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WO2016018115A1 - Méthode de diagnostic de la sensibilité à un agent thérapeutique pour un traitement de deuxième intention de patient cancéreux à l'aide de cellules tumorales circulantes - Google Patents

Méthode de diagnostic de la sensibilité à un agent thérapeutique pour un traitement de deuxième intention de patient cancéreux à l'aide de cellules tumorales circulantes Download PDF

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Publication number
WO2016018115A1
WO2016018115A1 PCT/KR2015/008025 KR2015008025W WO2016018115A1 WO 2016018115 A1 WO2016018115 A1 WO 2016018115A1 KR 2015008025 W KR2015008025 W KR 2015008025W WO 2016018115 A1 WO2016018115 A1 WO 2016018115A1
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Prior art keywords
pcr reaction
seq
reaction solution
standard
genomic dna
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English (en)
Korean (ko)
Inventor
신영기
오명렬
최준석
조상래
정경숙
변보현
김진주
김성수
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SNU R&DB Foundation
Gencurix Inc
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Seoul National University R&DB Foundation
Gencurix Inc
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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
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • 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

Definitions

  • the present invention relates to a method for diagnosing reactivity of a therapeutic agent for secondary treatment of cancer patients using circulating tumor cells, and more specifically, by detecting genetic variation information of cancer patients.
  • the present invention relates to a method suitable for automation as a method of calculating a treatment response predictive including multiple steps of detecting and detecting CTC from a patient's blood sample. .
  • Cancer is a group of abnormal cells caused by continuous division and proliferation due to the disruption of the balance between cell division and death by various causes, also called tumors or neoplasms. It usually occurs in more than 100 different parts of the body, including organs, white blood cells, bones, lymph nodes, etc., and develops into severe symptoms through infiltration into surrounding tissues and metastases to other organs.
  • the drug suitable for the patient can be selected in advance, thereby reducing the dropout rate caused by the wrong selection of the drug and increasing the compliance of the drug. There will be. It will also avoid the time it takes for the drug to take effect and the risk of side effects that the patient may experience.
  • the diagnostic methods of response to the therapeutic agents are generally less reliable than the highly experienced experimenter.
  • the patient's sample should be provided to the service provider according to a predetermined process in a central lab to obtain relatively meaningful results.
  • inter-laboratory variation inter-observer variation
  • day-to-day variation may raise questions about the reliability of the overall system.
  • the conventional method may cause an error in the diagnosis result according to the place, time, and experimenter.
  • a method or an automation process that requires the involvement of the experimenter is preferably excluded.
  • an object of the present invention is to provide a method for judging secondary treatment by detecting genetic mutation information of lung cancer patients. It is to provide a method for calculating a therapeutic response response comprising a.
  • the present invention provides the following steps from a blood sample of a patient in order to provide information for judging a second treatment in the genetic mutation information detection of lung cancer patients It provides a method of calculating the therapeutic response included.
  • (D) Standards comprising standard DNA, primers and probes specific for genomic DNA gene mutations, wherein the standard vector is processed to be linear DNA by restriction enzymes.
  • step (E) micronizing the PCR reaction solution of step (d) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively;
  • step (F) performing a PCR reaction such that PCR reaction is performed on each of the plurality of small droplets;
  • the present invention calculates a therapeutic response predictive value including the following step from a blood sample of a patient. Provide a method.
  • step (E) micronizing the PCR reaction solution of step (d) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively;
  • (F) PCR reactions are performed such that the PCR reaction is performed in each of the plurality of small rooms. Performing;
  • the method of the present invention is preferably an automated or semi-automated method. Automation in the above is the introduction of a sample (sample); Rearrangement or transfer with extraction, separation, reaction complete substrates (eg tubes, plates); Injection of reagents, buffers into stock, complement; This means that all or most of the process, except for the maintenance of equipment, is done by means other than humans (eg, robots).
  • Step (a) is the step of separating CTCs from the blood of the patient.
