WO2015105191A1 - Méthode d'évaluation des lésions lymphadénopathiques - Google Patents

Méthode d'évaluation des lésions lymphadénopathiques Download PDF

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WO2015105191A1
WO2015105191A1 PCT/JP2015/050552 JP2015050552W WO2015105191A1 WO 2015105191 A1 WO2015105191 A1 WO 2015105191A1 JP 2015050552 W JP2015050552 W JP 2015050552W WO 2015105191 A1 WO2015105191 A1 WO 2015105191A1
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seq
dna
lymphadenopathy
base
expression
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PCT/JP2015/050552
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Japanese (ja)
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総司 森下
真理人 荒木
則夫 小松
林崎　良英
昌可 伊藤
英哉 川路
寛子 大宮
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学校法人順天堂
国立研究開発法人理化学研究所
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Publication of WO2015105191A1 publication Critical patent/WO2015105191A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a technique for distinguishing neoplastic and non-neoplastic (reactive) lymphadenopathy lesions at the molecular level.
  • Malignant lymphoma is a general term for cancers of lymphoid tissues of various disease types, and many are accompanied by enlargement of lymph nodes.
  • lymph nodes often swell reactively due to infection or collagen disease (reactive lymphadenopathy), and are often difficult to differentiate from malignant lymphoma. Therefore, differentiation between neoplastic (malignant) and non-neoplastic (reactive) lymphadenopathy is important.
  • malignant lymphoma and reactive lymphadenopathy are histological information, cell morphology, flow cytometry using about 20 kinds of antibodies, histological information by tissue immunostaining based on the results of flow cytometry, etc.
  • tissue immunostaining based on the results of flow cytometry, etc.
  • a skilled specialist pathologist diagnoses. Specifically, a site exhibiting lymphatic swelling is excised, and the failure of the normal tissue is examined by histological analysis with Hematoxylin-Eosin (HE) staining (Non-patent Document 1). Since it is difficult to analyze histologically depending on the lesion, the presence or absence of tumor cells is examined mainly by analyzing the cell morphology by needle biopsy.
  • HE Hematoxylin-Eosin
  • a chromosomal abnormality is analyzed using the G-band method, and if there is a suspicion of an abnormality, or it is necessary to analyze the presence or absence of a chromosomal abnormality from a morphological analysis, the FISH method, Molecular biological analysis is performed on the presence or absence of 8-14 translocation, 11-14 translocation, 14-18 translocation, etc. by Southern blotting, PCR, etc. (Non-patent Document 1). Based on this information, a specialist pathologist diagnoses whether the lymphatic swelling is neoplastic or responsive, and finally the attending physician makes a diagnosis with other clinical findings. Yes.
  • the conventional methods require a lot of time and labor. For example, even if there is a single chromosomal abnormality, the result is not always useful for the intended diagnosis. Therefore, doctors often make subjective diagnosis based on intuition and experience, and the actual situation is that the diagnosis results may differ depending on the doctor.
  • RNA-seq Non-Patent Document 2
  • CAGE Cap Analysis Gene Expression: Non-Patent Document 3
  • the CAGE method has a feature that the activity of the transcription start site can be comprehensively quantified by selecting a long capped RNA such as mRNA and sequencing the 5 'end randomly and in large quantities.
  • the present invention relates to providing a method for objectively and quickly discriminating between malignant and benign lesions of lymphadenopathy with high accuracy.
  • the present inventors extract RNA from lesions of patients with malignant lymphoma and patients with reactive lymphadenopathy, and use the CAGE analysis method as an expression state in the vicinity of the transcription start region (Transcript Start Site; TSS) as sequence information.
  • TSS Transcript Start Site
  • the present invention relates to the following 1) to 4).
