WO2017114003A1 - Sonde de détection du gène alk et du gène eml4, procédé de préparation associé et kit de réactif - Google Patents

Sonde de détection du gène alk et du gène eml4, procédé de préparation associé et kit de réactif Download PDF

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WO2017114003A1
WO2017114003A1 PCT/CN2016/105703 CN2016105703W WO2017114003A1 WO 2017114003 A1 WO2017114003 A1 WO 2017114003A1 CN 2016105703 W CN2016105703 W CN 2016105703W WO 2017114003 A1 WO2017114003 A1 WO 2017114003A1
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gene
alk
eml4
ctd
alk gene
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Chinese (zh)
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何瑰
陈绍宇
欧焕金
张会清
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广州安必平医药科技股份有限公司
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    • 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
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention belongs to the field of biotechnology, and particularly relates to an ALK gene and EML4 gene detecting probe, a preparation method thereof and a kit.
  • lung cancer ranked first among the national incidence of malignant tumors (35.23/100,000, 19.59%) and mortality (27.93/100,000, 24.87%).
  • the occurrence of lung cancer is considered to be a multi-step process that is closely related to the amplification/activation of protooncogenes and/or the inactivation of tumor suppressor genes, and provides targets for patients as more and more cancer-driven factors are discovered.
  • the opportunity to treat The study found that 64% of patients with lung adenocarcinoma detected cancer-driven factors, of which 8% were ALK rearrangements.
  • ALK gene fusion is not a favorable prognostic factor for advanced NSCLC, but crizotinib improves survival in ALK-positive patients.
  • EML4-ALK echinoderma microtubule-associated protein-like 4-anaplastic lymphoma kinase
  • fusion gene mutation is the main type, accounting for about 5% of all NSCLC.
  • Other genes that are fused to the ALK gene in lung cancer include TFG, KIF5B, and the like.
  • the EML4-ALK fusion gene can be found in a variety of tumors, such as anaplastic large cell lymphoma, inflammatory myofibroblastoma, neuroblastoma, and NSCLC, which are caused by short arm insertion of chromosome 2.
  • the ALK fusion gene forms an intricate signal transduction network, which affects cell proliferation, differentiation and apoptosis through the activation and transmission of downstream substrate molecules and the mutual crossing and overlapping of transduction pathways.
  • the EML4-ALK fusion gene binds the kinase regions of two EML4-ALK molecules through the extracellular helical domain of the fusion partner to form a stable dimer, which activates downstream MAPK, PI3K/AKT, JAK/STAT3 by autophosphorylation. Wait for the pathway, causing the cells to metastasize.
  • ALK gene rearrangement was first discovered and named in anaplastic lymphoma, and was reported in lung cancer in 2007. Now, as a member of the tyrosine kinase signaling pathway, ALK gene abnormalities may be found in more cancerous species, and the indications for targeted drugs will continue to expand.
  • the probes used for hybridization can be roughly classified into three categories: 1) chromosome-specific repeat probes, such as alpha satellites, satellite class III probes, which often have a hybrid target of more than 1 Mb, do not contain scattered repeats, and bind tightly to the target. Strong hybridization signal, easy to detect; 2) whole chromosome or chromosomal region-specific probe consisting of a very different nucleotide fragment on a chromosome or a segment of a chromosome, which can be cloned into phage and plasmid A chromosome-specific large fragment is obtained; 3) a specific position probe consisting of one or several cloned sequences.
  • the fluorescein labeling of the probe can be performed by direct and indirect labeling.
  • the indirect labeling is a biotin-labeled DNA probe, which is detected by fluorescein avidin or streptavidin after hybridization, and the avidin-biotin-fluorescein complex can also be used to fluoresce signals. Amplification is performed so that a fragment of about 500 bp can be detected.
  • the direct labeling method is to directly bind fluorescein to the probe nucleotide or the pentose phosphate backbone, or to incorporate fluorescein nucleoside triphosphate in the nick translation labeling probe.
  • the direct labeling method has simple steps in detection and is convenient for clinical use.
  • One of the objects of the present invention is to provide an ALK gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting ALK gene rearrangement and identification, and has good specificity.
  • a method for preparing an ALK gene detection probe comprising the steps of:
  • the BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one as another set of probes;
  • the color of the fluorescein of the same group of probes is the same, the color of the fluorescein of different sets of probes is different, that is, the BAC clones are RP11-62B19, RP11-100C1 and CTD-2245E6.
  • the probe is different from the color of the fluorescein derived from the probes of the BAC clones CTD-2544I11, RP11-684O3 and RP11-134O13.
  • Another aspect of the present invention provides an ALK gene and EML4 gene detecting probe and a preparation method thereof, and the prepared probe can be used for detecting the ALK gene and the EML4 gene state, that is, for EML4/ALK gene fusion identification, which is excellent. Specificity.
  • a method for preparing an ALK gene and an EML4 gene detection probe comprising the steps of:
