JPWO2019082871A1 - 一標的細胞の染色体複数異常を同時検出する方法 - Google Patents
一標的細胞の染色体複数異常を同時検出する方法 Download PDFInfo
- Publication number
- JPWO2019082871A1 JPWO2019082871A1 JP2019551147A JP2019551147A JPWO2019082871A1 JP WO2019082871 A1 JPWO2019082871 A1 JP WO2019082871A1 JP 2019551147 A JP2019551147 A JP 2019551147A JP 2019551147 A JP2019551147 A JP 2019551147A JP WO2019082871 A1 JPWO2019082871 A1 JP WO2019082871A1
- Authority
- JP
- Japan
- Prior art keywords
- fish
- analysis method
- chromosomal abnormalities
- chromosomal
- fish analysis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 208000031404 Chromosome Aberrations Diseases 0.000 title claims abstract description 102
- 206010008805 Chromosomal abnormalities Diseases 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title description 4
- 238000011282 treatment Methods 0.000 claims abstract description 31
- 238000007901 in situ hybridization Methods 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 59
- 239000000523 sample Substances 0.000 claims description 53
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- 238000004458 analytical method Methods 0.000 claims description 36
- 210000004881 tumor cell Anatomy 0.000 claims description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 18
- 208000034578 Multiple myelomas Diseases 0.000 claims description 17
- 239000007850 fluorescent dye Substances 0.000 claims description 17
- 238000012360 testing method Methods 0.000 claims description 17
- 239000000427 antigen Substances 0.000 claims description 16
- 108091007433 antigens Proteins 0.000 claims description 16
- 102000036639 antigens Human genes 0.000 claims description 16
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 15
- 201000010099 disease Diseases 0.000 claims description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 13
- 238000010186 staining Methods 0.000 claims description 13
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 6
- 238000001228 spectrum Methods 0.000 claims description 6
- 238000011369 optimal treatment Methods 0.000 claims description 4
- 229940124597 therapeutic agent Drugs 0.000 claims description 4
- 238000010306 acid treatment Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 101500016898 Arabidopsis thaliana C-terminally encoded peptide 7 Proteins 0.000 claims description 2
- 101100082489 Arabidopsis thaliana CEP9 gene Proteins 0.000 claims description 2
- 102100022387 Transforming protein RhoA Human genes 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000004393 prognosis Methods 0.000 abstract description 14
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 208000019691 hematopoietic and lymphoid cell neoplasm Diseases 0.000 abstract 1
- 241001274197 Scatophagus argus Species 0.000 description 29
- 206010028980 Neoplasm Diseases 0.000 description 13
- 239000011521 glass Substances 0.000 description 13
- 230000005945 translocation Effects 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 101001063878 Homo sapiens Leukemia-associated protein 1 Proteins 0.000 description 10
- 102100030893 Leukemia-associated protein 1 Human genes 0.000 description 10
- 210000000349 chromosome Anatomy 0.000 description 10
- 210000001185 bone marrow Anatomy 0.000 description 9
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 8
- 101710160107 Outer membrane protein A Proteins 0.000 description 8
- 230000007812 deficiency Effects 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 102100024165 G1/S-specific cyclin-D1 Human genes 0.000 description 7
- 101000980756 Homo sapiens G1/S-specific cyclin-D1 Proteins 0.000 description 7
- 238000012217 deletion Methods 0.000 description 7
