WO2015105190A1 - 子宮体がんのリンパ節転移能の評価方法 - Google Patents
子宮体がんのリンパ節転移能の評価方法 Download PDFInfo
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- the present invention relates to a technique for discriminating between endometrial cancer and lymph node metastatic and non-metastatic at the molecular level.
- lymph node metastasis-positive cases may occur in spite of early cancer without muscle layer invasion.
- the cancer is negative for lymph node metastasis even if muscle layer infiltration is observed, and unnecessarily lymph node dissection is being performed.
- lymph node metastasis in endometrial cancer can be examined only by performing lymph node dissection and confirming pathologically whether there are cancerous tissues that have metastasized to the removed lymph nodes.
- no means for determining whether or not a lymph node metastatic uterine cancer has been obtained without lymph node dissection has been known.
- RNA-seq Non-Patent Document 1
- CAGE Cap Analysis Gene Expression: Non-Patent Document 2
- the CAGE method is characterized in 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 technique for discriminating between endometrial cancer and lymph node metastasis and non-metastasis at the molecular level.
- the present inventors extract RNA from cancer tissue with lymph node metastasis and cancer tissue without lymph node metastasis collected from endometrial cancer patients, and use transcription analysis region (Transcript Start) using CAGE analysis method.
- transcription analysis region Transcript Start
- TSS Site
- the expression level of DNA containing a specific transcription initiation region is significantly different between the two, and using this as an index, the lymph node metastatic uterus It was found that body cancer and non-metastatic endometrial cancer can be distinguished.
- the present invention relates to the following 1) to 4).
- 1) Endometrial cancer comprising a step of measuring the expression level of one or more expression products of DNA including a transcription initiation region for a biological sample derived from cancer tissue isolated from a patient with endometrial cancer
- a method for evaluating lymph node metastasis or lymph node metastasis ability of the DNA wherein the DNA is a base sequence represented by SEQ ID NOs: 1 to 277 and a base at an arbitrary position in the transcription initiation region and one or more bases downstream thereof DNA consisting of The evaluation method, wherein the transcription start region is a region whose both ends are defined by the first base of the base sequence represented by SEQ ID NOs: 1 to 277 and the 101st base from the 3 ′ end.
- Lymph node metastasis or lymph node metastasis capacity of endometrial cancer used in the method of 1) above, which contains an oligonucleotide that specifically hybridizes with the transcription product of DNA or an antibody that recognizes the translation product of DNA. Test kit for assessing.
- lymph node metastasis or lymph node metastasis ability of endometrial cancer classification of the same level or higher, without depending on the subjectivity of experts such as trained clinical technologists Can be performed objectively, and can be suitably used for tests (POCT: Point of Care Testing) performed by a medical worker alongside a patient from collection to analysis of a patient sample.
- POCT Point of Care Testing
- endometrial cancer means cancer that occurs in the uterine body part among uterine cancers. Endometrial cancer occurs in the endometrium, an epithelial tissue on the uterine cavity side, and is synonymous with endometrial cancer. Endometrial cancer is classified into stages I to IV according to the clinical stage classification of maternal women (2011) based on the size, spread, invasion and metastasis of cancer. In the present invention, the endometrial cancer to be evaluated is not particularly limited by the advanced stage, but is preferably a well-differentiated adenocarcinoma of endometrioid adenocarcinoma.
- lymph node metastasis in endometrial cancer means that endometrial cancer grows in lymph nodes such as in the pelvis and around the aorta.
- the evaluation of lymph node metastasis means evaluating or measuring the presence or absence of metastasis of endometrial cancer to the lymph node, and the evaluation of lymph node metastasis ability (lymph node metastasis) is the uterine body. It means to evaluate or measure whether cancer has the ability to metastasize to lymph nodes and proliferate.
- the biological sample used in the present invention is a pre-operative or intra-uterine endometrial cancer tissue separated from an endometrial cancer patient to be evaluated.
- the biological sample is appropriately prepared and processed for use in measurement.
- an RNA extract is prepared, and when the sample is subjected to 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.
- 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 277 is an endometrial cancer as shown in the Examples.