  • the BAC clones targeting the ALK gene were selected as at least one of RP11-62B19, RP11-100C1 and CTD-2245E6 as a set of probes, and the BAC clones were selected as CTD-2544I11, RP11-684O3 and RP11-134O13. At least one of the other set of probes; and the BAC clone selected for the EML4 gene is at least one of CTD-2358L8 and CTD-2547J15;
  • the color of the fluorescein labeled with the ALK gene and the detection probe for the EML4 gene is different, and for the ALK gene, the color of the fluorescein of the same group of probes is the same, different groups
  • the color of the fluorescein of the needle is different, that is, the probe derived from BAC clones as RP11-62B19, RP11-100C1 and CTD-2245E6 and the probe derived from BAC clones as CTD-2544I11, RP11-684O3 and RP11-134O13.
  • the color of fluorescein is not the same, that is.
  • the ALK rearrangement probe comprises two groups, the first group of BAC clones being RP11-62B19, RP11-100C1 and CTD-2245E6, respectively; the second group of BAC clones being CTD-2544I11, RP11- 684O3 and RP11-134O13.
  • the EML4 probe comprises a set and the third set of BAC clones are CTD-2358L8 and CTD-2547J15.
  • the labeled fluorescein selects a fluorescent dye known in the art, preferably fluorescein is Alexa FITC, Alexa Rhodamine, Texas Red, pacific DEAC.
  • the labeling of the gene probe can be performed by labeling the corresponding fluorescein to the double-stranded nucleic acid by methods in the prior art, including but not limited to: random primer method, nick translation, etc., marker gene probe
  • the needle can be used with a commercially available nick translation labeling kit and a random primer labeling kit, preferably abbott and Roche's Nick Translation Kit.
  • the plasmid DNA is preferably subjected to fluorescein labeling by a random primer method or a nick translation method.
  • the temperature of the label is between 15 ° C and 18 ° C and the time of labeling is between 8 and 12 hours.
  • Another object of the present invention is to provide an ALK gene and EML4 gene detecting kit.
  • a kit for detecting ALK gene and EML4 gene comprising the above ALK gene and EML4 gene detecting probe.
  • the present invention can detect unknown rearrangement associated with ALK or directly identify EML4/ALK rearrangement by screening for optimal EML4/ALK gene rearrangement detection probes and combinations thereof.
  • the signal counting is accurate and rapid, and the result is reproducible; supplementing the shortage of detection reagents in the clinic depends on the import, which is beneficial to the subsequent detection probe. It is used in clinical research for molecular typing and targeted therapy of lung cancer.
  • Fig. 1A is a schematic diagram showing the sequence of the ALK gene detecting probe in Example 1.
  • Fig. 1B is a schematic diagram showing the probe sequence of the EML4 gene detection in Example 1.
  • Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene detecting probe in Example 1.
  • Fig. 2A is a graph showing the results of FISH detection of the human peripheral blood culture cell sheet ALK gene and EML4 gene detection probe in Example 1.
  • Fig. 3A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F (RG adjacent or coincident), and the ALK gene is not rearranged.
  • FIG. 3B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 4A is a diagram showing the results of FISH detection of the ALK gene of the lung cancer tissue sample in Example 4, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement.
  • FIG. 4B is a diagram showing the results of FISH detection of the ALK gene and the EML4 gene in the lung cancer tissue sample of Example 4, wherein the detection signal type is 1F1A1G1RA, and the F signal is broken and the RA signal fusion occurs simultaneously with the normal signal type 2F2A.
  • ALK gene is broken and the rearrangement type is EML4/ALK.
  • GSP ALK includes two sets of probes:
  • the first set of probes included a first probe, a second probe, and a third probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the second set of probes included a fourth probe, a fifth probe, and a sixth probe, as specifically listed below, which were purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • Probe set 1 (red) BAC Insert start and end position First probe RP11-62B19 Chr2:29354267..29528536 Second probe RP11-100C1 Chr2:29210476..29383355 Third probe CTD-2245E6 Chr2:29066898..29207778
  • Probe set 2 (green) BAC Insert start and end position Fourth probe CTD-2544I11 Chr2:29923580..30121092 Fifth probe RP11-684O3 Chr2:30116506..30315853 Sixth probe RP11-134O13 Chr2:30282641..30436938
  • the preparation method of the EML4 detection probe of the present embodiment comprises the following steps:
  • GSP EML4 includes a set of probes including a first probe and a second probe, as shown in the following table, which was purchased from the Invitrogen RP11 BAC and CTD BAC clone libraries.
  • the labeled product was subjected to ethanol precipitation and concentration, and sodium acetate and absolute ethanol were sequentially added to a 1.5 ml centrifuge tube in the following manner, and protected from light and ice:
  • GSP ALK and GSP EML4 gene probe validation hybridization solution prepared using ALK probe set 1 + ALK probe set 2+ EML4 probe set 3, respectively, and ALK probe set 1 + ALK probe set 2 preparation
  • the hybridization solution is subjected to probe verification on human normal division metaphase lymphocyte droplets (detection method is referred to in Example 3).
  • the cultured cells contain metaphase or interphase chromosomal DNA. When fluorescent in situ hybridization, the chromosomal DNA appears as a morphologically recognizable chromosome or nucleus.
  • FISH hybridization of metaphase chromosomes as shown in Fig. 2A (experimental results applied to probe set 1 + probe set 2) and Fig.
  • FIG. 2A shows the upper metaphase chromosome of human peripheral blood cultured cells.