  • the DNA is a base sequence represented by SEQ ID NOs: 1 to 1037, and a base at an arbitrary position in the transcription start region and one base downstream thereof DNA consisting of the above
  • a method for evaluating a lymphadenopathy wherein the transcription initiation region is a region defined at both ends by the first base of the base sequence represented by SEQ ID NOs: 1 to 1037 and the 101st base from the 3 ′ end.
  • a test kit for evaluating a lymphadenopathy used in the method of 1) which comprises an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes the translation product of DNA.
  • the present invention it is possible to distinguish whether a lymphadenopathy lesion of a patient presenting with lymphadenopathy is neoplastic (malignant lymphoma) or non-neoplastic (reactive lymphadenopathy), thereby promptly Diagnosis is possible.
  • the differentiation between malignant lymphoma and reactive lymphadenopathy can be objectively differentiated even if it is not based on the subjectivity of an expert such as a trained clinical laboratory technician. It can be used effectively for testing (POCT: Point of Care Testing) performed by a healthcare professional alongside the patient from collection to analysis of patient specimens.
  • POCT Point of Care Testing
  • the lymphadenopathy refers to a lesion in which lymphocytes proliferate and lymph nodes such as the neck, axilla, elbow, groin, and popliteal swollen, and reactive swelling (non-neoplastic) And swelling due to tumor cell invasion and metastasis (malignant lymphoma).
  • Reactive lymphadenopathy includes swelling due to infection and swelling due to systemic lupus erythematosus, rheumatoid arthritis and sarcoidosis.
  • the evaluation of lymphadenopathy refers to differential evaluation or measurement of whether the lymphadenopathy is neoplastic or non-neoplastic.
  • Examples of the biological sample used in the present invention include lymphoid tissue collected from a lesioned part of a patient exhibiting lymphadenopathy to be evaluated or peripheral blood, lymph, bone marrow and the like containing cells derived from the lesioned part.
  • lymphocytes are separated from the sample as necessary, and when the sample is used for measurement at the nucleic acid level, an RNA extract is prepared, and when the sample is used for measurement at the protein level A protein extract is prepared.
  • Any known method can be used as a method for extracting RNA from a biological sample. Specific examples include Ambion RiboPure kit manufactured by Life Technologies, miRNeasy manufactured by Qiagen, and RNeasy manufactured by Qiagen. Among these, the miRNeasy kit manufactured by Qiagen is preferably used.
  • Examples of patients presenting with lymphadenopathy include patients with lymphadenopathy, but suspected malignant lymphoma.
  • nucleic acid or “polynucleotide” means DNA or RNA.
  • DNA includes not only double-stranded DNA but also single-stranded DNAs comprising a sense strand and an antisense strand constituting the DNA. Accordingly, the DNA includes double-stranded genomic DNA, single-stranded cDNA, single-stranded DNA having a sequence complementary to the DNA, and the like.
  • RNA includes any of total RNA, mRNA, rRNA, and synthetic RNA.
  • a transcription product of DNA consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 1037 is a sample that is a malignant lymphoma as shown in the Examples.
  • human genomic DNA consisting of a transcription initiation region and 100 nucleotides continuous downstream thereof
  • CAGE Cap Analysis Gene Expression
  • RNA RNA consisting of a base at an arbitrary position (transcription start point) in the transcription start region and one or more bases downstream thereof (hereinafter referred to as “sequence numbers 1 to 1037”).
  • An expression product (referred to as “expression product of the present invention”) (referred to as “expression product of the present invention”) (referred to as “DNA containing transcription start site”) is a biomarker for differential evaluation of malignant lymphoma and reactive lymphadenopathy That is, it can be a biomarker for differential assessment of lymphadenopathy, whether it is neoplastic or non-neoplastic.
  • the DNA expression product containing the transcription start point in SEQ ID NOs: 1 to 29 is a marker whose expression level is increased in malignant lymphoma, and the DNA expression product containing the transcription start point in SEQ ID NOs: 30 to 1037 is expressed in malignant lymphoma. A marker that lowers the level.
