Novel method for detecting abnormal cell having gene mutation

Abstract

Provided is a method for detecting an abnormal cell having a gene mutation, the method comprising: a step for amplifying a region corresponding to a region comprising a nucleotide sequence lying between a nucleotide located at position-1,316 and a nucleotide located at position-1,663 in SEQ ID NO:1 and / or a region corresponding to a region comprising a nucleotide sequence lying between a nucleotide located at position-1,931 and a nucleotide located at position-2,642 in SEQ ID NO: 1 to produce an amplification product, wherein SEQ ID NO:1 is included in genomic DNA derived from a peripheral blood mononuclear cell of the patient; and a step for determining the nucleotide sequence for the amplification product and detecting a nucleotide mutation. In the method, the presence of the lesion is indicated when the nucleotide mutation is detected, and the amplification is performed by a PCR using a primer having no additional sequence.

Description

A novel method for detecting abnormal cells with genetic mutations

[0001] The present invention relates to a novel method for detecting abnormal cells with genetic mutations. This application claims priority to Japanese Patent Application No. 2023-100764, filed on June 20, 2023, the contents of which are incorporated herein by reference.

[0002] Acute myeloid leukemia is a group of diseases that are subdivided into nearly 20 subgroups. Acute myeloid leukemia is classified into risk categories based on the patient's genetic background, and those in the intermediate- to high-risk group are recommended to undergo hematopoietic stem cell transplantation during first remission. Meanwhile, those in the low-risk group are recommended to undergo chemotherapy alone. However, there are cases of relapse even in the low-risk group.

[0003] Furthermore, even in the low-risk group, if minimal residual disease (MRD) is detected during the course of treatment, it is considered to be an independent risk of recurrence, and is a reason to select hematopoietic stem cell transplantation during the first remission phase.

[0004] There are various methods for detecting minimal residual disease. Known highly sensitive testing systems include a method for detecting WT1 mRNA (see, for example, Non-Patent Document 1) and FACS-MRD (see, for example, Non-Patent Document 2).

[0005] Inoue K., et al., Long-term follow-up of minimal residual disease in leukemia patients by monitoring WT1 (Wilms tumor gene) expression levels, Blood, 88 (6), 2267-2278, 1996.Campana D. and Coustan-Smith E., Detection of minimal residual disease in acute leukemia by flow cytometry, Cytometry, 38 (4),139-152, 1999. Yasuda T., et al., Clinical utility of target capture-based panel sequencing in hematological malignancies: A multicenter feasibility study, Cancer Science, 111, 3367-3378, 2020.

[0006] One type of acute myeloid leukemia with a good prognosis is known as acute myeloid leukemia with biallelic mutations in CEBPA (WHO 4th edition disease name) / acute myeloid leukemia with mutations in the bZip region of CEBPA (WHO 5th edition disease name) (hereinafter sometimes referred to as "CEBPA-acute myeloid leukemia (AML)" or "CEBPA-AML").

[0007] In CEBPA-AML, WT1 mRNA expression is low at the time of onset, and after one course of consolidation therapy, WT1 mRNA expression becomes negative in almost all cases. Furthermore, in the detection of minimal residual disease by FACS-MRD, although the recurrence rate differs between positive and negative cases, the false positive and false negative rates have been reported to be around 30-50%, resulting in low sensitivity and specificity.

[0008] A retrospective cohort study of CEBPA-AML conducted by the inventors also showed that neither the level of WT1 mRNA expression nor the detection of minimal residual disease by FACS-MRD was sufficiently sensitive or specific for predicting recurrence.

[0009] Therefore, an object of the present invention is to provide a technique for detecting lesions in patients with acute myeloid leukemia.

