WO2015105191A1 - Method for assessing lymphadenopathy lesions - Google Patents

Abstract

　Provided is a method for objectively and quickly differentiating with high accuracy between malignant and benign lymphadenopathy lesions.　This method for assessing lymphadenopathy lesions is a method for differentiating between neoplastic and non-neoplastic lesions, said method including a step for measuring, in a biological sample taken from a lesion in a patient presenting with lymphadenopathy, the level of expression of at least one expression product of DNA including a transcription start region, wherein said DNA comprises a base at any position in the transcription start region of a base sequence indicated by SEQ ID NO: 1-1037 and at least one base continuing downstream therefrom, and said transcription start region is a region in which both ends thereof are specified by the first base of the base sequence indicated by SEQ ID NO: 1-1037 and the 101th base from the 3' end.

Description

Method for evaluating lymphadenopathy

(Related applications and incorporation by reference)
This application claims the priority of Japanese Patent Application No. 2014-003192 filed on Jan. 10, 2014, the entire contents of which are hereby incorporated by reference.
Also, all documents cited herein are incorporated by reference in their entirety for all purposes. Citation of any document should not be construed as an admission that it is prior art with respect to the present invention.

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. On the other hand, 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.

Until now, 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. Based on molecular biological information such as the presence or absence of chromosomal translocation by FISH method, G-band method, Southern blot method, PCR method, 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.

When cells can be prepared from the specimen, (CD5 × CD20), (CD7 × TCR-alpha beta), (TCR-gamma delta × CD30), (CD19 × CD13), (CD45 × CD22), (CD4 XCD8), (CD3 * CD56), (CD10 * CD2), (lamda-ch * kappa-ch), etc. are combined to obtain information on the cell population in the tissue. Based on this information, further immunohistochemical staining using antibodies against CD5, CD10, CD20, CD23, CD56, Myc, Cyclin D, Bcl2, Bcl6, etc. is performed, and cells associated with morphological abnormalities in lymphoid tissues Information on the cell biological characteristics is acquired (Non-patent Document 1).

On the other hand, when the presence or absence of 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.

Thus, 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.

On the other hand, in recent years, gene expression analysis (expression) analysis), in which genes expressed in cells in a certain state are comprehensively analyzed by comparing gene expression states, and their types and expression levels are compared between cells. Techniques for have been developed. For example, RNA-seq (Non-Patent Document 2) and CAGE (Cap Analysis Gene Expression: Non-Patent Document 3) that comprehensively analyze the expression state of the gene at the transcription start site as sequence information are known. Among these, 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.

However, the relationship between the expression level of the transcription initiation region in the human genome and a specific disease has never been reported so far.

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. As a result of exhaustive analysis, it was found that the expression level of DNA including a specific transcription initiation region is significantly different between the two, and that malignant lymphoma and reactive lymphadenopathy can be discriminated using this as an index.

That is, the present invention relates to the following 1) to 4).
1) A step of measuring the expression level of one or more expression products of DNA containing a transcription initiation region for a biological sample collected from a lesion of a patient presenting with lymphadenopathy, wherein the lesion is neoplastic Wherein 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.
2) 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.
3) Use of one or more expression products of DNA containing a transcription initiation region as a marker for differentially evaluating whether a lymphadenopathy is neoplastic or non-neoplastic, In the base sequence represented by SEQ ID NOs: 1 to 1037, the DNA comprises a base at an arbitrary position in the transcription initiation region and one or more bases continuous downstream thereof,
Use of the expression product, 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.
4) A method for evaluating a lymphadenopathy, comprising a step of measuring the expression level of one or more proteins selected from FCRL3 and SYK for a biological sample collected from a lesion of a patient exhibiting lymphadenopathy.

According to 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. In addition, by using the present invention, 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.

In the present invention, 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.
In the present invention, 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. When the biological sample is used for measurement, 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.

In the present specification, the term “nucleic acid” or “polynucleotide” means DNA or RNA. In addition, “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.