  • the “transfer start region” refers to a region including a transfer start point.
  • the transcription start point from a specific promoter is not limited to a single base, and may be a base present at a plurality of positions downstream of the promoter on the genome.
  • a region including a plurality of these transfer start points is referred to as a transfer start region in this specification. More specifically, the transfer start area is an area between the transfer start point located on the most 5 ′ side and the transfer start point located on the most 3 ′ side among the plurality of transfer start points.
  • the transcription initiation region has a 5 ′ end corresponding to a region defined at both ends by the base at position 1 (5 ′ end) and the 101st base from the 3 ′ end. This is a base region to be formed.
  • each of the base sequences represented by SEQ ID NOs: 1 to 1037 shows a transcription start region and 100 bases following the transcription start point located on the most 3 ′ side in the transcription start region.
  • such a transcription start region is also referred to as “a transcription start region shown in SEQ ID NOs: 1 to 1037”.
  • the positions of the transcription start regions shown in SEQ ID NOs: 1 to 1037 on the genome, and related gene information, etc. are as shown in Tables 1-1 to 1-46 described later.
  • the DNA whose expression product expression level is measured is a base sequence at any position (transcription start point) in the transcription start region in the base sequence represented by SEQ ID NOs: 1 to 1037 and 1 downstream thereof. It is DNA consisting of a base sequence of bases or more.
  • the number of bases in the downstream base sequence may be any number that can identify the expression product. Examples of the number of bases include 1 base or more, 5 bases or more, 10 bases or more, 15 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, 40 bases or more, 50 bases or more.
  • the said base number 10 bases or less, 15 bases or less, 20 bases or less, 25 bases or less, 30 bases or less, 40 bases or less, 50 bases or less, 100 bases or less are mentioned, for example.
  • the number of bases on the downstream side is not particularly necessary in the case of measurement by the CAGE method, but in the case of measurement by hybridization or PCR, any part up to about 100 bases downstream is targeted in order to ensure the accuracy.
  • the DNA consisting of the transcription initiation region and 100 bases downstream thereof if it has a length of at least 20 bases, there is a high probability that it can be identified even in an experimental system targeting the entire genome.
  • the DNA also includes DNA having a base sequence substantially identical to the base sequence of the DNA as long as the expression product can be a biomarker for discriminating malignant lymphoma and reactive lymphadenopathy.
  • Such an expression product of the present invention can be distinguished from malignant lymphoma and reactive lymphadenopathy by grasping the expression level by combining one or two or more of them.
  • SEQ ID NO: 15 Transcription start in SEQ ID NO: 3, SEQ ID NO: 24, SEQ ID NO: 18, SEQ ID NO: 10, SEQ ID NO: 499, SEQ ID NO: 9, SEQ ID NO: 27, SEQ ID NO: 5, SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 23
  • Expression products of DNA containing dots can be classified with a specificity of 100% and a sensitivity of 100% when the threshold values shown in Table 2 are set. That is, these can be surely discriminated only by one expression level.
  • the number and content of the combination can be selected as appropriate, but suitable combinations (46 sets) when using at least two DNA expression products are listed below. It was shown in 3.
  • Such a combination is a malignant lymphoma or a reactive lymphadenopathy related to a sample for extracting a transcription start region, with the expression level as an explanatory variable for all two combinations selected from the above 1037 transcription start regions.
  • a logistic regression model for estimating whether or not a transcription start region extraction sample and a verification sample are extracted from those that can be classified with 100% specificity and 100% sensitivity. For the purpose of further improving the accuracy, these can be used in combination of two sets or three sets or more as appropriate.
  • the expression products of these two DNAs may be combined with the expression products of DNAs other than those shown in Table 3 among the DNAs containing the transcription start sites in SEQ ID NOS: 1 to 1037. May be combined with DNA expression products consisting of any other base sequence within a range that can contribute to the evaluation of the present invention.
