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WO2017207545A1 - Signature moléculaire du carcinome hépatocellulaire - Google Patents

Signature moléculaire du carcinome hépatocellulaire Download PDF

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Publication number
WO2017207545A1
WO2017207545A1 PCT/EP2017/062998 EP2017062998W WO2017207545A1 WO 2017207545 A1 WO2017207545 A1 WO 2017207545A1 EP 2017062998 W EP2017062998 W EP 2017062998W WO 2017207545 A1 WO2017207545 A1 WO 2017207545A1
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Prior art keywords
hcc
subject
markers
genes
expression
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PCT/EP2017/062998
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English (en)
Inventor
Cédric COULOUARN
Bruno Clement
Gaelle ANGENARD
Coralie ALLAIN
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
Universite De Rennes 1
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Publication of WO2017207545A1 publication Critical patent/WO2017207545A1/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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a set of markers of hepatocellular carcinoma (HCC) and to a method for determining whether a subject is at risk of having or has an HCC. It further relates to a method for determining a therapeutic regimen suitable for treating a subject suffering from an HCC.
  • HCC hepatocellular carcinoma
  • HCC Hepatocellular carcinoma
  • Liver carcinogenesis is a long process associated with multiple risk factors that contribute to HCC heterogeneity, e.g. viral hepatitis, alcohol abuse, metabolic disorders and obesity.
  • Most HCC develop in the setting of chronic liver disease during which cycles of tissue destruction and regeneration result in the activation of numerous signaling pathways and the accumulation of genomic alterations (e.g. chromosomal structural changes, mutations).
  • genomic alterations e.g. chromosomal structural changes, mutations.
  • next-generation sequencing approaches, including exome sequencing revealed the great diversity of mutational processes underlying the development of HCC.
  • Cell plasticity and tumor microenvironment remodeling also contribute to HCC heterogeneity.
  • HCC signature will help in the diagnosis of HCC and in the identification of combinations of treatments to target various signaling pathways altered in HCC.
  • the present invention thus relates to a set of markers of hepatocellular carcinoma (HCC), said set comprising or consisting of the group consisting in:
  • groups (a) and (b) are further defined as follows:
  • the present invention also relates to a set of HCC markers, said set comprising or consisting of the group consisting in:
  • the set of HCC markers as described herein further comprises at least one marker selected from the group consisting in the genes mentioned in Table 1 or transcription products and/or translation products thereof.
  • the set of HCC markers according to the invention comprises or consists in the group consisting in all the genes mentioned in Table 1 or transcription products and/or translation products thereof.
  • the present invention further relates to a method for determining whether a subject is at risk of having or has an HCC, said method comprising:
  • a level of expression of said markers as mentioned in Table 2 in comparison to the reference value is indicative of a subject at risk of having or having an HCC.
  • the present invention also relates to a method for monitoring the efficiency of an HCC therapy, said method comprising:
  • step b) repeating step a) on another biological sample from the same subject taken at a later point in time
  • a level of expression of said markers as mentioned in Table 3 in comparison to the reference value is indicative of the efficacy of said therapy.
  • the present invention further relates to a method of prognosing or classifying the outcome of an HCC in a subject undergoing a therapy, wherein said method comprises the step of determining the level of expression of the markers of the set of markers as described herein in a biological sample of said subject.
  • the invention also relates to a method for determining a therapeutic regimen suitable for treating a subject suffering from an HCC, wherein said method comprises the steps of :
  • the present invention further relates to a kit for determining whether a subject is at risk of having or has a HCC, said kit comprising means for detecting the markers of the set of HCC markers as described herein.
  • Set of HCC markers
  • HCC hepatocellular carcinoma
  • the set of HCC markers comprises or consists in the group (a) consisting in the genes MDK, SPINK1 , GPC3, BOLA1 , ADAMTS1 , IGFBP3 and DPT
  • said set further comprises at least one marker chosen from the genes COL15A1 , ANXA2, COL4A1 , KLKB1 , FGL2, CYR61 , F1 1 and CXCL12.
  • said set when said set comprises or consists in transcription products and/or translation products of the genes mentioned in the group (a) consisting in the genes MDK, SPINK1 , GPC3, BOLA1 , ADAMTS1 , IGFBP3 and DPT, said set further comprises at least one marker chosen from the transcription products and/or translation products of the genes COL15A1 , ANXA2, COL4A1 , KLKB1 , FGL2, CYR61 , F1 1 and CXCL12.
  • the set of HCC markers comprises or consists in the group (a) consisting in the genes MDK, SPINK1 , GPC3, BOLA1 , ADAMTS1 , IGFBP3 and DPT
  • said set further comprises all the following genes: COL15A1 , ANXA2, COL4A1 , KLKB1 , FGL2, CYR61 , F1 1 and CXCL12.
