EP1957672A2 - Verfahren zum nachweis von krebs - Google Patents

Verfahren zum nachweis von krebs

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Publication number
EP1957672A2
EP1957672A2 EP06831300A EP06831300A EP1957672A2 EP 1957672 A2 EP1957672 A2 EP 1957672A2 EP 06831300 A EP06831300 A EP 06831300A EP 06831300 A EP06831300 A EP 06831300A EP 1957672 A2 EP1957672 A2 EP 1957672A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acids
mammal
risk
cancer
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06831300A
Other languages
English (en)
French (fr)
Inventor
Alexander Krause
Philippe Leissner
Malick Paye
Bruno Mougin
Fabien Schweighoffer
Laurent Bracco
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biomerieux SA
ExonHit Therapeutics SA
Original Assignee
Biomerieux SA
ExonHit Therapeutics SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from FR0511080A external-priority patent/FR2892730A1/fr
Priority claimed from FR0602824A external-priority patent/FR2899239A1/fr
Application filed by Biomerieux SA, ExonHit Therapeutics SA filed Critical Biomerieux SA
Publication of EP1957672A2 publication Critical patent/EP1957672A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present application relates to methods and compositions useful for the detection of cancer in mammals, particularly humans.
  • it describes serum markers of cancers and their uses in diagnostic methods.
  • It also relates to tools and / or kits that can be used for the implementation of these methods (reagents, probes, primers, antibodies, chips, cells, etc.), their preparation and their uses.
  • the invention is useful for detecting the presence or the evolution of a cancer in mammals, in particular breast cancer, including in the early phase.
  • breast cancer The diagnosis of breast cancer is usually done by mammography. However, it is estimated that the minimum size of a mammogram-detectable tumor is 1 cm, which has an evolutionary history of 8 years on average during diagnosis. Smaller tumors are much less malignant than can be extrapolated from their sizes: the aggressiveness of large tumors does not only come from their size, but also from their "inherent aggression", which increases with age. a tumor (Bucchi et al., Br J Cancer 2005, pp. 156-161; Norden T, Eur J Cancer 1997, pp. 624-628).
  • the present application provides a set of biological markers that can be used, alone or in combination, preferably in combination, to reliably detect, characterize or track the presence or evolution of a cancer in a mammal, preferably breast cancer.
  • the invention is particularly advantageous insofar as it can be implemented from whole blood without the need for tissue biopsy or separation steps.
  • the present application results from the identification of serum genetic markers characteristic of human patients with breast cancer.
  • serum genetic markers characteristic of human patients with breast cancer.
  • These markers correspond for example to variations in splicing or gene expression levels, and may allow, alone or, advantageously, in combination, to detect in patients the presence or stage of evolution of a cancer. They advantageously make it possible to detect the presence of a breast cancer, as early as the early phases thereof (namely stages I and II, that is to say, in particular at a stage where a mammogram would be ineffective), reliably and simply, from a whole blood sample. They can also detect early breast cancers that can not be detected by mammography because it is below its detection limit.
  • An object of the present application relates to a method for detecting (in vitro or ex vivo) the presence or risk of developing cancer in a mammal, including determining the presence (or absence or amount of (relative) ), in a biological sample of the mammal, one, preferably several target molecules selected from: a) nucleic acids comprising a sequence selected from SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15 preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, b) the nucleic acids having a sequence complementary to a sequence according to a), c) the functional analogues of nucleic acids according to a) or b), or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence (or absence or (relative) amount) of such target molecules in the sample being indicative of the presence or of the risk of developing cancer at this mammal.
  • the method comprises the combined determination of the presence or absence or (relative) amount of at least 5, 10, 15, 20, 30, 40, 50, 60, 70 or more of the molecules. targets as defined above.
  • the "combined" determination refers to the fact that a hybridization profile (or signature) involving multiple markers is determined.
  • the combined determination is typically performed simultaneously, i.e., by overall measurement of an expression profile. Nevertheless, the combined determination can also be performed by parallel or sequential measurements of several markers, leading to the identification of a profile.
  • the invention makes it possible to establish and determine a hybridization profile (or a signature) on a set of markers, in order to evaluate the presence or risk of developing cancer in a mammal.
  • the hybridization profile is typically performed using a combination of several markers selected by the targets indicated above, for example containing all of these targets.
  • the method of the invention comprises determining the presence (or absence or (relative) quantity) in a biological sample of the mammal, at least 5 different target molecules selected from those defined above, preferably at least 10.
  • the method of the invention comprises the combined determination of the presence (or absence or (relative) amount) of a particular mammalian biological sample of particular subsets of target molecules selected from those defined above.
  • Such subsets, described in the examples, are particularly suitable for the detection, particularly in the early phase, of the presence of breast cancer in patients from a whole blood sample.
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids of the invention.
  • the set of a panel of targets (or signatures) comprising markers as defined in elements a) to d) above, preferably all the molecules of one of the panels 1 to 11 defined in the this request.
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of the Panel 1, comprising the sequences shown in Table 4, column 1, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a complementary sequence thereof and and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 2 a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a complementary sequence thereof and and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the Sequences represented in SEQ ID NOs: 18, 19, 23, 26, 27, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 161, 188, 225, 228, 240, 280 and 312 (PANEL 3) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a complementary sequence thereof and / or analogs functional groups thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 312 PANEL 312
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 13, 16-19, 23, 26-28, 47, 51-55, 58, 69, 80, 81, 89, 116, 121, 125, 145, 148, 158, 160, 161 , 164, 189, 190, 225, 229, 240, 248, 280, 281, 284, 299, 300, 310 and 312 (PANEL 4) or a distinctive fragment thereof having at least 15, preferably at least 16 , 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 4 distinctive fragment thereof having at least 15, preferably at least 16 , 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary there
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 7, 13, 14, 16-19, 23-28, 47, 51-55, 58, 69, 80, 81, 89, 116, 121, 125, 137, 139, 145, 148 , 158, 160, 161, 164, 189, 190, 225, 228, 229, 240, 245, 248, 252, 280, 281, 284, 290, 298-300, 310 and 312 (PANEL 5) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 5 distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NOs: 5, 7, 13, 14, 16-20, 23-28, 47, 51-55, 58, 64, 69, 80, 81, 88-90, 116, 121, 125, 137 , 139, 145, 148, 158, 160, 161, 164, 188-191, 208, 222, 225, 228, 229, 236, 240, 242, 245, 248, 252, 280, 281, 284, 290, 298 -300 and 309-312 (PANEL 6) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or having a sequence complementary to those and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 6 or a distinctive fragment
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 7, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the examples provided in the present application indeed show that this panel of markers makes it possible to predictively detect the presence, the risk of developing or the stage of evolution of a cancer.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 8, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, all the nucleic acids of Panel 9, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19 , 20, 25 or 30 consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or absence or quantity (relative)), in a biological sample of the mammal, of all the nucleic acids of Panel 10, comprising the sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the method further comprises the detection of one or more of the other target molecules as defined above.