  • the expression product of the present invention includes a transcription product and a translation product expressed from the DNA.
  • the transcription product include RNA generated by transcription from the DNA, preferably mRNA.
  • Specific examples of the translation product include a protein encoded by the RNA.
  • the protein expressed from the DNA containing the transcription start point in SEQ ID NO: 15 among the DNA expression products containing the transcription start point shown in Table 3 is “FCRL3” (Fc receptor-like protein 3; UniProtKB / Swiss -Prot: FCRL3_HUMAN, Q96P31), a protein expressed from DNA containing the transcription start site in SEQ ID NO: 23 has been identified as “SYK” (Spleen Tyrosine Kinase; UniProtKB / Swiss-Prot: KSYK_HUMAN, P43405).
  • the target of the measurement or detection of the expression product is cDNA artificially synthesized from the RNA, DNA encoding the RNA, protein encoded by the RNA, molecule interacting with the protein, and interaction with the RNA. Also included are molecules that act or molecules that interact with the DNA.
  • molecules that interact with RNA, DNA or protein DNA, RNA, protein, polysaccharide, oligosaccharide, monosaccharide, lipid, fatty acid, and their phosphates, alkylated products, sugar adducts, and the like, and Any of the above-mentioned complexes can be mentioned.
  • the expression level comprehensively means the expression level and activity of the expression product.
  • RNA, cDNA or DNA is targeted as a method for measuring the expression level, nucleic acid amplification represented by PCR method, real-time RT-PCR method, SmartAmp method, LAMP method, etc., using DNA hybridizing therewith as a primer Method, hybridization method using nucleic acids that hybridize to these as probes (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, northern blot, hybridization etc.), method for determining the base sequence, or these You can choose from a combination of methods.
  • the probe or primer used for the measurement that is, the primer for specifically recognizing and amplifying the expression product (transcription product) of the present invention or the nucleic acid derived therefrom, or the RNA or the nucleic acid derived therefrom is specific.
  • “specifically recognize” means that, for example, in the Northern blot method, substantially only the expression product (transcription product) of the present invention or a nucleic acid derived therefrom can be detected. This means that the detected product or product can be determined to be the transcript or the nucleic acid derived therefrom so that only the nucleic acid is produced.
  • an oligonucleotide containing a certain number of nucleotides complementary to DNA consisting of the base sequence represented by SEQ ID NOs: 1 to 1037 or a complementary strand thereof can be used.
  • the “complementary strand” refers to the other strand with respect to one strand of double-stranded DNA comprising A: T (U in the case of RNA) and G: C base pairs.
  • “complementary” is not limited to the case where the certain number of consecutive nucleotide regions are completely complementary sequences, preferably 80% or more, more preferably 90% or more, and still more preferably 95% or more. What is necessary is just to have the identity on arrangement
  • the identity of the base sequence can be determined by the algorithm such as BLAST.
  • BLAST oligonucleotide
  • Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are mentioned.
  • probe when used as a probe, it only needs to be capable of specific hybridization, and has at least a part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence represented by SEQ ID NOs: 1 to 1037.
  • a chain length of 10 bases or more, preferably 15 bases or more and, for example, 100 bases or less, preferably 50 bases or less, more preferably 25 bases or less is used.
  • the “oligonucleotide” may be DNA or RNA, and may be synthesized or natural.
  • the probe used for hybridization is usually labeled.
  • the probe DNA is first labeled with a radioisotope, a fluorescent substance, etc., and then the obtained labeled DNA is transferred to a nylon membrane or the like according to a conventional method. Hybridize with RNA. Thereafter, a method of detecting and measuring a signal derived from the labeled product of the formed duplex of labeled DNA and RNA can be used.
  • cDNA is prepared from RNA derived from a biological sample according to a conventional method so that the target expression product of the present invention (in this case, transcription product) can be amplified.