  • the set of HCC markers comprises or consists in transcription products and/or translation products of the genes mentioned in the group (a) consisting in the genes MDK, SPINK1 , GPC3, BOLA1 , ADAMTS1 , IGFBP3 and DPT
  • said set further comprises the transcription products and/or translation products of all the following genes: COL15A1 , ANXA2, COL4A1 , KLKB1 , FGL2, CYR61 , F1 1 and CXCL12.
  • the set of HCC markers comprises or consists in the group (b) consisting in the genes PPIA, FDPS, PSMD4, HSP90AB1 , FAM189B, ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A
  • said set further comprises at least one marker chosen from the genes CDK1 , NME1 , PEA15, ARPC1 A, RDBP, KPNA2, TBCE, SNRPE, IRAKI , ANXA2, PUF60, ETS2, CYR61 and C1 R.
  • said set when said set comprises or consists in transcription products and/or translation products of the genes mentioned in the group (b) consisting in the genes PPIA, FDPS, PSMD4, HSP90AB1 , FAM189B, ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A, said set further comprises at least one marker chosen from the transcription products and/or translation products of the genes CDK1 , NME1 , PEA15, ARPC1 A, RDBP, KPNA2, TBCE, SNRPE, IRAKI , ANXA2, PUF60, ETS2, CYR61 and C1 R.
  • the set of HCC markers comprises or consists in the group (b) consisting in the genes PPIA, FDPS, PSMD4, HSP90AB1 , FAM189B, ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A
  • said set further comprises all the following genes: CDK1 , NME1 , PEA15, ARPC1 A, RDBP, KPNA2, TBCE, SNRPE, IRAKI , ANXA2, PUF60, ETS2, CYR61 and C1 R.
  • the set of HCC markers comprises or consists in transcription products and/or translation products of the genes mentioned in the group (b) consisting in the genes PPIA, FDPS, PSMD4, HSP90AB1 , FAM189B, ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A
  • said set further comprises the transcription products and/or translation products of all the following genes: CDK1 , NME1 , PEA15, ARPC1 A, RDBP, KPNA2, TBCE, SNRPE, IRAKI , ANXA2, PUF60, ETS2, CYR61 and C1 R.
  • the set of HCC markers as described herein further comprises at least one marker selected from the group consisting in the genes mentioned in Table 1 or transcription products and/or translation products thereof.
  • the set of HCC markers as described herein further comprises or consists in the group consisting in all the genes mentioned in Table 1 or transcription products and/or translation products thereof.
  • At least one is meant 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or more markers according to the invention.
  • marker means a distinctive biological or biologically-derived indicator of a process, event, or condition.
  • the term marker as used herein refers to a gene or a transcription product or a translation product thereof i.e. DNA, mRNA, protein or peptide or variant that is differentially expressed in subjects with HCC.
  • the marker according to the invention is suitable to be used in methods of diagnosis (e.g. clinical screening, method for determining whether a subject is at risk of having or has an HCC), prognosis assessment; in monitoring the results of therapy, identifying subjects most likely to respond to a particular therapeutic treatment, drug screening and development. All the genes described herein as markers are known per se, and are listed in the Table 1 .
  • the term "gene” or “genes” relates to the nucleotidic sequences of the genes considered and does not relate to the genes as such.
  • Transcription products of the genes described herein refers to nucleic acids that are transcribed.
  • it comprises transcribed sequences that encode for a protein, polypeptide or peptide. It may comprise transcribed nucleic acid(s), regulatory sequences, coding sequences, or the like, isolated substantially away from other such sequences, such as other naturally occurring regulatory sequences, polypeptide or peptide encoding sequences, etc.
  • the transcribed nucleotide sequence comprises at least one functional protein, polypeptide and/or peptide encoding unit.
  • nucleic acid will generally refer to at least one molecule or strand of DNA, RNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine "A,” guanine “G,” thymine “T,” and cytosine “C”) or RNA (e.g. A, G, uracil “U,” and C).
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide.”
  • oligonucleotide refers to at least one molecule of between about 3 and about 100 nucleobases in length.
  • polynucleotide refers to at least one molecule of greater than about 100 nucleobases in length. These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double- stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence comprising a strand of the molecule.
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art.
  • Non-limiting examples of synthetic nucleic acid, particularly a synthetic oligonucleotide include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such described by Froehler et at., 1986 via deoxynucleoside H-phosphonate intermediates.
  • a non-limiting example of enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM or the synthesis of oligonucleotides.