  • the method of the invention comprises the combined determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising the sequences represented in SEQ ID NO: 23, 52, 53, 148 and 225 (PANEL 11), or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or Consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • PANEL 11 the sequences represented in SEQ ID NO: 23, 52, 53, 148 and 225
  • PANEL 11 a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or Consecutive bases, or having a sequence complementary thereto and / or functional analogues thereof from other species, and / or polypeptides encoded by these nucleic acids.
  • the examples provided in this application show in effect that this panel of markers makes it possible to predictive
  • the method of the invention comprises the determination of the presence (or the absence or the (relative) amount), in a biological sample of the mammal, of the nucleic acids comprising respectively the sequences represented in SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, or nucleic acids having a complementary sequence of these.
  • Another particular object of the invention thus lies in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and a set of probes specific for the following target molecules: a) the nucleic acids comprising the sequences represented in one of the PANELS 1 to 11 as defined above, or a fragment that is distinctive from those with at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of developing a breast cancer in this mammal.
  • this analysis reveals numerous genes involved in the signaling cascades used in the transduction of messages initiated by the stimulation of TLRs, in the secretion of cytokines or in the activation of T lymphocytes.
  • the invention thus demonstrates that alterations in these signaling cascades occur in cancer patients, and that any gene or RNA involved in these cascades or any deregulation in these genes or RNA may constitute a marker of the presence or predisposition to cancer. Such alterations can occur during the oxidative stress imposed by tumor cells on monocytes, macrophages and dendritic cells.
  • a particular object of the invention relates to a method for detecting (in vitro or ex vivo) the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a sample.
  • mammalian biology preferably in a sample (derived) of blood, an alteration in a gene or RNA participating in a signaling pathway involved in the immune response (innate or acquired), the presence of such alteration being indicative the presence or risk of developing cancer in this mammal.
  • the signaling pathway involved in the immune response is advantageously selected from TLR stimulation, cytokine secretion, or T cell activation.
  • TLRs toll-like receptors
  • Multiple TLRs react to different ligands that can be carried by pathogens or tumor cells and induce the production of various pro-inflammatory cytokines by dendritic cells. This phenomenon is accompanied by a presentation of antigens to na ⁇ ve T cells, thus initiating a specific acquired immune response.
  • the receivers we can preferentially mention the receivers, the adapters, enzymes, factors involved in the regulation of gene expression, chemokines, cytokines and interleukins.
  • a particular object of the invention is a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample of the mammal , preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the regulation of the signaling cascade controlling the phenomenon of innate immunity. More particularly, this phenomenon mobilizes the cascade initiated by TLRs and regulates the activity of macrophage and dendritic cells.
  • Another particular object of the invention resides in a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample of the mammal. mammal, preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the regulation of the signaling cascade controlling the phenomenon of acquired or adaptive immunity. More specifically, this phenomenon involves T cell receptors (TCRs).
  • TCRs T cell receptors
  • Yet another particular object of the invention is a method for detecting in vitro or ex vivo the presence or risk of developing cancer in a mammal, comprising determining the presence (or absence) in a biological sample. of the mammal, preferably in a sample (derived) of blood, an alteration in a gene or RNA involved in the transition, the coordination between the innate and acquired immunities. More particularly, these genes are involved in the biosynthesis of lipid molecules from precursors such as arachidonic acid.
  • a particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a sample.
  • mammalian biology preferably in a blood (derived) sample, an alteration in a gene or RNA involved in the stimulation of TLRs, in the secretion of cytokines, or in the activation of T lymphocytes, said gene or RNA being advantageously selected from receptors, adapters, enzymes, factors involved in the regulation of gene expression, chemokines, cytokines and interleukins, the presence of such alteration being indicative of the presence or risk of developing cancer in this mammal.
  • alteration in a gene or RNA denotes for the purposes of the invention any alteration of the expression, namely in particular a deregulation in the splicing leading to the appearance of particular spliced forms, or to a modification of the quantity ( relative) or the relationship between the different forms of splicing.
  • RNA sequences whose expressions are affected by splice impairment can be deposited or synthesized on any solid support and hybridized with nucleic probes derived from control blood and blood from cancer patients for selection. the most discriminating oligonucleotides. More broadly, the oligonucleotides can be chosen as representative of any mRNA encoding any protein involved in the innate and acquired immunities. More particularly, these oligonucleotides can derive from the genes indicated in Table 6. The alterations can also be detected in terms of the structure or levels of expression of the polypeptides encoded by these genes or RNAs, for example by means of specific antibodies. as will be described in detail in the rest of the text.
  • a particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a sample.
  • mammalian biology preferably in a sample (derived) blood, an alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 corresponding genes or RNA indicated in Table 5, in particular an alteration of the splicing of such a gene or RNA, the presence of such an alteration being an indication of the presence or risk of developing cancer in this mammal.
  • Another particular object of the invention therefore lies in a method for detecting (in vitro or ex vivo) the presence or the risk of developing a cancer in a mammal, comprising determining the presence (or absence) in a mammal.
  • biological sample of the mammal preferably in a sample (derived) from blood, an alteration in at least one, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 genes or
  • RNA shown in Table 6 in particular an alteration of the splicing of such a gene or RNA, the presence of such alteration being an indication of the presence or risk of developing cancer in this mammal.
  • the present invention is based on the demonstration and characterization of serum biological events characteristic of the presence of breast cancer in a human patient.
