WO2019082871A1 - Méthode de détection simultanée de multiples anomalies chromosomiques dans une cellule cible unique - Google Patents

Méthode de détection simultanée de multiples anomalies chromosomiques dans une cellule cible unique

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WO2019082871A1
WO2019082871A1 PCT/JP2018/039290 JP2018039290W WO2019082871A1 WO 2019082871 A1 WO2019082871 A1 WO 2019082871A1 JP 2018039290 W JP2018039290 W JP 2018039290W WO 2019082871 A1 WO2019082871 A1 WO 2019082871A1
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fish
analysis method
chromosomal
chromosomal abnormalities
fish analysis
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PCT/JP2018/039290
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Japanese (ja)
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智子 ▲高▼山
良子 國吉
康仁 照井
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公益財団法人がん研究会
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    • 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

Definitions

  • the present invention relates to methods of detecting chromosomal abnormalities in target cells using fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • the present invention relates to a method of simultaneously detecting multiple chromosomal abnormalities in one target cell, which is a new FISH method (Simultaneous Chromosomal Abnormalities in a target cell fluorescence in situ hybridization (SCAT FISH)).
  • Cancer is a disease that results from the generation of several genetic mutations in normal cells.
  • a gene mutation may be a mutation of one or several bases in a gene, such as a single base substitution or a single base deletion of a gene sequence, or a chromosomal translocation or deletion, etc.
  • large mutations occur in the chromosome.
  • chromosomal aberrations specific to the disease are known.
  • gene mutations including chromosomal abnormalities may be detected and targeted for treatment. Therefore, it is very important to determine what kind of gene mutation is occurring in diseases caused by gene mutation such as cancer, in setting a diagnosis and a treatment policy.
  • FISH method As a method for detecting chromosomal abnormalities, FISH method has been developed and used for diagnosis of cancer and chromosomal aberration syndrome.
  • FISH a probe having a sequence of a specific locus is labeled by an enzyme reaction or the like, and DNA in a chromosome or interphase nucleus to be examined is placed as single-stranded DNA, and the specific locus is hybridized. It is a method of observing under a microscope.
  • Hematological tumors are thought to progress by the accumulation of mutations called clonal evolution.
  • multiple chromosomal aberrations are induced in multiple steps in one cell, and are known to be correlated with the progress of the disease.
  • chromosomal findings indicate that the high-risk group, the medium-risk group, and the standard-risk group are classified, and it is known that there is a correlation between chromosomal mutation and prognosis. Therefore, detecting multiple chromosomal abnormalities occurring in tumor cells is also important for prognosis.
  • FISH which detects chromosomal abnormalities
  • detection is performed using two types of fluorescent dyes, only one or two types of chromosomal abnormalities can be detected simultaneously. That is, instead of simultaneously detecting a plurality of chromosomal abnormalities occurring in one cell, it is inferred what kind of chromosomal abnormality has occurred by performing a plurality of tests for detecting one chromosomal abnormality. is the current situation. Therefore, although the frequency of each chromosomal abnormality is known, it is not possible to obtain information on the frequency of overlapping chromosomal aberrations.
  • Patent Document 1 discloses qualitatively and quantitatively detecting the transcription of various target genes in cells by making them distinguishable barcodes by combining different fluorescent dyes. It is described that 31 targets can be distinguished theoretically by using 5 kinds of fluorescent dyes. Further, Non-Patent Document 1 discloses the result of analysis of expression of four different genes in Drosophila embryo by FISH.
  • Patent Document 1 and Non-patent Document 1 are methods for analyzing the expression of a large number of genes.
  • analyzing gene expression as described in Patent Document 1, it is possible to combine several types of fluorescent dyes into barcodes to identify many genes. Further, as in the method described in Non-Patent Document 1, it is also possible to analyze using a plurality of types of fluorescence having different fluorescence spectra. Either method is applicable to the case where multiple copies of mRNA binding to the probe are present in one cell, such as embryos and tissues, and even a relatively short probe can be expected to obtain a strong signal.