  • a non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells (see for example, Sambrook et al. 2000).
  • a nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al. 2000).
  • the nucleic acid molecule is preferably isolated, which means that it is essentially free of other nucleic acids. Essentially free from other nucleic acids means that the nucleic acid molecule is at least about 90%, preferably at least about 95% and, more preferably at least about 98% free of other nucleic acids.
  • the molecule is essentially pure, which means that the molecule is free not only of other nucleic acids, but also of other materials used in the synthesis and isolation of the molecule.
  • Materials used in synthesis include, for example, enzymes.
  • Materials used in isolation include, for example, gels, such as SDS-PAGE.
  • the molecule is at least about 90% free, preferably at least about 95% free and, more preferably at least about 98% free of other nucleic acids and such other materials.
  • Translation products of the genes described herein are polypeptides sequences encoded by said genes, or variants or fragments thereof.
  • polypeptide refers to any chain of amino acids linked by peptide bonds, regardless of length or post-translational modification.
  • Polypeptides include natural proteins, synthetic or recombinant polypeptides and peptides (i.e. polypeptides of less than 50 amino acids) as well as hybrid, post-translationally modified polypeptides, and peptidomimetic.
  • amino acid refers to the 20 standard alpha-amino acids as well as naturally occurring and synthetic derivatives.
  • a polypeptide may contain L or D amino acids or a combination thereof.
  • variants includes protein and nucleic acid variants.
  • Variant proteins may be naturally occurring variants, such as splice variants, alleles and isoforms, or they may be produced by recombinant means.
  • Variations in amino acid sequence may be introduced by substitution, deletion or insertion of one or more codons into the nucleic acid sequence encoding the protein that results in a change in the amino acid sequence of the protein.
  • the variation is by substitution of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids with any other amino acid in the protein. Amino acid substitutions may be conservative or non-conservative.
  • substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties. Additionally or alternatively, the variation may be by addition or deletion of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids within the protein. Amino acid substitutions may be conservative or non-conservative. Preferably, substitutions are conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and/or chemical properties. The substitution preferably corresponds to a conservative substitution as indicated in the table below.
  • fragments of the proteins and variant proteins disclosed herein are also encompassed by the invention. Such fragments may be truncated at the N-terminus or C- terminus, or may lack internal residues, for example, when compared with a full length protein. Certain fragments lack amino acid residues that are not essential for enzymatic activity. Preferably, said fragments are at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 150, 250, 300, 350, 400, 450, 500 or more amino acids in length.
  • Variant nucleic acid sequences include sequences capable of specifically hybridizing to the sequence of the genes described herein under moderate or high stringency conditions.
  • Stringent conditions or high stringency conditions may be identified by those that: (1 ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoll/0.1 % polyvinylpyrrolidone/5 OmM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution
  • Moderately stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and %SDS
  • moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C.
  • the invention further relates to a method for determining whether a subject is at risk of having or has an HCC, said method comprising:
  • the level of expression of said markers is determined by detecting transcription products and/or translation products of the genes described herein.
  • the term "gene” or “genes” relates to the nucleotidic sequences of the genes considered and does not relate to the genes as such.
  • sample means a substance of biological origin.
  • biological samples include, but are not limited to bodily fluids samples and biopsy. Bodily fluids include blood, urine, saliva or any other bodily secretion or derivative thereof. As used herein "blood” includes whole blood, plasma, serum, circulating epithelial cells, constituents, or any derivative of blood.
  • the biological sample according to the invention may be obtained from the subject by any appropriate means of sampling known from the person skilled in the art.
  • the term "subject” refers to a warm-blooded animal such as a mammal, animal or human, in particular a human, who is afflicted with, or has the potential to be afflicted with one or more diseases and conditions described herein, i.e. an HCC.
  • At risk means probability that an event will occur over a specific time period, as in the conversion to HCC, and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1 - p) is the probability of no event) to no- conversion.
  • a “reference value” denotes a level of expression of a marker as described herein in a control subject or group of control subjects.
  • control subject refers to a subject that has not shown any HCC symptoms and has not been diagnosed for this disease.
  • the reference value(s) may be determined as a single value or a range of values which is determined based on the level of expression of the markers as described herein measured in a population of control subjects.
  • the analysed population could be divided into quantiles based on the measured level of expression of the markers.
  • the reference value could be defined as the median, or the second tertile, or the second or third quartile, or the third or fourth quintile etc.
  • determining is meant measuring the level of expression of the markers described herein, or detecting a decrease or increase of the level of expression of the markers described herein.
  • decrease in the level of expression is meant a decrease of the level of expression of the marker in comparison to a reference or to a predetermined threshold value, for example a decrease of the level of expression of the marker of 5% or 10% in comparison to a reference or to a predetermined threshold value.