[0010] The present invention includes the following aspects: [1] A method for detecting a lesion in a patient with acute myeloid leukemia, comprising the steps of: amplifying a region corresponding to a region containing bases 1,316 to 1,663 of SEQ ID NO: 1 and / or a region corresponding to a region containing bases 1,931 to 2,642 of SEQ ID NO: 1 in genomic DNA derived from peripheral blood mononuclear cells of the patient to obtain an amplification product; and determining the base sequence of the amplification product to detect a base mutation, wherein detection of the base mutation indicates the presence of the lesion, and the amplification is carried out by a PCR reaction using primers without an additional sequence. [2] The detection method according to [1], wherein the base sequence of the amplification product is determined by next-generation sequencing. [3] The detection method according to [2], wherein the next-generation sequencing is carried out by paired-end sequencing. [4] A kit for detecting lesions in patients with acute myeloid leukemia, comprising primers for amplifying a region corresponding to a region containing the 1,316th to 1,663rd bases of the nucleotide sequence of SEQ ID NO: 1 and / or a region corresponding to a region containing the 1,931st to 2,642nd bases of the nucleotide sequence of SEQ ID NO: 1, wherein the primers do not contain any additional sequence.

[0011] According to the present invention, a technique for detecting minimal residual disease in acute myeloid leukemia patients after chemotherapy can be provided.

[0012] FIG. 1 is a schematic diagram of the CEBPA gene (genomic DNA).

[0013] [Method for detecting lesions in acute myeloid leukemia patients] In one embodiment, the present invention provides a method for detecting lesions in acute myeloid leukemia patients, comprising the steps of: amplifying a region corresponding to a region containing bases 1,316 to 1,663 of SEQ ID NO: 1 and / or a region corresponding to a region containing bases 1,931 to 2,642 of SEQ ID NO: 1 in genomic DNA derived from peripheral blood mononuclear cells of the patient to obtain an amplification product; and determining the base sequence of the amplification product and detecting a base mutation, wherein detection of the base mutation indicates the presence of the lesion, and the amplification is performed by a PCR reaction using primers without additional sequences. As described below in the Examples, the inventors have demonstrated that the method of this embodiment can detect lesions in acute myeloid leukemia patients.

[0014] Examples of acute myeloid leukemia include acute myeloid leukemia with biallelic mutations in CCAAT Enhancer Binding Protein Alpha (CEBPA) (WHO 4th Edition disease name) and acute myeloid leukemia with mutations in the bZip region of CEBPA (WHO 5th Edition disease name) (CEBPA-AML). The CEBPA gene (genomic DNA) corresponds to positions 33,299,934 to 33,302,534 on chromosome 19 of the human reference genome Hg38 (NCBI accession number: NC_000019.10).

[0015] In the method of this embodiment, a lesion in an acute myeloid leukemia patient refers to a cancerous cell that has a mutation in the CEBPA gene. That is, detecting a lesion in an acute myeloid leukemia patient means detecting the presence of a cell that has a mutation in the CEBPA gene.

[0016] In the method of this embodiment, a region corresponding to the region containing bases 1,316 to 1,663 of SEQ ID NO: 1 and / or a region corresponding to the region containing bases 1,931 to 2,642 of SEQ ID NO: 1 in genomic DNA derived from peripheral blood mononuclear cells of an acute myeloid leukemia patient is amplified to obtain an amplification product. As used herein, a region corresponding to a certain base sequence refers to a region that is highly homologous when the base sequence is subjected to homology analysis (alignment analysis) with genomic DNA, or a region that is mapped when the base sequence is mapped to genomic DNA. Here, "high homology" means, for example, that the sequence identity between the compared sequences is 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. A region corresponding to a certain base sequence can also be referred to as a region corresponding to a certain base sequence. SEQ ID NO: 1 is the base sequence of the genomic DNA of the CEBPA gene. The base sequence of SEQ ID NO: 1 corresponds to bases 33,299,934 to 33,303,000 of the base sequence of NCBI Accession Number: NC_000019.10.

[0017] Peripheral blood mononuclear cells can be collected from patient-derived blood by a conventional method, typically by density gradient centrifugation using a commercially available lymphocyte separation solution.

[0018] Figure 1 is a schematic diagram of the CEBPA gene (genomic DNA). In Figure 1, the numbers in the upper left and upper right corner indicate positions in the base sequence of NCBI accession number: NC_000019.10. Each row in Figure 1 corresponds to a CEBPA-AML patient case. The region corresponding to the region containing bases 1,316 to 1,663 of SEQ ID NO: 1 corresponds to region A, and the region corresponding to the region containing bases 1,931 to 2,642 of SEQ ID NO: 1 corresponds to region B.

