KR102506176B1 - Marker composition and primer set for Microsatellite Instability (MSI) Diagnosis - Google Patents

Abstract

The present invention relates to a marker composition and a primer set for diagnosing microsatellite instability, and more particularly, to a diagnostic use of microsatellite instability using a marker composition including BAT26 and CAT25 or a primer set thereof. In order to diagnose microsatellite instability, the present inventors selected PCR markers with the least polymorphism between race, gender, and group (BAT26 and CAT25), and compared the results of pentaplex PCR and NGS tests, which are conventional tests, when using the markers. By confirming that the sensitivity and specificity are 100%, a simple, low-cost and high-efficiency duplex microsatellite unstable PCR primer set was developed as a test method that can accurately determine whether or not MSI-high is present. The primer set according to the present invention is more efficient than a PCR test using five markers, and is expected to quickly and accurately derive microsatellite instability diagnosis results without PCR failure even in FFPE samples with poor DNA quality.

Description

Marker composition and primer set for microsatellite instability (MSI) Diagnosis}

The present invention relates to a marker composition and a primer set for diagnosing microsatellite instability, and more particularly, to a diagnostic use of microsatellite instability using a marker composition including BAT26 and CAT25 or a primer set thereof.

Microsatellite instability (MSI) is a defect in the DNA repair system due to methylation and mutation of genes related to the DNA mismatch repair system (MMR) in cancer cells, resulting in mono-, di-, tri-, tetra-nucleotide, etc. It refers to a state in which the number of microsatellite repetitions of is not maintained constant. MSI has been used to diagnose hereditary nonpolyposis colorectal cancer, predict treatment response, and survival rate through the NCI panel recommended by NIH/NCI. In addition, MSI has recently been identified in several tumors, has been used as a target for immuno-anticancer drugs since 2014, and MSI testing is widely used as a biomarker to predict the response of immuno-anticancer drugs.

Currently, the most useful method for confirming MSI is known as polymerase chain reaction (PCR), and the microsatellite instability test uses two mononucleotide microsatellites, BAT25 and BAT26, and three paired bases ( The Bethesda panel consisting of D5S346, D2S123, and D17S250, which are dinucleotide) microsatellites, has been mainly used. However, recently, research is being conducted to increase sensitivity and specificity compared to the existing Bethesda panel by presenting a new panel composed of single base microsatellites, BAT25, BAT26, NR21, NR24, and NR27. In particular, MSI-H (high) is defined as a case in which two or more microsatellites show instability among this panel, and MSI-L (low) is defined as a case in which only one microsatellite shows instability. A case without one is defined as MSS (stable), and a case of MSI-H is evaluated as having microsatellite instability. Since almost all patients are MSI-H, microsatellite instability testing can be used to screen for a population that requires additional genetic testing. In addition, MSI-H is emerging as a very important immunotherapy consideration in the response of immunotherapeutic agents, and active research on this is in progress (US Patent Registration 10/699802).

As for the current MSI-PCR test method, pentaplex PCR using BAT25, BAT26, NR21, NR24, and NR27, which are quasi-monomorphic markers showing an allele size difference of less than 2 bp, is known as the gold-standard, and this test method is different from the existing dinucleotide markers. It has the advantage of being able to diagnose the MSI phenomenon using only tumor tissue without comparing the results by PCR of paired-normals together. However, in the case of the recently increasing MSI test volume and the required amount of sample, the PCR failure rate is high due to poor DNA quality in the case of formalin-fixed paraffin embedded (FFPE) tissue. Therefore, it is necessary to develop a low-cost and high-efficiency test method to obtain a result with high sensitivity and specificity by discovering a more effective PCR marker set in a clinical diagnostic test.

In order to solve the above problems, the present inventors studied microsatellite instability diagnostic markers that can increase sensitivity and specificity. As a result, when using BAT26 and CAT25, even in FFPE samples with poor DNA quality, they can be quickly and accurately tested without PCR failure. The present invention was completed by confirming that microsatellite instability diagnosis results could be derived.

Accordingly, an object of the present invention is to provide a marker composition for diagnosis of microsatellite instability (MSI) comprising polynucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 or polynucleotides having sequences complementary thereto.

In addition, the present invention is a primer set for diagnosing microsatellite instability (MSI), including a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6, and the same Another object is to provide a composition comprising the composition and a kit comprising the composition.