  • Non-patent documents 2 and 3 disclose that chromosomes are stained with different fluorescent dyes to detect chromosomal abnormalities by M-FISH (Multicolor Fluorescence in situ hybridization) method.
  • M-FISH described in non-patent documents 2 and 3 is a method of staining an entire chromosome with a painting probe and identifying each chromosome by a combination of fluorescent dyes. By using this method, it is possible to identify the origin of each chromosome in a short time and detect the structural abnormality of the chromosome, but since it is necessary to use mitotic cells, cells of mitotic cells such as cerebrospinal fluid When the proportion is low, it is difficult to detect chromosomal abnormalities.
  • An object of the present invention is to provide a method and a probe for detecting a plurality of chromosomal abnormalities present in one cell at one time.
  • An object of the present invention is to simultaneously detect a plurality of chromosomal abnormalities present in one target cell after differentiating a tumor cell from a normal cell.
  • the present invention relates to the following test method and kit.
  • a fluorescence in situ hybridization (FISH) analysis method that simultaneously detects two or more chromosomal abnormalities in one target cell using three or more distinguishable labels.
  • the FISH analysis method according to (1) wherein fluorescence is separated and analyzed by image analysis software.
  • the FISH analysis method according to (1) or (2) wherein cells are fixed by formaldehyde fixation, methanol / acetic acid postfixation, and acetic acid treatment.
  • a plurality of fluorescent dyes selected from PF-405, PF-380-LSS, PF-395-LSS, Spectrum Green, PF-510-LSS, Spectrum Orange, ATTO Rho 12, Red 5-ROX, ATTO 620, PB 495, AF 594 The FISH analysis method according to any one of (1) to (4), wherein the FISH probe is labeled by (6)
  • the FISH analysis method according to (6) which detects a chromosomal abnormality caused by multiple myeloma.
  • the chromosomal aberration pattern of the patient obtained by the FISH analysis method according to any one of (1) to (10) and the treatment result of the patient are accumulated and analyzed using AI. How to choose the best treatment for you.
  • a method of simultaneously detecting multiple chromosomal aberrations in one target cell (Simultaneous Chromosomal Abnormalities in a Target cell Fluorescence in situ hybridization: SCAT FISH) using three or more labels that can be identified and fluorescence by image analysis software
  • a diagnostic method comprising separating and detecting, simultaneously detecting two or more chromosomal abnormalities in one cell, and classifying a disease type of disease by a chromosomal aberration pattern.
  • three or more distinguishable labels are used to separate and detect fluorescence by image analysis software, to simultaneously detect two or more chromosomal abnormalities in one cell, and by chromosomal aberration pattern
  • a therapeutic method characterized by selecting a therapeutic agent.
  • FIG. 1 The figure which shows the outline of SCAT FISH method.
  • FIG. 1 The microscope image which stained by the SCAT FISH method in Example 1.
  • the analysis method of the chromosomal aberration described below can be examined for any chromosomal aberration derived from any disease.
  • Hematological tumors include multiple myeloma, chronic myelogenous leukemia, acute lymphatic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, follicular lymphoma, diffuse large cell form There are B cell lymphoma, mantle cell lymphoma, MALT lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia and so on.
  • any sample may be used as a sample, as long as it is a cell in which a chromosomal abnormality may exist.
  • bone marrow fluid obtained by bone marrow aspiration or peripheral blood can be used as a sample. Samples should be used immediately after collection from patients or thawed at 37 ° C immediately after cryopreservation.
  • Bone marrow fluid includes leukocyte type cells such as bone marrow blasts, erythroblast type cells, bone marrow megakaryocytes, and plasma cells. Therefore, tumor cells can be selectively detected by separating positive cells using an antibody against a surface antigen expressed in tumor cells and further staining the surface antigens of the tumor cells.
  • an anti-CD138 antibody in the case of a tumor derived from plasma cells such as multiple myeloma
  • an anti-CD33 antibody in the case of myeloid leukemia an anti-CD19 antibody in the case of B cell lymphoma
  • an anti-CD20 antibody In the case of T cell lymphoma, an antibody corresponding to the origin of each tumor cell may be used, such as an anti-CD3 antibody, and in the case of an NK cell lymphoma, an anti-CD56 antibody.
  • positive cells expressing surface antigen are separated by magnetic force.