- 230000037430 deletion Effects 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 206010064571 Gene mutation Diseases 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 6
- 238000004140 cleaning Methods 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 238000009396 hybridization Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 102100027842 Fibroblast growth factor receptor 3 Human genes 0.000 description 5
- 101710182396 Fibroblast growth factor receptor 3 Proteins 0.000 description 5
- 101000962461 Homo sapiens Transcription factor Maf Proteins 0.000 description 5
- 101000613608 Rattus norvegicus Monocyte to macrophage differentiation factor Proteins 0.000 description 5
- 102100039189 Transcription factor Maf Human genes 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 5
- 210000004180 plasmocyte Anatomy 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000005856 abnormality Effects 0.000 description 4
- 239000006059 cover glass Substances 0.000 description 4
- 229960003957 dexamethasone Drugs 0.000 description 4
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000012758 nuclear staining Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 229960001467 bortezomib Drugs 0.000 description 3
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 231100000005 chromosome aberration Toxicity 0.000 description 3
- 238000002189 fluorescence spectrum Methods 0.000 description 3
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 201000005787 hematologic cancer Diseases 0.000 description 3
- 229960004942 lenalidomide Drugs 0.000 description 3
- GOTYRUGSSMKFNF-UHFFFAOYSA-N lenalidomide Chemical compound C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O GOTYRUGSSMKFNF-UHFFFAOYSA-N 0.000 description 3
- 208000003950 B-cell lymphoma Diseases 0.000 description 2
- 206010061764 Chromosomal deletion Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 208000034951 Genetic Translocation Diseases 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 230000005861 gene abnormality Effects 0.000 description 2
- 238000003505 heat denaturation Methods 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 230000000394 mitotic effect Effects 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000005259 peripheral blood Anatomy 0.000 description 2
- 239000011886 peripheral blood Substances 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 230000000306 recurrent effect Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- BUJRUSRXHJKUQE-UHFFFAOYSA-N 5-carboxy-X-rhodamine triethylammonium salt Chemical compound CC[NH+](CC)CC.[O-]C(=O)C1=CC(C(=O)[O-])=CC=C1C1=C(C=C2C3=C4CCCN3CCC2)C4=[O+]C2=C1C=C1CCCN3CCCC2=C13 BUJRUSRXHJKUQE-UHFFFAOYSA-N 0.000 description 1
- 241001316595 Acris Species 0.000 description 1
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 1
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 208000036170 B-Cell Marginal Zone Lymphoma Diseases 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 208000011691 Burkitt lymphomas Diseases 0.000 description 1
- 206010061765 Chromosomal mutation Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 206010065163 Clonal evolution Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 206010061850 Extranodal marginal zone B-cell lymphoma (MALT type) Diseases 0.000 description 1
- 206010066476 Haematological malignancy Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 201000003791 MALT lymphoma Diseases 0.000 description 1
- 208000025205 Mantle-Cell Lymphoma Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 201000007224 Myeloproliferative neoplasm Diseases 0.000 description 1
