WO2008029468A1 - Procédé et appareil pour microéchantillonnage multipoint - Google Patents

Procédé et appareil pour microéchantillonnage multipoint Download PDF

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Publication number
WO2008029468A1
WO2008029468A1 PCT/JP2006/317738 JP2006317738W WO2008029468A1 WO 2008029468 A1 WO2008029468 A1 WO 2008029468A1 JP 2006317738 W JP2006317738 W JP 2006317738W WO 2008029468 A1 WO2008029468 A1 WO 2008029468A1
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Prior art keywords
tissue
cell material
target cell
pieces
sampled
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PCT/JP2006/317738
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English (en)
Japanese (ja)
Inventor
Kikuya Kato
Takashi Kodama
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Neat Co., Ltd.
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Priority to PCT/JP2006/317738 priority Critical patent/WO2008029468A1/fr
Publication of WO2008029468A1 publication Critical patent/WO2008029468A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G01N2001/2886—Laser cutting, e.g. tissue catapult
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to a target cell identification method and apparatus for extracting a target cell material from a tissue, and in particular, diagnosis of the patient can be performed by identifying the target cell from a pathological tissue specimen obtained from the patient. It is about how to do it.
  • the laser capture microdissection technique is a very good isolation method as described above.
  • any of the conventional techniques it is necessary to extract the target cell along its contour, so that the operation is constant.
  • Technology is required.
  • cancer tissue generally contains a mixture of cancer cells and non-cancer cells. Therefore, in order to make an accurate diagnosis, skilled techniques for identifying and accurately cutting out only cancer cells are required.
  • the present invention has been made in view of such circumstances, and is intended to facilitate the extraction of the target cell material by a simpler method as compared with the conventional laser capture microdissection technique.
  • An object of the present invention is to provide a cell material identification method.
  • a further object of the present invention is to provide an effective method for various determinations / diagnosis suitable for the identification method described above.
  • a target cell material identification method for extracting a target cell material, the step of providing a target tissue, Arbitrary different potential forces in the tissue
  • a method comprising sampling a plurality of tissue pieces and identifying a target cell material from the sampled tissue pieces.
  • a sampled tissue piece is obtained by randomly sampling a statistically and empirically effective number of microtissue pieces from the target cell and surrounding tissue. Is classified as one of the following
  • the target cell can be identified by classifying the tissue piece into any one of the above (1) to (3), for example, by observing the expression of the gene, protein, etc. of the sampled tissue piece.
  • Random sampling of the minute tissue as described above can be performed by an automatic device.
  • tissue pieces are processed at the same time to observe gene expression or the like, target cells can be efficiently identified from the tissue pieces.
  • the present invention is applied to enzymes and antigens, and DNA, RNA (mRNA), lipids, and carbohydrates.
  • the step of sampling the tissue piece includes the step of: It is preferable that the plurality of tissue pieces are sampled by irradiating the tissue with laser light and cutting out the tissue force tissue pieces.
  • the plurality of tissue pieces are sampled in substantially the same area.
  • the tissue pieces may have any shape, but may have a diameter of 10 to 500 if approximated to a circle. It is desirable that the value be determined from m (about 1Z4 to 30,000 of main cells).
  • the thickness of the tissue piece to be sampled is 5 ⁇ m to 50 ⁇ m, more preferably 10 ⁇ m to 40 ⁇ m.
  • the number of the plurality of tissue pieces is at least 5 or more (in the range of 5 to preferably 192), and the most preferable number is 96.
  • the step of identifying the target cell material is performed by examining a purified product from each sampled tissue piece.
  • the purified product to be examined can be local tissue polypeptides, proteins such as enzymes and antigens, and DNA, RNA (mRNA), lipids, sugar chains, and other biological molecules and combinations thereof. preferable.
  • the step of identifying the target cell material is performed based on whether or not a specific gene is expressed for each tissue piece. In this case, Alternatively, it may be determined whether or not two or more specific genes are expressed for each tissue piece, and the target cell material may be identified based on the expression ratio of the two or more specific genes.
  • the step of sampling the plurality of tissue pieces drops the cut tissue piece into a container and transfers the container to the next process.