  • Tumor cells derived from plasma cells such as multiple myeloma often express CD138, so that tumor cells can be separated together with plasma cells.
  • tumor cells may be enriched by negative selection.
  • the method of concentration may be any method other than magnetic beads as long as tumor cells can be separated, such as separating tumor cells by FACS.
  • cell surface antigens specifically expressed in tumor cells are stained.
  • a biotin labeled antibody against cell surface antigen is bound and the cell surface antigen is stained using fluorescently labeled streptavidin.
  • the biotin-labeled antibody may be one against the same antigen as that used for separation above, or one against a different antigen.
  • direct cell staining may be performed using antibodies to fluorescently labeled cell surface antigens.
  • formaldehyde fixation and postfixation with methanol / acetic acid are performed according to a conventional method. After removing the supernatant by centrifugation, 10% acetic acid is added and the mixture is allowed to stand at room temperature for 10 minutes.
  • cell surface antigen staining may not be performed. If it is not necessary to stain the surface antigen, it will be possible to assign the dye to FISH and detect more chromosomal aberrations.
  • the air-dried slides may be hybridized immediately with the DNA probe or may be stored at -20 ° C until use.
  • the prepared slide glass is hybridized with a desired probe, an image is acquired, and analysis is performed.
  • a hybridization solution not containing a probe is dropped to a spot on which a cell is spotted on a slide glass, and antigen activation (heat denaturation) is performed by standing at 80 ° C. for 1 hour.
  • antigen activation heat denaturation
  • the FISH probe is used after heat denaturation together with or separately from the sample.
  • the probe and the sample are heat denatured together, they are denatured by treatment at 75 ° C. to 80 ° C. for 1 to 5 minutes.
  • the probe and the sample are separately heat treated, the probe is heated at 70 ° C. to 75 ° C. for 5 minutes and then quenched on ice, and the sample slide is immersed in a denaturing solution, and the temperature is 70 ° C.
  • the desired combination of labeled probes is mixed in the hybridization solution, heat denatured at 75 ° C. for 10 minutes, and kept at 45 ° C. until use.
  • the probe is mounted while holding the air-dried slide glass at 45 ° C., and hybridization is performed at 40 ° C. for 16 to 20 hours. After washing, the slide glass after hybridization is air dried and sealed for observation.
  • any software may be used as long as it is a software that processes an image in the same manner and separates and analyzes each fluorescence.
  • PB495 use laser 488 nm, maximum excitation wavelength 495 nm, maximum emission wavelength 517 nm, Lica Biosystems
  • AF594 use laser 561 nm, maximum excitation wavelength 590 nm, maximum emission wavelength instead of Red 5-ROX 617 nm, manufactured by Thermo Fisher Scientific. Any dye that is excited by each laser and exhibits a similar spectral pattern to the dyes listed in Table 1 can be used.
  • Example 1 ⁇ Surface antigen staining> After collecting bone marrow solution of multiple myeloma patients and adding PBS containing 2 mM EDTA and centrifuging at 1500 rpm for 5 minutes, MACS (registered trademark) buffer (the amount of precipitate is equal to the volume of the first sample) It suspended by Miltenyi (made by Biotec). Next, 1/20 volume of the sample was added magnetic beads (Miltenyi Biotec) bound with anti-CD138 antibody, reacted at 4 ° C for 15 minutes, and separated CD138 positive cells by AutoMACS (Miltenyi Biotec) . The supernatant was removed by centrifugation at 2000 rpm for 3 minutes to give 10 ⁇ l of cell suspension.
  • MACS registered trademark
  • the antigen activation treatment was performed for 60 minutes on a hot plate (StatSpin ThermoBrite, manufactured by ABBOTT) at 80 ° C.
  • the slide glass was removed from the cover glass in 2 ⁇ SSC at room temperature, immersed in 70%, 85%, and 100% ethanol for 1 minute each, air-dried, and kept at 45 ° C.
  • the probe was heat denatured by mixing the labeled probe in hybridization buffer and heating to 75 ° C. for 10 minutes.
  • the probes used to detect each chromosomal mutation and the fluorescent labels are as shown in Table 2.
  • the probes for MAF, FGFR3, BCL1, DLEU1 and 13q34 are BAC clones.
  • Empire Genomics IGH was purchased from Empire Genomics
  • Vysis p53 and Vysis CEP 17 were purchased from Vysis.
  • the fluorescent labeling of the probe was performed by a nick translation kit (manufactured by ABBOTT).
  • FIG. 2 shows the results of the eight probes shown in Table 2, CD138 as a cell surface antigen, and nuclear staining.
  • the upper left stained image is an image obtained by superimposing the stained image of a probe (see Table 2) for detecting a chromosomal aberration