In the present invention, a transcription product of DNA consisting of the nucleotide sequence shown in SEQ ID NOs: 1 to 1037 (human genomic DNA consisting of a transcription initiation region and 100 nucleotides continuous downstream thereof) is a sample that is a malignant lymphoma as shown in the Examples. As a result of comprehensive analysis of the expression state of DNA of 100 bases or more downstream including the transcription initiation region on the genome, using a CAGE (Cap Analysis Gene Expression) analysis method, There was a significant difference in the expression level (transcriptional activity) between lymphoma and reactive lymphadenopathy. Specifically, regarding the transcriptional activity of RNA, a differential analysis between a clinical specimen-derived profile group obtained from “malignant lymphoma” and a clinical specimen-derived profile group obtained from “reactive lymphadenopathy” was performed by R / Bioconductor. An edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40) was used, and FDR (false discovery rate) 5% was set as a threshold value and extracted.
Therefore, in the base sequences shown in SEQ ID NOs: 1 to 1037, DNA 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.

In the present invention, 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. In each of the nucleotide sequences represented by SEQ ID NOs: 1 to 1037, 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. In other words, 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. In the present specification, 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.

In the present invention, 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.
Here, 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. Moreover, as 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. In 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. . Here, the substantially identical base sequence is, for example, using the homology calculation algorithm NCBI BLAST, expectation value = 10; allow gap; filtering = ON; match score = 1; mismatch score = −3 When searching, it means that there is 90% or more, preferably 95% or more, and even more preferably 98% or more identity with the base sequence shown in SEQ ID NOs: 1 to 227.

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. Among these, 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.

In addition, when the expression level is confirmed by combining a plurality of expression products, 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. Further, in addition to the combination of the expression products of these two DNAs, it 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. Specific examples of 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. For example, 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. Here, as 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.

When 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.

Here, 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. This corresponds to the probes for detection in the above, and these can be designed based on the nucleotide sequences represented by SEQ ID NOs: 1 to 1037. Here, “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.
Specifically, 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. Here, 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. In addition, “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 | sequence. The identity of the base sequence can be determined by the algorithm such as BLAST.
When such an oligonucleotide is used as a primer, it only needs to be capable of specific annealing and chain extension, and is usually 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and for example 100 bases or less. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are mentioned. Further, 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.
Here, the “oligonucleotide” may be DNA or RNA, and may be synthesized or natural. Moreover, the probe used for hybridization is usually labeled.

For example, when using the Northern blot hybridization method, 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.

When using the RT-PCR method, first, 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. Thereafter, PCR is performed according to a conventional method, and the obtained amplified double-stranded DNA is detected. For 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.

When measuring the expression level of mRNA in a sample using a DNA microarray, at least one nucleic acid (cDNA or DNA) derived from the expression product of the present invention (in this case, a transcription product) is immobilized on a support. Using the array, 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. For example, the expression product of the present invention (transcription) The product may be a nucleic acid having the entire sequence or a partial sequence. Here, the “partial sequence” includes a nucleic acid consisting of at least 15 to 25 bases.
Here, 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%. As 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.
When the expression level is measured using the CAGE method, it can be carried out in accordance with the method described in Examples below.

When measuring a protein (translation product) encoded by DNA containing the transcription start site in SEQ ID NOs: 1 to 1037, a molecule that interacts with the protein, a molecule that interacts with RNA, or a molecule that interacts with DNA Protein chip analysis, immunoassay (for example, ELISA etc.), 1-hybrid method (PNAS 100, 12271-12276 (2003)) and 2-hybrid method (Biol. Reprod. 58, 302-311 (1998)) Such a method can be used and can be appropriately selected depending on the object.
For example, when a protein is used as a measurement target, an antibody against the expression product of the present invention (in this case, a translation product) is brought into contact with a biological sample, the polypeptide in the sample bound to the antibody is detected, and the level is detected. This is done by measuring. For example, according to Western blotting, after using the above-described antibody as a primary antibody, 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.
On the other hand, 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)).
When immunohistochemical analysis is performed, 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. As the secondary antibody, 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.

