WO2010142751A1 - Méthode de diagnostic/pronostic in vitro et kit d'évaluation du rejet chronique médié par anticorps dans la transplantation rénale - Google Patents

Méthode de diagnostic/pronostic in vitro et kit d'évaluation du rejet chronique médié par anticorps dans la transplantation rénale Download PDF

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WO2010142751A1
WO2010142751A1 PCT/EP2010/058126 EP2010058126W WO2010142751A1 WO 2010142751 A1 WO2010142751 A1 WO 2010142751A1 EP 2010058126 W EP2010058126 W EP 2010058126W WO 2010142751 A1 WO2010142751 A1 WO 2010142751A1
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expression profile
rejection
genes
graft
kidney
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WO2010142751A8 (fr
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Marina Guillet
Alessandra Cervino
Joanna Ashton-Chess
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Tc Land Expression
Institut National De La Sante Et De La Recherche Medicale (Inserm)
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention refers to the field of human medicine, and specifically to the diagnosis of the rejection state in kidney transplant recipients. More precisely, the present invention concerns a method for the in vitro diagnosis of a kidney graft chronic antibody mediated rejection (then referred as ABMR) or non-rejection phenotype, comprising: (a) determining from a kidney grafted subject biological sample an expression profile comprising the 45 genes of Table 1 or subgroups of this list of genes as described in Tables 2, 3 and 4, (b) comparing the obtained expression profile with at least one reference expression profile, and (c) determining the graft rejection or graft non-rejection phenotype from said comparison.
  • the present invention also relates to kits and nucleic acid microarrays for performing said method.
  • the present invention also relates to methods of treatment of kidney transplant recipients.
  • the diagnosis of "Suspicious for chronic AMBR” is given in the case of morphologic evidence of tissue injury where C4d is positive but donor specific antibodies (DSA) are absent, or if DSA are present but C4d is negative.
  • DSA donor specific antibodies
  • the diagnosis of chronic AMBR requires both an invasive biopsy and a non- invasive blood test.
  • the biopsy serves to identify the presence of specific lesions and complement deposit by specific histology and immunohistology techniques.
  • the blood test serves to detect the presence of circulating donor-specific antibodies using high-definition in vitro methods. As the incidence of chronic AMBR appears to increase throughout the post transplant course, the majority of cases are detected upon biopsy for graft deterioration i.e. biopsy for cause.
  • kidney AMBR kidney graft rejection or non-rejection phenotype
  • the invention also relates to a method for designing a kidney transplant recipient immunosuppressive treatment, said method comprising:
  • the invention also relates to a method for adapting the immunosuppressive treatment of a kidney transplant recipient, said method comprising: (a) Determining from a biological sample of said kidney transplant recipient an expression profile comprising or consisting of the 45 genes of following Table 1, or of the subsets of 29, 15 or 14 genes following Tables 2, 3 or 4, or equivalents thereof,
  • the invention is also drawn to a method for treating of a kidney transplant recipient, comprising:
  • step (b) adapting the immunosuppressive treatment in function of the result of step (a).
  • Said adaptation of the immunosuppressive treatment may consist in: a modification of said immunosuppressive treatment if the subject has been diagnosed as graft rejection, or a maintenance of said immunosuppressive treatment if the subject has been diagnosed as graft non rejection.
  • the present invention presents a major interest. It permits to diagnose or prognose (i.e. to identify), among patients under immunosuppressive treatment, those who are in the process of rejecting their graft and who could thus benefit from an adapted immunosuppressive treatment dedicated to stop or at least slow down the rejection process. Due to the non reversible aspects of the damages caused by chronic rejection and the current late diagnosis of such phenotype, this achievement is really crucial and would allow a better management of the patients.
  • a "kidney transplanted subject” or a “kidney transplant recipient” is a subject that was grafted with a non syngeneic, including allogenic or even xenogenic, kidney. Said kidney transplanted subject may further have been grafted with another organ of the same donor providing the kidney.
  • a "graft rejection phenotype” is defined as a state of chronic antibody mediated rejection of a subject to his graft.
  • a “state of rejection” means that this subject (referred to as a “graft rejecting subject”) is rejecting his graft according to the Banff classification.
  • Such phenotype can correspond either to a strict chronic antibody mediated rejection definition or a "suspicious for ABMR" definition.
