WO2023152133A1 - Procédé de diagnostic du cancer colorectal - Google Patents

Procédé de diagnostic du cancer colorectal Download PDF

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Publication number
WO2023152133A1
WO2023152133A1 PCT/EP2023/052997 EP2023052997W WO2023152133A1 WO 2023152133 A1 WO2023152133 A1 WO 2023152133A1 EP 2023052997 W EP2023052997 W EP 2023052997W WO 2023152133 A1 WO2023152133 A1 WO 2023152133A1
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Prior art keywords
colorectal cancer
subject
plasma
timp
clu
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PCT/EP2023/052997
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English (en)
Inventor
Philippe Blache
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Institut Régional Du Cancer De Montpellier
Unicancer
Université De Montpellier
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Publication of WO2023152133A1 publication Critical patent/WO2023152133A1/fr

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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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

Definitions

  • the invention relates to a method for diagnosing a colorectal cancer in a subject in need thereof comprising i) determining in a sample obtained from the subject the expression levels of at least one biomarker selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs.
  • the present invention relates to a method for diagnosing a colorectal cancer in a subject in need thereof comprising i) determining in a sample obtained from the subject the expression levels of at least one biomarker selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs.
  • the invention is defined by its claims.
  • a first aspect of the invention relates to a method for diagnosing a colorectal cancer in a subject in need thereof comprising i) determining in a sample obtained from the subject the expression levels of at least one biomarker selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs ii) comparing the expression level determined at step i) with its predetermined reference value and iii) concluding that the subject in need thereof has a colorectal cancer when the expression level determined at step i) is higher than its predetermined reference value, or concluding that the subject in need thereof has not a colorectal cancer when the expression level determined at step i) is lower than its predetermined reference values.
  • the expression levels of 1, 2, 3 or 4 biomarkers selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs can be determined according to the method of the invention.
  • the expression levels of the 4 biomarkers can be determined.
  • the invention relates to a method for diagnosing a colorectal cancer in a subject in need thereof comprising i) determining in a sample obtained from the subject the expression levels of the biomarkers selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs ii) comparing the expression levels determined at step i) with their predetermined reference values and iii) concluding that the subject in need thereof has a colorectal cancer when the expression levels determined at step i) are higher than their predetermined reference values, or concluding that the subject in need thereof has not a colorectal cancer when the expression levels determined at step i) are lower than their predetermined reference values.
  • a step of addition of the expression levels of the 4 biomarkers of the invention can be realized to obtain the RBTC index.
  • the invention also relates to the invention relates to a method for diagnosing a colorectal cancer in a subject in need thereof comprising i) determining in a sample obtained from the subject the expression levels of the biomarkers selected from the group consisting of RPS28, B2M, TIMP-1 and CLU mRNAs, ii) adding the expression levels of the 4 biomarkers to obtain the RBTC index iii) comparing RBTC index at step ii) with a predetermined reference values and iv) concluding that the subject in need thereof has a colorectal cancer when the RBTC index determined at step ii) is higher than its predetermined reference value, or concluding that the subject in need thereof has not a colorectal cancer when the RBTC index determined at step ii) is lower than its predetermined reference value.
  • the expression levels of B2M, TIMP-1 and CLU mRNAs can be determined according to the method of the invention.
  • the inventors show that if a high expression of the 3 biomarkers B2M, TIMP-1 and CLU mRNAs levels is measured, thus the subject is having all stage, in particular stage 4, of colorectal cancer.
  • high expression levels of TIMP-1 and CLU mRNAs levels in a subject suffering from colorectal compared to the expression levels of TIMP-1 and CLU mRNAs levels from healthy individuals (HI) indicates all stage, in particular stage 4, of colorectal cancer.
  • the expression levels of TIMP-1 and CLU mRNAs can be determined according to the method of the invention.
  • the inventors show that if a high expression of the 2 biomarkers TIMP-1 and CLU mRNAs levels is measured, thus the subject is having all stage, in particular stage 4 of colorectal cancer.
  • high expression levels of TIMP-1 and CLU mRNAs levels in a subject suffering from colorectal compared to the expression levels of TIMP-1 and CLU mRNAs levels from healthy individuals (HI) indicates all stage, in particular stage 4, of colorectal cancer.
  • the methods of the invention are in-vitro methods.
  • the colorectal cancer is a metastatic colorectal cancer.
  • sample denotes, blood, peripheral-blood, serum or plasma. Particularly, the sample is plasma.
  • the plasma can be collected in Streck Cell-free DNA BCT® tube or in tube with EDTA.
  • the inventors show that in a tube streck, high expression of RPS28 B2M, TIMP-1 and CLU is equal to a stage 4 of colorectal cancer.
  • the inventors show that in a EDTA tube, high expression of TIMP-1 and CLU is equal to all stage of colorectal cancer.