Thus, 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.
Here, the “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
For example, the expression level of the expression product in the lesion of the target patient 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).

Further, 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. In this case, as 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).

In accordance with the method of the present invention, lymph node swelling lesions are evaluated based on the provided information in combination with other methods (CT, MRI, PET-CT, etc.) as necessary. When it is determined that 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. Specifically, a 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. And 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.
In addition to the antibody and nucleic acid, 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.

(2) Storage and preparation of sample The extracted 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.

(3) Preparation of RNA The sample subjected to the crushing and extraction treatment 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. In addition, 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.

(4) CAGE library preparation 5 μg 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)”). Specifically, 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. After digesting and purifying the single-stranded RNA portion with RNase I, only the 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. After adapters necessary for sequencing were connected to both ends of the collected cDNA, 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.

(5) RNA expression analysis i) Preparation of reference transcription initiation region Project to profile the activity of transcription initiation sites measured in genome-wide for a total of about 1000 human samples including human primary cultured cells, cell lines, and tissues. Among the transcription initiation regions identified in “FANTOM5” (submitting paper), about 180,000 transcription initiation regions defined on the human reference genome hg19 were used as reference transcription initiation regions.

ii) Quantification of transcriptional activity The reads obtained by sequencing and the human reference genome (hg19) were aligned using bwa (Bioinformatics. 2009 Jul 15; 25 (14): 1754-60). Only alignments with a mapping quality of 20 or more and an alignment start position within the reference transcription start region were selected, and the number of reads for each transcription start region was counted. Using the total number of reads in each library and the library size estimated by the RLE (Genome Biol. 2010; 11 (10): R106) method, the count is converted to counts per million. .

(6) Results (A) Extraction of transcription start regions with different activities Regarding the transcriptional activity in each sample for extracting transcription start regions quantified above, a profile group derived from clinical specimens obtained from “malignant lymphoma”, “Reaction Difference analysis between clinical specimen-derived profile groups obtained from “sympathetic lymphadenopathy” was performed using the R / Bioconductor edgeR package (Bioinformatics. 2010 Jan 1; 26 (1): 139-40). In other words, whether the average expression level is different between the two groups (if the average hypothesis is equal to the null hypothesis and this null hypothesis is true, the probability that the measurement result will occur by chance Is calculated statistically. When 5% FDR (false discovery rate) was set as a threshold, 1037 DNAs containing a transcription initiation region smaller than this were identified (Table 1). This criterion is statistically estimated that 95% of the candidates extracted by the corresponding threshold are truly differential in expression, and the commonly used P value (assuming there is no differential expression) This is a stricter standard than when the probability of accidental occurrence is 5%.

(B) Selection of transfer start region for high-precision prediction (1)
Consider whether it is possible to classify malignant lymphoma or reactive lymphadenopathy using only one expression level among the transcription initiation regions identified in (A) above. By setting some threshold for each, it was confirmed that both the transcription start region extraction sample and the verification sample can be classified with 100% specificity and 100% sensitivity. Table 2 shows examples of the threshold values (when the maximum value in one group is smaller than the minimum value in the other group, the average of these values is shown in Table 2).

(C) Selection of transfer start region for performing highly accurate prediction (2)
Consider whether it is possible to classify malignant lymphoma or reactive lymphadenopathy using a plurality of the expression levels of 1037 transcription initiation regions identified above. As an example, consider the construction of a logistic regression model, which is a kind of generalized linear model. This is a dependent variable (Y) indicating whether it is malignant lymphoma or reactive lymphadenopathy, and is an explanatory variable (Xi, logarithm of the above counts per million) that is the expression level of the transcription initiation region. When considering probabilistic prediction, it is one of the simplest models.