  • increase in the level of expression is meant an increase of the level of expression of the marker in comparison to a reference or to a predetermined threshold value, for example an increase of expression level of the marker of 5% or 10% in comparison to a reference or to a predetermined threshold value.
  • predetermined threshold for one marker may refer to the median value of the level of expression of the marker in biological samples of a control subject. The skilled person can easily determine such a predetermined threshold using methods well- known in the art.
  • a level of expression of the markers of the set of markers as described herein as mentioned in Table 2 in comparison to the reference value is indicative of a subject at risk of having or having an HCC.
  • "Up in HCC” refers to an increase of the level of expression of the marker whereas “Down in HCC” refers to a decrease of the level of expression of the marker.
  • Table 2 mentions if the level of expression of the genes part of the different set of markers described herein is increased or decreased in comparison to a reference value. It has to be understood that the increase or decrease mentioned in relation to the genes part of the different set of markers described herein also apply to the corresponding transcription products and translation products.
  • an increase of the level of expression of the genes MDK, SPINK1 , GPC3 and BOLA1 or of transcription products and/or translation products thereof and a decrease of the level of expression of the genes ADAMTS1 , IGFBP3 and DPT, or of transcription products and/or translation products thereof, in comparison to the reference value, is indicative of a subject at risk of having or having an HCC.
  • FDPS FDPS
  • PSMD4 HSP90AB1 and FAM189B or of transcription products and/or translation products thereof and a decrease of the level of expression of the genes ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A, or of transcription products and/or translation products thereof, in comparison to the reference value, is indicative of a subject at risk of having or having an HCC.
  • Level of expression of the markers as described herein can be determined by methods well known by a man skilled in the art. It can be performed by immunoassay or immunoblots or by analytical methods, like for example mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS, LC-MS/MS), quantitative methods with isotopic labeling (stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags (ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), label-free methods like selective reaction monitoring (SRM) or multiple reaction monitoring (MRM) assays, or bio- molecular interaction analysis/surface plasmon resonance (BIA/SPR) technologies encompassing methods with calibration and without calibration as calibration free concentration analysis for example.
  • MS mass spectrometry
  • CE-MS capillary electrophoresis-mass spectrometry
  • LC-MS liquid chromat
  • immunoassay includes competition, direct reaction, or sandwich type assays.
  • assays include, but are not limited to, agglutination test, enzyme-labelled and mediated immunoassays, such as ELISA, biotin/avidin type assay, radioimmunoassay, Immunoelectrophoresis, and immunoprecipitation.
  • Mass spectrometry (MS), capillary electrophoresis-mass spectrometry (CE-MS), liquid chromatography coupled to mass spectrometry (LC-MS/MS), stable isotope labeling by amino acids in cell culture (SILAC), isotope coded affinity tags(ICAT), isobaric tag for relative and absolute quantitation (ITRAQ), selective reaction monitoring (SRM) assays, multiple reaction monitoring (MRM) assays, bio-molecular interaction analysis/surface plasmon resonance (BIA SPR) technologies, calibration free concentration analysis, are all analytical methods very well known by the man skilled in the art which are suitable to carry out the measure of the cerebellin-2 protein level according to the invention.
  • SILAC isotope coded affinity tags
  • ITRAQ isobaric tag for relative and absolute quantitation
  • SRM selective reaction monitoring
  • MRM multiple reaction monitoring
  • BIOSPR bio-molecular interaction analysis/surface plasmon resonance
  • Such methods to determine the level of expression of the markers as described herein also include methods for measuring the quantity of mRNA.
  • the nucleic acid contained in the biological sample may be 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 may be then detected by hybridization (e. g., Northern blot analysis).
  • the extracted mRNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that enable amplification of a region in said genes.
  • RT-PCR polymerase chain reaction
  • Extracted mRNA may be reverse-transcribed and amplified, after which amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art.
  • Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • the invention further relates to a method for monitoring the efficiency of an HCC therapy, said method comprising:
  • step b) repeating step a) on another biological sample from the same subject taken at a later point in time
  • An alteration of the level of expression of the markers by comparing its levels of expression in the subject biological sample to another one of the same subject at a late point in time is indicative that the subject is sensitive or resistant to therapy.
  • alteration refers to a statistically significant difference in the level of expression of said marker measured between two samples such as for example, the subject biological sample and the biological sample of the same subject taken at a later point in time.
  • the alteration of the expression can be compared using the ratio of the level of expression of a given marker or markers as compared with the expression level of the given marker or markers(s) of another sample, wherein the ratio is not equal to 1 .