[0019] As shown in Figure 1, two types of mutations, mutations in region A and mutations in region B, exist in each CEBPA-AML patient case, and the nature of these mutations varies from case to case. This makes it difficult to design primers for quantitative real-time PCR to detect lesions in acute myeloid leukemia patients.

[0020] Furthermore, the A and B regions of the genomic DNA of the CEBPA gene are GC-rich. Therefore, PCR amplification of these regions results in the generation of abnormal PCR products. PCR amplification often involves adding additional base sequences for molecular barcoding or secondary PCR to primers. In response to this, the inventors discovered that PCR amplification of the region corresponding to the region containing bases 1,316 to 1,663 of SEQ ID NO: 1 (region A) and / or the region corresponding to the region containing bases 1,931 to 2,642 of SEQ ID NO: 1 (region B) can be suppressed by PCR using primers without additional sequences. A primer without additional sequences refers to a primer without an additional sequence. In other words, the entire primer is complementary to the genomic DNA. After amplification of the genomic DNA, PCR amplification using primers with additional sequences may be performed. Examples of additional sequences include adapter sequences for next-generation sequencing, as described below.

[0021] The inventors also found that mutations observed in cases of CEBPA-AML patients are often deletion mutations or insertion mutations, and that these mutations are rarely observed in abnormal PCR products. Therefore, by performing PCR amplification of genomic DNA using primers without additional sequences and determining the nucleotide sequence of the amplified product, it is possible to determine the nucleotide sequence of the CEBPA gene in genomic DNA derived from peripheral blood mononuclear cells of CEBPA-AML patients and detect nucleotide mutations in regions A and B, or in either region A or B.

[0022] The method of this embodiment may be used for diagnosing acute myeloid leukemia (CEBPA-AML). As will be described later in the Examples, the method of this embodiment enables diagnosis of acute myeloid leukemia with higher accuracy than the conventional capture panel method.

[0023] Alternatively, the method of this embodiment may be used to detect lesions in patients with acute myeloid leukemia after chemotherapy. Here, the lesion may be minimal residual disease (MRD). As will be described later in the Examples, the results of detecting minimal residual disease after chemotherapy using the method of this embodiment correlate with the relapse rate of acute myeloid leukemia. In other words, the method of this embodiment can predict the relapse of acute myeloid leukemia, and therefore can provide information for determining whether hematopoietic stem cell transplantation should be performed during the first remission phase.

[0024] In the method of this embodiment, the base sequence of the amplification product is preferably determined by next-generation sequencing, which can significantly reduce the labor required for sequencing.

[0025] Next-generation sequencing is preferably performed by paired-end sequencing. Paired-end sequencing allows for more accurate sequencing because the amplified product is sequenced from both ends and then spliced ​​together. Furthermore, to reduce sequencing errors, it is preferable to programmatically remove base sequences that cannot be sequenced properly.

[0026] The length of the base sequence that can be determined by next-generation sequencing (read length) is approximately 200 bp. Therefore, it is preferable to select primers so that the length of the amplified product of region A and / or region B is 200 bp or less. When paired-end sequencing is performed, it is preferable to select primers so that the length of the amplified product of region A and / or region B is 400 bp or less.

[0027] The region corresponding to the region containing bases 1,316 to 1,663 of SEQ ID NO: 1 (region A) may be amplified by PCR using one set of primers. On the other hand, the region containing bases 1,931 to 2,642 of SEQ ID NO: 1 (region B) is 600 bp or longer, so it is preferably amplified by dividing it into two or more regions. Furthermore, PCR amplification of region A and / or region B may be performed using multiple primer sets, respectively, to confirm base mutations multiple times.

[0028] The primer set for amplifying region A is not particularly limited as long as it can amplify this region, and specific examples include a primer set of 5'-GGCCAAGAAGTCGGTGGACA-3' (SEQ ID NO: 2) and 5'-CTCGGGCAAGCCTCGAGAT-3' (SEQ ID NO: 3). The primer set of SEQ ID NO: 2 and SEQ ID NO: 3 can amplify the region from bases 33,301,249 to 33,301,596 in the nucleotide sequence of the genomic DNA of the CEBPA gene (nucleotide sequence of NCBI accession number: NC_000019.10).