  • the process to perform is included.
  • the container is preferably a well plate having a number of wells corresponding to the number of tissue pieces to be sampled.
  • the identifying step can be performed by simultaneously processing each tissue piece held in each well of the well plate in units of the well plate.
  • a method for performing a specific determination on a patient using a tissue from which patient force is also separated, and a plurality of different determinations from any arbitrary position in the tissue Sampling a piece of tissue and a purpose from the plurality of sampled tissue pieces
  • a method comprising the steps of: identifying a cellular material; and performing a specific determination regarding the patient based on the identification of the target cellular material.
  • the patient can be diagnosed based on the ability to refine a specific product related to a specific disease for each yarn and woven piece.
  • the specific determination may be a determination of sensitivity to a drug.
  • the cell material that does not react with the cell material that reacts with the drug is identified. It is preferable to determine the sensitivity based on the ratio of the materials without reacting.
  • the drug is gefitinib, and the identification of the cell material that does not react with the cell material that reacts with the drug is performed based on the amount of EGFR mutation in the cell material.
  • a target cell material sampling apparatus for extracting a target cell material from yarn and weave, the means for holding the target tissue, and the tissue And a means for cutting out and sampling a plurality of tissue pieces from arbitrary different positions in the tissue, and a means for holding the plurality of sampled tissue pieces.
  • the plurality of tissue pieces cut out by the means for sampling the cellular material have a constant area
  • the means for holding the tissue removes the tissue pieces from the tissue piece cut out by a laser beam trajectory. It is preferable to have an opening for dropping the holding means.
  • the means for holding the tissue and the means for holding the plurality of sampled tissue pieces are at least horizontally and vertically with respect to the irradiation head that irradiates the laser beam.
  • the tissue piece is relatively movable, and the tissue piece is cut out from different positions of the tissue, and the cut tissue piece is held at different positions of the means for holding the plurality of sampled tissue pieces.
  • an effective number of tissue pieces with a small area can be randomly obtained from a tissue based on statistics or experience.
  • the means for holding the tissue and the means for holding the plurality of sampled tissue pieces can be moved relatively at least in the horizontal and vertical directions with respect to the irradiation head that irradiates the laser light.
  • the present invention relates to a method for extracting and identifying tissue, especially pathological tissue force, for the purpose of analyzing cellular material at the molecular or genetic level.
  • the gist of the present invention is to cut out a small section of tissue force including target cells randomly and in large numbers, and individually purified products (local tissue polypeptides, proteins such as enzymes and antigens, DNA, RNA (mRNA), lipids)
  • the target cell material is identified by examining sugar chains added to protein lipids, and other biological molecules and combinations thereof.
  • this concept is referred to as “multi-point minute sampling”.
  • the size of the tissue section cut out from the pathological tissue force is desirably sampled in substantially the same area, and the tissue piece may be any shape but approximated to a circle. It is desirable that the value be determined from a diameter of 10 to 600 m (about 1Z4 to 50,000 main cells).
  • the thickness of the tissue piece to be sampled is 5 to 50 ⁇ m, more preferably 10 to 40 ⁇ m.
  • the number of the plurality of tissue pieces is at least 5 or more (in the range of 5 to preferably 192), and the most preferable number is 96.
  • the size and number (and pitch) of the tissue sections are set so as to always include a desired pattern according to statistics or experience, as will be described later. Specifically, a large number of obtained tissue pieces include only those other than the target cell material, those that include both the target cell material and the other, and those that include only the target cell material. It is set to a size and number that are empirically or statistically valid so that each pattern is always included.
  • telomere sequence there are various methods for identifying a target cell by this multipoint micro-sampling. Can be adopted. For example, if a large number of obtained tissue pieces are considered to contain one or more DNA or RNA of interest, the extracted sample may be subjected to polymerase chain reaction (PCR) amplification followed by, for example, hybridization. , And Southern and Northern plots, sequencing, etc. can be left as desired.