  • a pair of prepared primers (a normal strand that binds to the cDNA ( ⁇ strand) and a reverse strand that binds to the + strand) are hybridized therewith.
  • PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected.
  • detection of the amplified double-stranded DNA use a method of detecting the labeled double-stranded DNA produced by performing the above PCR using a primer previously labeled with RI, a fluorescent substance, etc. Can do.
  • nucleic acid derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support.
  • a nucleic acid derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support.
  • labeled cDNA or cRNA prepared from mRNA is bound on the microarray, and the label on the microarray is detected, whereby the mRNA expression level can be measured.
  • the nucleic acid immobilized on the array may be any nucleic acid that hybridizes specifically (ie, substantially only to the target nucleic acid) under stringent conditions.
  • the expression product of the present invention transcription
  • the product may be a nucleic acid having the entire sequence or a partial sequence.
  • the “partial sequence” includes a nucleic acid consisting of at least 15 to 25 bases.
  • stringent conditions can usually include washing conditions of about “1 ⁇ SSC, 0.1% SDS, 37 ° C.”, and more stringent hybridization conditions include “0.5 ⁇ SSC, 0.1%.
  • a more stringent hybridization condition a condition of “0.1 ⁇ SSC, 0.1% SDS, 65 ° C.” can be mentioned.
  • Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001) and the like.
  • Examples of the method for determining the base sequence include the CAGE method, the TSS-seq method, the RNA-seq method, the DGE method, and the SAGE method, and the CAGE method is preferable.
  • the expression level is measured using the CAGE method, it can be carried out in accordance with the method described in Examples below.
  • an antibody against the expression product of the present invention in this case, a translation product
  • a biological sample an antibody against the expression product of the present invention
  • the polypeptide in the sample bound to the antibody is detected, and the level is detected.
  • This is done by measuring.
  • an antibody that binds to a primary antibody labeled with a radioisotope, a fluorescent substance, an enzyme, or the like as a secondary antibody is used. Labeling is performed, and signals derived from these labeling substances are measured with a radiation measuring instrument, a fluorescence detector or the like.
  • the antibody against the translation product may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to a known method. Specifically, the polyclonal antibody is used to immunize non-human animals such as rabbits by using a protein expressed and purified in E. coli or the like according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method.
  • a monoclonal antibody is obtained by immunizing a non-human animal such as a mouse with a protein expressed and purified in Escherichia coli according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from the prepared hybridoma cells. Monoclonal antibodies may also be prepared using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108 (7)).
  • a biological sample isolated from a patient is fixed in formalin by a conventional method, embedded in paraffin, sliced into tissue pieces, and pasted on a slide glass. It is preferably used as a section sample.
  • an enzyme-labeled antibody such as alkaline phosphatase or peroxidase can be used, but it is preferable to perform highly sensitive detection using the ABC method of Vector or the EnVision detection system of DAKO.
  • the expression level of the expression product of the present invention in a biological sample collected from a lesion part of a patient exhibiting lymphadenopathy is measured, and based on the expression level, the lesion part is neoplastic or non-neoplastic. Is identified. Specifically, the expression level of the detected expression product of the present invention is evaluated by comparing it with a control level.
  • control level means, for example, the expression level of the expression product in a diseased tissue isolated from a patient with reactive lymphadenopathy or a normal tissue isolated from a patient exhibiting lymphadenopathy, or lymphadenopathy
  • the expression level of the expression product in a group of healthy people who have not developed is within the range of the expression level close to the expression level in a diseased tissue, normal tissue or tissue derived from a healthy person isolated from a patient with reactive lymphadenopathy Or significantly higher (or lower) than the expression level, it can be evaluated that the lymph node lesion of the patient is unlikely to be neoplastic (malignant lymphoma).
  • the evaluation of the lymphadenopathy in the present invention can also be performed by increasing / decreasing the expression level of the expression product of the present invention.
