs, which can simultaneously quantitatively analyze 754 Personal miRNAs and three endogenous control groups; and experiments were performed using the ABI 7900 real-time PCR detection system. The above PCR analysis was carried out by using their respective miRNAs and their corresponding unique primers (Applied Biosystems Inc.). Taking miR-125b and miR-550 as examples, the primers (Applied Biosystems Inc.) are hsa-miR-125b (Assay ID 000449) and hsa-miR-550 (Assay ID 002410), and the target sequences are each SEQ. ID NO: 1 (sequence of miR-125b) and SEQ ID NO: 2 (sequence of miR-550) were amplified as templates, and all samples were double-repeatd. The data collection and processing of the present invention is analyzed by the SDS 2.3 program and the software using RealTime StatMiner software. The relative expression of miRNA is presented in ΔCt mode. Ct is the threshold cycle number in the PCR system. The calculation equation is △Ct=(Ct _miR -Ct _U6 ), which is the Ct value of the specific miRNA minus the housekeeping miRNA ( Housekeeping miRNAs) The Ct value of U6, in addition, when the Ct value of miRNA is greater than 34, it will be considered undetectable; and further standardized by the equation log ₁₀ (2 ^{- ΔCt} ), calculate log ₁₀ (2 ^{- △ Ct} ) Mean and standard deviation (SD) were used to compare the change in miRNA expression between SVR responders and non-SVR responders. When the miRNA expression volume change is greater than 4 times (ie log 2 rate value) 2 or -2) will be selected into the verification group.

(5) Verification of miRNAs in the group by real-time quantitative RT-PCR

Analytical validation of miRNA experiments in groups of 5 μL of RNA was quantified using TaqMan real-time quantitative reverse transcriptase-PCR (qRT-PCR), and the primers and reagents required for the experiments were from Applied Biosystems Inc. (ABI) (Foster). City, CA) obtained. Taking miR-125b and miR-550 as examples, the primers (Applied Biosystems Inc.) are hsa-miR-125b (Assay ID 000449) and hsa-miR-550 (Assay ID 002410), and the target sequences are each SEQ. ID NO: 1 and SEQ ID NO: 2 were amplified as templates, and all samples were double-repeatd. The expression of miRNA is represented by ΔCt. The Ct value refers to the number of cycles of detecting fluorescent signals exceeding the threshold in the PCR system, and U6 RNA is used as a reference for the internal control group to standardize and quantify miRNAs. The amount of performance, in addition, when the Ct value of the miRNA is greater than 34, will be considered undetectable.

(6) Interleukin-28B (IL28B) genotype

Studies have shown that there is a strong correlation between the single nucleotide polymorphism (SNP) of IL28B rs8099917 and the therapeutic effect of interferon antiviral drugs in patients with hepatitis C. The results show that patients with hepatitis C are With the TT genotype of IL28B rs8099917, patients have a high rate of HCV clearance in serum and therefore a higher success rate after receiving interferon-related therapy (Huang CF et al., Host interleukin-28B genetic variants versus Viral kinetics in determining responses to standard-of-care for Asians with hepatitis C genotype 1. Antiviral Res , 2012; 93: 239-44). Accordingly the present invention using ABI TaqMan ^® SNP genotyping techniques, to identify the genotype.

(7) Statistical analysis

Student's t test and chi-square test ( X ² ) are used to analyze the parameters of the patient; Multivariate logistic regression model is used to judge the correlation factors of the results; and the recipient is used. The area under the curve (AUC) of the receiver-operating characteristics (ROC), and in the ROC analysis, a suitable clinical cut-off point is needed to distinguish the miRNA expression between patients. The amount is used to predict whether the miRNA expression is related to the SVR response, and the cut-off point is determined based on the point on the ROC curve that is closest to the (0, 1) distance. The calculation of AST-to-platelet ratio index (APRI) by glutamic acid transaminase (AST) is based on the following formula: [(AST performance/upper limit of normal AST range) / platelet count (10 ⁹ / L)] × 100, the index can show the severity of liver fibrosis. The calculation of the P value is based on two-tailed statistics and is judged by 0.05 as the level of significant difference.