[0029] The primer set for amplifying region B is not particularly limited as long as it can amplify this region, but specific examples include a combination of a primer set of 5'-TCCATCGACATCAGCGCCTA-3' (SEQ ID NO: 4) and 5'-GGCGGCTGGTAAGGGAAGAG-3' (SEQ ID NO: 5), and a primer set of 5'-GGCCTGCCGGGTATAAAAGC-3' (SEQ ID NO: 6) and 5'-GCCAGGAACTCGTCGTTGAA-3' (SEQ ID NO: 7).

[0030] The set of primers of SEQ ID NO: 4 and SEQ ID NO: 5 can amplify a region from base 33,301,864 to base 33,302,234 in the nucleotide sequence of the genomic DNA of the CEBPA gene (nucleotide sequence of NCBI accession number: NC_000019.10).

[0031] The set of primers of SEQ ID NO: 6 and SEQ ID NO: 7 can amplify the region from bases 33,302,179 to 33,302,575 in the genomic DNA base sequence of the CEBPA gene (base sequence of NCBI accession number: NC_000019.10). That is, the set of primers of SEQ ID NOs: 4, 5, 6, and 7 amplifies region B by dividing it into two regions.

[0032] [Kit for detecting lesions in acute myeloid leukemia patients] In one embodiment, the present invention provides a kit for detecting lesions in acute myeloid leukemia patients, the kit comprising primers for amplifying a region corresponding to a region containing base sequence from bases 1,316 to 1,663 of SEQ ID NO: 1 and / or a region corresponding to a region containing base sequence from bases 1,931 to 2,642 of SEQ ID NO: 1, wherein the primers do not contain any additional sequence. The kit of this embodiment allows the above-described method for detecting lesions in acute myeloid leukemia patients to be preferably carried out.

[0033] In the kit of this embodiment, the acute myeloid leukemia patients, the lesions, the primers not containing additional sequences, etc. are the same as those described above.

[0034] The primer set for amplifying the region corresponding to the region containing the 1,316th to 1,663rd bases of the base sequence of SEQ ID NO: 1 (region A) is not particularly limited as long as it can amplify this region, and specific examples include the primer set of SEQ ID NO: 2 and SEQ ID NO: 3 described above.

[0035] The specific primer set for amplifying the region corresponding to the region containing the 1,931st to 2,642nd bases of SEQ ID NO: 1 (region B) is not particularly limited as long as it can amplify this region, and specific examples include a combination of the primer set of SEQ ID NO: 4 and SEQ ID NO: 5 described above, and the primer set of SEQ ID NO: 6 and SEQ ID NO: 7 described above.

[0036] The kit of this embodiment may include either a primer set for amplifying a region corresponding to region A or a primer set for amplifying a region corresponding to region B, or may include both a primer set for amplifying a region corresponding to region A and a primer set for amplifying a region corresponding to region B.

[0037] [Other Embodiments] In one embodiment, the present invention provides a method for treating a patient with acute myeloid leukemia, comprising: (a) amplifying a region corresponding to a region including base sequences from bases 1,316 to 1,663 of SEQ ID NO: 1 and / or a region corresponding to a region including base sequences from bases 1,931 to 2,642 of SEQ ID NO: 1 in genomic DNA derived from peripheral blood mononuclear cells of the patient to obtain an amplification product, wherein the amplification is performed by a PCR reaction using primers without additional sequences; (b) determining the base sequence of the amplification product and detecting a base mutation, wherein detection of the base mutation indicates the presence of a lesion in the acute myeloid leukemia patient; and (c) if the base mutation is detected, performing hematopoietic stem cell transplantation to the patient in the first remission phase.

[0038] In the treatment method of this embodiment, the acute myeloid leukemia patient, the lesion, the primer not containing an additional sequence, etc. are the same as those described above.