In addition, another object of the present invention is to provide an information providing method for diagnosing microsatellite instability (MSI).

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Accordingly, in order to achieve the above object, the present invention provides a marker composition for diagnosis of microsatellite instability (MSI) comprising polynucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 or polynucleotides having sequences complementary thereto. do.

In addition, the present invention provides a primer set for diagnosing microsatellite instability (MSI), including a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 do.

In addition, the present invention provides a composition for diagnosing microsatellite instability (MSI) comprising the primer set and a kit for diagnosing microsatellite instability (MSI) including the same.

In addition, the present invention provides an information providing method for diagnosing microsatellite instability (MSI), which includes the following steps.

(a) isolating DNA from the biological sample;

(b) performing a polymerase chain reaction (PCR) using the isolated DNA as a template and using the primer set; and

(c) confirming the PCR product amplified in step (b) by electrophoresis.

In one embodiment of the present invention, diagnosing the microsatellite instability may be for predicting the prognosis of cancer.

In another embodiment of the invention, the prognosis may be recurrence, survival, or disease-free survival.

In another embodiment of the present invention, the biological sample may be any one or more selected from the group consisting of tissue, blood, plasma, serum, nasal mucus, saliva, urine, sputum, and lymph.

In order to diagnose microsatellite instability, the present inventors selected PCR markers with the least polymorphism between race, gender, and group (BAT26 and CAT25), and compared the results of pentaplex PCR and NGS tests, which are conventional tests, when using the markers. By confirming that the sensitivity and specificity are 100%, a simple, low-cost and high-efficiency duplex microsatellite unstable PCR primer set was developed as a test method that can accurately determine whether or not MSI-high is present. The primer set according to the present invention is more efficient than a PCR test using five markers, and is expected to quickly and accurately derive microsatellite instability diagnosis results without PCR failure even in FFPE samples with poor DNA quality.

Figure 1 shows the results of PCR using the pentaplex primer set (NR27, NR21, BAT26, BAT25, NR24) primers.
Figure 2a shows the results of PCR-genotyping using a pentaplex primer set (NR27, NR21, BAT26, BAT25, NR24) or a duplex primer set (BAT26, CAT25) in the case of microsatellite instability.
Figure 2b shows the results of PCR-genotyping using a pentaplex primer set (NR27, NR21, BAT26, BAT25, NR24) or a duplex primer set (BAT26, CAT25) when the microsatellite is stable.
Figure 3 shows the results of confirming PCR-genotyping using the BAT26 primer set according to the present invention in normal control and MSI-high sample patients.
Figure 4 shows the results of confirming PCR-genotyping using the CAT25 primer set according to the present invention in normal control and MSI-high sample patients.

The present inventors studied microsatellite instability diagnostic markers that can increase sensitivity and specificity. As a result, when BAT26 and CAT25 are used, microsatellite instability diagnosis results can be derived quickly and accurately without PCR failure even in FFPE samples with poor DNA quality. The present invention was completed by confirming that it could be.

Accordingly, the present invention provides a marker composition for diagnosis of microsatellite instability (MSI) comprising polynucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 or polynucleotides having sequences complementary thereto.

The marker composition represented by the nucleotide sequence of SEQ ID NO: 1 according to the present invention refers to the CAT25 marker, which is the 3'UTR sequence of the CASP2 (caspase 2) gene, and the marker composition represented by the nucleotide sequence of SEQ ID NO: 2 is the intron of the MSH2 gene 5 sequence, BAT26 marker. At this time, the marker composition is 80% or more, preferably 90% or more, more preferably 95%, 96%, 97%, 98%, 99% or more of the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 It may contain nucleotide sequences having homology.

In addition, the present invention provides a primer set for diagnosing microsatellite instability (MSI), including a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6 do.

The primers represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 according to the present invention are forward and reverse primer sets targeting the T25 region located in the 3'UTR of the CASP2 gene, and the bases of SEQ ID NO: 5 and SEQ ID NO: 6 Primers represented by sequence are primer sets targeting the A25 region located in intron 5 of the MSH2 gene. At this time, the primer set is 80% or more, preferably 90% or more, more preferably 95%, 96%, 97%, 98%, 99% or more of the nucleotide sequence represented by SEQ ID NO: 3 to SEQ ID NO: 6 It may contain nucleotide sequences having homology.