  • the lower left image is an image of cell surface antigen (CD138) and nuclear staining
  • the upper right image is a chromosome It shows an image obtained by superposing the CEP 17 with the images of P53, 13q34 and DLEU1 in which an abnormality is detected.
  • Example 2 FISH which detects the abnormality of one chromosome which is a conventional method, and the result comparison by SCAT FISH are shown (Table 3).
  • the testing company was asked to test the bone marrow fluid of 10 multiple myeloma patients (specimen P1 to P10), and the ratio of CD138 positive cells to the total cells by FACS (FACS result of testing company), DLEU1 (RB1) defect , P53 deletion, BCL1 translocation, FGFR3 translocation, and FISH examination of MAF translocation were requested.
  • FACS FACS result of testing company
  • DLEU1 (RB1) defect DLEU1
  • P53 deletion P53 deletion
  • BCL1 translocation FGFR3 translocation
  • FISH examination of MAF translocation were requested.
  • the same samples were subjected to FISH by SCAT FISH using the same method as in Example 1, and the rates at which chromosomal abnormalities were detected were summarized.
  • Table 3 Shown in Table 3 is the percentage of chromosomal abnormalities detected in each sample. Since false positives are observed even in healthy individuals, peripheral blood samples of 5 healthy individuals were analyzed by SCAT FISH. The column of healthy individuals shows the mean (AVE) of false positives detected in healthy individuals and the standard deviation (SD). The shaded columns in the table indicate that SCAT FISH detected a chromosomal aberration at a frequency that was significantly (P ⁇ 0.05) higher than that of healthy individuals. Furthermore, because false positives also occur in the analysis of the testing company, DLEU1 (RB1) deficiency is judged as 5% or less, P53 deficiency is 2% or less, and BCL1, FGFR3 and MAF translocation is judged as false positive as 1% or less. . "None" indicates that the inspection company has not requested FISH inspection.
  • DLEU1 (RB1) deficiency is relatively high at 18.6% in healthy individuals, false positives are observed, but all patient samples show significantly higher values than the average value of healthy individuals, and DLEU1 deficiency is present. It became clear. According to the analysis results of the testing company, half of the patients did not have DLEU1 deficiency, indicating that SCAT FISH is a very sensitive method.
  • High sensitivity is particularly pronounced in patients with a low percentage of CD138 positive cells.
  • the ratio of CD138 to all cells was very low at 1.3%, so that no FISH test performed by a testing company found any chromosomal abnormality.
  • SCAT FISH DLEU1 (RB1) deficiency was recognized at 24.6%, which is significantly higher than that of healthy individuals.
  • DLEU1 (RB1) of specimens P1, P7, P9 and P10, BCL1 translocation of P5 to P7, FGFR3 translocation of P1 and MAF translocation of P2 to P6 are detected only by SCAT FISH.
  • SCAT FISH collects and stains CD138-positive cells expressed in plasma cells, so that chromosomal aberrations can be detected with high sensitivity. Thus, tumors can be detected early and prognostic typing can be performed.
  • Example 3 An example of detecting and analyzing a plurality of chromosomal abnormalities in one cell by SCAT FISH is shown below.
  • SCAT FISH was performed in the same manner as in Example 1 in two multiple myeloma relapse cases, and analysis was performed to determine what complex chromosomal aberration occurred in one cell (Table 4 to Table 7). .
  • Table 4 analyzes the percentage of each chromosomal abnormality in the patient P8 of relapse cases and shows the percentage. Three years and five months from P8-1 to P8-2, one year and eight months from P8-2 to P8-3, and two years from P8-3 to P8-4.
  • MP indicates melphalan
  • prednisolone indicates lenalidomide
  • dexamethasone indicates lenalidomide
  • BD indicates bortezomib
  • dexamethasone over a multiple myeloma chemotherapy regimen indicates progressive Disease
  • NC No Change.
  • Table 5 shows the results of SCAT FISH analysis of overlapping chromosomal abnormalities occurring in one cell for the same sample. Table 5 shows, from the top, chromosomal abnormalities detected in one cell in patient P8 at each stage.
  • SCAT FISH allows to examine chromosomal abnormalities and to select the best treatment for a particular pattern of chromosomal abnormalities.
  • SCAT FISH is effective in detecting multiple chromosomal aberrations that overlap in various hematological malignancies, as well as multiple myeloma.
  • Detailed chromosomal aberration information obtained using SCAT FISH can be used for selection of treatment methods and prognosis.
  • chromosomal abnormalities and probes for detecting them are summarized in Table 8 (Table 8-1 and Table 8-2).