[0039] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0040] Experimental Example 1 (Detection of Lesions Using Samples from Acute Myeloid Leukemia Patients) Peripheral blood mononuclear cells were collected from a blood sample of a CEBPA-AML patient (at the time of initial onset), and DNA was extracted. Subsequently, PCR reactions were performed using the primer set of SEQ ID NO: 2 and SEQ ID NO: 3, the primer set of SEQ ID NO: 4 and SEQ ID NO: 5, and the primer set of SEQ ID NO: 6 and SEQ ID NO: 7, and amplification products were obtained. The primers of SEQ ID NOs: 2, 3, 4, 5, 6, and 7 had the same base sequence as the genomic DNA and contained no additional sequence.

[0041] As described above, PCR using the primer set of SEQ ID NO: 2 and SEQ ID NO: 3 can amplify the region from base 33,301,249 to base 33,301,596 in the nucleotide sequence of the genomic DNA of the CEBPA gene (the nucleotide sequence of NCBI accession number: NC_000019.10) (the region from base 1,316 to base 1,663 in SEQ ID NO: 1, a region including region A).

[0042] Furthermore, by PCR using the primer set of SEQ ID NO: 4 and SEQ ID NO: 5, a region from base 33,301,864 to base 33,302,234 in the base sequence of the genomic DNA of the CEBPA gene (base sequence of NCBI accession number: NC_000019.10) (a region from base 1,931 to base 2,301 in SEQ ID NO: 1, a part of region B) can be amplified.

[0043] Furthermore, by PCR using the primer set of SEQ ID NO: 6 and SEQ ID NO: 7, a region from base 33,302,179 to base 33,302,575 in the base sequence of the genomic DNA of the CEBPA gene (base sequence of NCBI accession number: NC_000019.10) (a region including bases 2,246 to 2,642 in the sequence of SEQ ID NO: 1, a part of region B) can be amplified.

[0044] Next, a sequencing adapter sequence was added to the amplified product using a commercially available kit (product name: "Kapa Hyper Prep Kit" from Kapa Biosystems), followed by paired-end sequencing using a sequencer (product name: "MiSeq" from Illumina).

[0045] Tables 1 and 2 below show the case numbers, mutation locations, mutation details, and mutation detection results for CEBPA-AML patients. (A) after the case number indicates that the mutation is in region A. Also, (B) after the case number indicates that the mutation is in region B.

[0046] The mutation location indicates the location on chromosome 19 of the human reference genome Hg38 (NCBI accession number: NC_000019.10). The mutation details indicate the specific mutation at the mutation location. The detection results are shown as the number of paired-end sequenced reads in the denominator, and the number of reads containing the CEBPA mutation for each case in the numerator.

[0047] In this experiment, samples from the initial onset of CEBPA-AML were used, and therefore, reads having mutations were detected at a rate of approximately 50%.

[0048]

[0049]

[0050] Conventional gene mutation testing performed at the initial diagnosis of acute myeloid leukemia includes the capture panel method (see, for example, Non-Patent Document 3). The capture panel method involves fragmenting genomic DNA to approximately 150-250 bases, recovering gene regions of interest using complementary probes, and sequencing them using a next-generation sequencer. In principle, the capture panel method makes it difficult to detect insertions or deletions of long base sequences. The inventors found a mutation only in the B region of CEPBA in gene mutation testing using the capture panel method. However, in one case where the clinical course and surface phenotype suggested CEBPA-AML with mutations in both the A and B regions, the present experimental method confirmed that the patient had a 45-base insertion mutation in the A region. This result demonstrates that the present experimental method can diagnose CEBPA-AML with higher accuracy than the capture panel method.

[0051] The above results indicate that lesions in acute myeloid leukemia patients could be detected by amplifying the genomic DNA of acute myeloid leukemia patients through PCR reactions using primers without additional sequences and sequencing the amplified products with a next-generation sequencer.