  • These events constitute (bio) markers, the detection of which in a patient makes it possible, preferably in combination, to determine, even at an early stage, the risk of developing such a cancer or the presence of such a cancer.
  • markers and transcripts are used interchangeably, except when the context gives them a specific meaning.
  • the identified biological events typically correspond to changes in the regulation of gene expression. It may be a partial or total inhibition of the expression of genes or RNA, or certain forms of genes or RNA, an increase in the expression of genes or certain forms of genes or RNA, the appearance or disappearance of gene splicing forms, etc.
  • the invention is therefore based on the detection, in a sample, of one or more target molecules representative of the biological events thus identified.
  • these target molecules can be chosen from: a) nucleic acids comprising a sequence selected from SEQ ID NOs: 1-437 or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, b) the nucleic acids having a sequence complementary to a sequence according to a), c) the functional analogues of nucleic acids according to a) or b), or d) the polypeptides encoded by the nucleic acids according to a) to c).
  • the target molecule may be the full sequence of the gene or I 1 RNA or protein corresponding to the sequences SEQ ID NOs: 1-437, or a distinctive fragment thereof, that is to say, a fragment including the sequence is specific for said gene or RNA, or said protein, and / or comprises a domain of variability (splicing, deletion, polymorphism, etc.) representative of the biological event to be detected.
  • the complete list of markers and the corresponding genes are shown in Table 1.
  • the term "functional analog” refers to an analogue from another mammalian species. Indeed, the sequences SEQ ID NOs: 1-437 have been identified from human subjects, and these sequences constitute effective markers adapted to the detection of cancer in human patients. Nevertheless, for an application of the methods of the invention to other mammalian species, it is generally preferable to use functional analogues of these sequences, characterized in the species under consideration. These analogs can be identified by any technique known to those skilled in the art, in particular in view of the sequences provided in the application and the names of the corresponding genes.
  • the method comprises determining the presence of at least one nucleic acid according to a) to c).
  • the method is used to detect cancer in a human subject and includes determining the presence of at least one nucleic acid according to a) or b). More preferably still, the method comprises the combined detection of the presence (or absence) or the quantity (relative or absolute) of a panel of target markers, as defined in this application (Panels 1 to 11 or Tables 5 and 6).
  • a particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount (relative or absolute) in a sample mammalian blood, a set of molecules comprising at least the following target molecules: a) nucleic acids comprising the sequences of PANEL 1 or 11 shown in Table 4, or a distinctive fragment thereof having at least 15 preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) nucleic acids having a sequence complementary sequence according to a), and / or c) functional analogues of nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence of such target molecules in the sample being an indication of the presence or the risk of developing breast cancer in this mammal.
  • a particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount, in a mammalian blood sample, the following target molecules: a) nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228 , 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c ) the presence of such target molecules in the sample being an indication of the presence or risk of developing breast cancer in that
  • a particular embodiment of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising the combined determination of the presence or amount, in a mammalian blood sample, the following target molecules: a) the nucleic acids comprising the PANEL sequences shown in Table 4, or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or consecutive bases, and / or b) nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence of such target molecules in the sample being an indication of the presence or risk of developing breast cancer in this mammal.
  • Another specific object of the invention is a method for detecting the presence or risk of developing breast cancer in a mammal, comprising detecting, in a blood sample of the mammal, the following target molecules: a) the acids nucleic acids comprising the sequences shown in SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 (PANEL 2) or a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c) the functional analogues of the nucleic acids according to a) or b) from another species, and / or d) the polypeptides encoded by the nucleic acids according to a) to c), the presence, absence or (relative) amount of these target molecules in the sample being an indication of the presence or risk of developing breast
  • the invention also allows the definition of additional panels, comprising at least some markers as defined above, which may optionally be combined with other markers.
  • additional panels can be obtained by testing the presence or absence of these markers in patient samples, to define other predictive combinations, where appropriate specific pathologies.
  • RNA samples are useful in the present invention, such as, for example, Northern blotting, selective hybridization, the use of probed oligonucleotide-coated supports, the amplification of nucleic acid, nucleic acid such as, for example, by RT-PCR, quantitative PCR or ligation-PCR, etc.
  • a nucleic probe eg an oligonucleotide
  • a nucleic probe capable of selectively or specifically detecting the target nucleic acid in the sample.
  • the amplification may be carried out according to various methods known per se to those skilled in the art, such as PCR, CSF, transcription-mediated amplification (TMA), strand displacement amplification (SDA), NASBA, the use of allele-specific oligonucleotides (ASO), allele-specific amplification, Southern blotting, SSCA conformational analysis, in situ hybridization (eg, FISH), gel migration, heteroduplex analysis, etc.
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA the use of allele-specific oligonucleotides (ASO), allele-specific amplification, Southern blotting, SSCA conformational analysis, in situ hybridization (eg, FISH), gel migration, heteroduplex analysis, etc.
  • the method comprises the detection of the presence or absence or the (relative) amount of a nucleic acid according to a) to c) by selective hybridization or selective amplification.
  • Selective hybridization is typically performed using nucleic probes, preferably immobilized on a support, such as a solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic probes.
  • a support such as a solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic probes.
  • Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, etc.
  • the nucleic probes may be any nucleic acid (DNA, RNA, PNA, etc.), preferably single-stranded, comprising a sequence specific for a target molecule as defined in a) to c) above.
  • the probes typically comprise from 5 to 400 bases, preferably from 8 to 200, more preferably less than 100, and even more preferentially less than 75, 60, 50, 40 or even 30 bases.
  • the probes may be synthetic oligonucleotides, produced on the basis of the target molecule sequences of the invention according to conventional synthesis techniques. Such oligonucleotides typically have from 10 to 50 bases, preferably from 20 to 40, for example about 25 bases.
  • oligonucleotides are used to detect the same target molecule. They may be oligonucleotides specific for different regions of the same target molecule, or centered differently on the same region. It is also possible to use pairs of probes, one of which is perfectly matched to the target molecule, and another has a mismatch, thus making it possible to estimate the background noise. In the following examples, 6 to 11 pairs of 25 base oligonucleotides were used for each target molecule.
  • the probes may be synthesized beforehand and then deposited on the support, or synthesized directly in situ, on the support, according to methods known per se to those skilled in the art.