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009104 chemotherapy regimen Methods 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000013135 deep learning Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 201000003444 follicular lymphoma Diseases 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000003703 image analysis method Methods 0.000 description 1
- 230000016507 interphase Effects 0.000 description 1
- 229960003648 ixazomib Drugs 0.000 description 1
- MXAYKZJJDUDWDS-LBPRGKRZSA-N ixazomib Chemical compound CC(C)C[C@@H](B(O)O)NC(=O)CNC(=O)C1=CC(Cl)=CC=C1Cl MXAYKZJJDUDWDS-LBPRGKRZSA-N 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000010801 machine learning Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000001167 myeloblast Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000002071 myeloproliferative effect Effects 0.000 description 1
- 210000000822 natural killer cell Anatomy 0.000 description 1
- 210000003924 normoblast Anatomy 0.000 description 1
- 238000010422 painting Methods 0.000 description 1
- 229960005184 panobinostat Drugs 0.000 description 1
- FWZRWHZDXBDTFK-ZHACJKMWSA-N panobinostat Chemical compound CC1=NC2=CC=C[CH]C2=C1CCNCC1=CC=C(\C=C\C(=O)NO)C=C1 FWZRWHZDXBDTFK-ZHACJKMWSA-N 0.000 description 1
- 229960005205 prednisolone Drugs 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical compound O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000010187 selection method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Sustainable Development (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
(1)識別可能な3以上の標識を用いて、一標的細胞中の2以上の染色体異常を同時検出するFISH(fluorescense in situ hybridization)解析法。
(2)画像解析ソフトによって蛍光を分離して解析する(1)記載のFISH解析法。
(3)ホルムアルデヒド固定、メタノール/酢酸後固定、酢酸処理を行い細胞を固定する(1)又は(2)記載のFISH解析法。
(4)細胞表面抗原を染色することにより腫瘍細胞を識別し、染色体異常を検出する(1)〜(3)いずれか1つ記載のFISH解析法。
(5)PF−405、PF−380−LSS、PF−395−LSS、SpectrumGreen、PF−510−LSS、SpectrumOrange、ATTO Rho12、Red5−ROX、ATTO 620、PB495、AF594から選択される複数の蛍光色素によってFISHプローブを標識する(1)〜(4)いずれか1つ記載のFISH解析法。
(6)表8のいずれか1つの疾患を検出するために用いる(1)〜(5)いずれか1つ記載のFISH解析法。
(7)多発性骨髄腫を原因とする染色体異常を検出する(6)記載のFISH解析法。
(8)表8に記載のプローブを用いて、del13q34、del17p13、t(11;14)(q13;q32)、t(4;14)(p16;q32)、t(14;16)(q32;q23)、t(6;14)(p25;q32)、t(6;14)(p21;q32)、t(8;14)(q24;q32)、t(14;20)(q32;q11)、1q21増幅、del1p32.3、CEP7、CEP9のうちのいずれか2つ以上の染色体異常を同時に検出する(7)記載のFISH解析法。
(9)さらに、CD138、及び/又は核を染色する(7)、又は(8)記載のFISH解析法。
(10)多発性骨髄腫の治療薬を選択するために用いる(7)〜(9)いずれか1つ記載のFISH解析法。
(11)(1)〜(10)いずれか1つ記載の血液腫瘍の解析法に用いる検査キットであって、表8に記載のプローブとFISHに必要な試薬を含むことを特徴とする検査キット。
(12)前記プローブが蛍光標識されたものである(11)記載の検査キット。
(13)(1)〜(10)いずれか1つ記載のFISH解析法で得られた患者の染色体異常パターンと、患者の治療成績を蓄積し、AIを用いて解析することによって染色体異常パターンごとの最適な治療法を選択する方法。
(14)一標的細胞の染色体複数異常を同時検出する方法(Simultaneous Chromosomal Abnormalities in a Target cell Fluorescence in situ hybridization:SCAT FISH)によって、識別可能な3以上の標識を用いて、画像解析ソフトによって蛍光を分離して検出し、1細胞中の2以上の染色体異常を同時に検出し、染色体異常パターンによって疾患の病型を分類することを特徴とする診断方法。
(15)SCAT FISH解析法によって、識別可能な3以上の標識を用いて、画像解析ソフトによって蛍光を分離して検出し、1細胞中の2以上の染色体異常を同時に検出し、染色体異常パターンによって治療薬を選択することを特徴とする治療方法。
(1)方法
試料としては、染色体異常が存在する可能性のある細胞であれば、どのような試料を用いてもよい。血液腫瘍であれば、骨髄穿刺によって得られた骨髄液、あるいは末梢血を試料として用いることができる。試料は患者から採取後すぐに、あるいは凍結保存したものを37℃で速やかに解凍して使用する。骨髄液には、骨髄芽球などの白血球系の細胞、赤芽球系の細胞、骨髄巨核球、形質細胞が含まれる。そこで、腫瘍細胞において発現している表面抗原に対する抗体を用いて陽性細胞を分離し、さらに、腫瘍細胞の表面抗原を染色することによって腫瘍細胞を選択的に検出することができる。
本方法では、複数の蛍光色素で標識されたプローブを用いてFISHを行うので、各スペクトルに重複する波長領域が生じる。そこで、ラムダスキャン(Lambda scan、Leica Microsystems社)によって画像を取得し、蛍光スペクトルを分離し解析を行う。種々の蛍光色素を検討した結果、現在ラムダスキャンによって画像取得後に蛍光スペクトルを分離し、最も明瞭に解析を行うことができるのは、以下の10の蛍光色素であった(表1)。なお、DRAQ5はDNAに結合する試薬であり、核染色に用いている。
<表面抗原染色>
多発性骨髄腫患者の骨髄液を採取し、2mM EDTAを含むPBSを加え1500rpm5分間の遠心処理を行った後、沈殿を最初の試料の量と同じ量になるようにMACS(登録商標)バッファー(Miltenyi Biotec社製)で懸濁した。次に、抗CD138抗体が結合した磁気ビーズ(Miltenyi Biotec社製)を試料の1/20量加え、4℃で15分間反応させた後、AutoMACS(Miltenyi Biotec社製)によりCD138陽性細胞を分離した。2000rpm3分間の遠心により上清を除き、10μlの細胞懸濁液とした。ビオチン標識抗CD138抗体(Acris社製)を10μl添加し、4℃で10分インキュベートした。次に、1.2ml MACSバッファーを加え、2000rpm3分間の遠心後、上清を除き、10μlの細胞懸濁液とした。10倍希釈したATTO 620−ストレプトアビジン(ATTOTECH社製)を1μl加え、4℃で15分間反応させた。次に、1.2ml MACSバッファーを加え、2000rpm3分の遠心を2回繰り返し洗浄後、MACSバッファーで50μlの細胞懸濁液とした。