  • CTCs are tumor cells found in the peripheral blood of malignant tumor patients. Since CTC plays an important role in the cancer metastasis process, CTC is considered to be very important in cancer research and diagnosis.However, the number of circulating tumor cells in peripheral blood is very rare, and dozens or less mixed with millions of normal blood cells There is a need for a detection system that requires a sensitivity that is capable of detecting tumor cells.
  • Step (b) is the step of separating genomic DNA from CTC cells.
  • the nucleic acid to be isolated from the CTC of the present invention is preferably genomic DNA, more preferably genomic DNA assumed to carry mutations.
  • Step (c) is a step of preparing a PCR reaction solution containing primers and probes specific for the DNA and genomic DNA gene mutations.
  • PCR premixes may include DNA polymerase for PCR reactions (eg Tag polymerase), dyes for quantitative detection of PCR reactions (eg fluorescent dyes), buffers suitable for PCR reactions, dNTPs, and the like. .
  • step (d) is to prepare a standard PCR reaction solution containing primers and probes specific for genomic DNA gene mutations, such as standard DNA genomic DNA gene mutation to the standard DNA vector (restriction enzyme) to be a linear DNA Step.
  • step (d) the step (c) is used except that a standard material vector is used instead of genomic DNA in step (c) as a template for PCR amplification.
  • Restriction enzymes to be treated in the standard vector can be selected from any of the restriction enzymes present in the vector if the vector can be linearized, Clal was used in the embodiment of the present invention.
  • the level after PCR amplification of the detection target in the present invention may vary widely depending on the target sample, a criterion for determining whether to amplify by primer / probe specific to the mutation is necessary.
  • the standard vector is for this purpose
  • 100-350 bp of polynucleotides covering DNA gene mutations can be used transformed into a conventional vector.
  • the standard vector of the present invention may be used by inserting about 300bp into the pIDTSmart Amp vector by mutating the exon of the EGFR, that is, the probe position in the center.
  • the standard vector of the present invention may be a vector containing a DNA fragment of about 300 bp with each mutation in the exon of the EGFR, that is, the probe position in the center, which is applied to a host cell such as E. coli. After transformation, amplification and extraction can be used. More preferably, the standard vector of the present invention is 1597 of the EGFR gene (genbank accession no.
  • NG_007726 for axon 18 to 100 to 350 bp of polynucleotide at base 100100, for the axon 19, EGFR gene Of polynucleotides 100 to 350 bp at 160501 base in 160501, 100 to 350 bp polynucleotide at 167101 to 167500 base in EGFR gene for axon 20, and 177551 to 177930 bases for EGFR gene in axon 21 for axon 21 100 to 350 bp poly
  • the nucleotide DNA fragment may be inserted into the pIDTSmart Amp vector.
  • primer refers to an oligonucleotide, a nucleic acid chain
  • the primer can serve as a starting point for the synthesis under conditions in which the synthesis of primer extension products complementary to (template) is induced, i.e., the presence of polymerizers such as nucleotides and DNA polymerases, and conditions of suitable temperature and pH.
  • the primer is deoxyribonucleotide and single chain.
  • Primers used in the present invention may include natural ly occur ing dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides.
  • the primer may also include ribonucleotides.
  • the primer should be long enough to prime the synthesis of the extension product in the presence of the polymerizer. Suitable lengths of primers are typically 15-30 nucleotides, depending on a number of factors, such as silver, application, and source of the primer. Short primer molecules generally require lower temperatures to form a more complex stable complex with the template.
  • annealing or “priming” refers to the juxtaposition of oligodioxynucleotides or nucleic acids to a template nucleic acid, where the polymerase polymerizes the nucleotides to complement the template nucleic acid or portion thereof. To form nucleic acid molecules.
  • probe is designed as a kind of taqman probe used for quantitative PCR
  • the probe is attached with fluorescent material (HEX, VIC, F ⁇ dye), TAMRA can be used as a quencher on the 3 'side.
  • TaqMan probes are generally ol igonuc leotides tagged with the 5' end as the fluorescent material and the 3 'end as the quencher material, and the TaqMan probe is the anneal ing step.