  • the probes can also be manufactured by genetic techniques, for example by amplification, recombination, ligation, etc.
  • the probes thus defined constitute another object of the present application, as well as their uses (essentially in vitro) for the detection of cancer in a subject.
  • a set of nucleic probes comprising all or a fragment of at least 15 consecutive bases, preferably 17, is used.
  • Hybridization can be carried out under standard conditions known to those skilled in the art and adjustable by it (Sambrook, Fritsch, Maniatis (1989) Molecular Cloning, CoId Spring Harbor Laboratory Press). In particular, the hybridization can be carried out under conditions of high, medium or low stringency, depending on the desired level of sensitivity, the quantity of available material, etc. For example, appropriate hybridization conditions include a temperature of 55 to 63 ° C for 2 to 18 hours on low density media. Other hybridization conditions may be required for high density media, such as a temperature. hybridization between 45 and 55 ° C.
  • washes can be performed to remove unhybridized molecules, typically in SSC buffers comprising SDS, such as a buffer comprising 0.1 to 10 X SSC and 0.5-0.01% SDS.
  • SSC buffers comprising SDS
  • Other wash buffers containing SSPE, MES, NaCl or EDTA may also be used.
  • the nucleic acids are prehybridized in hybridization buffer (Rapid Hybrid Buffer, Amersham) typically containing 100 ⁇ g / ml of salmon sperm DNA. 65 ° C for 30 min.
  • hybridization buffer Random Hybrid Buffer, Amersham
  • the nucleic acids of the sample are then contacted with the probes
  • the nucleic acids of the sample are marked beforehand with any known labeling (radioactive, enzymatic, fluorescent, luminescent, etc.).
  • the supports are then washed in 5 ⁇ SSC buffer, 0.1% SDS at 65 ° C. for 30 minutes, then in a 0.2 ⁇ SSC buffer, 0.1% SDS.
  • the hybridization profile is analyzed according to conventional techniques, for example by measuring the marking on the support by means of a suitable instrument (for example Instantlmager, Packard Instruments).
  • Hybridization conditions can naturally be adjusted by those skilled in the art, for example by modifying the hybridization temperature and / or the saline concentration of the buffer, as well as by adding auxiliary substances such as formamide or simple DNA. strand.
  • a particular object of the invention thus lies in a method for detecting the presence or the risk of developing breast cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and from a set of probes specific for the following target molecules: at least: a) nucleic acids comprising the sequences of one of the panels 1 to 11 defined above, or a fragment characterized by them having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of developing breast cancer in this mammal.
  • a particular object of the invention thus lies in a method for detecting the presence or risk of developing breast cancer in a mammal, comprising bringing into contact, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a mammalian blood sample and a set of probes specific for the following target molecules: a) the nucleic acids comprising the sequences represented in one of the PANELS 1, 2 or 11 as defined above, or a a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and or c) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or the risk of developing oppose breast cancer in this mammal.
  • the methods of the invention also use other target molecules and / or other probes, especially the subsets of target molecules mentioned in the present application.
  • another particular object of the invention resides in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising contacting, under conditions allowing hybridization between complementary sequences, nucleic acids derived from a sample of mammalian blood and a set of probes specific for at least two distinct molecules selected from the following targets: a) nucleic acids comprising the sequences shown in SEQ ID NOs: 1-437 or a distinctive fragment of these having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, and / or b) the nucleic acids having a sequence complementary to sequences according to a), and / or c ) the functional analogues of the nucleic acids according to a) or b) from another species, to obtain a hybridization profile, the hybridization profile being characteristic of the presence or risk of development to develop cancer in
  • the hybridization profile can be compared to one or more reference profiles, including a reference profile characteristic of healthy subjects and / or subjects with cancer, the comparison making it possible to determine the probability or the risk that the tested patient is reached. of a cancer.
  • the comparison is made using computer programs known per se to those skilled in the art.
  • the selective amplification is preferably performed using a primer or pair of primers for amplifying all or part of one of the target nucleic acids in the sample, when present therein.
  • the primer may be specific for a target sequence according to SEQ ID NO: 1-437, or a region flanking the target sequence in a nucleic acid of the sample.
  • the primer typically comprises a single-stranded nucleic acid, preferably between 5 and 50 bases long, preferably between 5 and 30.
  • Such a primer constitutes another object of the present application, as well as its use (essentially in in vitro) for the detection of cancer in a subject.
  • another object of the invention is the use of a nucleotide primer or a set of nucleotide primers for the amplification of all or part of one or, preferably, several genes or RNAs comprising a target sequence according to SEQ ID NO: 1-437, for the detection of a cancer in a mammal, preferably breast cancer, particularly in a human being. human.
  • Another particular object of the invention resides in a method for detecting the presence or the risk of developing a cancer in a mammal, comprising bringing into contact, under conditions allowing amplification, nucleic acids derived from a blood sample.
  • amplification profile being characteristic of the presence or risk of develop cancer in this mammal.
  • the method comprises determining the presence of a polypeptide according to d).
  • the detection of a polypeptide in a sample can be carried out by any technique known per se, such as in particular by means of a specific ligand, for example an antibody or an antibody fragment or derivative.
  • the ligand is an antibody specific for the polypeptide, or a fragment of such an antibody (for example an Fab, Fab ', CDR, etc.), or a derivative of such an antibody (for example a single antibody). chain, ScFv).
  • the ligand is typically immobilized on a support, such as a slide, ball, column, plate, etc.
  • the presence of the target polypeptide in the sample can be detected by demonstrating a complex between the target and the ligand, for example using a labeled ligand, using a second revealing ligand marked, etc.
  • Immunological techniques that can be used and are well known are ELISA, RIA, etc.
  • Antibodies specific for the target polypeptides may be produced by conventional techniques, including immunizing a non-human animal with an immunogen comprising the polypeptide (or an immunogenic fragment thereof), and recovering antibodies (polyclonal) or producing cells (to produce monoclonals). Techniques for producing poly- or monoclonal antibodies, ScFv fragments, human or humanized antibodies are described for example in Harlow et al., Antibodies: A laboratory Manual, CSH Press, 1988; Ward et al., Nature 341 (1989) 544; Bird et al., Science 242 (1988) 423; WO94 / 02602; US5,223,409; US5,877,293; WO93 / 01288.