(8) Results (1) Patient analysis

Comparing the demographic, virological and clinical parameters of the patient, including body weight, basic viral load, AST and alanine aminotransferase (ALT) performance and APRI, the above parameters can be observed in the training group and Verify that there are no differences between the groups, except that the patients in the training group are younger than the verification group, see Table 1 below.

(2) Identification of miRNAs associated with interferon therapeutic response

Using 754 miRNA probes to detect PBMC in the training group through the microarray, 207 candidate miRNAs were detected; in the training group, there were 9 miRNA expressions between SVR responders and non-SVR patients. Significant differences; among the SVR responders, the miR-151-3p, miR378 and miR-93 ^* miRNAs showed higher ploidy changes than non-SVR responders, while the other 6 miRNAs, such as miR-125b and miR-19b- 1 ^* , miR-505 ^* , miR-142-3p, miR-19a and miR-550, are lower than non-SVR patients, see Table 2. Further comparison with the validation cohort, and in the 6 low-expression miRNAs, the expression levels of miR-125b and miR-550 were significantly different, and the P values were 0.04 and 0.03, respectively (Fig. 1). From the above results, it was revealed that miR-125b and miR-550 have a significant correlation with the SVR reaction.

Univariate analysis was used to compare patient parameters such as gender, age, pre-treatment ALT performance, basic viral load, IL28B rs8099917 genotype, and miR-125b and miR-550 in PBMC. three. When other covariates (sex, age, ALT, and IL28B rs8099917 genotypes) were significantly different between SVR responders and non-SVR responders, miR-125b and miR-550 were also significantly different. The two miRNAs were independent of the IL28B rs8099917 genotype factor and reached significant differences between the two groups. The P values were 0.03 and less than 0.01, respectively.

Using ROC curve analysis, the dCt (delta Ct) of miR-125b-U6 was the best cutoff value in 12.3 cycles, and the patient was divided into the high-performance group and the low-performance group of miR-125b. Using the AUC of miR-125b to predict the SVR response value of 0.78 ( P = 0.0004), in the low-expression group of miR-125b, approximately 79% of patients achieved SVR response (30 of 38 patients), significantly higher than miR 30% of the -125b high performance group (3 out of 10 patients) (the adjusted odds ratio was 16.5, the confidence interval was 1.95-266, P = 0.0087) (Figure 2). In addition, the dCt cutoff value of miR-550-U6 was 13.6 times, and the patient was divided into the high performance group and the low performance group of miR-550. In the miR-550 low-performance group, approximately 94% of patients achieved an SVR response (32 of 34 patients), significantly higher than 43% of the miR-550 high-performance group (6 of 14 patients) (6 of 14 patients) Figure 3).

Figure 1. Verification of the relative performance of each miRNA in patients with SVR response and non-SVR response in the cohort (analyzed by Mann-Whitney U, ^* represents a P value of less than 0.05).

Figure 2. Proportion of patients with high-low miR-125b and viral persistent response (SVR).

Figure 3. The proportion of miR-550 performance and the proportion of patients with viral persistent response (SVR).

<110> Kaohsiung Medical University

<120> Methods and kits for predicting the efficacy of interferon

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Claims (7)

A method for predicting the response of an individual with hepatitis C infection to an interferon in vitro comprises: (a) providing a sample of the individual; (b) detecting the microRNA-125b (miR-125b) expression of the sample And (c) detecting the expression of miR-125b in a control group of hepatitis C-infected individuals presenting a viral persistent response (SVR); wherein if the miR-125b expression of the sample is lower than the sustained viral response The amount of miR-125b expression in the control group of hepatitis C infected individuals (SVR) indicates that the individual is suitable for treatment with interferon. The method of claim 1, wherein the system is a mammal or a human. The method of claim 1, wherein the test system is blood, plasma, serum or body fluid. For example, the method of claim 1, wherein the blood mononuclear cells are surrounded by the system. A kit for predicting the therapeutic response of interferon in the treatment of hepatitis C, comprising: a nucleotide primer for detecting miR-125b, which is used to detect the amount of miR-125b expression. The kit of claim 5, wherein the nucleotide primer for detecting miR-125b is heterozygous for SEQ ID NO: 1. For example, the kit of claim 5, wherein the nucleotide primer for detecting miR-125b can be further immobilized on a detection wafer.