A probe, a kit and its application for detecting microsatellite instability

Abstract

The present invention relates to a biomarker combination, kit, and application for detecting microsatellite instability, belonging to the field of biotechnology. The present invention provides a diagnostic reagent for detecting microsatellite instability, comprising probes having nucleotide sequences represented by SEQ ID No. 1 to SEQ ID No. 224. The present invention also provides a kit for detecting microsatellite instability, as well as the application of the probes in an analytical method for detecting microsatellite instability. The biomarker combination for detecting microsatellite instability of the present invention covers a wide range of loci, provides more accurate detection, and has high sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to a probe, a kit and an application for detecting microsatellite instability, and belongs to the technical field of biomedicine. Background Art

[0002] Microsatellite instability (MSI) refers to any change in microsatellite length caused by insertions or deletions of microsatellite repeat units in tumors compared to normal tissue, resulting in the appearance of new microsatellite alleles. Both the NCCN and CSCO guidelines indicate that MSI may be a prognostic factor for CRC and is associated with the efficacy of chemotherapy or immunotherapy in CRC patients. Studies have shown that patients with stage II colorectal cancer with dMMR / MSI-H have a favorable prognosis and do not benefit from single-agent adjuvant chemotherapy with fluoropyrimidines. Patients with advanced / metastatic colorectal cancer with dMMR / MSI-H may benefit from immunotherapy (anti-PD-1 monoclonal antibodies). MSI can also be used as an aid in the diagnosis of Lynch syndrome in colorectal cancer. Due to functional inactivation of the MMR gene, patients with Lynch syndrome often exhibit a dMMR and / or MSI-H phenotype. Studies have shown that over 90% of Lynch syndrome cases are MSI-H. In clinical practice, MSI H has been used as an important molecular marker for the prognosis and formulation of adjuvant treatment plans for colorectal cancer and other solid tumors: On May 23, 2017, the FDA approved Keytruda for the treatment of unresectable or metastatic adult and pediatric tumors with microsatellite instability-high (MSI H) or mismatch repair deficiency (dMMR) molecular characteristics; On July 11, 2018, the FDA accelerated the approval of nivolumab combined with low-dose ipilimumab for the treatment of adult or pediatric (≥12 years old) metastatic colorectal cancer (mCRC) patients with high microsatellite instability (MSI H) or mismatch repair deficiency (dMMR) whose disease progressed after treatment with fluorouracil, oxaliplatin, and irinotecan.

[0003] Currently, there are two main types of MSI detection technologies: 1. Immunohistochemistry (IHC): This method relies on protein level detection using immunohistochemistry to detect the expression of MMR-related mismatch repair genes MLH1, MSH2, MSH6, and PMS2 in tumor tissues. The disadvantages of the immunohistochemistry method are slightly lower detection sensitivity and large performance differences between different detection kits. 2. Fluorescence PCR-capillary electrophoresis: This method uses fluorescently labeled primers and capillary electrophoresis to determine the fragment length polymorphism of five sites: BAT-25, BAT-26, D2S123, D5S346, and D17S250. The PCR-capillary electrophoresis method requires simultaneous detection of the test sample and the normal control sample, resulting in high detection costs and workload. The detection process is cumbersome, the sample throughput is limited, and it is difficult to complete the detection quickly. High-throughput sequencing technology (NGS) can accurately detect changes in DNA sequences and has become a new tool for identifying MSI detection. However, due to the large differences in different detection and analysis methods, no universal detection standard has yet been formed, and currently no microsatellite instability detection kit based on high-throughput sequencing technology has been approved for marketing by the NMPA. Summary of the Invention

[0004] The problem to be solved by the present invention is that: in the current microsatellite detection, different detection and analysis methods vary greatly, and no universal detection standard has yet been formed.

[0005] The present invention solves the above technical problems through the following technical solutions:

[0006] In a first aspect of the present invention, a diagnostic reagent for detecting microsatellite instability is provided, wherein the diagnostic reagent comprises probes having nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 224. The probes are as follows:

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[0018] In a second aspect of the present invention, a kit for detecting microsatellite instability is provided, wherein the kit comprises probes having nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 224.

[0019] A third aspect of the present invention provides a method for detecting microsatellite instability using the aforementioned probe, which comprises the following steps:

[0020] Step 1: Extract DNA from FFPE samples, fragment, end-repair, linker-ligate, and PCR enrich the genomic DNA to prepare a pre-library;

[0021] Step 2: hybridizing and capturing the probe according to any one of claims 1 or 2 with the pre-library, obtaining a sequencing library after purification with magnetic beads, and sequencing the sequencing library using a gene sequencer to obtain sequencing data;

[0022] Step 3: Use the analysis tool to calculate the number of MSI-positive sites in all 224 microsatellite loci. Figure 1 ), it is determined to be microsatellite unstable; otherwise, it is microsatellite stable.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. Microsatellite instability biomarkers have wide coverage and improved detection performance

[0025] The microsatellite instability biomarker combination includes 224 microsatellite loci, covering a wide range of loci and providing more accurate detection. Through experimental verification, an MSI-H judgment threshold was established, that is, if the number of MSI-positive loci is ≥22, it is judged as microsatellite instability (MSI-H), and vice versa, it is microsatellite stable (MSS). Based on this microsatellite instability biomarker combination and MSI-H discrimination, the method for detecting MSI has high sensitivity and specificity.