  • the inventors show that in a EDTA tube, high expression of B2M, TIMP-1 and CLU is equal to a stage 4 of colorectal cancer.
  • biomarkers of the inventions Measuring the expression level of the 4 biomarkers mRNAs of the invention can be performed by a variety of techniques well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis and/or amplification (e.g., RT-PCR).
  • hybridization e. g., Northern blot analysis and/or amplification (e.g., RT-PCR).
  • LCR ligase chain reaction
  • TMA transcription- mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
  • the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected either directly or indirectly.
  • a label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • detectable labels include fluorescent molecules (or fluorochromes).
  • fluorescent molecules or fluorochromes
  • Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies).
  • fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No.
  • fluorophores include thiol -reactive europium chelates which emit at approximately 617 nm (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof.
  • fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos.
  • a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138).
  • Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties.
  • Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos.
  • quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).
  • Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • radioisotopes such as 3 H
  • metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+
  • liposomes include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
  • Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • enzymes for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
  • an enzyme can he used in a metallographic detection scheme.
  • SISH silver in situ hybridization
  • Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate.
  • Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate.
  • an oxido-reductase enzyme such as horseradish peroxidase
  • Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
  • ISH procedures for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)
  • CGH comparative genomic hybridization
  • ISH In situ hybridization
  • a sample containing target nucleic acid sequence e.g., genomic target nucleic acid sequence
  • a metaphase or interphase chromosome preparation such as a cell or tissue sample mounted on a slide
  • a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence).
  • the slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization.
  • the sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids.
  • the probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium).
  • the chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
  • a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase.
  • fluorescein-labeled avidin or avidin-alkaline phosphatase For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin.
  • FITC fluorescein isothiocyanate
  • samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer).
  • AP alkaline phosphatase
  • Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties.
  • probes labeled with fluorophores including fluorescent dyes and QUANTUM DOTS®
  • fluorophores including fluorescent dyes and QUANTUM DOTS®
  • the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety.
  • a hapten such as the following nonlimiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyl
  • Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • a labeled detection reagent such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand.
  • the detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
  • the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH).
  • the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
  • multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample).
  • a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP.
  • the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 nm).
  • a first specific binding agent in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 nm
  • a second specific binding agent in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®,
  • Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500.
  • Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified.
  • the probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC.
  • SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit.
  • a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR (or q RT-PCR).
  • the expression level of the mRNA is expressed as absolute expression level (in copy genomic DNA equivalent/ml of plasma).
  • a reference value (or cut-off or threshold) can be determined.
  • the cut-off value is 101516 for the RBTC index after a second centrifugation of the plasma at 160G and 546.5 for B2M after a second centrifugation of the plasma at 16000G, in absolute expression level.
  • Methods of the invention may comprise a step consisting of comparing the mRNA of the invention with a control value.
  • expression levels of an mRNA refers to an amount or a concentration of the mRNA, for instance the biomarkers of the invention.
  • a level of a mRNA can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example.
  • Predetermined reference values used for comparison of the expression levels may comprise “cut-off’ or “threshold” values that may be determined as described herein.
  • Each reference (“cut-off’) value for the biomarkers levels may be predetermined by carrying out a method comprising the steps of: a) providing a collection of samples from subjects suffering of a colorectal cancer; b) determining the level of the biomarkers of the invention for each sample contained in the collection provided at step a); c) ranking the tumor tissue samples according to said level or combine all the expression levels of the biomarkers of the invention to obtain a score; d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level, e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding colorectal cancer subject; f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve; g) for each pair of subsets of samples calculating the statistical