As used herein, the term "primer" refers to a short nucleic acid sequence capable of forming a base pair with a complementary template and serving as a starting point for template strand replication.

The term “microsatellite instability (MSI)” used in the present invention is a state in which intracellular genes are easily mutated, and DNA repair system due to methylation and mutation of genes related to DNA mismatch repair system (MMR) in cancer cells. It means a state in which the number of repeats of microsatellites such as mono-, di-, tri-, tetra-nucleotide, etc. is not maintained constant due to a defect in , MSH-H is known to have a better cancer prognosis than MSS or MSI-L, and therefore, diagnosing microsatellite instability according to the present invention may be for predicting cancer prognosis.

As used in the present invention, the term “prognosis” refers to prediction of disease progression and recovery, and refers to prospect or preliminary evaluation. In the present invention, prognosis means cancer recurrence, survival (overall survival), or disease-free survival (disease-free survival), but is not limited thereto.

The present inventors confirmed through specific examples that the novel primer set used in the present invention is superior in sensitivity and specificity to PCR using a prior art pentaplex primer set.

According to an embodiment of the present invention, it was confirmed that the polymorphism between groups was lowest and the PCR success efficiency was high when the Duplex PCR of the present invention was performed compared to Pentaplex PCR, and the PCR-genotyping of each primer set was confirmed. As a result, when checking BAT26 or CAT25 alone, it was confirmed that the normal control group and the MSI-high sample patients #1 and #2 showed significantly different deletion phenomena (see Example 2).

Therefore, in selecting MSI-high patients, it can be inferred that using a duplex PCR primer set is a much more efficient, low-cost, highly sensitive and specific method than a test method using a pentaplex PCR primer set.

Accordingly, as another aspect of the present invention, the present invention provides a composition for diagnosing microsatellite instability (MSI) including the primer set and a kit including the composition.

The kit of the present invention may optionally include reagents necessary for conducting a target amplification PCR reaction (eg, PCR reaction) such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate. In addition, the kit may include various polynucleotide molecules, reverse transcriptase, buffers and reagents, and antibodies that inhibit DNA polymerase activity. In addition, the optimal amount of reagents used in a specific reaction of the kit can be easily determined by a person skilled in the art who has learned the disclosure herein. Typically, the kit may be made into a separate package or compartment containing the aforementioned ingredients.

By using the kit, PCR can be easily performed, and microsatellite instability can be diagnosed by reducing the failure rate and increasing specificity even in a small amount of FFPE tissue.

As another aspect of the present invention, the present invention provides an information providing method for diagnosing microsatellite instability (MSI) comprising the following steps.

(a) isolating DNA from the biological sample;

(b) performing a polymerase chain reaction (PCR) using the isolated DNA as a template and using the primer set; and

(c) confirming the PCR product amplified in step (b) by electrophoresis.

The biological sample according to the present invention may be any one or more selected from the group consisting of tissue, blood, plasma, serum, nasal mucus, saliva, urine, sputum, and lymph, but is not limited thereto.

DNA extraction from the biological sample may be performed according to a method commonly used in the art, and may be performed using a commercially available DNA extraction kit. Specifically, a PCR reaction solution can be prepared by mixing reaction buffer (2X), primer set according to the present invention, tertiary distilled water, and genomic DNA extracted from an individual, and PCR can be performed using DNA polymerase and dNTP together. can

A method for confirming the PCR product in step (c) by electrophoresis may use agarose gel or polyacrylamide gel electrophoresis depending on the size of the amplified product.

In the present invention, PCR-genotyping was confirmed using a primer set including a primer pair of SEQ ID NO: 3 and SEQ ID NO: 4 and a primer set including a primer pair of SEQ ID NO: 5 and SEQ ID NO: 6, and one of the panels MSI-H (high) was defined as the case in which the above microsatellite showed instability, and MSS (microsatellite stable) was defined as the case without microsatellite instability.

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

[Example]

Example 1. Preparation of a novel primer set

1-1. CAT25 primer set manufacturing

A primer set targeting the T25 region located in the 3' UTR of the CASP2 gene (CAT25F: 5'-CCCTGCTTATCTGAAACTTCCC-3' (SEQ ID NO: 3), CAT25R: 5'-GGAGTTGGAGCTTGCAGTGA-3' (SEQ ID NO: 4)) was prepared. .