[0026] 2. Low testing cost

[0027] Single-sample testing only requires testing tumor tissue samples, without the need for normal sample controls, to accurately determine the microsatellite instability status of the sample, reducing the amount of testing and lowering the testing cost.

[0028] 3. Simple bioinformatics analysis process

[0029] The process from off-machine data to MSI detection and analysis can be completed in one step. The automated analysis process does not require human intervention in the intermediate process, and the analysis time is greatly shortened, which saves patients valuable detection and analysis time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The distribution of the number of MSI-positive sites in 365 clinical samples;

[0031] Figure 2 ROC curves were used to evaluate the quality of MSI detection in 123 clinical samples. DETAILED DESCRIPTION

[0032] In order to make the present invention more clearly understood, preferred embodiments are described in detail as follows:

[0033] Example 1 Determination of the discrimination value of the MSI-H state

[0034] In a tumor sample, the probability of observing n positive sites from 224 microsatellite sites is Poisson distribution.

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[0036] Where n = 1, 2, 3, ..., e is the Euler constant (e = 2.71828 ...), n! is the factorial of n, and λ is the expected value of the variable X of the number of positive sites.

[0037] The λ parameter in the Poisson model was estimated using the observed values of the number of MSI-positive sites in 365 clinical samples. Figure 1 ), Figure 1 The gray distribution graph is calculated using the present invention to detect the number of MSI positive sites; the blue line is calculated according to formula 1; the red line is the theoretical threshold of Poisson distribution, k0 = 21, Pr (k>k0) =4.52×10 -6 .

[0038] To obtain a robust λ parameter, the median is used to estimate the λ parameter, which is 7. Therefore, λ=7.

[0039] Based on λ=7, the distribution of the theoretical number of positive sites of the Poisson distribution is consistent with the distribution of the actual observed values in 365 clinical samples. Therefore, the theoretical discrimination value of the MSI-H status in the detection of the present invention is set to ≥22 positive sites.

[0040] Example 2 Sample DNA extraction, library preparation and panel capture sequencing analysis

[0041] DNA is extracted from FFPE samples, and the genomic DNA (gDNA) is fragmented, end-repaired, adapter-ligated, and PCR enriched to prepare a pre-library. A probe combination that detects microsatellite instability biomarkers is then hybridized and captured with the pre-library, and the sequencing library is obtained after magnetic bead purification. The sequencing library is sequenced on a NextSeq 550Dx / Novaseq 6000 gene sequencer to obtain sequencing data.

[0042] Use analysis software to analyze the original offline data. The bioinformatics analysis process can be divided into the following steps:

[0043] 1. Data Preprocessing: Data were analyzed using software to measure the sequencing quality parameter, Q30 base ratio. Quality control passed if the Q30 ratio was ≥ 60%; otherwise, it failed. The resulting BCL files were converted to Fastq files using Illumina software. Adapter sequences and low-quality base fragments introduced during library construction were removed using Trimmomatic-0.36 software.

[0044] 2. Data alignment: Use bwa Version: 0.7.10-r806 to align the fragments to the hg19 reference genome;

[0045] 3. Data Quality Control: The sample sequencing quality is determined based on parameters such as the sample Q30 base ratio, the sequence alignment ratio to the reference genome, and the average sequencing depth of the target region. If Q30 is ≥60%, the sequence alignment ratio to the reference genome is ≥90%, and the average sequencing depth is ≥300X, the sample sequencing data quality control passes. Otherwise, the test is considered failed and requires resequencing.

[0046] 4. Mutation analysis: MSI detection was performed using the MSIsensor2 software, and the analysis parameters were set using the default parameters.

[0047] 5. If the number of MSI microsatellite positive sites is ≥22, it is judged as microsatellite instability (MSI-H); otherwise, it is microsatellite stable (MSS).

[0048] Example 3 Microsatellite instability detection in 123 clinical samples

[0049] 123 colorectal cancer clinical samples were collected and the MSI status was determined by a reference method (multiplex fluorescence PCR-capillary electrophoresis) as the gold standard for reference analysis. The samples were sequenced and analyzed using this kit to determine the number of MSI-positive sites. The verification results are detailed in Table 1 and Figure 2 .

[0050] Table 1. MSI validation experimental analysis results

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[0052] Based on the theoretically calculated threshold for the number of positive sites, the MSI status of 123 samples analyzed using this kit was classified based on the number of positive sites. The results were compared with the gold standard, as shown in Table 2. The results showed a sensitivity of 100% (CI 95%: 88.41%-100%) and a specificity of 100% (CI 95%: 99.12%-100%).

[0053] Table 2. Scores and MSS / MSI status of 123 clinical samples obtained by this kit

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[0058] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A diagnostic reagent for detecting microsatellite instability, characterized in that The diagnostic reagent includes a probe having a nucleotide sequence as shown in SEQ ID No. 1 to SEQ ID No.

224.

2. A kit for detecting microsatellite instability, characterized in that: The kit includes probes with nucleotide sequences as shown in SEQ ID No. 1 to SEQ ID No. 224.

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