- CAGE Cap Analysis Gene Expression
- RNA transcriptional activity a differential analysis between a clinical specimen-derived profile group obtained from “with lymph node metastasis” and a clinical specimen-derived profile group obtained from “without lymph node metastasis”
- the bioconductor edgeR package Bioinformatics. 2010 Jan 1; 26 (1): 139-40
- FDR false discovery rate
- the expression product (referred to as “the expression product of the present invention”) (referred to as “the expression product of the present invention”) (referred to as “DNA containing the transcription start point”) It can be a biomarker for distinguishing cancer.
- the DNA expression product containing the transcription start point in SEQ ID NOs: 1 to 275 is a marker that increases the expression level when there is lymph node metastasis
- the DNA expression product containing the transcription start point in SEQ ID NOs: 276 to 277 is It is a marker that decreases the expression level when there is lymph node metastasis.
- 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.
- each of the base sequences shown in SEQ ID NOs: 1 to 277 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 277 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 277”.
- the positions of the transcription start regions shown in SEQ ID NOs: 1 to 277 on the genome and the gene information related thereto are as shown in Tables 1-1 to 1-12 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 277 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 downstream base 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 has a length of at least 20 bases out of DNA consisting of the transcription start region and 100 bases downstream from it, there is a high probability that it can be identified even in an experimental system for the entire genome.
- the DNA has substantially the same base sequence as the DNA as long as the expression product can be a biomarker for distinguishing lymph node metastatic endometrial cancer from non-metastatic endometrial cancer.
- DNA having the nucleotide sequence of is also included.
- Such expression products of the present invention can be distinguished from lymph node metastatic endometrial cancer and non-metastatic endometrial cancer by grasping the expression level by combining one or more kinds.
- classification can be made with specificity 100% and sensitivity 100%. That is, these can be surely discriminated only by one expression level.
- the number and the content of the combination can be selected as appropriate, but suitable combinations (434 sets) when using at least two DNA expression products are described in the table below. It was shown in 3.
- Such a combination is a logistic regression model for estimating the presence or absence of lymph node metastasis in the transcription start region extraction sample with the expression level of all the two combinations selected from the 277 transcription start regions as explanatory variables.
- both the transcription start region extraction sample and the verification sample are extracted from those that can be classified with a specificity of 100% and a sensitivity of 100%. For the purpose of further improving the accuracy, these can be used in combination of two sets or three sets or more as appropriate.
- DNA expression products in addition to the combinations of the two DNA expression products, among DNAs containing the transcription start sites in SEQ ID NOS: 1 to 277, DNA expression products other than those shown in Tables 3-1 to 3-6 In addition, DNA expression products consisting of any other base sequences may be combined 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: 264 is “TACC2” (Transforming, Acidic Coiled-Coil Containing Protein 2; UniProtKB / Swiss-Prot: TACC2_HUMAN, O95359).
- 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 to these primers Methods, hybridization methods using nucleic acids that hybridize to these as probes (DNA chips, DNA microarrays, dot blot hybridizations, slot blot hybridizations, northern blot hybridizations, etc.), methods for determining base sequences, or combinations thereof You can choose from different 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 277 or its complementary strand 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 a nucleotide sequence
- 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.
- oligonucleotide 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 consisting of the base sequence represented by SEQ ID NOs: 1 to 277 (or its complementary strand), for example, 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 highly sensitive detection is performed using a three-step method such as ABC method or LSAB method, or the DAVision EnVision detection system. Is preferred.
- the expression level of the expression product of the present invention in a biological sample derived from cancer tissue isolated from a patient with endometrial cancer is measured, and based on the expression level, lymph node metastasis or lymph node metastasis ability is measured. Presence or absence is evaluated. 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, expression of the expression product in endometrial cancer tissue isolated from endometrial cancer patients without lymph node metastasis or normal tissue isolated from endometrial cancer patients Level, or the expression level of the expression product in a group of healthy individuals who do not develop endometrial cancer.
- the expression level of the expression product in the cancer tissue of the target patient is the expression level in an endometrial cancer tissue, normal tissue, or tissue derived from a healthy person isolated from a patient with endometrial cancer that does not have lymph node metastasis. Close, within the range of the expression level, or significantly higher (or lower) than the expression level, the patient's endometrial cancer can be evaluated as having no lymph node metastasis or low lymph node metastasis .