スライドガラス上の細胞をスポットした箇所に、ハイブリバッファー(Lica Biotechnology社製)3μlを載置し、カバーガラスを載せ、ペーパーボンド(KOKUYO社製)によって封入した。80℃のホットプレート(StatSpin ThermoBrite、ABBOTT社製)上で60分間、抗原賦活化処理を行った。スライドガラスは、室温の2×SSC中でカバーガラスをはずし、70%、85%、100%エタノールに各1分ずつ浸漬し、風乾後45℃に保温した。
(1)50% ホルムアミド/2×SSC、45℃、10分、3回
(2)2×SSC 45℃、10分
(3)0.1%NP40/2×SSC、45℃、5分
(4)2×SSC すすぎ
次に、従来法である1つの染色体の異常をそれぞれ検出するFISHと、SCAT FISHによる結果比較を示す(表3)。検査会社に、10名の多発性骨髄腫患者(検体P1〜P10)の骨髄液の検査を依頼し、FACSによるCD138陽性細胞の全細胞に対する割合(検査会社のFACS結果)、DLEU1(RB1)欠損、P53欠損、BCL1転座、FGFR3転座、MAF転座のFISHによる検査を依頼した。同じ試料をSCAT FISHによって、実施例1と同様の方法を用いてFISHを行い、染色体異常が検出された割合をまとめた。
SCAT FISHにより一細胞中の染色体異常を複数検出し、解析した例を次に示す。2例の多発性骨髄腫再発例において、実施例1と同様にSCAT FISHを行い、一細胞中でどのような染色体異常が複合して生じているか、解析を行った(表4〜表7)。
[実施例4]
Claims (13)
- 識別可能な3以上の標識を用いて、
一標的細胞中の2以上の染色体異常を同時検出するFISH(fluorescense in situ hybridization)解析法。 - 画像解析ソフトによって蛍光を分離して解析する請求項1記載のFISH解析法。
- ホルムアルデヒド固定、メタノール/酢酸後固定、酢酸処理を行い細胞を固定する請求項1又は2記載のFISH解析法。
- 細胞表面抗原を染色することにより腫瘍細胞を識別し、
染色体異常を検出する請求項1〜3いずれか1項記載のFISH解析法。 - PF−405、PF−380−LSS、PF−395−LSS、SpectrumGreen、PF−510−LSS、SpectrumOrange、ATTO Rho12、Red5−ROX、ATTO 620、PB495、AF594から選択される複数の蛍光色素によってFISHプローブを標識する請求項1〜4いずれか1項記載のFISH解析法。
- 表8のいずれか1つの疾患を検出するために用いる請求項1〜5いずれか1項記載のFISH解析法。
- 多発性骨髄腫を原因とする染色体異常を検出する請求項6記載のFISH解析法。
- 表8に記載のプローブを用いて、
del13q34、del17p13、t(11;14)(q13;q32)、t(4;14)(p16;q32)、t(14;16)(q32;q23)、t(6;14)(p25;q32)、t(6;14)(p21;q32)、t(8;14)(q24;q32)、t(14;20)(q32;q11)、1q21増幅、del1p32.3、CEP7、CEP9のうちのいずれか2つ以上の染色体異常を同時に検出する請求項7記載のFISH解析法。 - さらに、CD138、及び/又は核を染色する請求項7、又は8記載のFISH解析法。
- 多発性骨髄腫の治療薬を選択するために用いる請求項7〜9いずれか1項記載のFISH解析法。
- 請求項1〜10いずれか1項記載の解析法に用いる血液腫瘍の検査キットであって、
表8に記載のプローブと
FISHに必要な試薬を含むことを特徴とする検査キット。 - 前記プローブが蛍光標識されたものである請求項11記載の検査キット。
- 請求項1〜10いずれか1項記載のFISH解析法で得られた患者の染色体異常パターンと、患者の治療成績を蓄積し、
AIを用いて解析することによって染色体異常パターンごとの最適な治療法を選択する方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2017204711 | 2017-10-23 | ||
JP2017204711 | 2017-10-23 | ||
PCT/JP2018/039290 WO2019082871A1 (ja) | 2017-10-23 | 2018-10-23 | 一標的細胞の染色体複数異常を同時検出する方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
JPWO2019082871A1 true JPWO2019082871A1 (ja) | 2020-11-12 |
JP7324711B2 JP7324711B2 (ja) | 2023-08-10 |
Family
ID=66247438
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2019551147A Active JP7324711B2 (ja) | 2017-10-23 | 2018-10-23 | 一標的細胞の染色体複数異常を同時検出する方法 |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP7324711B2 (ja) |
WO (1) | WO2019082871A1 (ja) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004022741A1 (ja) * | 2002-09-03 | 2004-03-18 | Japan Science And Technology Agency | 哺乳類人工染色体 |
JP2012103077A (ja) * | 2010-11-09 | 2012-05-31 | Olympus Corp | 遺伝子異常細胞の解析方法 |
WO2016106298A1 (en) * | 2014-12-22 | 2016-06-30 | Sigma-Aldrich Co. Llc | Visualizing modified nucleotides and nucleic acid interactions in single cells |
-
2018
- 2018-10-23 WO PCT/JP2018/039290 patent/WO2019082871A1/ja active Application Filing
- 2018-10-23 JP JP2019551147A patent/JP7324711B2/ja active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004022741A1 (ja) * | 2002-09-03 | 2004-03-18 | Japan Science And Technology Agency | 哺乳類人工染色体 |
JP2012103077A (ja) * | 2010-11-09 | 2012-05-31 | Olympus Corp | 遺伝子異常細胞の解析方法 |
WO2016106298A1 (en) * | 2014-12-22 | 2016-06-30 | Sigma-Aldrich Co. Llc | Visualizing modified nucleotides and nucleic acid interactions in single cells |
Non-Patent Citations (5)
Title |
---|
'ATTO DYES FOR SUPERIOR FLUORESCENT IMAGING', [ONLINE], 2017.9.24 公開, SIGMA-ALDRICH, [2019.1.11 検, JPN6019001855, ISSN: 0005004543 * |
BLOOD, vol. 109, JPN6019001853, 2007, pages 2089 - 2099, ISSN: 0005004541 * |