  • there is an alteration of the expression of a marker if the ratio of the level of expression in a first sample as compared with a second sample is greater than or less than 1 .0.
  • the alteration of the expression is measured using p-value.
  • an alteration of the expression of a marker is as between a first sample and a second sample or a control sample when the p- value is less than 0.1 , preferably less than 0.05, more preferably less than 0.01 , even more preferably less than 0.005, the most preferably less than 0.001 .
  • the "alteration of the expression” means an increase or a decrease of the level of expression of a marker as described herein by comparing its level of expression in a biological sample of the subject following treatment with the therapy to its level of expression in a biological sample of the subject prior to treatment with the therapy.
  • therapeutic regimen refers to a course of treatment intended to reduce or eliminate the affects or symptoms of a disease or to prevent progression of a disease from one state to a second, more detrimental state.
  • a therapeutic regimen can comprise a prescribed drug, surgery or radiation treatment.
  • the therapy or therapeutic regimen mentioned in the methods according to the invention refers to the treatment with histone deacetylase (HDAC) inhibitors (in particular trichostatin A and/or vorinostat), and/or PI3K inhibitor (in particular LY294002) and/or mTOR inhibitor (in particular sirolimus also known as rapamycin) and/or alpha-estradiol and/or resveratrol.
  • HDAC histone deacetylase
  • PI3K inhibitor in particular LY294002
  • mTOR inhibitor in particular sirolimus also known as rapamycin
  • a level of expression of the markers of the set of markers as described herein as mentioned in Table 3 in comparison to the reference value is indicative of the efficacy of said therapy.
  • Table 3 mentions if the level of expression of the genes part of the different set of markers described herein is increased or decreased in comparison to a reference value. It has to be understood that the increase or decrease mentioned in relation to the genes part of the different set of markers described herein also apply to the corresponding transcription products and translation products.
  • a decrease of the level of expression of the genes MDK, SPINK1 , GPC3 and BOLA1 or of transcription products and/or translation products thereof and an increase of the level of expression of the genes ADAMTS1 , IGFBP3 and DPT or of transcription products and/or translation products thereof, is indicative of the efficacy of said therapy.
  • a decrease of the level of expression of the genes PPIA, FDPS, PSMD4, HSP90AB1 and FAM189B or of transcription products and/or translation products thereof and an increase of the level of expression of the genes ETFDH, ADAMTS1 , RND3, IGFBP3 and GPM6A or of transcription products and/or translation products thereof is indicative of the efficacy of said therapy.
  • the invention further relates to a method of prognosing or classifying the outcome of an HCC in a subject undergoing a therapy, wherein said method comprises the step of determining the level of expression of the markers of the set of markers according to the invention in a biological sample of said subject.
  • prediction refers to the prediction of the likelihood of benefit from therapy.
  • prediction or predicting refers to the likelihood that a subject will respond either favourably or unfavourably to a particular therapy.
  • prediction or predicting relates to the extent of those responses.
  • the prediction or predicting relates to whether and/or the probability that a subject will survive or improve following treatment, for example treatment with a particular therapeutic agent, and for a certain period of time without disease recurrence.
  • the predictive methods of the invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular subject.
  • the predictive methods of the present invention are valuable tools in predicting if a subject is likely to respond favourably to a treatment regimen, such as a given therapeutic regimen, or whether long-term survival of the subject following a therapeutic regimen is likely.
  • the invention further relates to a method for determining a therapeutic regimen suitable for treating a subject suffering from an HCC, wherein said method comprises the steps of :
  • the method for determining a therapeutic regimen suitable for treating a subject suffering from an HCC according to the invention will aid a physician in selecting a course of treatment for the HCC subject.
  • the subject will be determined to be a therapy-sensitive subject on the basis of a probability, and the subject will be subsequently treated with that therapy alone, optionally in combination with other chemotherapeutic drugs.
  • the subject will be determined to be therapy-resistant, thereby allowing the physician to exclude that candidate treatment for the subject, thereby sparing the subject the unnecessary toxicity.
  • the methods as described herein are in vitro methods.
  • the present invention also relates to a method for treating a subject suffering from an HCC, wherein said method comprises the steps of:
  • treat refers to therapeutic treatment wherein the object is to eliminate or lessen symptoms.
  • beneficial or desired clinical results include, but are not limited to, elimination of symptoms, alleviation of symptoms, diminishment of extent of condition, stabilized (i.e., not worsening) state of condition, delay or slowing of progression of the condition, to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof.
  • the terms refer to the treatment with or administration of a compound provided herein prior to the onset of symptoms.
  • the terms encompass the inhibition or reduction of a symptom of the particular disease.