1-2. Preparation of the BAT26 primer set

A primer set targeting the A25 region located in intron 5 of the MSH2 gene (BAT26F: 5'-ACTGACTACTTTTGACTTCAGCC-3' (SEQ ID NO: 5), BAT26R: 5'-TTTTAACCATTCAACATTTTTAACCCT-3' (SEQ ID NO: 6)) was prepared.

1-3. New primer set PCR conditions

In order to react under the same PCR conditions during 1 tube-multiplex PCR, PCR efficiency was increased by designing the Tm value of the primer to 58 ° C.

In addition, since a high concentration of template DNA and a large amount of primer set are put into one tube and reacted, problems such as reduced PCR amplification efficiency and formation of non-specific amplification products due to interference and inhibition between primers may occur. Solgent's 2X multiplex PCR Multiplex PCR was performed using hot-start PCR using enzyme, enabling highly specific amplification even in a small amount of FFPE tissue with poor DNA quality.

Example 2. Confirmation of PCR sensitivity and specificity of the new primer set

As shown in Figure 1, in the case of pentaplex PCR, PCR is performed using 5 primers. In this case, interference between primers occurs, and in the case of poor DNA, PCR failure is generally very high, so two markers with high sensitivity and specificity When duplex PCR was performed by selecting , the PCR success rate was very high, and the polymorphism between groups was the lowest and the PCR success efficiency was high compared to pentaplex PCR (NR27, NR21, BAT26, BAT25, NR24).

More specifically, duplex MSI PCR markers using BAT26 and CAT25 were developed, and the sensitivity and specificity of the PCR markers with conventional pentaplex PCR (NR27, NR21, BAT26, BAT25, NR24) and NGS tests were compared.

As a result, when duplex PCR was performed using BAT26 and CAT25, as shown in FIG. 2a, deletions were observed in both markers with high sensitivity as the result of MSI-H, BAT26 and CAT25, and as shown in FIG. 2b, MSS As a result, both BAT26 and CAT25 markers were observed as normal peaks.

Furthermore, as a result of confirming PCR-genotyping of each primer set, as shown in FIG. 3, in the case of BAT26, the normal control group showed a high peak at about 125 values, but MSI-high sample patients #1 and #2 showed about 110 It showed a high peak around 115 to 115, confirming a significantly different deletion phenomenon from the normal control group.

In addition, as shown in FIG. 4, in the case of CAT25, the normal control group showed a high peak at a value of 139 to 142, but MSI-high sample patients #1 and #2 had a value of 131 to 133 or high at 128 and 140, respectively. A peak was shown, and a significantly different deletion phenomenon from the normal control group was confirmed.

From the above results, in selecting patients with MSI-high, which is known as a biomarker for immunotherapy, the use of duplex PCR primers enables much higher efficiency, lower cost diagnosis, and higher sensitivity and specificity than the test method using pentaplex PCR primers. diagnosis can be inferred.