  • the p-value can be calculated as well as others parameters (AIC, BIC, LLR, etc.).
  • the expression level of the biomarkers of the invention has been assessed for 100 colorectal cancer samples of 100 subjects.
  • the 100 samples are ranked according to their expression level.
  • Sample 1 has the best expression level and sample 100 has the worst expression level.
  • a first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples.
  • the next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100.
  • Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.
  • the reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest.
  • the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.
  • the reference value (cut-off value) may be used in the present method to discriminate colorectal cancer samples and therefore the corresponding subjects.
  • Kaplan-Meier curves of percentage of survival as a function of time are commonly used to measure the fraction of subjects living for a certain amount of time after treatment and are well known by the man skilled in the art.
  • kits for performing the methods of the invention wherein said kits comprise means for measuring the expression level of the biomarkers of the invention.
  • kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as above defined, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be prelabelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the inventors have designed an efficient, rapid and cost-effective RT-QPCR based- method to purify and analyze mRNA from small volumes of blood plasma (see the results part).
  • the invention also relates to a method to analyse mRNA from a sample obtained from a subject comprising i) preparing a blood sample obtained from said subject to obtained plasma ii) doing a supplemental centrifugation of 160G or 16000G iii) applying an optional freezing step of the plasma obtained in ii), iv) purifying the mRNA and v) doing a PCR assay like qPCR assay.
  • a supplementation centrifugation of 160G or 16000G is done after the freezing.
  • the supplemental centrifugation is of 160G.
  • the invention also relates to a method to analyse mRNA from a sample obtained from a subject comprising i) preparing a blood sample obtained from said subject to obtained plasma ii) doing a centrifugation of 160G or 16000G iii) applying a freezing step of the plasma obtained in ii), iv) doing a supplementation centrifugation of 160G or 16000G after the freezing, v) purifying the mRNA and vi) doing a PCR assay like qPCR assay.
  • the preparation of blood sample in made by well-known techniques like decantation or centrifugation.
  • the purification of the mRNA is made thank two different columns: the first to remove the residual gDNA and the second for purifying the total mRNA.
  • a Silicon-Carbide based technology column can be used.
  • the PCR assay can be a qPCR or RTPCR.
  • the invention also relates to a method for treating a colorectal cancer in a subject diagnosed as having a colorectal cancer as described above comprising the administration to said subject of an anti -colorectal cancerous agent.
  • Anti -colorectal cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).
  • Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5 -fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, lev
  • additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.
  • Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.
  • Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.
  • the further therapeutic active agent can be an antiemetic agent.
  • Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron.
  • the further therapeutic active agent can be an hematopoietic colony stimulating factor.
  • Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.
  • the other therapeutic active agent can be an opioid or nonopioid analgesic agent.
  • opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene.
  • Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.
  • the further therapeutic active agent can be an anxiolytic agent.
  • Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.
  • the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.
  • the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • CTL4 cytotoxic T lymphocyte-associated protein 4
  • PDCD1 programmed cell death 1
  • NK cells like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).
  • the checkpoint blockade cancer immunotherapy agent is an antibody.
  • the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti- BTLA antibodies, and anti-B7H6 antibodies.
  • the invention in another embodiment, relates to a method for treating a colorectal cancer in a subject diagnosed as having a colorectal cancer as described above comprising the use to said subject of radiotherapy, heavy ion treatment, brachy -radiotherapy or radioimmunotherapy.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 RPS28, B2M, TIMP-1 and CLU mRNA levels are increased in the plasma of metastatic colorectal cancer patients.
  • Each dot represented as one case and mean ⁇ SEM are indicated.
  • Table 3 Statistics regarding the analysis of plasma B2M mRNA in different stages of colorecta cancer.
  • Table 4 Statistics regarding the analysis of plasma TIMP-1 mRNA in different stages of colorectal cancer.
  • Table 5 Statistics regarding the analysis of plasma CLU mRNA in different stages of colorectal cancer.
  • PANIRINOX Blood samples from patients with metastatic colorectal cancer we analyzed were obtained from a part of samples from PANIRINOX clinical trial that is still the in course.
  • PANIRINOX is a Phase II randomized study comparing efficacy of FOLFIRINOX + Panitumumab versus mFOLFOX6 + Panitumumab in metastatic colorectal cancer patients selected by RAS and B-RAF status from circulating DNA analysis (Protocol n° UC-0110/1608. EudraCT n°: 2016-001490-33).
  • RNA isolation, reverse transcription and quantitative real-time PCR qRT-PCR