  • the quencher at the 3' end of the probe does not fluoresce when light is applied, but in the next step, the extension step 5 ' ⁇ 3' exonuc 1 possessed by Taq DNA polymerase.
  • the fluorescence is quantitatively determined by PCR reaction based on the principle that the fluorescent material is separated from the probe, released by the quencher, and fluoresced. Will be released.
  • primers and probes specific for genomic DNA gene mutations are used in the same sequence for the PCR reaction solution and the standard PCR reaction solution, each independently a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 2, and SEQ ID NO: 9
  • Step (e) is a step of micronizing the PCR reaction solution of step (e) and the standard PCR reaction solution of step (f) into a plurality of droplets, respectively.
  • each PCR reaction solution is divided into a plurality of small droplets.
  • This micronization process allows each small chamber to be subjected to a subsequent PCR reaction to target each droplet.
  • the micronized microdroplets of the present invention may be about lnl in size, and may be micronized to 10, 000 to 25, 000 for convenience of measurement of PCR reaction and reaction.
  • Step (f) is a step of performing a PCR reaction so that PCR reaction is performed on each of the plurality of small drops.
  • PCR reactions are performed using the sample genomic DNA or the DNA of the standard material vector as a template.
  • PCR reaction can be carried out according to methods known in the art, generally should be carried out under conditions that do not cross the primer / probe cross-linking, according to the method of the present invention based on a standard vector (vector) Because the value can be set, the PCR reaction can be performed even under some conditions where crosslinking is allowed.
  • PCR reaction conditions for example at 95 ° C for 10 minutes at the enzyme activation reactions and, 94 ° C 30 seconds, and the cool ing process of one minutes to 40 cycles, 98 ° at C 10 seconds and 4 ° C at 60 ° C PCR can be performed via.
  • (G) is a step of measuring the PCR reaction in all or part of each micronized droplet.
  • the determination of the PCR reaction can be performed according to methods known in the art, but can be measured by an optical quantitative analysis system using a probe labeled with a reporter fluorescent dye and / or a quencher fluorescent dye. And preferably, by measuring the fluorescence value for the PCR reaction of each micronized droplet.
  • the FAM, HEX, VIC fluorescent dye (fluorescent material) or EvaGreen fluorescent dye is used in combination with the probe, it may be performed by measuring the fluorescence thereof. This process can be performed by a commercially available detection device (e.g., Biorad's Droplet Reader), which detects the droplet fluorescence signal of each sample and the number of posi- tive and negative drop let, respectively. The count can be automatically completed until the analysis.
  • a commercially available detection device e.g., Biorad's Droplet Reader
  • the probe added to the PCR reaction solution and the probe added to the standard PCR reaction solution for detection may be associated with different fluorescent materials. .
  • Step (h) is a step of calculating the mutation rate (% mutat ion) from the measurement of the PCR reaction in the PCR reaction solution and the standard PCR reaction solution.
  • the mutation rate (% mutat ion) is calculated by comparing the measured values of PCR reactions in the PCR reaction solution and the standard PCR reaction solution by using a mutation rate at a ratio equal to or higher than the threshold value corresponding to the measurement value in the standard PCR reaction solution. (% mutat ion) can be calculated
  • step (i) the predictive value predicts that the higher the mutation rate, the higher the therapeutic responsiveness.
  • the therapeutic responsiveness in the present invention can be defined as "banung” for a therapeutic agent if lung cancer growth rate is inhibited as a result of contact with the therapeutic agent as compared to its growth not in contact with the therapeutic agent.
  • the growth of lung cancer can be measured in various ways, for example, the expression of tumor markers appropriate to the size of the tumor or its tumor type can be measured.
  • the "banungseong” may indicate a significant increase in the survival time on the survival curve.
  • Lung cancer is associated with the treatment compared to its growth rate where the growth rate is not in contact with the treatment. As a moist result, it is "non-fungling" for the therapeutic agent if it is inhibited or not to a very low degree.