  • the immunogen may be synthetically manufactured, or by expression, in a suitable host, of a target nucleic acid as defined above.
  • a suitable host of a target nucleic acid as defined above.
  • Such an antibody, monoclonal or polyclonal, as well as its derivatives having the same antigenic specificity, are also an object of the present application, as well as their use for detecting cancer.
  • the method of the invention is applicable to any biological sample of the mammal under test, in particular any sample comprising nucleic acids or polypeptides.
  • a sample of blood, plasma, platelet, saliva, urine, stool, etc. may be advantageously mentioned, more generally any tissue, organ or, advantageously, biological fluid comprising nucleic acids or polypeptides.
  • the sample is a blood or plasma sample.
  • the invention results indeed from the identification of blood markers of cancer, and thus allows detection of these pathologies without tissue biopsy, but only from blood samples.
  • the sample can be obtained by any technique known per se, for example by sampling, by non-invasive techniques, from collections or banks. samples, etc.
  • the sample may also be pre-treated to facilitate the accessibility of the target molecules, for example by lysis (mechanical, chemical, enzymatic, etc.), purification, centrifugation, separation, etc.
  • the PaxGene system is used (Feezor et al., Physiol Genomics 2004, pp. 247-254).
  • the sample can also be labeled, to facilitate the determination of the presence of the target molecules (fluorescent, radioactive, luminescent, chemical, enzymatic labeling, etc.).
  • the biological sample is a whole blood sample, that is to say having not undergone a separation step, which may optionally be diluted.
  • the invention is applicable to any mammal, preferably humans.
  • the method of the invention is particularly useful for the detection of breast cancer, including the presence, risk of development or stage of development of breast cancer in humans.
  • the data provided in the examples show that the invention makes it possible to detect the presence of breast cancer with a sensitivity greater than 92% and a specificity greater than 86%.
  • It is particularly suitable for screening for breast cancer at early stages, that is, at stages I or II (as defined in the classification of malignant tumors TNM ("Classification of Malignant Tumors") developed and maintained by UICC ("International Union against Cancer").
  • TNM classification is also used by the AJCC (American Joint Committee on Cancer) and FIGO (International Federation of Gynecology and Obstetrics).
  • a particular object of the present application relates to a method for detecting the presence, evolution or risk of developing breast cancer in a human subject, comprising the combined determination of the presence (or absence or quantity thereof ( relative)), in a biological sample of the human subject, target molecules selected from: a) nucleic acids comprising a sequence selected from SEQ ID NO: 1-
  • the method comprises the combined determination of the presence, absence or quantity of 5, 10, 20, 30, 40, 50 or 60 target molecules as defined above.
  • Another particular object of the present application relates to a method for detecting the presence, evolution or risk of developing breast cancer in a human subject, comprising contacting a biological sample of the subject containing nucleic acids with a product comprising a support on which are immobilized nucleic acids comprising a sequence complementary to and / or specific to one or, preferably, several target molecules chosen from (i) nucleic acids comprising a sequence chosen from SEQ ID NO: 1- 437 or a fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases and (ii) the nucleic acids having a sequence complementary to a sequence according to ), and determining the hybridization profile, the profile indicating the presence, the stage or the risk of developing breast cancer in said human subject.
  • the product comprises distinct nucleic acids comprising a complementary and / or specific sequence of at least 5, 10, 20, 30, 40, 50, 60 or more different target molecules as mentioned above.
  • nucleic acids comprising a complementary and / or specific sequence of one or, preferably, several target molecules selected from (i) nucleic acids comprising a sequence selected from SEQ ID NO: 1-437 or a fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases and (ii) the nucleic acids having a sequence complementary to a sequence according to (i).
  • the product comprises distinct nucleic acids comprising a complementary and / or specific sequence of at least 5, 10, 20, 30, 40, 50, 60 or more different target molecules as mentioned above.
  • it comprises distinct nucleic acids comprising a sequence complementary and / or specific to one of the panels of markers as defined in the present application.
  • Another object of the present application relates to a product comprising a support on which is immobilized at least one, preferably several, nucleic acids comprising a sequence chosen from SEQ ID NO: 1-437, or a functional analogue thereof.
  • the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more different nucleic acids selected from the nucleic acids mentioned above.
  • the product each comprises nucleic acids of sequence SEQ ID NO: 1-376.
  • a product comprising a support on which at least one ligand of a polypeptide encoded by a target nucleic acid as defined above, that is to say a nucleic acid comprising a sequence selected from SEQ ID NO: 1-437, a distinctive fragment thereof having at least 15, preferably at least 16, 17, 18, 19, 20, 25 or 30 consecutive bases, a nucleic acid having a complementary sequence thereof or a functional analogue thereof.
  • the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more different polypeptide ligands selected from the polypeptides mentioned above.
  • the support can be any solid or semi-solid support having at least one surface, flat or not, allowing the immobilization of nucleic acids or polypeptides.
  • Such supports are for example a blade, ball, membrane, filter, column, plate, etc. They can be made of any compatible material, such as glass, silica, plastic, fiber, metal, polymer, polystyrene, teflon, etc.
  • the reagents can be immobilized on the surface of the support by known techniques, or, in the case of nucleic acids, synthesized directly in situ on the support. Immobilization techniques include passive adsorption (Inouye et al., J. Clin Microbiol 28 (1990) 1469), the covalent bond.
  • the product of the invention comprises a plurality of synthetic oligonucleotides, of a length of between 5 and 100 bases, specific for one or more target nucleic acids defined in a) to c).
  • the products of the invention typically comprise control molecules for calibrating and / or normalizing the results.
  • kits comprising a compartment or container comprising at least one, preferably several, nucleic acids comprising a complementary and / or specific sequence of a target nucleic acid as defined above and / or a , preferably several ligands of a target polypeptide as defined above.
  • the product comprises at least 5, 10, 20, 30, 40, 50, 60 or more nucleic acids and / or different ligands selected from the nucleic acids and ligands mentioned above.
  • the product each comprises nucleic acids of sequence SEQ ID NO: 1-437 or a ligand for each of the target polypeptides as defined above.