- the lymph node metastasis or lymph node metastasis ability of endometrial cancer in the present invention can also be evaluated by increasing / decreasing the expression level of the expression product of the present invention.
- a control level for example, based on the expression level of the expression product derived from normal tissue, endometrial cancer tissue isolated from endometrial cancer patients without lymph node metastasis or healthy tissue, This can be done by setting a value (threshold level) and comparing the expression level of the expression product in a patient-derived biological sample with a standard value (for example, the range of ⁇ 2SD is allowed).
- the endometrial cancer of the patient can be evaluated as having no lymph node metastasis or having a low lymph node metastasis ability.
- the possibility of lymph node metastasis or the ability of lymph node metastasis is determined based on the information provided in combination with other methods (CT, MRI, PET-CT, etc.) as necessary.
- CT computed tomography
- MRI magnetic resonance imaging
- PET-CT magnetic resonance imaging
- the criteria for performing lymph node biopsy and lymph node dissection are left to the judgment of the doctor, but if it is determined that there is a possibility of lymph node metastasis or high lymph node metastasis capacity, for example, lymph node dissection is performed. be able to. On the other hand, if it is determined that there is no possibility of lymph node metastasis or lymph node metastasis ability is low, it is considered unnecessary to perform lymph node dissection.
- the test kit for evaluating lymph node metastasis or lymph node metastasis ability of endometrial cancer 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.
- 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 that enables discrimination between lymph node metastatic endometrial cancer and non-metastatic endometrial cancer (1) Obtaining specimen samples Patients who received comprehensive consent before surgery 5 mm each of endometrial cancer portions of endometrial cancer (well-differentiated adenocarcinoma) with a myometrial invasion of 1/2 or less were collected. The samples used were 10 specimens for extracting the transcription start region (including 3 specimens with lymph node metastasis and 7 specimens without lymph node metastasis), and 5 specimens (of which 2 specimens with lymph node metastasis). 3 specimens without lymph node metastasis).
- 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.
- a model was constructed, and samples that can be classified with 100% specificity and 100% sensitivity were selected for both the transcription start region extraction sample and the verification sample (Tables 3-1 to 3-6).
- a logistic regression model which is one of the simplest machine learning devices, is adopted.
- Example 2 Using 23 surgically-extracted specimens (9 specimens with lymph node metastasis, 14 specimens without lymph node metastasis) different from the specimens used in Example 1, a CAGE library was prepared and lymph nodes were prepared in the same manner as in Example 1. Transcription initiation regions with different activities were extracted in the group with node metastasis and the group without lymph node metastasis. As a result, the transcriptional activity level of the transcription initiation region represented by SEQ ID NOs: 35, 36, 63, 73, 140, 161, 189, 193, 205 and 219 is significantly different between the groups with and without metastasis. It was confirmed (Table 4).
- Example 3 Discrimination between lymph node metastatic endometrial cancer and non-metastatic endometrial cancer using TACC2 as a marker
- (1) specimen Surgical excision specimen of a patient who received comprehensive consent before surgery (myometrium) Samples that were diagnosed as having lymph node metastasis or no lymph node metastasis were collected in 5 mm each of endometrial cancer sections of endometrial cancer (well-differentiated adenocarcinoma) with invasion of 1/2 or less. .
- TACC2 The expression level of the TACC2 gene was corrected with the housekeeping gene, SUDS4, and calculated as a relative change amount with respect to the control. The results are shown in FIG. From FIG. 2, in the real-time PCR performed using this primer, the expression level of TACC2 is different between with and without lymph node metastasis, and it can be considered that TACC2 can be used as a marker.
- the present invention it is possible to objectively determine the presence or absence of lymph node metastasis of endometrial cancer and the prediction of its occurrence by examining the primary endometrial lesions collected before or during surgery. Thereby, unnecessary lymph node dissection can be avoided, and complications such as postoperative lymphedema can be reduced.