  • the figure shows that the ALK gene (2p23) segment of chromosome 2 shows a red-green fusion signal (yellow), the other segments have no hybridization signal point, and the ALK probe group 1 and probe Group 2 has excellent specificity.
  • Fig. 2B shows the upper metaphase chromosome of human peripheral blood culture cells. The red-green fusion signal of 2p23 segment (yellow), the cyan signal of 2p21 segment, the hybridization signal point of other segments, ALK probe group 1, ALK probe group are shown in the figure. 2 and EML4 probe set 3 have superior specificity.
  • Example 2 Kit for ALK gene detection, ALK gene and EML4 gene detection
  • This embodiment provides two kits, namely an ALK gene detection kit, and an ALK gene and EML4 gene detection kit.
  • the ALK gene rearrangement detection kit includes two components of a hybridization solution and a DAPI counterstain, wherein the hybridization solution comprises the GSP ALK two-set probe (two-color) described in Example 1, and is used in a hybrid environment (promoting hybridization). Buffer components, closed repeats of COT Human DNA, and the like.
  • DAPI counterstaining agent is mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, so that the nucleus shows blue fluorescence, and the counterstaining agent containing p-phenylenediamine can maintain fluorescence stability.
  • the ALK gene and EML4 gene detection kit includes two components of ALK/EML4 hybridization solution and DAPI counterstaining agent, wherein the ALK and EML4 hybridization solution comprises the set of GSP EML4 gene probes described in Example 1 and two sets of GSP ALK. A combination of gene probes.
  • the ALK gene has two sets of detection probes, and the EML4 gene has a set of detection probes.
  • the ALK gene and the EML4 gene detection kit are: ALK (group 1+group 2)+EML4 (group 3) Combinations, buffer components for hybrid environment (promoting hybridization), CDM Human DNA such as closed repeats, DAPI counterstains are mainly used for counterstaining of cells after hybridization, in which DAPI binds to DNA, allowing the nucleus to show Blue fluorescence, a counterstain containing p-phenylenediamine, maintains fluorescence stability.
  • the relevant fluorescence and DAPI need to be observed with a suitable filter block.
  • Each negative control panel randomly counts the complete 100 cells, counts the number and percentage of abnormal signal cells in each sample, the average and standard deviation of the statistical percentage, and the negative threshold is set to the mean + 3 standard deviation .
  • the two detection kits described in Example 2 one is a combination of (ALK (Group 1) + ALK (Group 2) + EML4 (Group 3) (hereinafter referred to as EML4/ALK rearrangement test kit), and another kit for combination of ALK (group 1) + ALK (group 2) (hereinafter referred to as ALK rearrangement detection kit), for 20 parts
  • Clinical samples which were confirmed by pathological examination, the specimens were formalin-fixed paraffin-embedded tissue samples, see the table below), and were tested separately. The experiments of the two kits were repeated three times, and the test results were repeated. The same, the detection consistency is good.
  • FIG. 3A is a diagram of the FISH detection result of the lung tissue sample of the ALK rearrangement detection kit, wherein the detection signal type For 2F (RG adjacent or coincident), the ALK gene is not rearranged.
  • FIG. 3A is a diagram of the FISH detection result of the lung tissue sample of the ALK rearrangement detection kit, wherein the detection signal type For 2F (RG adjacent or coincident), the ALK gene is not rearranged.
  • FIG. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples by the EML4/ALK rearrangement detection kit, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 3B is a diagram showing the results of FISH detection of lung cancer tissue samples in the examples, wherein the detection signal type is 2F2A, the ALK gene is not rearranged, and no EML4/ALK rearrangement occurs.
  • FIG. 4A is a diagram showing the results of FISH detection of lung cancer tissue samples by ALK rearrangement detection kit, wherein the detection signal type is 1F1G1R, and the F signal is broken compared with the normal signal type 2F, and the detection result is: ALK gene rearrangement.
  • Figure 4B is a diagram showing the results of FISH detection of lung cancer tissue samples by EML4/ALK rearrangement detection kit.
  • the detection signal type is 1F1A1G1RA. Compared with the normal signal type 2F2A, F signal rupture occurs and RA signal fusion occurs at the same time.
  • the detection result is : ALK gene is broken and the rearrangement type is EML4/ALK.
  • the detection kits of the EML4 gene and the ALK gene gene rearrangement and fusion type detection are performed using three sets of probes, respectively.
  • the length of the probe group 3 of the EML4 gene, the probe set 1 of the ALK gene, and the probe set of the probe set 2 of the ALK gene of the present invention are 261 Kb, 462 Kb, and 513 Kb, respectively.
  • the ALK rearrangement partner has many genes, and EML4/ALK rearrangement is one of them.
  • the ALK gene rearrangement detection probe (including the ALK two-group probe) can detect ALK rearrangement, and breakage means that rearrangement occurs, but the rearrangement type cannot be judged.
  • the EML4/ALK rearrangement detection probe (including the ALK two-set probe and the EML4 set of probes) can detect ALK rearrangement and simultaneously determine whether EML4/ALK rearrangement has occurred.
  • the samples can be molecularly classified according to the detection results, and used for clinical treatment plan formulation, drug selection and efficacy judgment according to the significance of the detection indicators.