  • RNA is prepared from 200pl using Norgen Plasma/Serum RNA/DNA Purification Mini Kit columns (reference 55200). The first column run is used to remove contaminating DNA, the second column is used to purify the total RNA which is eluted in 12 pl. QuantiTect Reverse Transcription Kit from Qiagen (reference 20531) was used for reverse transcription reaction. Briefly, purified RNA is incubated in gDNA wipeout buffer at 42°C for 5 minutes to thoroughly eliminate contaminating genomic DNA. Then, the reverse transcription reaction was conducted in a final volume of 20pl during 30 min.
  • Quantitation of mRNA was carried out by qRT-PCR using iQTM SYBR® Green Supermix (BIO-RAD) according to the manufacturer’s instructions with a LightCycler® 480 (Roche). Absolute quantification was performed with human genomic DNA from Promega (reference G1471). The results are therefore expressed in genomic DNA copy equivalent considering that the weight of one copy of human male DNA is 3.3 pg [5], All values below 100 copies per ml which corresponds to one copy in the QPCR assay are considered to be equal to zero copies detected.
  • Primer sequences were used to amplify genomic DNA and the biomarkers of the invention.
  • Blood samples from healthy individuals (HI) and from mCRC patients were collected in identical conditions (Streck cell-free DNA BCT® tubes). These types of sampling tubes are now widely used to analyze circulating DNA from blood samples that have been in shipment for several days [6,7], After preparation of the plasma as recommended by the tube supplier, and despite the fact that about 1 ml of plasma above the buffy coat is removed, the presence of residual cells in the collected plasma cannot be completely excluded if the buffy coat has been accidentally disturbed during the pipetting process.
  • RNA - a 160G centrifugation that is classically used in cell culture to pellet cells without breaking them in order to avoid they release their RNA.
  • the plasmas that have undergone a second centrifugation are immediately frozen at - 20°C.
  • the analyses by QPCR can be temporally deferred.
  • RNA Ribosomal Protein S28
  • GUS beta-glucuronidase
  • B2M Beta2- microglobulin
  • TMP-1 tissue inhibitor of metalloproteinase 1
  • CLU Clusterin
  • TIMP-1 mRNA level there is also a highly significant difference in TIMP-1 mRNA level when plasmas are centrifuged at 160 G ( Figure IE) and for centrifugation at 16000G (Table 1, Figure IF).
  • the difference of CLU mRNA level is significant between the plasmas of healthy individuals and patients when the second centrifugation occurred at 160G (Table 1, Figure 1G), however when the second centrifugation occurred at 16000G the difference is not significant (Table 1, Figure 1H).
  • the plasma level of the four mRNAs in each patient was represented ( Figure 2A and 2B) and the dots representing each mRNA for each patient are connected by a line. We observed that the lines of the graph often intersect.
  • the AUC of RBTC index is higher than the AUCs of the four mRNAs (RPS28, B2M, TIMP-1, and CLU) calculated individually (Table 2).
  • a RBTC index level cutoff of 101516 was determined using the Youden’s index; it has 81.6% sensitivity and 90.6% specificity to discriminate CRCm from healthy controls.
  • the ROC analysis of the four RNAs and RBTC index when the second centrifugation is performed at 16000G shows that the best AUC (0.736) is for B2M ( Figure 2E and Table 2).
  • the inventors show that the B2M mRNA levels (Figure 3A and table 3), the TIMP-1 mRNA levels ( Figure 4A and table 4), the CLU mRNA levels ( Figure 5A and table 5) and the sum of the plasma levels of TIMP-1 and CLU mRNAs levels ( Figure 6A and table 6) in plasma samples from patients with stage 4 CRC were higher than respectively B2M mRNA levels, TIMP-1 mRNA levels, CLU mRNA levels and sum of the plasma levels of TIMP-1 and CLU mRNAs levels in plasma samples from healthy individuals (HI).
  • the inventors also show that the B2M mRNA levels (Figure 3B), the TIMP-1 mRNA levels ( Figure 4B), the CLU mRNA levels ( Figure 5B) and the sum of the plasma levels of TIMP-1 and CLU mRNAs levels (Figure 6B) in plasma samples from patients with all stage of CRC patients were higher than respectively B2M mRNA levels, TIMP-1 mRNA levels, CLU mRNA levels and sum of the plasma levels of TIMP-1 and CLU mRNAs levels in plasma samples from healthy individuals (HI).
  • the inventors show that the B2M mRNA levels is not higher in stage 1, 2 and 3 CRC patient plasma samples than in healthy individual plasma samples ( Figure 3A) whereas the TIMP-1 and CLU mRNAs levels is higher in stage 1, 2 and 3 CRC patient plasma samples than in healthy individual plasma samples. ( Figures 4A and 5A).
  • Bossard, C. et al. HLA-E/P2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression. Int. J. Cancer 131, 855-863 (2012).
  • TIMP-1 overexpression promotes tumorigenesis of MDA-MB-231 breast cancer cells and alters expression of a subset of cancer promoting genes in vivo distinct from those observed in vitro.
  • Tissue inhibitor of metalloproteinase- 1 (TIMP-1) as a prognostic biomarker in gastrointestinal cancer: a meta-analysis. PeerJ 9, el0859 (2021).

Abstract

La présente invention concerne le diagnostic du cancer colorectal. Les inventeurs ont ainsi conçu un procédé efficace, rapide et rentable pour purifier et analyser l'ARNm à partir de petits volumes de plasma sanguin. Ils ont constaté que les niveaux de RPS28, B2M, TIMP-1 et CLU étaient significativement plus élevés chez les patients atteints de cancer colorectal métastatique. Ainsi, la présente invention concerne un procédé pour diagnostiquer un cancer colorectal chez un sujet en ayant besoin, comprenant i) l'établissement, dans un échantillon prélevé sur le sujet, des niveaux d'expression d'au moins un biomarqueur choisi dans le groupe constitué des ARNm RPS28, B2M, TIMP-1 et CLU.
PCT/EP2023/052997 2022-02-08 2023-02-07 Procédé de diagnostic du cancer colorectal WO2023152133A1 (fr)

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