  • PCR polymerase chain reaction
  • the extracted tissue piece includes, or appears to contain, the protein or polypeptide of interest
  • the extracted sample is, for example, an enzyme using one or more labeled substrates. It can be subjected to zymography, immunoassay using labeled antibodies or functional fragments thereof, biochemical assays and similar means.
  • the present invention includes, in its technical scope, a combination of the above-mentioned multipoint microsampling and a known analysis technique optimized for this multipoint sampling.
  • the present invention is also applicable to a specific determination for a patient, for example, a pathological diagnosis of a specific disease or determination of drug adaptability through the analysis using the multi-point micro sampling.
  • the present invention is further directed to a specific determination regarding a patient using the multipoint micro sampling.
  • multipoint microsampling is also applied to a dedicated device for performing the sampling.
  • the apparatus includes a means for holding a target pathological tissue, a means for irradiating the pathological tissue with laser light, cutting out a plurality of tissue pieces from different arbitrary positions in the pathological tissue, and sampling. Means for holding a plurality of tissue pieces.
  • cancer tissue is generally a mixture of cancer cells and non-cancer cells.
  • the purpose of this embodiment is to isolate and detect cancer cells by multipoint microsampling.
  • FIG. 1 is an example of a cancer tissue.
  • This tissue includes cells other than cancer and other tissues.
  • the part indicated by A in the figure is cancer cells
  • B is lymphocytes
  • C is stroma. This Since such contamination with other cells deteriorates the diagnostic accuracy, it is necessary to cut out only cancer cells. In other words, in the examination using the whole tissue, the profile of the cancer cell is diluted, and it is difficult to detect the initial cancer in particular. To accurately identify the outline of a cancer cell, image processing is often used, but it is not possible to accurately handle all types of cancer cells! Required.
  • microtissue pieces are randomly obtained from cancer tissue using multipoint microsampling, and these are individually detected. Then, by using gene A that is specifically expressed in cancer cells and gene B that is specifically expressed in non-cancer cells, a minute sample is formed!
  • cancer cells can be detected by using the two genes A and B described above, and cancer cells can be isolated and detected by collecting samples containing only cancer cells. It is.
  • a slice of cancer tissue was fixed on a slide glass for microdissection.
  • the thickness of this cancer tissue is 35 ⁇ m.
  • the cancer tissue was fixed with ethanol by a usual method and stained with hematoxylin.
  • Steps 1-3 using a laser beam microdissection device (in this example, a Leica device), 100 ⁇ 100 / zm tissue sections were randomly cut, and 351 RLT buffer (Qiagen's It was immersed in RNeasy micro kit).
  • a laser beam microdissection device in this example, a Leica device
  • 351 RLT buffer Qiagen's It was immersed in RNeasy micro kit.
  • RNA is purified from the excised tissue section.
  • a Qiagen RNeasy micro kit was used.
  • RNA can be obtained in a state dissolved in distilled water of 12 1
  • RNA was converted to DNA by the following protocol.
  • the reaction cycle is 35 cycles of (94 degrees for 30 seconds, 55 degrees for 30 seconds, 72 degrees for 1 minute).
  • the reaction product was confirmed by / 0- agarose gel electrophoresis.
  • Step 1-7 Judgment
  • the sample contains only cancer cells.
  • the sample contains only non-cancerous cells.
  • both the gene A and the gene B can be detected as amplification products, both cancer cells and non-cancer cells are included.
  • the sample contains cells.
  • cancer cells can be collected by collecting samples that can detect only the amplification product of gene A.
  • tissue piece to be cut out has a small area defined by the relationship with cancer cells, and statistically or empirically includes only cancer cells. Obtaining a tissue piece with randomness suitable for obtaining the tissue piece.
  • a tissue piece of a predetermined area is cut out at random, so that a specialized and special technique like a pathologist is not required for cutting out. For this reason, this multi-point micro sampling is very suitable for automation.
  • analysis of the cut tissue piece can be performed automatically. That is, by using a primer suitable for the desired pathological diagnosis, the purified product of the tissue piece is analyzed, and the pathological diagnosis regarding various diseases can be performed only by classifying the tissue piece. It is also useful to prepare individual test kits for diagnosing various diseases.
  • FIG. 4 is a flowchart showing an outline of the automatic sampling diagnosis process.
  • Step 2-1 a large number of tissue pieces of about 50 ⁇ m to 200 m are cut out from the pathological yarn and weave with an automatic sampling device.
  • the pathological specimen is a frozen one having a size of about 0.3 to 0.5 cm and sliced into a thin piece having a thickness of about 10 m.
  • step 2-3 if even one of the small pieces shows cancer-specific gene expression, it is judged as cancer.
  • FIG. 5 is a schematic configuration diagram showing an example of an automatic multipoint micro-sampling apparatus that can be used for the automatic diagnosis as described above.
  • the apparatus includes a base 10, a well plate holding mechanism 12 that is installed on the base 10 and holds a 96-well well plate 11 for holding the sampled tissue piece.
  • Laser light is applied to the pathological tissue holding mechanism 14 for holding the pathological tissue held in the pathological tissue holder 22 provided on the base 10 and the pathological tissue 13 held in the pathological tissue holding mechanism 14 from above.
  • a laser irradiation unit 15 for irradiating the light.
  • the well plate holding mechanism 12 also works with the fine movement X stage 16, the fine movement Y stage 17 and the holding base 18 so as to fix and hold the 96-well well plate 11 on the holding base 18. ing.
  • the well plate used in this embodiment is a known 96-well plate, but other shapes can also be used as the tissue piece holder of the present invention.
  • the well plate holding mechanism 12 includes the fine movement X stage 16, the fine movement Y stay. By operating the screw 17, the respective wells of the 96-well well plate 11 are made to face the laser irradiation position by the laser irradiation unit 15, and the tissue piece cut out from the pathological tissue 13 is received in the well. And then speak.
  • the pathological tissue holding mechanism 14 similarly includes a fine movement X stage 19, a fine movement Y stage 20, and a holding base 21.
  • the pathological tissue 13 is held by a pathological tissue holder 22 as shown in FIG. 6 for convenience of handling.
  • the cage 22 is configured so that the pathological tissue 13 is sandwiched and fixed between the lower plate 23 and the upper plate 24.
  • the upper plate 23 and the lower plate 24 have an upper force and a laser beam L. Slits 23a and 24a for passing the cut and cut pieces of the fabric down are formed at positions facing each other.
  • the lower plate 24 is formed longer than the upper plate 23 so as to protrude in one horizontal direction, and an identification symbol description part 25 for indicating a patient name and a sample name is provided at the protruding portion.
  • an identification symbol description part 25 for indicating a patient name and a sample name is provided at the protruding portion.
  • the pathological tissue holding mechanism 14 can position and hold the pathological tissue holder 22 on the holding base 21. Then, as shown in FIG. 6, the position to be cut out positioned in the slits 23a and 24a is opposed to the irradiation position of the laser beam L, so that the above-described cutting is performed, and the cut tissue piece is shown in FIG. It is designed to be dropped into the wall 11a of the 96-hole well plate 11 shown in enlargement.
  • the well is filled with, for example, an RLT buffer solution, and the excised tissue piece is immersed in the buffer solution.
  • the laser irradiation unit 15 is held via a Z-direction fine movement stage 26, and is configured to perform laser irradiation in conjunction with the operation of each of the holding mechanisms 12 and 14.
  • This Z-direction fine stage 26 is for controlling the cut-out thickness of the tissue piece. In this embodiment, it is installed on the laser irradiation unit 15 side, but is provided on the pathological tissue holding mechanism 14 side. May be.
  • Each of the above mechanisms 12, 14, and 15 is connected to a control unit indicated by 28 in FIG. 5 and is controlled by this control unit.
  • control by the control unit 28 will be described with reference to the flowchart shown in FIG.
  • step 3-1 the pathological tissue holder 22 is attached to the pathological tissue holding mechanism 14 holding key. Set to 21.
  • the control unit 28 inputs and stores the slice thickness of the pathological tissue 13, the number of cuts in the Z direction, the cut shape in the XY direction, the cut pitch, and the number of cuts (step 3-2).