  • the kit may furthermore comprise reagents for a hybridization or immunological reaction, as well as, if appropriate, controls and / or instructions.
  • Another subject of the invention relates to the use of a product or kit as defined above for the detection of a cancer in a mammalian subject, preferably a human subject, in particular breast cancer.
  • Another subject of the invention relates to a nucleic acid with a sequence chosen from SEQ ID NO: 1-437, or a distinctive fragment thereof comprising at least 15 consecutive bases, preferably at least 16, 17, 18, 19, 20, 25 or 30, or a nucleic acid having a sequence complementary thereto, or a functional analogue thereof.
  • the invention also relates to a cloning vector or expression comprising these nucleic acids, as well as any recombinant cell comprising such a vector or nucleic acid.
  • Another subject of the invention relates to the use of a nucleic acid comprising a sequence chosen from SEQ ID NO: 1-437, or a distinctive fragment thereof comprising at least 15 consecutive bases, preferably at least 16, 17, 18, 19, 20, 25 or 30, or a nucleic acid having a complementary sequence thereof, or a functional analogue thereof, for the detection (essentially in vitro) of cancer in a mammalian subject .
  • a blood sample is taken from a mammal to be tested.
  • the blood sample is optionally processed to make the nucleic acids more accessible, and these are labeled.
  • the nucleic acids are then applied to a product as defined above and the hybridization profile is determined, to diagnose the presence or absence of cancer in the subject.
  • the method of the invention is simple, practiced ex vivo, and allows the early detection of cancer from a blood sample.
  • Figure 1 PCR amplification of DATAS cDNAs derived from Profiling # 1 (PBMN) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage I & II cancers versus controls and controls against stage I & II cancers). A positive control using cDNA from HepG2 cells is included.
  • Figure 2 PCR amplification of DATAS cDNAs derived from Profiling # 2 (PBMO) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage III & IV cancers versus controls and controls against stage III & IV cancers). A positive control using cDNA from HepG2 cells is included
  • Figure 3 PCR amplification of DATAS cDNAs derived from Profiling # 3 (PBMP) using 10 pairs of semi-degenerate primers. DATAS profiling was performed in both directions (stage I to IV cancers versus controls and controls against stage I to rV cancers). A positive control using cDNA from HepG2 cells is included.
  • Figure 4 Specific configuration of probes to measure the expression of variants generated by alternative splicing.
  • Probe A common to both isoforms, measures the expression of both variants.
  • Probe B specific for the additional sequence of the long form, as well as probes C and D, specific for the junction sequences around this additional sequence measure the expression of the long isoform.
  • the E probe specific for the junction resulting from the absence of the additional sequence, measures the expression of the short isoform.
  • Figure 5 Definition of the "target” sequences. 15 nucleotides on either side of each junction and up to 500 nucleotides for the additional and common upstream sequences are captured.
  • Figure 6 Sequences "targets and associated data. Description of the columns: idn °: identification number of the splicing event; Description: type of splicing event; long form: accession number of the long form; short form: accession number of the short form; target A to E: Sequences of targets A to E; long. : size of the sequences of the previous column.
  • Figure 7 Hierarchical clustering on 37 controls and 55 patients using 100 oligonucleotides.
  • RNA samples 5 ml of whole blood taken from two PaxGene tubes. These samples included 37 blood samples from healthy control patients (S, obtained from the French Blood Establishment) and 55 samples from patients with stage I / II breast cancer (CI / II). 2. Extraction of total RNA from the blood sample
  • RNA samples were collected directly into PAXGene -TM Blood RNA tubes (PreAnalytix, Hombrechtikon, CH). After the blood sample collection step and in order to obtain a total lysis of the cells, the tubes were left at ambient temperature for 4 h and then stored at -20 ° C. until the biological material was extracted. Specifically, in this protocol, total RNAs were extracted using PAXGene Blood RNA® kits (PreAnalytix) according to the manufacturer's recommendations. Briefly, the tubes were centrifuged (15 min, 3000 g) to obtain a nucleic acid pellet. This pellet was washed and taken up in a buffer containing proteinase K necessary for protein digestion (10 min at 55 ° C.).
  • RNAse free DNAse set Qiagen, Hilden, Germany.
  • the quality of the total RNAs was analyzed by the AGILENT 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany).
  • DATAS is a technology for profiling gene expression between two samples that allows characterizing qualitative differences in messenger RNAs, such as those generated by alternative splicing. This proprietary technology is described in US Patent 6,251,590.
  • RNAs corresponding to the two situations, one normal (mN) and the other pathological (mP) are isolated from the blood samples using the PAXgene system (PreAnalytix) described above. These RNAs (50 ⁇ g per group) are converted to complementary DNAs (cN) and (cP) using reverse transcriptase (RT) (Invitrogen) and a biotinylated oligodT25 oligonucleotide (Invitrogen). Samples contributing to 50 ⁇ g per group are shown in Tables A to F.
  • Hybrids mN / cP and cN / mP are then made in the liquid phase. After ethanol precipitation of mN and cP and cN and mP, the precipitates are taken up in 30 ⁇ l of hybridization solution containing 80% formamide and 0.1% SDS for incubation at 40 ° C overnight. The heteroduplexes are then captured using magnetic beads Streptavidin (Dynal). A magnet keeps the balls in the tube during rinsing operations. The beads / heteroduplexes are then taken up in 50 .mu.l of RNAseH buffer and incubated with RNaseH (Invitrogen) for 30 minutes at 37.degree. The supernatant is recovered after a new application of the magnet.
  • RNaseH Invitrogen
  • RNAsel action is removed by DNAsel action (Ambion). After inactivation of the enzyme, the fragments of RNAs are precipitated with ethanol and taken up in DEPC treated water supplemented with RNAse out (Ambion). The RNA fragments are then reverse transcribed (TaqMan Reverse transcription kit, Applied Biosystem) using random hexamer oligonucleotides. The complementary DNAs obtained are then amplified by PCR using semi-degenerate primers. 10 pairs of primers are generally used. The amplicons obtained can be visualized by agarose gel electrophoresis (FIGS. 1, 2 and 3). There are heterogeneous amplified populations of nature and intensity that vary according to the primers and the samples used.