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Abstract
Description
本出願は、2014年1月10日に出願した日本国特願2014-003191の優先権を主張するものであり、その全内容は本明細書において参照として援用される。
また、本明細書に引用される全ての文献は、あらゆる目的から全体として参照により援用される。いずれの文献の引用も、それが本発明に関する先行技術であることを認めるものと解釈されてはならない。
1)子宮体がん患者から分離されたがん組織由来の生体試料について、転写開始領域を含むDNAの1種又は2種以上の発現産物の発現レベルを測定する工程を含む、子宮体がんのリンパ節転移又はリンパ節転移能を評価する方法であって、該DNAが配列番号1~277で示される塩基配列における、転写開始領域の任意の位置の塩基とその下流に連続する1塩基以上からなるDNAであり、
該転写開始領域が、配列番号1~277で示される塩基配列の1番目の塩基と3’末端から101番目の塩基によって両端が規定される領域である、前記評価方法。
2)前記DNAの転写産物と特異的にハイブリダイズするオリゴヌクレオチド、又は前記DNAの翻訳産物を認識する抗体を含有する上記1)の方法に用いる子宮体がんのリンパ節転移又はリンパ節転移能を評価するための検査用キット。
3)転写開始領域を含むDNAの1種又は2種以上の発現産物の、子宮体がんのリンパ節転移又はリンパ節転移能を評価するためのマーカーとしての使用であって、該DNAが配列番号1~277で示される塩基配列における、転写開始領域の任意の位置の塩基とその下流に連続する1塩基以上からなるDNAであり、
該転写開始領域が、配列番号1~277で示される塩基配列の1番目の塩基と3’末端から101番目の塩基によって両端が規定される領域である、前記発現産物の使用。
4)子宮体がん患者から分離されたがん組織由来の生体試料について、TACC2のRNA又はタンパク質の発現レベルを測定する工程を含む、子宮体がんのリンパ節転移又はリンパ節転移能を評価する方法。
生体試料からRNAを抽出する方法は、公知の任意の方法を用いることができる。具体的には、ライフテクノロジーズ社製Ambion RiboPureキット、キアゲン社製miRNeasy、同社製RNeasyが例示できるが、これらのうちキアゲン社製miRNeasyキットが好適に用いられる。
したがって、配列番号1~277で示される塩基配列における、転写開始領域の任意の位置(転写開始点)の塩基とその下流に連続する1塩基以上からなるDNA(以下、「配列番号1~277における転写開始点を含むDNA」と称する)の(又はそれによってコードされる)発現産物(「本発明の発現産物」と云う)は、リンパ節転移性の子宮体がんと非転移性の子宮体がんを判別するためのバイオマーカーとなり得る。尚、配列番号1~275における転写開始点を含むDNAの発現産物は、リンパ節転移がある場合に発現レベルが上がるマーカーであり、配列番号276~277における転写開始点を含むDNAの発現産物はリンパ節転移がある場合に発現レベルが下がるマーカーである。
配列番号1~277において示される転写開始領域のゲノム上の位置、及びそれに関連する遺伝子情報等は後記表1-1~表1-12に示すとおりである。
ここで、下流に続く塩基配列の塩基数は、発現産物を特定できる数であればよい。当該塩基数としては、例えば1塩基以上、5塩基以上、10塩基以上、15塩基以上、20塩基以上、25塩基以上、30塩基以上、40塩基以上、50塩基以上が挙げられる。また、当該塩基数としては、例えば10塩基以下、15塩基以下、20塩基以下、25塩基以下、30塩基以下、40塩基以下、50塩基以下、100塩基以下が挙げられる。
下流側の塩基は、CAGE法による測定の場合には特に必要ないが、ハイブリダイゼーションやPCRによる測定の際にはその精度を担保するために下流100塩基程度までの何れかの部分を対象とすることができ、転写開始領域とその下流100塩基からなるDNAのうち、少なくとも20塩基以上の長さのものであればゲノム全体を対象にした実験系であっても特定できる確率が高い。
また、発現レベルとは、当該発現産物の発現量や活性を包括的に意味する。