[0052] [Experimental Example 2] (Study of Lesion Detection Accuracy) A sample derived from an acute myeloid leukemia patient was diluted to study the lesion detection accuracy. Specifically, peripheral blood mononuclear cells were collected from a blood sample of a CEBPA-AML patient (Case 107, at the time of initial onset) and mixed with peripheral blood mononuclear cells derived from a healthy subject so that the patient-derived cells were present at 10%, 1%, 0.1%, and 0.01%. As a negative control, peripheral blood mononuclear cells derived from a healthy subject that had not been mixed with patient-derived cells were used.

[0053] Subsequently, DNA was extracted from each cell, and the B region of the genomic DNA of the CEBPA gene was amplified by PCR using the primer set of SEQ ID NO:6 and SEQ ID NO:7.

[0054] Next, a sequencing adapter sequence was added to the amplified product using a commercially available kit (product name: "Kapa Hyper Prep Kit" from Kapa Biosystems), followed by paired-end sequencing using a sequencer (product name: "MiSeq" from Illumina).

[0055] Table 3 below shows the results of sequencing and analyzing approximately 20,000 reads to detect mutations. In Table 3, the detection results are shown as the number of reads for which paired-end sequencing was possible in the denominator, and the number of reads containing CEBPA mutations in the numerator. As a result, the number of detected mutations decreased in accordance with the mixing ratio of patient-derived cells.

[0056]

[0057] Next, samples containing 0.1% and 0.01% patient-derived cells were sequenced and analyzed using approximately 400,000 reads to detect mutations.

[0058] The results are shown in Table 4 below. In Table 4, the detection results are shown as the number of reads for which paired-end sequencing was possible in the denominator, and the number of reads among them that had a CEBPA mutation in the numerator.

[0059]

[0060] As a result, no mutations were detected in the negative control sample derived from a healthy individual, whereas mutations were detected at rates of 0.02% and 0.002% in samples containing 0.1% and 0.01% patient-derived cells.

[0061] This result indicates that this detection system can detect mutations with high specificity down to a rate of about 0.01% of mutant cells.

[0062] [Experimental Example 3] (Study of the prognosis of acute myeloid leukemia patients after chemotherapy) Using the same method as in Experimental Example 1, minimal residual disease was detected in acute myeloid leukemia patients during chemotherapy and at the end of chemotherapy. As a result, it was possible to track the time of disappearance of minimal residual disease and the persistence of persistent residual disease during the course of treatment. This indicates that detection during treatment makes it possible to determine whether additional treatment is necessary. Furthermore, while many patients who were positive for minimal residual disease after the final chemotherapy relapsed, very few patients who were negative for minimal residual disease after the final chemotherapy relapsed, and the relapse-free survival rate between the two groups was calculated to be significantly different at P<0.01 using the Wilcoxon method.

[0063] The present invention provides a technique for detecting lesions in patients with acute myeloid leukemia. This detection technique enables diagnosis of acute myeloid leukemia with CEBPA mutations with higher accuracy than conventional methods (capture panel methods). Furthermore, it enables prediction of the prognosis of acute myeloid leukemia patients after chemotherapy, and can be used as information for deciding on additional treatments such as hematopoietic stem cell transplantation.

Claims

1. A method for detecting a lesion in a patient with acute myeloid leukemia, comprising the steps of: amplifying a region corresponding to the region including base sequences from base 1,316 to base 1,663 of SEQ ID NO:1 and / or a region corresponding to the region including base sequences from base 1,931 to base 2,642 of SEQ ID NO:1 in genomic DNA derived from peripheral blood mononuclear cells of the patient to obtain an amplification product; and determining the base sequence of the amplification product and detecting base mutations, wherein detection of the base mutations indicates the presence of the lesion, and the amplification is performed by a PCR reaction using primers without additional sequences.

2. The detection method according to claim 1, wherein the base sequence of the amplification product is determined by next-generation sequencing.

3. The detection method according to claim 2, wherein the next-generation sequencing is performed by paired-end sequencing.

4. A kit for detecting lesions in patients with acute myeloid leukemia, comprising primers for amplifying a region corresponding to a region including the 1,316th to 1,663rd bases of the nucleotide sequence of SEQ ID NO:1 and / or a region corresponding to a region including the 1,931st to 2,642nd bases of the nucleotide sequence of SEQ ID NO:1, wherein the primers do not contain an additional sequence.