HAEMATOLOGICA, vol. 97, no. 8, JPN6019001854, 2012, pages 1272 - 1277, ISSN: 0005004542 * |
LEUKEMIA, vol. 30, JPN6019001852, 2016, pages 1197 - 1201, ISSN: 0005004540 * |
ONCOL. REP., vol. 18, JPN6019001851, 2007, pages 1099 - 1106, ISSN: 0005004539 * |
Also Published As
Publication number | Publication date |
---|---|
JP7324711B2 (ja) | 2023-08-10 |
WO2019082871A1 (ja) | 2019-05-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Feldman et al. | Recurrent translocations involving the IRF4 oncogene locus in peripheral T-cell lymphomas | |
US7964345B2 (en) | Methods of analyzing chromosomal translocations using fluorescence in situ hybridization (FISH) | |
US20070020670A1 (en) | Methods for detecting and confirming minimal disease | |
DK2191023T3 (en) | DIAGNOSTICS OF B-CELL Lymphoma | |
Hartmann et al. | Detection of genomic abnormalities in multiple myeloma: the application of FISH analysis in combination with various plasma cell enrichment techniques | |
Zhang et al. | The role of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fine‐needle aspiration | |
Sebastián et al. | High-resolution copy number analysis of paired normal-tumor samples from diffuse large B cell lymphoma | |
Hui et al. | Imaging flow cytometry to assess chromosomal abnormalities in chronic lymphocytic leukaemia | |
WO2014160120A1 (en) | Compositions and methods for detecting and determining a prognosis for prostate cancer | |
Dowd et al. | Detection of chromosome 13 (13q14) deletion among Sudanese patients with multiple myeloma using a molecular genetics fluorescent in situ hybridization technique (FISH) | |
Koessler et al. | Implementing circulating tumor DNA analysis in a clinical laboratory: a user manual | |
JP2018504115A (ja) | サイズに基づいて希少細胞を回収するためのカラムベースのデバイスおよび方法ならびにその使用 | |
WO2016057852A1 (en) | Markers for hematological cancers | |
US9096902B2 (en) | Genetic barcodes | |
JP2016067268A (ja) | 胎児の染色体異数性の非侵襲的判別方法 | |
Bouvier et al. | Deletions of chromosomes 1p and 19q are detectable on frozen smears of gliomas by FISH: usefulness for stereotactic biopsies | |
Slovak et al. | Targeting plasma cells improves detection of cytogenetic aberrations in multiple myeloma: phenotype/genotype fluorescence in situ hybridization | |
Hussein et al. | Practical diagnostic approaches to composite plasma cell neoplasm and low grade B‐cell lymphoma/clonal infiltrates in the bone marrow | |
JP7324711B2 (ja) | 一標的細胞の染色体複数異常を同時検出する方法 | |
Salvianti et al. | Evaluation of the liquid biopsy for the detection of brafv600e mutation in metastatic melanoma patients | |
Robetorye et al. | Profiling of lymphoma from formalin-fixed paraffin-embedded tissue | |
WO2014078571A1 (en) | Methods of detecting 14q32 translocations | |
Murase et al. | Immunohistochemistry for identification of CCND1, NSD2, and MAF gene rearrangements in plasma cell myeloma | |
Rosolen et al. | Efficacy of two fluorescence in situ hybridization (FISH) probes for diagnosing malignant pleural effusions | |
Hotinski et al. | The future of laboratory testing in chronic lymphocytic leukaemia |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20211015 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20221003 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221129 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20230306 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230508 |
|
TRDD | Decision of grant or rejection written | ||
A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230724 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230731 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 7324711 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |