EP4090765A1 - Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé - Google Patents

Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé

Info

Publication number
EP4090765A1
EP4090765A1 EP21704397.5A EP21704397A EP4090765A1 EP 4090765 A1 EP4090765 A1 EP 4090765A1 EP 21704397 A EP21704397 A EP 21704397A EP 4090765 A1 EP4090765 A1 EP 4090765A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
allele frequency
sample
mutation rate
determining
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21704397.5A
Other languages
German (de)
English (en)
Inventor
Andreas Bollmann
Björn Nowack
Eileen STROH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sensid GmbH
Original Assignee
Sensid GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sensid GmbH filed Critical Sensid GmbH
Publication of EP4090765A1 publication Critical patent/EP4090765A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/6876—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
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention relates to a new method for determining the allele frequency and / or mutation rate in nucleic acids, in particular in tumor nucleic acids in the context of an iwS. Polymerase chain reaction (PCR) and diagnostics for this, using at least one reference nucleic acid (RN) and a mutation sequence for the reference nucleic acid.
  • PCR polymerase chain reaction
  • RN reference nucleic acid
  • This reference nucleic acid and mutation sequence allows the validation of methods for polymerase chain reaction (PCR), in particular as a function of device parameters and sample preparation.
  • the invention comprises an associated diagnosis and prognosis method, in particular for tumor diagnosis in the course of a liquid biopsy.
  • tumor markers in particular oncogenes, have been determined in body fluids such as blood, urine, sputum or tissue samples. These are components of tumor cells that are formed in increased or decreased form and whose changes can be detected in body fluids such as blood, plasma or serum.
  • these tumor markers do not allow general screening for tumor diseases, since their diagnostic specificities and sensitivities are usually too low. Although these tumor markers could often indicate malignant changes at an earlier stage than imaging methods, they have so far not allowed adequate detection of malignant changes.
  • nucleic acids ribonucleic acids, RNA and deoxyribonucleic acids, DNA
  • PCR polymerase chain reaction
  • such cell material is provided for diagnostics in the following steps, such as sample preparation, extraction, concentration, isolation, purification, reverse transcription, amplification and detection.
  • qPCR real-time quantitative polymerase chain reaction
  • ddPCR digital droplet PCR
  • NGS next generation sequencing
  • Parallel sequencing such as “Massive Parallel Sequencing” the so-called non-invasive liquid biopsy, has been established in tumor diagnostics in recent years, whereby circulating free DNA is examined in a sample.
  • circulating free DNA can originate, for example, from a cancer / tumor cell (so-called: ctDNA).
  • ctDNA makes up e.g. 1% of the cfDNA (0.01-90% of the normal cfDNA, depending on the stage and location of the tumor).
  • the inventors were able to determine that for such a standard the use of a human serum or plasma sample, which, however, is free of DNA, allows improved validation and diagnosis.
  • the object of the present invention is therefore to provide an improved method for the detection of an allele frequency and / or mutation rate in a sample nucleic acid including the associated diagnosis, whereby a suitable standard is to be used.
  • the object is achieved by a method for validating a polymerase chain reaction (PCR) method by determining the allele frequency and / or mutation rate in nucleic acids with the steps: a.) Providing at least one human
  • a aforementioned "certain allele frequency" is equal to or greater than 0% and can in particular assume values such as 0.01%, 0.1%, 0.5%, 1%, 2.5, 5% and many more.
  • reference nucleic acid means any known human wild-type sequence of an allele.
  • alleles can be databases such as COSMIC: https: //cancer.stician.ac.uk/cosmic,
  • Targeted Cancer Care http: // targetedcancercare .massgeneral.org / My-Trial- Guide / Diseases / Lung-Cancer / KRAS / G12C- (C-34G-T) .aspx, or OncoKB: https://oncokb.org /, My Cancer Genome: https://www.mycancergenome.org/.
  • the content of a reference nucleic acid is preferably 20-600 ng, in particular 400 ng DNA in serum or plasma, such as e.g.
  • 400 ng / 5ml corresponds to 80 ng / ml, corresponds to 0.08 ng / m2 DNA in serum or plasma. It is also preferred that the reference nucleic acid has a size of 50 bp to 500 bp.
  • “Mutation sequence or mutation nucleic acid” is a sequence which has one or more mutations compared to a human wild-type sequence of an allele as reference nucleic acid.
  • known mutation sequences can be used or artificial mutations can be introduced compared with the human wild-type sequence of an allele It is essential that this reference sequence and mutation sequence are known in the method according to the invention (e.g. described with reference to the nucleic acid sequence).
  • the mutation sequence has at least one tumor marker, e.g. oncogene, with known mutations.
  • tumor marker sequences or oncogenes have other artificial mutations.
  • the content of a mutation sequence is less than the content of a reference sequence. It is also preferred that the mutation sequence has a size of 50 bp to 500 bp.
  • reference nucleic acid and mutation sequence are each present in a specific concentration.
  • allele frequency or mutation rate means the ratio of reference nucleic acid to mutation sequence (RN / MS), for example via the number of copies of reference nucleic acid present to mutation sequence or that Ratio based on the concentration of reference nucleic acid to the mutation sequence.
  • mutated allele 200 mutation sequences
  • wild-type allele a reference nucleic acid
  • this is 4.76%, i.e. the frequency of the allele mutation or mutation sequence is 4.76% or the mutation rate is 4.76%.
  • validation samples according to the invention can be provided which have a specific or set allele frequency (allele frequency) or mutation rate.
  • validation means that the determined or set allele frequency or mutation rate can be detected in the course of a polymerase chain reaction (PCR) using the specified validation samples. This can depend on the one hand on device parameters and on the other hand on the implementation of the polymerase chain reaction (PCR) and in particular on the sample preparation.
  • the validation samples can be provided by means of a kit.
  • calibration or validation curves can be created.
  • sample nucleic acid is cfDNA or ctDNA from a patient or test person.
  • the invention therefore relates to a method for determining the allele frequency and / or mutation rate of at least one sample nucleic acid by means of a PCR method, the validation method according to the invention having been carried out.
  • a quantitative determination of the sample nucleic acid can be carried out particularly advantageously on the basis of the calibration or validation.
  • Another particular embodiment of the invention relates to a method for diagnosis or prognosis of a tumor disease, with a change in the allele frequency and / or mutation rate of a sample nucleic acid from a first sample and a second and / or further sample on the early detection and detection, for assessing the severity and for the therapy-accompanying assessment of the course leaves, a calibration having been carried out with the aid of the validation method according to the invention.
  • the second or further sample can be taken from a patient at a respective later point in time.
  • a particularly advantageous application therefore relates to the validation of devices for carrying out a polymerase chain reaction (PCR), in particular next generation sequencing (NGS), the method according to the invention being carried out.
  • PCR polymerase chain reaction
  • NGS next generation sequencing
  • an exactly predetermined amount of DNA material or concentration of the standard according to the invention can particularly advantageously be introduced into a sample, this sample largely simulating a patient serum or plasma sample and being highly suitable for performing tumor diagnostics.
  • the concentration of mutation nucleic acid is preferably 4 ⁇ 10E-05 fg / m ⁇ to 0.03 fg / m ⁇ .
  • the mutation sequence according to the invention preferably has a tumor marker, in particular an oncogene selected from the group MTOR, MPL, NRAS, PARP1, AKT3, DNMT3A, MSH2, IDH1, VHL, MLH1, MYD88, CTNNB1, ATR, PIK3CA, FGFR3, PDGFRA, KIT, FBXW7, APC, GABRG2, NPM1, EGFR, MET, BRAF, EZH2, JAK2, GNAQ, RET, PTEN, ATM, KRAS, PTPNII, FLT3, RB1, PARP2, ARHGAP5, AKT1, RAD51, IDH2, TR53, NF1, SMAD4, AKT2, ERCC1, GNAS, ERBB2, FOXL2, NOTCH1, NTKR.
  • an oncogene selected from the group MTOR, MPL, NRAS, PARP1, AKT3, DNMT3A, MSH2, IDH1, VHL, MLH1, MYD88,
  • Diagnosis in the context of this invention means that conclusions can be drawn about tumor activity from the mutation rate. The greater the mutation rate, the greater the rate Tumor activity, especially the likelihood of metastasis. Accordingly, the invention relates to the diagnosis of cancer or a tumor.
  • diagnosis includes medical diagnostics and related examinations, in particular in-vitro diagnostics and laboratory diagnostics. The statement about a disease or a condition of a patient is preferred.
  • kits containing a.) Part (eg tube) with at least one human reference nucleic acid and one nucleic acid having one or more mutations to the reference sequence in a DNA free serum or plasma sample, with a predetermined allele frequency being set, and optionally b.) part with a human reference nucleic acid in a DNA-free serum or plasma sample, and optionally c.) part with a DNA-free serum or plasma sample, for Carrying out one of the methods described above or the use of such a kit for carrying out one of the methods described above.
  • the invention is illustrated below by means of examples. However, the invention is not restricted to the examples, but can basically be used universally.
  • Example 1 Implementation of the method according to the invention with provision of a kit:
  • DNA nucleic acids
  • cfDNA nucleic acids
  • commercial kits such as QIAamp ccfDNA / RNA kit (Qiagen®), PME free-circulating DNA extraction kit (AnalytikJena®), MagMAX TM Cell-Free DNA Isolation Kit (ThermoFisher®).
  • a qualitative analysis of the nucleic acids, in particular cfDNA can be carried out, such as a fragment length analysis with a bioanalyzer (Agilent®), fragment analyzer (Agilent®) or pulse field gel electrophoresis.
  • a bioanalyzer Agilent®
  • fragment analyzer Agilent®
  • pulse field gel electrophoresis a qualitative analysis of the nucleic acids, in particular cfDNA
  • the nucleic acid obtained, in particular cfDNA, is then fed to a PCR.
  • ddPCR Using the example of a QX200 ddPCR system from BioRad®: Using ddPCR, a defined volume of the nucleic acid to be examined is distributed into thousands of individual reaction spaces. According to the Poisson distribution, the DNA sequences to be examined are distributed over these reaction spaces. The nucleic acids are amplified in the reaction spaces if the nucleic acid is present. The mutation sequence triggers a color-coded PCR reaction from the reference sequence (wild-type sequence). Using relevant software, the results can be evaluated and displayed in a plot, in particular a 2D amplitude plot:
  • the events that were detected in the FAM channel (blue fluorescence) (channel 1 amplitude) are plotted on the Y axis.
  • the events that were detected in the HEX / VIC channel (green fluorescence) (channel 2 amplitude) are plotted on the X axis.
  • Each plotted point represents a reaction space by either adding a reaction with a reference sequence
  • the tubes of the kit (respective standard sample with preset allele frequency, e.g.
  • FIG. 1 A calibration curve can be created from the calculated values (FIG. 2, table) and the validation can be completed (FIG. 3).
  • patient or test subject samples can be determined in parallel or offset and a diagnosis can be carried out.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
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  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un nouveau procédé de détermination de la fréquence allélique et/ou du taux de mutation dans des acides nucléiques, en particulier dans des acides nucléiques tumoraux dans le cadre d'une réaction en chaîne par polymérase (PCR), ainsi qu'un diagnostic associé, au moins un acide nucléique de référence (RN) et une séquence de mutation par rapport à l'acide nucléique de référence étant utilisés. Cet acide nucléique de référence et cette séquence de mutation permettent la validation de procédés de réaction en chaîne par polymérase (PCR), notamment en fonction de paramètres d'appareil et de la préparation d'échantillons. L'invention concerne en outre un procédé de diagnostic et de pronostic correspondant, en particulier pour le diagnostic de tumeurs au cours d'une biopsie liquide.
EP21704397.5A 2020-01-17 2021-01-18 Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé Pending EP4090765A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20152341.2A EP3851540A1 (fr) 2020-01-17 2020-01-17 Nouveau procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic
PCT/EP2021/050962 WO2021144471A1 (fr) 2020-01-17 2021-01-18 Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé

Publications (1)

Publication Number Publication Date
EP4090765A1 true EP4090765A1 (fr) 2022-11-23

Family

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Family Applications (2)

Application Number Title Priority Date Filing Date
EP20152341.2A Withdrawn EP3851540A1 (fr) 2020-01-17 2020-01-17 Nouveau procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic
EP21704397.5A Pending EP4090765A1 (fr) 2020-01-17 2021-01-18 Procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic associé

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP20152341.2A Withdrawn EP3851540A1 (fr) 2020-01-17 2020-01-17 Nouveau procédé de détermination de la fréquence allélique / du taux de mutation et diagnostic

Country Status (6)

Country Link
US (1) US20230056502A1 (fr)
EP (2) EP3851540A1 (fr)
JP (1) JP2023510600A (fr)
CN (1) CN115135773A (fr)
CA (1) CA3166647A1 (fr)
WO (1) WO2021144471A1 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004036285A1 (de) * 2004-07-27 2006-02-16 Advalytix Ag Verfahren zum Bestimmen der Häufigkeit von Sequenzen einer Probe
WO2015073080A1 (fr) * 2013-11-12 2015-05-21 Life Technologies Corporation Réactifs et méthodes de séquençage
US11208679B2 (en) * 2016-05-31 2021-12-28 The Translational Genomics Research Institute Method for validating assays of biological samples
EP3541934B1 (fr) 2016-11-17 2024-04-10 LGC Clinical Diagnostics, Inc. Procédés de préparation d'un matériau de référence d'adn et témoins

Also Published As

Publication number Publication date
CN115135773A (zh) 2022-09-30
CA3166647A1 (fr) 2021-07-22
JP2023510600A (ja) 2023-03-14
EP3851540A1 (fr) 2021-07-21
WO2021144471A1 (fr) 2021-07-22
US20230056502A1 (en) 2023-02-23

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