  • amplified populations are cloned into a TOPO TA cloning vector (Invitrogen) to be transformed into a strain of competent E. coli bacteria (Invitrogen).
  • the colonies are subcultured into 96-well plates and grown overnight in 2XTY supplemented with ampicillin. A glycerol stock (50%) is then made.
  • 1728 clones were sequenced in the framework of DATAS profiling No. 1, 1920 were cloned and sequenced in the framework of DATAS profiling n ° 2 and 1920 were cloned and sequenced in the framework of DATAS profiling n ° 3.
  • Table G summarizes the number of clones characterized in the three profiling banks.
  • a so-called “singleton” clone means that the sequence of this clone has been identified only once in the bank and that no other clone covers this sequence.
  • a “cluster” is a group of clones whose sequences overlap. The three DATAS profilings generated 1741 non-redundant clones.
  • the 1741 DATAS clones could be associated with 1170 different genes, some non-overlapping DATAS fragments being associated with different regions of the same gene.
  • microarray splice variants require the use of a particular probe configuration.
  • Any reference messenger RNA / splice variant pair can be modeled as long isoform / short isoform ( Figure 4).
  • a splice variant associated with an exon skip will be the short isoform relative to the reference variant.
  • a splice variant with a new exon or intron retention will be the long isoform relative to the reference variant.
  • Other alternative splicing events (uses of 5 'or 3' splicing sites) can also be modeled in the same way.
  • the set of probes necessary for measuring the expression of splice variants is also indicated in FIG. 4.
  • This set consists of two traditional "exon" A and B probes and three exon-exon junction probes. or exon-intron C, D and E.
  • Probe A measures the expression of both isoforms
  • probes B, C and D express the long isoform
  • probe E expresses the short form.
  • the target sequences corresponding to the junction probes C, D and E are defined by a length of 30 nucleotides, 15 nucleotides on either side of the junction. It is thus possible to "cover" any junction by probes of 25 nucleotides of the type: 10/15, 11/14, 12/13, 13/12, 14/11 and 15/10 (the sign / representing the zone of junction).
  • the constraints are less strong on the "exonic" probes A and B.
  • the additional sequence, specific for the long form (sequence 2 of FIG.
  • sequence 1 of FIG. 4 defines the target sequences for these probes with however a ceiling of 500 nucleotides for sequences that may exceed this size.
  • the definition of the target sequences is summarized in Figure 5.
  • the 1170 genes corresponding to the 1741 DATAS clones were used to identify, for each of them, the cDNAs and ESTs represented in the public sequence data banks, potentially having qualitative differences. of sequences, source of splicing events. The events retained are located within 100 nucleotides with respect to the 5 'or 3' ends of the DATAS fragments.
  • This information includes, for a given event: an identification number of the event, the nature of the event, the access numbers of the long and short forms, the target sequences A to E and the size of these target sequences.
  • each event was characterized by its five A-E target sequences.
  • a and B 11 pairs of 25 nucleotide probes were designed, whereas the C, D, or E target sequences were detected with 6 pairs of 25 nucleotide probes.
  • pair of probes is meant a first probe that hybridises perfectly (this is called PM probes or perfect match) with one of the cDNAs from a target transcript, and a second probe, identical to the first probe at the same time. except for a mismatch (this is called a MM or mismatched probe) in the center of the probe.
  • PM probes or perfect match a first probe that hybridises perfectly
  • second probe identical to the first probe at the same time. except for a mismatch (this is called a MM or mismatched probe) in the center of the probe.
  • MM probe was used to estimate the background noise corresponding to a hybridization between two nucleotide fragments of non-complementary sequence.
  • the complementary DNAs (cDNAs) of the mRNAs contained in the total RNAs as purified above were obtained from 400 ng of total RNAs by using of a Klenow 3'-5'-exonuclease enzyme, 100 units of SuperScript II reverse transcription enzyme (Invitrogen), 10 units of RNAse H Superase-IN inhibitor (Ambion, Huntigdon, UK) and 200 pmol of "random" primer containing the T7 promoter (RP-T7-primer, Eurogenetec, Seraing, Belgium).
  • Reverse transcription was performed with 800 units of SuperScriptII (Invitrogen) and 10 units of an RNAse H inhibitor, in the presence of Klenow enzyme, for 1 h at 37 ° C.
  • the double-stranded cDNA resulting from this approach was then purified using the QIAquick PCR Purification Kit (Qiagen) and quantified by spectrophotometry.
  • the fragmented cDNAs were then labeled with biotin using 330 units of terminal transferase (Roche Molecular Biochemicals, Meylan, France) and 1 ⁇ l of DNA Labeling Reagent (DLR-Ia, 5mM) [Affymetrix] per microgram cDNA for 60 min at 37 ° C.
  • the entire fragmented and labeled cDNA was finally hybridized on the custom DNA chip (labeled "A520138F", cf Example 6) according to a standard hybridization protocol adapted to 11 ⁇ m chips.
  • RNA variants representing about 800 genes
  • hybridization buffer Affymetrix
  • oligonucleotides oligo B2
  • the biotinylated and hybrid cDNAs on the chip were revealed by the use of streptavidin-phycoerythrin solution and the signal was amplified by the use of anti-streptavidin antibodies.
  • Hybridization was performed in a GeneChip Hybridization oven (Affymetrix), and the Euk GE-WS2 protocol of Affymetrix was followed. The washing and revelation steps were carried out on a "Fluidics Station 450" station (Affymetrix). Each chip was then analyzed on an Affymetrix G3000 GeneArray Scanner scanner at a resolution of 1.5 microns to identify hybridized areas on the chip.
  • This scanner allows the detection of the signal emitted by the fluorescent molecules after excitation by an argon laser using the epifluorescence microscope technique. For each position, a signal is obtained which is proportional to the quantity of cDNAs fixed. The signal was then analyzed by GeneChip Operating Software (GCOS 1.2, Affymetrix). In order to prevent the variations obtained by the use of different chips, a standardization approach using the "Bioconductor” tool has been carried out, which makes it possible to harmonize the average distribution of the raw data obtained for each chip. The results obtained on one chip can then be compared to the results obtained on another chip.
  • the GCOS 1.2 software also included the inclusion of a statistical algorithm to consider whether a transcript was expressed or not.