  • a control level for example, a standard value (threshold level) is set based on the expression level of the expression product derived from a normal tissue, a diseased tissue isolated from a patient with reactive lymphadenopathy, or a healthy tissue. Then, the expression level of the expression product in the patient-derived biological sample can be compared with a standard value (for example, a range of ⁇ 2SD is set as an allowable range). For example, when the expression level of the expression product in a patient-derived biological sample is higher or lower than a threshold level, it can be evaluated that the lymph node lesion of the patient is unlikely to be neoplastic (malignant lymphoma).
  • lymph node swelling lesions are evaluated based on the provided information in combination with other methods (CT, MRI, PET-CT, etc.) as necessary.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET-CT PET-CT
  • the lesion may be neoplastic (malignant lymphoma), for example, steroid administration, chemotherapy, biological preparation administration, radiation therapy, and the like can be appropriately combined. If the lesion is determined to be reactive lymphadenopathy, it may not be necessary to perform these treatments.
  • the test kit for evaluating the lymphadenopathy of the present invention contains a test reagent for measuring the expression level of the expression product of the present invention in a biological sample separated from a patient.
  • a test reagent for nucleic acid amplification or hybridization containing an oligonucleotide that specifically binds (hybridizes) to the expression product (transcription product) or the like of the present invention, or the expression product (translation product) of the present invention.
  • a reagent for immunological measurement including an antibody that recognizes Oligonucleotides, antibodies and the like included in the kit can be obtained by known methods as described above.
  • the test kit can contain a labeling reagent, a buffer solution, a chromogenic substrate, a secondary antibody, a blocking agent, instruments and controls necessary for the test, and the like.
  • Example 1 Extraction and verification of transcription initiation region enabling discrimination between malignant lymphoma and reactive lymphadenopathy (1) Obtaining specimen sample The specimen (sample) is surgically excised or needle biopsyed from the lymphadenopathy site Etc. The samples used were 9 samples (including 7 samples for malignant lymphoma and 2 samples for reactive lymphadenopathy) as samples for extracting the transcription start region, and 5 samples (of which 3 samples for malignant lymphoma were reactive). There are 2 specimens of lymphadenopathy.
  • tissue pieces were appropriately frozen and stored at -80 ° C.
  • the preserved tissue piece is placed in a 2 mL microtube so that the tissue piece is 50 mg or less, QIAzol manufactured by Qiagen is added, and one zirconia bead is sealed and crushed by osmosis treatment using TissueLyser manufactured by Qiagen. did.
  • RNA was prepared for RNA according to the attached protocol using miRNeasy mini kit manufactured by Qiagen.
  • the prepared RNA was measured for ultraviolet absorption (230, 260, 280 nm) with a spectrophotometer to calculate 260/230, 260/280 ratios, and the quality of the RNA was tested.
  • electrophoresis was performed using BioAnalyzer RNA nano chip manufactured by Agilent, and the RIN value indicating the degree of RNA degradation was calculated to test the degree of RNA degradation.
  • CAGE library preparation 5 ⁇ g of purified RNA was prepared, and unamplified untagged CAGE method (“Cell Engineering, separate volume, Advanced Method for Next-Generation Sequencer Purpose”, Juno Kanno, Satoshi Suzuki, Gakken Medical Shujunsha, 2012 (Published on September 19), CAGE library was prepared by Chapter 3 3, “Exhaustive promoter analysis (non-amplified CAGE method using Illumina sequencer)”).
  • CAGE library was prepared by Chapter 3 3, “Exhaustive promoter analysis (non-amplified CAGE method using Illumina sequencer)”.
  • the purified RNA was subjected to reverse transcription reaction and purified, and then aldehyde was formed by participation of a ribose diol with sodium periodate, and biotin hydrazide was added to add biotin to the aldehyde group.
  • RNA / cDNA double strand biotinylated with avidin magnetic beads was bound to the bead surface, and the cDNA was released and recovered by RNase H digestion and heat treatment.