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Marker composition and primer set for Microsatellite Instability (MSI) Diagnosis <130> PD20-213 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1124 <212> DNA <213> CASP2 3′ UTR <400> 1 gagaaggcca ggaagaatgg tgtgtttccc tagactctgt aaccacctct ctgtcttttt 60 ccttcctgag aaacgtccat ctctctccct tactattccc actttcattc aatcaacctg 120 cacttcatat ctagatttct agaaaagctt cctagcttat ctccctgctt catatctctc 180 ccttctttac cttcatttca tcctgttggc tgctgccacc aaatctgtct agaatcctgc 240 tttacaggat catgtaaatg ctcaaagatg taatgtagtt ctttgttcct gctttctctt 300 tcagtattaa actctccttt gatattatgt ggcttttatt tcagtgccat acatgttatt 360 gttttcaacc tagaaacctt tatccctgct tatctgaaac ttcccaactt ccctgttctt 420 taagactttt tttttttttt tttttttttt tgagacagag tctcgctctg tcgcccaggc 480 tggagggcag tggcacgatc tcagctcact gcaagctcca actcccgggt tcacgccatt 540 ctcctgcctc agccttccaa gtagctggga ctacaggtgc ccgccaccgt gcccggctaa 600 tttttttgta tttttagtag agacagggtt tcaccatgtt agccgggatg gtcttgatct 660 cctgacctca tgatccaccc acctcagcct cccaaagtgt tgggattaca ggcgtgagcc 720 actgcgcccg ggcaagacct ttttttaaaa aaaaaaaaaa aaaaacttcc attctttctt 780 cctccagtct gttctcacat aacagagtag ttttggtttt taattttttt tggttgtttg 840 ctgttttttg ttttttaagg tgagttctca ctatgtttct cagactggtc tcgaactcct 900 ggcctcaagc catcttcccg cctcagcctc tcaaatagct gggcttacag gcatgagcca 960 ccacacctgg ccaggatttg gttgtttaaa tataaatctg atcacccccc tgcttagaac 1020 ccttctgctt tctattaccc ctcatttaaa atgtaaactc ttcaccttgg tttatgagaa 1080 ctggttcttg ccttcccctt gaacctcatt aaatggtgat ttct 1124 <210> 2 <211> 450 <212> DNA <213> MSH2 intron5 <400> 2 gttgcagttt catcactgtc tgcggtaatc aagtttttag aactcttatc agatgattcc 60 aactttggac agtttgaact gactactttt gacttcagcc agtatatgaa attggatatt 120 gcagcagtca gagcccttaa cctttttcag gtaaaaaaaa aaaaaaaaaa aaaaaaaaag 180 ggttaaaaat gttgaatggt taaaaaatgt tttcattgac atatactgaa gaagcttata 240 aaggagctaa aatattttga aatattatta tacttggatt agataactag ctttaaatgg 300 ctgtattttt ctctcccctc ctccactcca ctttttaact tttttttttt taagtcagag 360 tctcacttgt tccctaggcc agagtgcagt ggcacaatct cagcccactc taacctccac 420 ctcccaagta gttgggatta cagttgcctg 450 <210> 3 <211> 22 <212> RNA <213> artificial sequence <220> <223> CAT25 F primer <400> 3 ccctgcttat ctgaaacttc cc 22 <210> 4 <211> 20 <212> RNA <213> artificial sequence <220> <223> CAT25 R primer <400> 4 ggagttggag cttgcagtga 20 <210> 5 <211> 23 <212> RNA <213> artificial sequence <220> <223> BAT26 F primer <400> 5 actgactact tttgacttca gcc 23 <210> 6 <211> 27 <212> RNA <213> artificial sequence <220> <223> BAT26 R primer <400> 6 ttttaaccat tcaacatttt taaccct 27

Claims (12)

A marker composition for diagnosis of microsatellite instability (MSI), comprising polynucleotides represented by SEQ ID NO: 1 and SEQ ID NO: 2 or polynucleotides having sequences complementary thereto. According to claim 1,
Diagnosing the microsatellite instability is characterized in that the use for predicting the prognosis of cancer, the marker composition. According to claim 2,
The prognosis is characterized in that recurrence, survival, or disease-free survival, marker composition. A primer set for diagnosing microsatellite instability (MSI), comprising a primer pair represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a primer pair represented by SEQ ID NO: 5 and SEQ ID NO: 6. According to claim 4,
Diagnosing the microsatellite instability is characterized in that the use for predicting the prognosis of cancer, the primer set. According to claim 5,
The prognosis is characterized in that recurrence, survival, or disease-free survival, the primer set. A composition for diagnosis of microsatellite instability (MSI), comprising the primer set of any one of claims 4 to 6. A kit for diagnosing microsatellite instability (MSI), comprising the composition of claim 7. A method for providing information for diagnosing microsatellite instability (MSI), including the following steps.
(a) isolating DNA from the biological sample;
(b) using the isolated DNA as a template and performing a polymerase chain reaction (PCR) using the primer set of claim 4; and
(c) confirming the PCR product amplified in step (b) by electrophoresis. According to claim 9,
Diagnosing the microsatellite instability is an information providing method, characterized in that for predicting the prognosis of cancer. According to claim 10,
The prognosis is characterized in that recurrence, survival, or disease-free survival, information providing method. According to claim 9,
The biological sample is characterized in that at least one selected from the group consisting of tissue, blood, plasma, serum, mucus, saliva, urine, sputum, and lymph fluid, information providing method.

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