  • Subjects with familial history of a disease are candidates for treatment regimens in certain embodiments. Examples of modes of administration include parenteral e.g. subcutaneous, intramuscular, intravenous, intradermal, as well as oral administration.
  • kits that are useful in the above methods. Indeed is provided a kit for determining whether a subject is at risk of having or has a HCC, said kit comprising means for detecting the set of HCC markers as described herein.
  • said kit consists of means for detecting the set of HCC markers as described herein.
  • kits can be used, e.g. for determining whether a subject is at risk of having or has a HCC, for monitoring the efficiency of an HCC therapy, for prognosing or classifying the outcome of an HCC in a subject undergoing a therapy or for determining a therapeutic regimen suitable for treating a subject suffering from an HCC.
  • the kit further comprises a sample of reference value indicative of the amount and/or level of expression of the markers of the set of markers described herein.
  • the kit consists of means for detecting the set of HCC markers as described herein and a sample of reference value indicative of the amount and/or level of expression of said markers.
  • kits according to the invention may for example comprise, in addition to the means for detecting the amount and/or level of expression of the markers of the set of markers described herein, one of (i) to (iii) below:
  • a positive reference value sample indicative of the amount and/or level of expression of the markers of the set of markers described herein in a subject suffering from HCC;
  • a negative reference value sample indicative of the amount and/or level of expression of the markers of the set of markers described herein in a control subject
  • kits for determining whether a subject is at risk of having or has a HCC, for monitoring the efficiency of an HCC therapy, for prognosing or classifying the outcome of an HCC in a subject undergoing therapy or for determining a therapeutic regimen suitable for treating a subject suffering from an HCC.
  • kit may for example comprise (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii).
  • the level of expression of the markers of the set of markers according to the invention can be determined by measuring the quantity of translation products described herein or the quantity of transcription products described herein, preferably mRNA.
  • Means for detecting mRNA include real-time quantitative-PCR.
  • the kit may further comprise a second reagent, labeled with a detectable compound, which binds to said mRNA, such as e.g. SYBER GREEN reagents.
  • Means for measuring the level of expression of the translations products include antibodies specifically binding to these products. Such means can be labeled with detectable compound such as fluorophores or radioactive compounds.
  • the antibody specifically binding to the translation product may be labeled with a detectable compound.
  • the kit may further comprise a secondary antibody, labeled with a detectable compound, which binds to an unlabelled antibody specifically binding to the translation product.
  • the antibody may be polyclonal or monoclonal, preferably monoclonal.
  • Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • Antibodies useful in practicing the present invention also include anti-biomarkers fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to markers of the invention.
  • phage display of antibodies may be used.
  • single- chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13.
  • a suitable host e.g., mouse
  • the coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence.
  • a suitable carrier e. g., bacteria
  • Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay. Instructions for using the kit according to methods of the invention may comprise instructions for processing the biological sample obtained from the subject and/or for performing the test, or instructions for interpreting the results. A kit may also contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products.
  • the kit may further comprise one or more of: extraction buffer and/or reagents, western blotting buffer and/or reagents, and detection means.
  • Protocols for using these buffers and reagents for performing different steps of the procedure may be included in the kit.
  • kits of the invention may be supplied in a solid (e.g. lyophilized) or liquid form.
  • kits of the present invention may optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer and/or reagent.
  • Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form.
  • Other containers suitable for conducting certain steps of the disclosed methods may also be provided.
  • the individual containers of the kit are preferably maintained in close confinement for commercial sale. The invention will be further illustrated by the following figures and examples.
  • Figure 1 Large scale meta-analysis of genomic data identifies core transcriptional hallmarks in human HCC. (a) Analytical workflow of the meta-analysis that led to the identification of a core gene expression signature in human HCC (gene HCC signature).
  • Each bar represents cell viability (mean ⁇ SD) in the investigated cell lines (SNU-475, SNU-449, SNU-423, SNU-387, HepG2/C3A, SK-Hep-1 , from left to right).
  • a two-tailed non- parametric Mann-Whitney test was used for the comparison between experimental groups for each cell line (DMSO vs. untreated control and drug vs. DMSO). * denotes a P value ⁇ 0.05.
  • GSEA Gene set enrichment analysis
  • S1 and S2 HCC subtypes are associated with a poor prognosis and included highly proliferative and poorly differentiated tumors.
  • S1 subtype is particularly associated with the activation of a pro-metastatic TGF3 signaling and the S2 subtype included tumors with a progenitor-like phenotype.
  • S3 subtype is associated with a better prognosis, low proliferation and includes well-differentiated tumors that retained a hepatocyte-like phenotype.