  • step 3-3 when a cutout start button (not shown) is pressed (step 3-3), the cutout sequence after step 3-4 is started, and multipoint microsampling is automatically executed.
  • Step 3-4 the remaining number of cutouts in the XY directions is confirmed. If it is not 0, the pathological tissue holding mechanism 14 is driven, and a predetermined number according to the number of cutouts of the pathological tissue 13 is determined. The cut-out position is made to face the laser irradiation position by the laser irradiation unit 15 (step 3-5), and the number of XY cut-outs is incremented by 1 (step 3-6).
  • the remaining number of cutouts in the Z direction is confirmed. If it is not 0, the fine movement Z stage 26 is driven, and the cutting thickness by the laser irradiation unit 15 is set to a predetermined value.
  • the laser irradiation unit 15 is driven, and the pathological tissue holding mechanism 14 is driven in the XY direction while irradiating the pathological tissue 13 with the laser L.
  • the pathological tissue is cut into a cutout shape (predetermined area), and a microtissue piece 13a is cut out.
  • the laser drive by the laser irradiation unit 15 is stopped, and the number of times of cutting in the Z direction is incremented and decreased by one.
  • the wel plate 11 is also driven in conjunction with the positioning in the XY directions so that each wel 11a receives the cut yarn and woven piece 13a. Sequentially, it is stored in each of the holes 11a of the 96 holes.
  • the wall plate 11 is transferred to a real-time PCR apparatus, and a predetermined test is performed on the tissue piece 13a in each of the wells 11a.
  • This example is an example in which multipoint microsampling is used to determine drug adaptability. [0089] First, the background power will be described.
  • cancer tissues also have cancer cell power with different genetic changes. This heterogeneity within the tumor may be a potential problem for chemotherapy. In other words, the sensitivity to drugs varies among cancer cells in a single tumor tissue.
  • the recently available new U a type of anticancer drug, molecular target drug, is designed to inhibit specific molecules important for cancer cell growth. Therefore, the target molecule may be used as a marker to distinguish sensitive and resistant cells.
  • gefitiv is designed to inhibit the phosphorylation activity of epidermal growth factor receptor (EGFR), which is a non-small cell lung cancer.
  • EGFR epidermal growth factor receptor
  • NSCLC non-small cell lung cancer
  • somatic mutations introduced into the phosphate chain of EGFR are important for the effects of Gefitiv. This means that tumors with mutations are sensitive to gefitib.
  • Several conflicting studies exist and more hopeful ones are needed to establish, but the correlation between mutations and the effects of gefitinib has gained consensus at least among the Japanese people! /
  • the inventors pay attention to Geifib, and verify that the above correlation at the tissue level extends to the cellular level, and that the EGFR mutation can distinguish cells. It was.
  • NSCLC tissue primary lesions that also had EGFR mutations were collected from patients who were administered gevitib, and multipoint microsampling was performed from each tissue, Mutation status was examined. 14 tissues have EGFR mutations Only included cancer cells! /, But 6 tissues contained cancer cells with and without mutations. And both duration and overall survival were shorter in the latter group. In this way, by performing multipoint microsampling, the cell composition of NSCLC tissue was found, and thereby it was possible to more accurately determine the sensitivity to getivib.
  • NSCLC tissue was obtained from 21 patients by surgical disserction. After the surgical incision, the patient was first treated with conventional chemotherapy, followed by administration of gefitib. All cancer tissues were confirmed to have reported EGFR mutations by direct sequencing of PCR products.
  • Genomic DNA was purified from tumor tissue obtained from patients by using a Qiagen genomic DNA purification kit.
  • the EGFR gene was amplified by PCR, and the amplified product was direct sequenced using the BigDye terminator kit.
  • the tissue pieces from which the tumor tissue force was also cut out are about 60 pieces having a size force of 00 X 100 X 35 ⁇ m.
  • Genomic DNA was purified from each sample using the Qiagen DNA purification kit. Mutation status was measured using SNaPshot assay (Applied Biosystems). In SNaPshot, oligonucleotide primers were designed next to the mutant nucleotides to be analyzed based on the results of direct sequencing of PCR products. Using fluorochrome labeled dideoxynucleotides, one nucleotide is added by DNA polymerase. The state of the mutation can be determined by the inserted nucleotide.