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Abstract

La détection simultanée de multiples sortes d'anomalies chromosomiques se produisant dans une cellule cible unique, si possible, permet une classification de type de tumeur sanguine avec une précision élevée et, par conséquent, contribue au diagnostic, au traitement et à la prédiction pronostique associés. Une méthode de détection simultanée de multiples anomalies chromosomiques dans une cellule cible unique à l'aide de 3 marqueurs identifiables ou plus (anomalies chromosomiques simultanées dans une cellule cible par hybridation in situ à fluorescence : SCAT FISH) a été établie.
PCT/JP2018/039290 2017-10-23 2018-10-23 Méthode de détection simultanée de multiples anomalies chromosomiques dans une cellule cible unique WO2019082871A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022741A1 (fr) * 2002-09-03 2004-03-18 Japan Science And Technology Agency Chromosome mammifere artificiel
JP2012103077A (ja) * 2010-11-09 2012-05-31 Olympus Corp 遺伝子異常細胞の解析方法
WO2016106298A1 (fr) * 2014-12-22 2016-06-30 Sigma-Aldrich Co. Llc Visualisation de nucléotides modifiés et d'interactions concernant des acides nucléiques dans des cellules isolées

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022741A1 (fr) * 2002-09-03 2004-03-18 Japan Science And Technology Agency Chromosome mammifere artificiel
JP2012103077A (ja) * 2010-11-09 2012-05-31 Olympus Corp 遺伝子異常細胞の解析方法
WO2016106298A1 (fr) * 2014-12-22 2016-06-30 Sigma-Aldrich Co. Llc Visualisation de nucléotides modifiés et d'interactions concernant des acides nucléiques dans des cellules isolées

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ATTO DYES FOR SUPERIOR FLUORESCENT IMAGING, 24 September 2017 (2017-09-24), Retrieved from the Internet <URL:https://web.archive.org/web/20170924053652/https://www.sigmaaldrich.com/life-science/cell-biology/detection/learning-center/atto.html> *
CHENG S.H. ET AL.: "4q loss is potentially an important genetic event in MM tumorigenesis: identification of a tumor suppressor gene regulated by promoter methylation at 4ql3.3, platelet factor 4", BLOOD, vol. 109, 2007, pages 2089 - 2099 *
LI F. ET AL.: "Identification of characteristic and prognostic values of chromosome lp abnormality by multi- gene fluorescence in situ hybridization in multiple myeloma", LEUKEMIA, vol. 30, 2016, pages 1197 - 1201, XP055595252 *
ROSS F.M. ET AL.: "Report from the European Myeloma Network on interphase FISH in multiple myeloma and related disorders", HAEMATOLOGICA, vol. 97, no. 8, 2012, pages 1272 - 1277, XP055595254 *
SAEZ B. ET AL.: "Multicolor interphase cytogenetics for the study of plasma cell dyscrasias", ONCOL. REP., vol. 18, 2007, XP055595248 *

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