具体的には、配列番号1~277で示される塩基配列からなるDNA又はその相補鎖に相補的な一定数のヌクレオチドを含むオリゴヌクレオチドを利用することができる。ここで「相補鎖」とは、A:T(RNAの場合はU)、G:Cの塩基対からなる2本鎖DNAの一方の鎖に対する他方の鎖を指す。また、「相補的」とは、当該一定数の連続したヌクレオチド領域で完全に相補配列である場合に限られず、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上の塩基配列上の同一性を有すればよい。塩基配列の同一性は、前記BLAST等のアルゴリズムにより決定することができる。
斯かるオリゴヌクレオチドは、プライマーとして用いる場合には、特異的なアニーリング及び鎖伸長ができればよく、通常、例えば10塩基以上、好ましくは15塩基以上、より好ましくは20塩基以上、かつ例えば100塩基以下、好ましくは50塩基以下、より好ましくは35塩基以下の鎖長を有するものが挙げられる。また、プローブとして用いる場合には、特異的なハイブリダイゼーションができればよく、配列番号1~277で示される塩基配列からなるDNA(又はその相補鎖)の少なくとも一部若しくは全部の配列を有し、例えば10塩基以上、好ましくは15塩基以上、かつ例えば100塩基以下、好ましくは50塩基以下、より好ましくは25塩基以下の鎖長のものが用いられる。
なお、ここで、「オリゴヌクレオチド」は、DNAあるいはRNAであることができ、合成されたものでも天然のものでもよい。また、ハイブリダイゼーションに用いるプローブは、通常標識したものが用いられる。
前記アレイに固定化される核酸としては、ストリンジェントな条件下に特異的(すなわち、実質的に目的の核酸のみに)にハイブリダイズする核酸であればよく、例えば、本発明の発現産物(転写産物)の全配列を有する核酸であってもよく、部分配列からなる核酸であってもよい。ここで、「部分配列」とは、少なくとも15~25塩基からなる核酸が挙げられる。
ここでストリンジェントな条件は、通常「1×SSC、0.1%SDS、37℃」程度の洗浄条件を挙げることができ、より厳しいハイブリダイズ条件としては「0.5×SSC、0.1%SDS、42℃」程度、さらに厳しいハイブリダイズ条件としては「0.1×SSC、0.1%SDS、65℃」程度の条件を挙げることができる。ハイブリダイズ条件は、J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001)などに記載されている。
CAGE法を用いて、発現レベルを測定する場合、後記実施例に記載した方法に準じて実施することができる。
例えば、測定対象としてタンパク質が用いられる場合は、本発明の発現産物(この場合、翻訳産物)に対する抗体を生体試料と接触させ、当該抗体に結合した試料中のポリペプチドを検出し、そのレベルを測定することによって実施される。例えば、ウェスタンブロット法によれば、一次抗体として上記の抗体を用いた後、二次抗体として放射性同位元素、蛍光物質又は酵素等で標識した一次抗体に結合する抗体を用いて、その一次抗体を標識し、これら標識物質由来のシグナルを放射線測定器、蛍光検出器等で測定することが行われる。
尚、上記翻訳産物に対する抗体は、ポリクローナル抗体であっても、モノクローナル抗体であってもよい。これらの抗体は、公知の方法に従って製造することができる。具体的には、ポリクローナル抗体は、常法に従って大腸菌等で発現し精製したタンパク質を用いて、あるいは常法に従って当該タンパク質の部分ポリペプチドを合成して、家兎等の非ヒト動物に免疫し、該免疫動物の血清から常法に従って得ることが可能である。
一方、モノクローナル抗体は、常法に従って大腸菌等で発現し精製したタンパク質又は該タンパク質の部分ポリペプチドをマウス等の非ヒト動物に免疫し、得られた脾臓細胞と骨髄腫細胞とを細胞融合させて調製したハイブリドーマ細胞から得ることができる。また、モノクローナル抗体は、ファージディスプレイを用いて作製してもよい(Griffiths, A.D.; Duncan, A.R., Current Opinion in Biotechnology, Volume 9, Number 1, February 1998 , pp. 102-108(7))。
また、免疫組織化学分析法を行う場合には、患者から分離した生体試料を常法によりホルマリン固定をした後、パラフィンに包埋して組織片に薄切し、スライドガラスに貼り付けたものを切片試料として使用するのが好ましい。二次抗体としては、アルカリホスファターゼやペルオキシダーゼ等の酵素標識抗体を用いることができるが、ABC法やLSAB法等の三段階法、またDAKO社のEnVision検出システム等を用いて高感度な検出を行うのが好ましい。
ここで、「対照レベル」とは、例えば、リンパ節転移がない子宮体がん患者から分離された子宮内膜がん組織若しくは子宮体がん患者から分離された正常組織における当該発現産物の発現レベル、又は子宮体がんを発症していない健常人群における当該発現産物の発現レベルが挙げられる。
例えば、対象患者のがん組織の当該発現産物の発現レベルが、リンパ節転移がない子宮体がん患者から分離された子宮内膜がん組織、正常組織或いは健常人由来の組織における発現レベルに近い、当該発現レベルの範囲内に属する、或いは当該発現レベルより有意に高い(又は低い)場合には、当該患者の子宮体がんはリンパ節転移がない又はリンパ節転移能が低いと評価できる。
また、当該検査用キットには、上記抗体や核酸の他、標識試薬、緩衝液、発色基質、二次抗体、ブロッキング剤や、試験に必要な器具やコントロール等を含むことができる。
(1)検体試料の入手
術前に包括的同意を頂いた患者の手術摘出検体で、子宮筋層浸潤が1/2以下の子宮体がん(高分化型腺癌)の子宮内膜癌部を5mm各採取した。使用したサンプルは、転写開始領域抽出用サンプルとして10検体(うち、リンパ節転移ありが3検体、リンパ節転移なしが7検体)、検証用サンプルとして5検体(うち、リンパ節転移ありが2検体、リンパ節転移なしが3検体)である。
摘出された組織片は、適宜冷凍処理されて-80℃で保存した。保存組織片は、2mLマイクロチューブに組織片を50mg以下になるように入れてキアゲン社製QIAzolを添加して、ジルコニアビーズを1個入れて密閉し、キアゲン社製TissueLyserを用いて浸透処理により破砕した。
破砕・抽出処理を行った試料は、キアゲン社製miRNeasy mini kitにより、添付されたプロトコルに従ってRNA調製を行った。調製後のRNAは、分光高度計による紫外吸収(230、260、280nm)を測定して、260/230、260/280比を算出し、そのRNAの質を検定した。また、アジレント社製BioAnalyzer RNA nano chipにより電気泳動を行い、RNA分解度を示すRIN値を算出して、RNAの分解度合いを検定した。
精製RNAを5μg用意し、非増幅非タグ化CAGE法(「細胞工学別冊 次世代シークエンサー目的別アドバンストメソッド」、菅野純夫、鈴木穣監修、学研メディカル秀潤社、2012年09月19日発行)内、第3章3、“網羅的プロモーター解析(イルミナシーケンサーを用いた非増幅CAGE法)”参照)により、CAGEライブラリーを調製した。具体的には、精製RNAを逆転写反応に供して精製後、過ヨウ素酸ナトリウムによりリボースのジオールを参加してアルデヒド化し、ビオチンヒドラジドを添加してアルデヒド基にビオチンを付加した。RNaseIにより一本鎖RNA部分を消化・精製後、アビジン磁気ビーズによりビオチン化されたRNA/cDNA二本鎖のみをビーズ表面に結合させ、RNaseH消化及び熱処理によりcDNAを遊離させて回収した。回収したcDNAの両端にシーケンスに必要なアダプターを連結させた後、イルミナ社製HiSeq2500によりシーケンスを行った。なお、本工程において精製・緩衝液置換等に用いるAMPure XP(ベックマン・コールター社製)の標準的な条件では、二本鎖の場合で100塩基以上の長さの核酸が回収される条件であり、これを採用した本工程により生産されるCAGEライブラリーは100塩基以上の鎖長をもつ二本鎖DNAからなる。
i)リファレンス転写開始領域の準備
ヒトの初代培養細胞や細胞株、さらに組織等を含め合計約1000ものヒトサンプルについて転写開始点の活性がゲノムワイドに測定されたプロファイルするプロジェクトである「FANTOM5」(論文投稿中)において同定された転写開始領域のうち、ヒトリファレンスゲノムhg19上に定義された約18万の転写開始領域をリファレンス転写開始領域とした。
シーケンシングにより得られたリードとヒトのリファレンスゲノム(hg19)のアラインメントをbwa(Bioinformatics. 2009 Jul 15;25(14):1754-60)を用いて行った。マッピングクオリティが20以上、かつアラインメントの開始位置が、リファレンス転写開始領域内に位置するようなアラインメントだけを選択し、各転写開始領域ごとのリード数を数え上げた。各ライブラリーの総リード数と、RLE(Genome Biol. 2010;11(10):R106)法により推定されたライブラリサイズを用いて、カウントを100万あたりのリード数(counts per million)に変換する。
(A)活性の異なる転写開始領域の抽出
上記で定量された、転写開始領域抽出用各サンプルでの転写活性について、「リンパ節転移あり」から得られた臨床検体由来プロファイル群、「リンパ節転移なし」から得られた臨床検体由来プロファイル群の間における差分解析をR/Bioconductor edgeRパッケージ(Bioinformatics. 2010 Jan 1;26(1):139-40)を用いて行った。すなわち、二群間で発現量の平均が異なるかどうか(発現量の平均が等しいことを帰無仮説とし、この帰無仮説が真であることを仮定した場合、測定結果が偶然に起きる確率を計算する)を統計的に検定するものである。閾値としてFDR(false discovery rate)5%を設定したところ、これよりも小さな転写開始領域を含むDNAを277個同定した(表1-1~表1-12)。この基準は、該当する閾値により抽出される候補のうち95%は真に発現差があると統計的に推定されたものであり、通常広く使われるP値(発現差が無いことを仮定した場合に偶然起きる確率)を5%とする場合よりも厳しい基準である。
上記(A)で同定された転写開始領域のうち、一つの発現レベルのみを用いてリンパ節転移の有無を分類できるかどうかを考える。それぞれについて、何らかの閾値を設定することで、転写開始領域抽出用サンプル、検証用サンプル共に特異度100%・感度100%で分類できることを確認した。表2に、その閾値の例を示す(ある群における最大値の方が、その他の群における最小値よりも小さい場合、これらの平均を表2に示している)。
上記で同定された277個の転写開始領域の発現レベルのうち、複数を用いてリンパ節転移の有無を分類できるかどうかを考える。例としてここでは、一般化線形モデルの一種であるロジスティック回帰モデルを構成することを考える。これは、リンパ節転移の有無を示す従属変数(Y)を、転写開始領域の発現レベルである説明変数(Xi、上記counts per millionの対数をとったもの)で確率的に予測することを考える場合、最もシンプルなモデルの一つである。
ここでは、機械学習器の中でも最も単純なものの一つであるロジスティック回帰モデルを採用しているが、当該TSSの発現を測定する方法や、他の遺伝子等の発現レベルや遺伝子型との組み合わせ等によって、他の数理モデルを適切に利用することで、より頑健な予測が可能になる。
実施例1で用いた検体とは別の手術摘出検体23検体(リンパ節転移あり9検体、リンパ節転移なし14検体)を用いて、実施例1と同様に、CAGEライブラリーを調製してリンパ節転移ありの群とリンパ節転移なしの群で活性の異なる転写開始領域を抽出した。その結果、配列番号35、36、63、73、140、161、189、193、205及び219で示される転写開始領域の転写活性レベルが、転移あり/なしの群の間で優位に異なることが確認された(表4)。
(1)検体
術前に包括的同意を頂いた患者の手術摘出検体(子宮筋層浸潤が1/2以下の子宮体がん(高分化型腺癌)の子宮内膜癌部を5mm各採取)で、リンパ節転移あり又はリンパ節転移なしと診断がなされている検体を使用した。
患者から分離した生体試料を常法によりホルマリン固定をした後、パラフィンに包埋して組織片に薄切し、スライドガラスに貼り付けたものを切片試料とした。次いで切片試料を、熱処理(121℃、10分)して抗原を賦活化した。次いで、抗TACC2抗体(EMD Millipore社、「Polyclonal Rabbit anti-TACC2 antibody」)(一次抗体、濃度0.5μg/mL)を一晩(4℃)反応させた。緩衝液にて十分に洗浄後、二次抗体としてAnti-Rabbit Immunoglobulins (DAKO #E432)を用い、30分間(20℃)反応させた。緩衝液にて十分に洗浄後、標識試薬Peroxidase-conjugated streptavidin (DAKO #P0397)を用い、30分間(20℃)反応させた(ABC法)。緩衝液にて十分に洗浄後、DABを用いて発色させた。その標本を光学顕微鏡下で陽性・陰性を観察した。その一例を図1に示す。
その結果、リンパ節転移あり/なしの間で染色パターンが異なり、TACC2をマーカーとして両者を区別可能であることが確認された。
検体組織からRNA抽出キット(QIAGEN、RAase Plus Mini Kit (#74134))を用いて全RNA抽出した。RNAからcDNAへの逆転写反応は、TAKARA、PrimeScriptII 1st strand cDNA Synthesis Kit(#6210A)により行った。リアルタイムPCRは、ABI、Fast SYBR Green Master Mix (#4385612)を用い、以下に示すプライマーを用いて、ABI 7500 Fast Real Time PCR Systemで実施した。
Primer F: CCAgTTgCTgAAgggCAgAA(配列番号278)
Primer R: gCggACCTTggAgTCTgAg(配列番号279)
尚、TACC2遺伝子の発現レベルは、ハウスキーピング遺伝子、SUDS4で補正し、対照に対する相対変化量として算出した。結果を図2に示す。
図2より、このプライマーを用いて行ったリアルタイムPCRでは、リンパ節転移あり/なしの間ではTACC2の発現レベルが異なり、TACC2をマーカーとして両者を区別することは可能であると考えられる。
Claims (9)
- 子宮体がん患者から分離されたがん組織由来の生体試料について、転写開始領域を含むDNAの1種又は2種以上の発現産物の発現レベルを測定する工程を含む、子宮体がんのリンパ節転移又はリンパ節転移能を評価する方法であって、該DNAが配列番号1~277で示される塩基配列における、転写開始領域の任意の位置の塩基とその下流に連続する少なくとも1塩基以上からなるDNAであり、
該転写開始領域が、配列番号1~277で示される塩基配列の1番目の塩基と3’末端から101番目の塩基によって両端が規定される領域である、前記評価方法。 - 配列番号1~277で示される塩基配列が、配列番号208、配列番号264、配列番号237、配列番号270、配列番号57、配列番号274、配列番号108及び配列番号182で示される塩基配列である、請求項1記載の方法。
- さらに、前記DNAの発現産物の発現レベルを対照レベルと比較する工程を含む、請求項1~3のいずれか1項記載の方法。
- さらに、前記DNAの発現産物の発現レベルを閾値レベルと比較する工程を含む、請求項1~3のいずれか1項記載の方法。
- 発現産物の発現レベルの測定が、転写産物の量又は翻訳産物の量を測定することによって行われる、請求項1~5のいずれか1項記載の方法。
- 前記DNAの転写産物と特異的にハイブリダイズするオリゴヌクレオチド、又は前記DNAの翻訳産物を認識する抗体を含有する請求項1~6のいずれか1項記載の方法に用いる子宮体がんのリンパ節転移又はリンパ節転移能を評価するための検査用キット。
- 転写開始領域を含むDNAの1種又は2種以上の発現産物の、子宮体がんのリンパ節転移又はリンパ節転移能を評価するためのマーカーとしての使用であって、該DNAが配列番号1~277で示される塩基配列における、転写開始領域の任意の位置の塩基とその下流に連続する1塩基以上からなるDNAであり、
該転写開始領域が、配列番号1~277で示される塩基配列の1番目の塩基と3’末端から101番目の塩基によって両端が規定される領域である、前記発現産物の使用。 - 子宮体がん患者から分離されたがん組織由来の生体試料について、TACC2のRNA又はタンパク質の発現レベルを測定する工程を含む、子宮体がんのリンパ節転移又はリンパ節転移能を評価する方法。
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