For all two combinations (537,166) selected from the above 1037 transcription initiation regions, these expression levels are used as explanatory variables, and the sample for extracting transcription initiation region is malignant lymphoma or reactive lymphadenopathy A logistic regression model was constructed to estimate whether or not the transcription start region extraction sample and the verification sample were classified with 100% specificity and 100% sensitivity (Table 3).
Here, a logistic regression model, which is one of the simplest machine learning devices, is adopted. However, a method for measuring the expression of the TSS, the expression level of other genes, combinations with genotypes, etc. By using other mathematical models appropriately, more robust prediction is possible.

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.

(2) Detection of protein by immunochemical staining Using FCRL3 and SYK as malignant lymphoma markers, the expression of each protein was evaluated by immunochemical staining using these antibodies.
i) Antibody • Anti-FCRL3 antibody (Abcam, “ab121471”)
Anti-SYK antibody (Santa Cruz, “sc-1240”)

ii) Immunochemical staining A biological sample separated from a patient was fixed in formalin by a conventional method, embedded in paraffin, sliced into tissue pieces, and pasted on a slide glass to obtain a slice sample. Next, the section sample was heat-treated (121 ° C., 10 minutes, citrate buffer solution (pH 6.0)) to activate the antigen. Subsequently, an antibody against each marker protein (primary antibody, concentration 0.01 to 4 μg / mL) was reacted overnight (4 ° C.). After thoroughly washing with a buffer solution, a reaction was carried out for 30 minutes (20 ° C.) using an avidin-labeled goat anti-mouse IgS or goat anti-rabbit IgG as a secondary antibody, and then a biotin-peroxidase complex was reacted. After thoroughly washing with a buffer solution, color was developed using DAB. Finally, after nuclear staining with a hematoxylin solution, it was washed with distilled water, and the specimen was observed for positive / negative under an optical microscope.
The pathologist defined the tumor cells in the specimen based on the size of the nucleus and the tissue structure, and determined the ratio of the antibody-positive cells in the tumor cells. The results are shown in Table 4.

In accordance with the present invention, 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 利用 Care Testing) performed by a medical worker from the patient's sample collection to analysis.

Claims (9)

1. A biological sample collected from a lesion of a patient exhibiting lymphadenopathy, the step of measuring the expression level of one or more expression products of DNA including a transcription initiation region, wherein the lesion is tumorous A method for differentially evaluating whether the DNA is non-tumor or not, wherein the DNA is a base sequence represented by SEQ ID NOs: 1 to 1037 and a base at an arbitrary position in the transcription initiation region and at least 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.
2. The nucleotide sequences represented by SEQ ID NO: 1 to 1037 are SEQ ID NO: 15, 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. The method according to claim 1, wherein the nucleotide sequence is represented by SEQ ID NO: 22, SEQ ID NO: 21 and SEQ ID NO: 23.
3. The method according to claim 1, wherein the base sequences represented by SEQ ID NOs: 1 to 1037 are combinations of base sequences represented by SEQ ID NOs: 1) to 46) below.
   

   
4. The method according to any one of claims 1 to 3, further comprising a step of comparing the expression level of the expression product of the DNA with a control level.
5. The method according to any one of claims 1 to 3, further comprising a step of comparing the expression level of the expression product of the DNA with a threshold level.
6. The method according to any one of claims 1 to 5, wherein the expression level of the expression product is measured by measuring the amount of the transcription product or the amount of the translation product.
7. The lymphadenopathy used in the method according to any one of claims 1 to 6, comprising an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes the translation product of DNA. Inspection kit for.
8. Use of one or more expression products of DNA containing a transcription initiation region as a marker for differential evaluation of whether a lymphadenopathy is neoplastic or non-neoplastic, Is a DNA comprising a base at an arbitrary position in the transcription initiation region and one or more bases continuous downstream thereof in the base sequence represented by SEQ ID NOs: 1 to 1037,
   Use of the expression product, 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.
9. A method for evaluating lymphadenopathy, comprising a step of measuring the expression level of one or more proteins selected from FCRL3 and SYK for a biological sample collected from a lesion of a patient exhibiting lymphadenopathy.