  • the growth of lung cancer can be measured in a variety of ways, eg, the expression of tumor markers appropriate to the size of the tumor or its tumor type can be measured. Non-ungular measures may be assessed using additional criteria beyond the growth size of the tumor, including patient quality of life and metastasis.
  • the treatment response to the lung cancer treatment agent may be treatment responsiveness to an inhibitor of epidermal growth factor receptor (EGFR).
  • EGFR epidermal growth factor receptor
  • EGFR is a protein product of the oncogene erbB or ErbBl.
  • erbB or ErbBl is part of the ERBB family of protooncogenes known to be important factors in numerous cancer developments.
  • EGFR target drugs have been developed for the treatment of epithelial cell carcinoma such as lung cancer.
  • OSI Pharmaceuticals, Inc. trade name "TARCEVA”
  • Zephytinib and erlotinib are quinazoline compounds that inhibit cell growth by inhibiting tyrosine kinase activity of EGFR to inhibit phosphorylation.
  • the method of the present invention calculates the therapeutic responsiveness using the blood of the patient who is difficult to obtain the FFPE tissue sample, and can predict and diagnose the patient's therapeutic agent. It can be useful for purposes such as determining the necessity of administration and providing clues about the direction of future treatment.
  • 1 shows a vector map of a pIDTSmart Amp back jump.
  • FIG. 2 is a conceptual diagram of a CTC separation method of the method of the present invention.
  • CTCs were obtained by CTC separation apparatus based on magnetophores i s blood samples obtained from cancer patients.
  • the conceptual diagram of the CTC separation method by magnetophoresis is shown in FIG.
  • the separation method of the CTC will be described in more detail as follows.
  • a microfluidic chip (mi crofizidics chip) prepared for CTC separation was installed in a blood and buffer-injectable CTC separation device. Blood was flowed through the inlets at both ends while flowing the buffer into the center channel. In the blood, an antibody (EpCam ant ibody) attached with a magnetic substance was added to the CTC to specifically bind to the CTC so that the CTC was specifically magnetized.
  • the ferromagnetic material pre-installed on the microfluidics chip is magnetized by the permanent magnet installed in the CTC separation device, and the CTC cells are moved to the central CTC separation channel by the magnetic force generated from the magnetized ferromagnetic material. After the flow of blood, CTC collected on the side of the CTC separation channel was used for subsequent experiments.
  • a standard vector (named mini-clone) was constructed to validate the designed primers and probes, and to make the standards needed to perform ddPCR.
  • mini-clone about 300bp was synthesized by mutating the exon of each EGFR.
  • the synthesized DNA fragment was inserted between the universal link sequence of the pIDTSmart Amp vector (see FIG. 1), and the produced clone was transformed into E. coli DH5a cells.
  • Standard Vector Miniclone DNA
  • Clal restriction enzyme was reacted at 37 ° C for 30 minutes, and the reaction product was quantified and stored at -20 ° C until use.
  • Probes were designed as taqman probes by screening those that met conditions.
  • HEX / VIC reporter fluorescence was attached to 5 'wild type probe, and FAM dye was attached to 5' mutant probe to detect amplification afterwards.
  • TAMRA was used as quencher on the 3 'side of all probes. 4, 31, 8, and 4 probes were designed and synthesized at EGFR exon 18, 19, 20, and 21, respectively. Probes designed by the inventors had allele specifics and almost all probes had cosmic numbers.
  • WT wildtype
  • mt mutant (hereinafter ⁇ same as Table 3)
  • Samples adjusted by dilution in 7 steps up to 0.02% and 0.01% were simultaneously measured by the method according to the Covas EGFR gene mutation test kit and the method in the present invention. Covas EGFR gene mutation testing was performed according to the manufacturer's instructions.
  • the minimum measurement result of the Cobas EGFR mutation test was 0.5% to 5%, whereas the method of the present invention In this case, it was confirmed that the test can be performed from 0.02% to 0.1%, and the results of particular note are that the mutation position of 2239_2257> GT cannot be measured by the COVAS EGFR gene mutation test 0.05% according to the method of the present invention. Sensitivity of the result was confirmed to have more than equivalent results compared to the approved method of COVA EGFR gene mutation test.