  • the inventors have also studied the simultaneous expression of 100 nucleotide sequence transcripts chosen from the sequences presented in Table 2 to obtain an expression profile.
  • 89% of patients were correctly classified. More specifically, 31 of the 37 controls and 51 of 55 patients were correctly classified, which corresponds to a sensitivity of 92.7% and a specificity of 83.7%.
  • hierarchical clustering In this unsupervised analysis, a Control is positioned among the patients (to the left of the red dotted line, see Figure 7), and 10 patients are among the healthy controls (to the right of the red dotted line).
  • Figure 7 shows the hierarchical clustering analysis of blood samples obtained from 55 patients with early stage cancer (CI / II, also called D) and 37 patients (healthy donors) using the expression of 100 genes identified by algorithmic analysis.
  • the hierarchical clustering function of the Spotfire software organizes the CI / II patients and controls in columns, and genes in rows so as to obtain adjacent patients or genes with comparable expression profiles.
  • the Pearson correlation coefficient was used as a similarity index for genes and patients.
  • the results correspond to the Affymetrix fluorescence level normalized by the "bioconductor" tool.
  • the expression levels of each gene were normalized by calculating a reduced centered variable.
  • White represents low levels of expression, gray intermediate levels and black high levels.
  • the height of the branches of the dendrogram indicates the index of similarity between the expression profiles.
  • the inventors have demonstrated a combination of 66 markers, based on the sequences SEQ ID Nos: 5, 7, 13, 14, 16-20, 23-28, 47, 51-55, 58, 64, 69, 80, 81, 88-90, 116, 121, 125, 137, 139, 145, 148, 158, 160, 161, 164, 188-191, 208, 222, 225, 228, 229, 236, 240, 242, 245, 248, 252, 280, 281, 284, 290, 298-300 and 309-312 (see Table 3). This combination correctly classifies more than 80% of the samples.
  • the inventors have also demonstrated a combination of 53 markers, based on the sequences SEQ ID Nos: 7, 13, 14, 16-19, 23-28, 47, 51-55, 58, 69, 80, 81, 89 , 116, 121, 125, 137, 139, 145, 148, 158, 160, 161, 164, 189, 190, 225, 228, 229, 240, 245, 248, 252, 280, 281, 284, 290, 298-300, 310 and 312.
  • This combination also makes it possible to correctly classify more than 80% of the samples.
  • the inventors have also demonstrated a combination of 42 markers, based on the sequences SEQ ID Nos: 13, 16-19, 23, 26-28, 47, 51-55, 58, 69, 80, 81, 89, 116 , 121, 125, 145, 148, 158, 160, 161, 164, 189, 190, 225, 229, 240, 248, 280, 281, 284, 299, 300, 310 and 312.
  • This combination also makes it possible to classify correctly more than 80% of the samples.
  • the inventors have demonstrated a combination of 22 markers, based on the sequences SEQ ID Nos: 18, 19, 23, 26, 27, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 161, 188, 225, 228, 240, 280 and 312. This combination correctly classifies 76% of the samples.
  • the inventors have also demonstrated a combination of 18 markers, based on the target sequences SEQ ID NOs: 18, 19, 23, 26, 51, 52, 53, 54, 55, 69, 80, 125, 145, 148, 225, 228, 240 and 312 shown in Table 3. This combination correctly classifies 76% of the samples.
  • Panel 1 includes all the sequences common to Panels 7-9.
  • Panel 11 comprises all the sequences common to all Panels 1 to 10, i.e. the nucleic acids comprising the sequences shown in SEQ ID NO: 23, 52, 53, 148 and 225 (see Table 4).
  • Table C Selected Samples for the Late Breast Cancer Group for DATAS # 2 (PBMO)
  • Table D Samples selected for the control group for DATAS # 2 (PBMO)
  • Table E Samples selected for the all-stage breast cancer group for DATAS # 3 (PBMP).
  • Table 1- List of 437 transcripts expressed differentially during the development of breast cancer.
  • Table 5 List of genes / transcripts identified from DATAS libraries derived from profiling the blood of breast cancer patients as being associated with immunity signaling pathways.
  • IFIT1 interferon-induced protein with tetratricopeptide repeats 1
  • NM 001008540 (CXCR4), variant 1 transcript, mRNA. 11/2005
  • LSP1 lymphocyte-specific protein 1
  • nuclear receptor subfamily 3 group C, member 1 (glucocorticoid receptor) (NR3C1),
  • ANXA11 Homo sapiens annexin A11 (ANXA11), transcript
  • NM 001175 beta (ARHGDIB), mRNA. 10/2005
  • IL8RB interleukin 8 receptor beta
  • NM 001629 activating protein (ALOX5AP), mRNA. 11/2005
  • CD97 antigen CD97
  • FTH 1 Homo sapiens ferritin, heavy polypeptide 1 (FTH 1),
  • MAP2K3 mitogen-activated protein kinase kinase 3
  • variant A transcript variant A transcript
  • NM 002985 (CCL5), mRNA. 11/2005
  • SP2 transcription factor SP2 transcription factor
  • tumor necrosis receptor factor superfamily member 14 (herpesvirus entry mediator)
  • NM 003820 (TNFRSF14), mRNA. 11/2005
  • NM 003853 protein (IL18RAP), mRNA. 10/2005
  • NM 004120 inducible GFP2
  • mRNA 9/2005 Homo sapiens colony stimulating factor receptor 3
  • CSF3R granulocyte
  • variant 4 transcript mRNA
  • NM 203346 vigilin (vigilin) (HDLBP), mRNA. 10/2005

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EP06831300A 2005-10-28 2006-10-26 Verfahren zum nachweis von krebs Withdrawn EP1957672A2 (de)

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FR0511080A FR2892730A1 (fr) 2005-10-28 2005-10-28 Methode pour detecter la presence ou le risque de developper un cancer
FR0602824A FR2899239A1 (fr) 2006-03-31 2006-03-31 Procede de detection du cancer
PCT/FR2006/051108 WO2007048978A2 (fr) 2005-10-28 2006-10-26 Procede de detection du cancer

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JP2009513125A (ja) 2009-04-02
WO2007048978A2 (fr) 2007-05-03
WO2007048978A3 (fr) 2007-09-07

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