  • the S3 subtype is enriched in tumors that exhibit activating mutations in CTNNB1 gene encoding ⁇ -catenin.
  • HCC stratification into homogeneous subtypes opened new avenues for personalized targeted therapies.
  • the highlighting of core transcriptional hallmarks in human HCC suggests that efficient therapies should consider drugs targeting common HCC hallmarks together with drugs targeting specific HCC subtypes.
  • CTNNB1 mut mutated beta-catenin gene
  • HCC hepatocellular carcinoma
  • microarray platforms were re-annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https ://david . ncifcrf.gov/) ( Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4, 44-57 (2009)). In total, 24,085 non-redundant annotated genes were present at least in one out of 28 retrieved microarray datasets. Statistical analysis of microarray data was performed using R-based BRB-ArrayTools as previously described (Coulouarn, C, et al.
  • Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma.
  • Cancer Res 72, 2533-2542 (2012) Median-based normalization was applied and differentially expressed genes between the tumor and surrounding non-tumor tissues were identified by a 2- sample univariate f test (P ⁇ 0.01 ) and a random variance model as described (Coulouarn, C, et al. Hepatocyte-stellate cell cross-talk in the liver engenders a permissive inflammatory microenvironment that drives progression in hepatocellular carcinoma. Cancer Res 72, 2533-2542 (2012)). Permutation P values for significant genes were computed on the basis of 10,000 random permutations.
  • GSEA Gene set enrichment analysis
  • a panel of 6 liver cancer cell lines from ATCC (http://www.lqcstandards-atcc.org) was used to evaluate the impact of selected molecules, including SNU-475 (ATCC® CRL- 2236TM, grade ll-IV/V), SNU-449 (ATCC® CRL-2234TM, grade ll-lll/IV), SNU-423 (ATCC® CRL-2238TM, grade lll/IV), SNU-387 (ATCC® CRL-2237TM, grade IV/V), HepG2/C3A (ATCC® CRL-10741TM) and SK-HEP-1 (ATCC® HTB-52TM). ATCC performed cell lines authentication by STR DNA profiling. The impact of selected molecules was evaluated within 6 months after receipt.
  • the concentration of each molecule used to treat the cells was initially determined based on the results of the connectivity map algorithm.
  • Cell viability was evaluated using a PrestoBlue® cell viability reagent (Invitrogen) after 48h and 72h of treatment with DMSO, trichostatin A (1 ⁇ ), alpha-estradiol (1 ⁇ ), vorinostat (50 ⁇ ), sorafenib (2 ⁇ ), rapamycin (2 ⁇ ), LY294002 (50 ⁇ ) and resveratrol (500 ⁇ ).
  • DMSO trichostatin A
  • alpha-estradiol 1 ⁇
  • vorinostat 50 ⁇
  • sorafenib sorafenib
  • rapamycin rapamycin
  • LY294002 50 ⁇
  • resveratrol 500 ⁇ .
  • the concentrations were optimized to induce 50% cell mortality after a 72h drug exposure, in order to allow the extraction of nucleic acids from the remaining viable cells.
  • the significance of differences in cell viability between experimental conditions was determined by a two-tailed non-parametric Mann-Whitney test.
  • the concentrations were optimized to induce 50% cell mortality after a 72-hour drug exposure, to allow the extraction of nucleic acids from the remaining viable cells.
  • cells were treated with trichostatin A (0.6 mmol/L), a-estradiol (1 mmol/L), vorinostat (3.7 mmol/L), sorafenib (0.65 mmol/L), rapamycin (2 mmol/L), LY294002 (45 mmol/L), and resveratrol (166 mmol/L).
  • RNA amplification yield was 7.0 ⁇ 1 ⁇ g cRNA and specific activity was 18.2 ⁇ 2.3pmol Cy3 per ⁇ g of cRNA.
  • Gene expression data were processed using Feature Extraction and GeneSpring softwares (Agilent Technologies). Microarray data have been deposited in NCBI's GEO and are accessible through GEO Series accession numbers GSE79246 and GSE85257. Results
  • the initial step of the study was to assemble well-annotated human HCC gene expression profiles.
  • Gene Expression Omnibus and ArrayExpress 28 liver-oriented datasets were retrieved including 1 ,657 HCC gene expression profiles from a total of 3,047 ( Figure 1 a).
  • Out of 28 datasets 15 were relevant to derive a universal HCC signature defined as a set of genes differentially expressed between HCC and the surrounding non-tumor tissue (ST).
  • the gene expression profiles were generated from various microarray platforms (i.e. from academic or industrial sources, including several updated contents). Consequently, variations in both the number and the nature of interrogated gene features between platforms greatly impede the integrative analysis of the datasets.