  • Cobas EGFR mutat ion kit uses 50ng (1.5x10 copies) as template
  • ⁇ i60> Secondly, the sensitivity analysis was performed for the correlation test using DNA extracted from Hor izon's FFPE tissue and Horizon's mutant genomic DNA. Although measured by the method of the present invention according to the position of the minimum measured value was determined to 0.02% ⁇ 0.5% value. This indicates that at least 10 times the sensitivity is shown in the licensed product comparison analysis.
  • Cobas EGFR mutat ion kit uses 50ng (1.5xl0 copies) as template
  • EGFR gene mutations were measured in DNA isolated from human CTC cells. DNA was isolated from the CTC isolated in Example 4, and the mutation was confirmed according to the method of the present invention using the primer / probe of the present invention.
  • the method of the present invention calculates the anticoagulant predictive value using the blood of the patient who is difficult to obtain the FFPE tissue sample, and thus predicts the responsiveness and diagnosis of the patient's therapeutic agent. It can be useful for the purpose of presenting clues about the direction of future treatment, including the necessity of administering anticancer drugs.

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Abstract

La présente invention concerne une méthode permettant de diagnostiquer la sensibilité à un agent thérapeutique pour un traitement de deuxième intention d'un patient cancéreux à l'aide de cellules tumorales circulantes. Plus précisément, la présente invention concerne une méthode pour calculer une valeur prédite de sensibilité à un traitement qui comprend plusieurs étapes de séparation et de détection des CTC à partir d'un échantillon de sang d'un patient afin de fournir des informations pour la détermination du traitement de deuxième intention par l'intermédiaire de la détection d'informations d'une mutation génique chez le patient cancéreux, le procédé convenant pour une automatisation. La méthode de la présente invention peut être utile à des fins d'orientation d'un traitement futur, comprenant la détermination de la nécessité de l'administration d'un agent thérapeutique anticancéreux, étant donné qu'il est possible de prédire et de diagnostiquer la sensibilité d'un patient à l'agent thérapeutique par le calcul de la valeur prédite de sensibilité à un traitement à l'aide du sang d'un patient qui est soumis au traitement de deuxième intention et à partir duquel l'obtention d'un échantillon de tissu FFPE est difficile.
PCT/KR2015/008025 2014-08-01 2015-07-31 Méthode de diagnostic de la sensibilité à un agent thérapeutique pour un traitement de deuxième intention de patient cancéreux à l'aide de cellules tumorales circulantes Ceased WO2016018115A1 (fr)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DIDELOT, A. ET AL.: "Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples", CLIN. CHEM., vol. 59, no. 5, 12 February 2013 (2013-02-12), pages 815 - 823, XP055197965, DOI: doi:10.1373/clinchem.2012.193409 *
HUBERS, A. J. ET AL.: "EGFR mutation analysis in sputum of lung cancer patients: a multitechnique study", LUNG CANCER, vol. 82, no. 1, 5 August 2013 (2013-08-05), pages 38 - 43 *
LEWANDOWSKA, M. A. ET AL.: "Application of PCR methods to evaluate EGFR, KRAS and BRAF mutations in a small number of tumor cells in cytological material from lung cancer patients", ONCOL. REP., vol. 30, no. 3, 1 June 2013 (2013-06-01), pages 1045 - 1052 *
OXNARD, G. R. ET AL.: "Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell -free plasma DNA", CANCER RES., vol. 20, no. 6, 15 January 2014 (2014-01-15), pages 1698 - 705 *
PUNNOOSE, E. A. ET AL.: "Evaluation of circulating tumor cells and circulating tumor DNA in non-small cell lung cancer: association with clinical endpoints in a phase II clinical trial of pertuzumab and erlotinib", CLIN. CANCER RES., vol. 18, no. 8, 5 April 2012 (2012-04-05), pages 2391 - 2401, XP055073510, DOI: doi:10.1158/1078-0432.CCR-11-3148 *

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KR20160016034A (ko) 2016-02-15

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