  • sequencing was performed using HiSeq 2500 manufactured by Illumina.
  • the standard conditions of AMPure XP (manufactured by Beckman Coulter) used for purification, buffer replacement, etc. in this step are conditions for recovering nucleic acids having a length of 100 bases or more in the case of double strands.
  • the CAGE library produced by this process using this is composed of double-stranded DNA having a chain length of 100 bases or more.
  • Example 2 Differentiation between malignant lymphoma and reactive lymphadenopathy using protein expression as an index (1) Specimen A specimen (sample) was obtained from a site of lymphadenopathy by surgical resection or needle biopsy. Among them, malignant lymphoma (diffuse large B cell lymphoma, follicular lymphoma, MALT lymphoma, Burkitt lymphoma, mantle cell lymphoma, Hodgkin lymphoma, T cell lymphoma, etc.) or reactive lymphadenopathy (Kikuchi disease, Forty-five or sixteen patients diagnosed with Castleman's disease and others) were used.
  • malignant lymphoma diffuseuse large B cell lymphoma, follicular lymphoma, MALT lymphoma, Burkitt lymphoma, mantle cell lymphoma, Hodgkin lymphoma, T cell lymphoma, etc.
  • reactive lymphadenopathy Karl-five or sixteen patients diagnosed with Castleman's disease and others
  • an expert such as a trained clinical technician, can determine whether a patient's lymphadenopathy is neoplastic (malignant lymphoma) or non-neoplastic (reactive lymphadenopathy). Even if it is not based on the subjectivity, it can be performed objectively and quickly. In other words, it can be suitably used for tests (POCT: Point of ⁇ Care Testing) performed by a medical worker from the patient's sample collection to analysis.
  • POCT Point of ⁇ Care Testing

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Abstract

　Cette invention concerne une méthode pour différencier de manière objective et rapide à une précision élevée les lésions lymphadénopathiques malignes des lésions lymphadénopathiques bénignes. La méthode d'évaluation des lésions lymphadénopathiques selon l'invention est une méthode de différenciation entre les lésions néoplasiques et non néoplasiques, ladite méthode comprenant une étape consistant à mesurer, dans un échantillon biologique prélevé sur une lésion chez un patient souffrant de lymphadénopathie, le niveau d'expression d'au moins un produit d'expression de l'ADN comprenant une région début de transcription, ledit ADN comprenant une base à une position quelconque dans la région début de transcription d'une séquence de bases représentée par SEQ ID No : 1-1037 et au moins une base à sa suite dans le sens aval, et ladite région début de transcription étant une région dont les deux extrémités sont spécifiées par la première base de la séquence de bases représentée par SEQ ID No : 1-1037 et la 101ème base à partir de l'extrémité 3'.
PCT/JP2015/050552 2014-01-10 2015-01-09 Méthode d'évaluation des lésions lymphadénopathiques WO2015105191A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021092440A1 (fr) * 2019-11-07 2021-05-14 Icahn School Of Medicine At Mount Sinai Arn modifié synthétique et ses utilisations

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006000090A (ja) * 2004-06-21 2006-01-05 Toru Takano 再構成された遺伝子の単クローン性を判定する方法、及びこれを利用した腫瘍性病変の鑑別方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006000090A (ja) * 2004-06-21 2006-01-05 Toru Takano 再構成された遺伝子の単クローン性を判定する方法、及びこれを利用した腫瘍性病変の鑑別方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KATSUMI HIGASHI: "Lymph Shu no Rinsho Kensa", THE JAPANESE SOCIETY FOR LABORATORY HEMOTOLOGY ZASSHI, vol. 11, no. 2, 2010, pages 242 - 245 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021092440A1 (fr) * 2019-11-07 2021-05-14 Icahn School Of Medicine At Mount Sinai Arn modifié synthétique et ses utilisations

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