  • the fluorescent dyes used for dideoxynucleotides are (A, dR6G, C, dTAMARA, G, dR110, T, dROX). Fragments were detected using the ABI 3100 DNA analyzer. Judging from the inserted nucleotides, each sample was classified as either wild-type EGFR, mutant EGFR (heterozygote), or mutant EGFR (homozygote). Of all small areas By measuring the state of the mutation in it, the ratio of each cell type is measured. In this way, the cellular composition for EGFR mutations is analyzed.
  • the tumor tissue consists of cancer cell forces with different EGFR mutation states, you can distinguish those differences by analyzing the mutation states in many small regions
  • the number of genotype regions is counted as the ratio of getivib-sensitive cells (positive for EGFR mutation) to gefitib-insensitive cells (negative for EGFR mutation and wild type).
  • Measure with Sampling is a force that should be taken care of non-cancerous cell contamination This is accomplished by performing accurate laser beam microdissection.
  • SNaPshot which is a primer extension analysis
  • oligonucleotide primers were designed next to abruptly mutated nucleotides to be analyzed based on the results of direct sequencing of PCR products.
  • One nucleotide was added by DNA polymerase using fluorescent dye-labeled dideoxynucleotides. The state of the mutation can be measured by the inserted nucleotide.
  • the sample size was determined to be 100 x 100 x 35 m. This region contains 30-60 cells.
  • FIG. 9 shows an example of EGFR gene mutation diversity in lung cancer tissue.
  • the left side of Fig. 9 is a histological image of gastric cancer, and the sample areas excised and collected by laser dissection are indicated by symbols 1 to 4, and the right side of the figure is the SNap shot results for each collected sample area. is there.
  • the tumor is a mixture of cancer cells with and without EGFR mutation.
  • the tissues of 21 patients were examined. A total of 60 regions were collected from each tumor tissue and their EGFR mutation status was measured. Cancer tissues are divided into two groups: EGFR mutated cells only (15 cases, group I) and EGFR mutated cells and EGFR mutated cells (6 cases, group II) It was classified into two. In the eight tissues of group I, as shown in Figure 10, the gene dosage of the mutated EGFR gene and the mutated ⁇ EGFR gene are almost the same, indicating that these cells are heterozygous. It suggests that there is. It is speculated that the difference in the ratio between the areas is mainly caused by the introduction under analysis.
  • the other 8 tissues in group I also consist of mutated and non-mutated genes, but there are many mutated genes, as shown in Figure 11. This is either due to the specific amplification or ploidy of the mutated EGFR gene, ie the dominance of the chromosome with the mutated gene.
  • the remaining 6 yarns and weaves consist not only of cells having mutant genes but also of wild type cells.
  • the composition of wild-type cancer cells varies from 5% to 100%.
  • the patient numbers 4-21 have no cells with the mutated gene. Only However, the direct sequence of the PCR product revealed the presence of the mutation. This is because of the heterogeneity between these samples because the tissue regions used for the sequence were from different parts of the same tumor tissue.
  • the present invention relates to a novel multipoint microsampling method.
  • the present invention is an apparatus and a diagnostic method that can be applied to this multipoint microsampling.
  • the cells to be identified currently include, but are not limited to, kidney glomeruli, primary breast cancer, prostate epithelial tumor, lymphoid nodule, and the like.
  • the size of the pathological tissue, the size, shape, number, and pitch of the tissue pieces to be cut out are not limited to the above embodiment, but are appropriately selected according to the size of the cell and the specifications of the measuring instrument. Is possible.
  • the target tissue is a force that was a pathological tissue.
  • the normal tissue that is not limited to this may be sampled.
  • FIG. 1 A diagram for explaining cells constituting a cancer tissue based on a micrograph of the cancer tissue.
  • FIG. 2 is an explanatory diagram for explaining the concept of multipoint micro sampling.
  • FIG. 3 is a flowchart showing an example of target cell identification using multipoint microsampling.