  • the gene HCC signature covers well-established cancer hallmarks
  • RAD21, RUVBL2) and nucleotide excision repair e.g. ERCC3, FEN1, OGG1 were induced.
  • DNA glycosylase OGG1 is involved in the excision of 8-oxoguanine, a mutagenic base byproduct which occurs as a result of exposure to reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • key regulators of redox homeostasis were repressed in the 935-gene HCC signature (e.g. CTH, CBS, NFE2L2, PRDX4).
  • CRADD, DAPK1 genes encoding proteins that prevent apoptotic cell death were induced (e.g. BIRC5, DAD1).
  • Metabolic reprogramming Deregulation of metabolism-associated genes (e.g. lipid, carbohydrate and amino acid metabolisms) was a prominent feature in the 935-gene HCC signature, in agreement with metabolic changes observed in cancer cells during tumor onset and progression. This was particularly noticeable for down-regulated genes involved in liver specific metabolisms (Figure 1 d), including those encoding acute phase plasma proteins (e.g. A2M, ALB, CP), components of complement and coagulation cascades (e.g. C5-9, CFB, F2) or detoxication enzymes (e.g. ADH1A, CYP2E1).
  • acute phase plasma proteins e.g. A2M, ALB, CP
  • components of complement and coagulation cascades e.g. C5-9, CFB, F2
  • detoxication enzymes e.g. ADH1A, CYP2E1
  • ACLY and CS were recurrently up-regulated in HCC.
  • ACLY encodes ATP citrate lyase, the primary enzyme for the synthesis of acetyl-CoA, a major intermediate for biosynthetic pathways including lipogenesis.
  • CS encodes the citrate synthase, a key enzyme in the tricarboxylic acid cycle that contributes to lipogenesis by enhancing the conversion of glucose to lipids.
  • the 935-gene HCC signature included several angiogenesis-related genes.
  • PLG encoding plasminogen was strongly repressed in almost all HCC (Table 2).
  • plasminogen is activated by proteolysis and converted to plasmin, an activator of matrix metalloproteases, and angiostatin, a potent inhibitor of angiogenesis.
  • AMOTL2 encoding an angiomotin like 2 protein was repressed.
  • Angiomotin is known to mediate the inhibitory effect of angiostatin on tube formation. While these angiogenesis inhibitors were repressed, endothelial cell markers were induced (e.g. ESM1).
  • SMAD2 that mediates TGF3 signals was either induced (80% datasets) or repressed, highlighting the functional duality of the TGF3 pathway, acting as a tumor promoting or a tumor suppressing factor in cancer, including HCC 15 .
  • SFRP1 acting as a negative modulator of the Wnt/3-catenin pathway frequently activated in HCC microenvironment, was repressed.
  • PSMA 1, PSMA6, PSMD2, PSMD4) were similarly overexpressed, evocative of a proteotoxic stress associated with a protein hyper-production and/or mysfolding.
  • the second prominent promising HCC hallmark is the up-regulation of numerous genes acting at the epigenetic level (Table 2).
  • the 935-gene HCC signature included important regulators of chromatin assembly and remodeling (e.g. CHAF1A, HDAC1, HDAC5, HMGB2), components of the polycomb-repressive complex 2 (e.g. EZH2, SUZ12) that catalyzes the trimethylation of H3K27 (H3K27me3), and master regulators of microRNA processing (e.g. DROSHA).
  • the gene HCC signature highlights drug candidates for systemic therapies
  • HCC histone deacetylase
  • TCB histone deacetylase
  • LY294002 PI3K inhibitor
  • mTOR inhibitor sirolimus also known as rapamycin
  • alpha-estradiol alpha-estradiol
  • resveratrol Figure 2a

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Abstract

La présente invention décrit un ensemble de marqueurs du carcinome hépatocellulaire (HCC) et un procédé de détermination du fait qu'un sujet présente un risque de présenter ou d'avoir un HCC. Comme dans de nombreux cancers, l'hétérogénéité de la tumeur dans le HCC empêche le développement de thérapies personnalisées. En exécutant une méta-analyse d'ensembles de données sur le HCC humain qui ont été produits sur une période de plus de 10 ans, en commençant à partir des données brutes de puces à ADN et en utilisant les mêmes algorithmes d'analyse pour contourner les variabilités techniques, les inventeurs ont défini une signature transcriptionnelle universelle et complète concernant le HCC et ont déterminé si cette signature pourrait être utile pour identifier des candidats médicaments cliniquement pertinents. L'invention concerne en outre ainsi un procédé de détermination d'un régime thérapeutique convenant au traitement d'un sujet souffrant d'un HCC.
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