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Abstract

La présente invention concerne une sonde de détection du gène ALK, une sonde de détection du gène ALK et du gène EML4 et son procédé de préparation. Le procédé comprend les étapes suivantes : sélectionner au moins un clone parmi RP11-62 B19, RP11-100 C1 et CTD-2245E6 comme clone BAC pour un gène ALK, et sélectionner au moins un clone parmi CTD-2544I11, RP11-684O3 et RP11-134O13 comme clone BAC ; sélectionner au moins un clone parmi CTD-2358L8 et CTD-2547J15 comme clone BAC pour un gène EML4 ; obtenir un ADN plasmidique ; et effectuer le marquage. La présente invention concerne également un kit de réactif comprenant la sonde de détection du gène ALK et du gène EML4.
PCT/CN2016/105703 2015-12-30 2016-11-14 Sonde de détection du gène alk et du gène eml4, procédé de préparation associé et kit de réactif WO2017114003A1 (fr)

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WO2009102446A2 (fr) * 2008-02-12 2009-08-20 The Brigham And Women's Hospital, Inc. Dosage fish pour détecter la fusion entre eml4 et alk dans le cancer du poumon
CN105543353A (zh) * 2015-12-30 2016-05-04 广州安必平医药科技股份有限公司 Alk基因和eml4基因检测探针及其制备方法和试剂盒

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Publication number Priority date Publication date Assignee Title
WO2009102446A2 (fr) * 2008-02-12 2009-08-20 The Brigham And Women's Hospital, Inc. Dosage fish pour détecter la fusion entre eml4 et alk dans le cancer du poumon
CN105543353A (zh) * 2015-12-30 2016-05-04 广州安必平医药科技股份有限公司 Alk基因和eml4基因检测探针及其制备方法和试剂盒

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Title
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JOKOJI R ET AL.: "Combination of morphological feature analysis and immunohistochemistry is useful for screening of EML4-ALK-positive lung adenocarcinoma", J CLIN PATHOL, vol. 63, 9 October 2010 (2010-10-09), pages 1066 - 1070 *

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