  • FIG. 4 Flow chart for automatic multi-point micro sampling and automatic diagnosis.
  • FIG. 5 is a schematic configuration diagram showing an automatic multipoint micro sampling device.
  • FIG. 6 is a schematic configuration diagram showing a pathological tissue holder.
  • FIG. 7 is a flowchart showing the control of the automatic multipoint micro sampling device.
  • FIG. 8 is a table showing oligonucleotide primer sequences used for SNap shot analysis.
  • FIG. 9 Figure showing an example of EGFR gene mutation diversity in lung cancer tissue: The left side of the figure is a histological image of gastric cancer, showing areas 1 to 4 excised and collected by laser dissection, in the figure The right side is the result of SNap shot for each area.
  • FIG. 10 A graph showing in-tumor heterogeneity of EGFR gene dosage in group I tissue: vertical axis
  • the percentage of mutated EGFR genes is indicated by white columns, and the percentage of mutated EGFR genes in each of the approximately 60 regions is listed in the left force lateral direction from the smallest.
  • FIG. 11 A diagram showing in-tumor heterogeneity of EGFR gene dosage in the same group I tissue.
  • FIG. 12 is a graph showing in-tumor heterogeneity of EGFR gene dosage in group II tissues.
  • FIG. 13 Graph showing intra-tumor heterogeneity of EGFR gene dosage in the same group II tissue
  • FIG. 14 A graph showing force plan Meyer analysis of patients in group I and group II, a) no recurrence period after gefitinib administration, b) overall survival after gefitinib administration, In both cases, the vertical axis shows the proportion of patients with no recurrence (a) or surviving patients (b), and the horizontal axis shows the number of days elapsed.

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Abstract

Le problème à résoudre dans le cadre de la présente invention consiste à proposer un procédé d'identification d'une matière cellulaire cible moyennant quoi ladite matière peut être facilement extraite par une procédure pratique par rapport à la technique anatomique de microspectroscopie par piégeage laser existante. La solution proposée consiste en un procédé d'identification d'une matière cellulaire cible, qui vise à identifier la matière cellulaire cible à partir d'un tissu, comprenant l'étape consistant à fournir le tissu sujet, l'étape consistant à échantillonner plusieurs morceaux de tissu à partir de sites différents arbitraires dans le tissu, et l'étape consistant à identifier le matériau cellulaire cible parmi les morceaux de tissu ainsi échantillonnés.
PCT/JP2006/317738 2006-09-07 2006-09-07 Procédé et appareil pour microéchantillonnage multipoint WO2008029468A1 (fr)

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PCT/JP2006/317738 WO2008029468A1 (fr) 2006-09-07 2006-09-07 Procédé et appareil pour microéchantillonnage multipoint

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PCT/JP2006/317738 WO2008029468A1 (fr) 2006-09-07 2006-09-07 Procédé et appareil pour microéchantillonnage multipoint

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WO2008029468A1 true WO2008029468A1 (fr) 2008-03-13

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CN105229442A (zh) * 2013-05-15 2016-01-06 皇家飞利浦有限公司 组织从样本分离
WO2016207986A1 (fr) * 2015-06-24 2016-12-29 株式会社日立製作所 Système d'inspection, dispositif d'inspection et procédé d'inspection

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105229442A (zh) * 2013-05-15 2016-01-06 皇家飞利浦有限公司 组织从样本分离
JP2016522900A (ja) * 2013-05-15 2016-08-04 コーニンクレッカ フィリップス エヌ ヴェＫｏｎｉｎｋｌｉｊｋｅ Ｐｈｉｌｉｐｓ Ｎ．Ｖ． 試料からの組織分離
WO2016207986A1 (fr) * 2015-06-24 2016-12-29 株式会社日立製作所 Système d'inspection, dispositif d'inspection et procédé d'inspection
JPWO2016207986A1 (ja) * 2015-06-24 2017-12-28 株式会社日立製作所 検査システム、検査装置、及び検査方法
CN107614674A (zh) * 2015-06-24 2018-01-19 株式会社日立制作所 检查系统、检查装置以及检查方法

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