  • a "graft non-rejection phenotype” refers to the absence in said subject of a state of rejection, meaning that said subject (referred to as a "graft non- rejecting subject”) would, at the time of the diagnosis, not reject his/her graft.
  • the population of graft rejecting subjects only includes subjects in a state of rejection to their graft
  • the population of graft non-rejecting subjects thus includes all other subjects and is composed of a variety of different states such as patients with signs of toxicity of Calcineurin inhibitors, patients with signs of interstitial fibrosis and/or tubular atrophy without C4d deposit nor anti-HLA antibodies but also stable patients with or without anti-HLA antibodies, who cannot at this time be considered as rejecting but who may later develop a graft rejection phenotype.
  • the mechanisms of rejection are complex and still not elucidated, and the cellular and molecular processes of rejection induction may require a prolonged laps of time.
  • the population of graft rejecting subjects only includes subjects who have already reached a state of ABMR or suspicion of ABMR to their graft
  • the population of graft non-rejecting subjects is heterogeneous and includes all other subjects, i.e. both subjects with renal failure for other causes than ABMR and subjects with stable graft function.
  • a “biological sample” may be any sample that may be taken from a grafted subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a biopsy.
  • Preferred biological samples for the determination of an expression profile include a blood sample, a lymph sample, or a biopsy.
  • the biological sample is a blood sample, more preferably a peripheral blood sample comprising peripheral blood mononuclear cells (PBMC) or whole blood.
  • PBMC peripheral blood mononuclear cells
  • expression profile is meant the expression levels of a group of genes comprising the 45 genes of Table 1 or a subgroup thereof.
  • the expression profile consists of the 45 genes of Table 1, of the 29 genes of Table 2, of the 15 genes of Table 3 or of the 14 genes of Table 4 or Equivalent Expression Profiles thereof, since these expression profiles have been demonstrated to be particularly relevant for assessing kidney graft rejection/non-rejection.
  • Equivalent Expression Profile is meant expression profiles of tables 1, 2, 3 or 4 wherein the addition, deletion or substitution of some of the genes does not change significantly the reliability of the test and is considered as an "acceptable expression profile".
  • the addition of other genes already described as significant in chronic AMBR diagnostic, such as TRIB 1 (WO 2007/138011) or PSMBlO (EP08300084) should be considered as an equivalent.
  • the addition or substitution of some of the genes of the sets described in the present invention by other genes belonging to the same metabolic pathway should also be considered as an equivalent expression profile.
  • Acceptable Expression Profile an expression profile which is able to correctly classify at least 60% of the analyzed samples, preferably 65%, and more preferably 70%, has a sensitivity of at least 60% preferably 65%, and more preferably 70% and has a negative predictive value of at least 70%, preferably 80%, and more preferably 90%.
  • the sensitivity value is defined as the ratio of the number of patients actually clinically rejecting their graft due to chronic ABMR and classified as rejecting using the test according to the invention amongst all patients analysed displaying clinical signs of ABMR.
  • the negative predictive value is defined as the ratio of the number of patients clinically defined as non rejecting ones and classified as non- rejecting using the test according to the invention amongst the total number of patients classified as non-rejecting using the test according to the invention.
  • Best Expression Profile an expression profile which is able to correctly classify at least 70% of the analyzed samples, has a sensitivity of at least 70%, and has a negative predictive value of at least 90%.
  • Figure 1 or 2 respectively, or by substitution of one or more genes of the genes sets of Table 3 or 4 by one or more genes depicted in Figure 1 or 2 respectively.
  • Figures 1 and 2 or by substitution of one or more genes of the genes sets of Table 1 or 2 by one or more genes of the same metabolic pathway as depicted in Figures 1 and 2.
  • the determination of the presence of a graft rejection or graft non-rejection phenotype is carried out thanks to the comparison of the obtained expression profile with at least one reference expression profile in step (b).
  • a “reference expression profile” is a predetermined expression profile, obtained from a biological sample from a subject with a known particular graft state.
  • the reference expression profile used for comparison with the test sample in step (b) may have been obtained from a biological sample from a graft rejecting subject ("rejecting reference expression profile"), and/or from a biological sample from a graft non-rejecting subject ("non-rejecting reference expression profile").
  • At least one reference expression profile is a rejecting reference expression profile.
  • at least one reference expression profile may be a non- rejecting reference expression profile.
  • the determination of the presence or absence of a graft rejection phenotype is carried out by comparison with at least one rejecting and at least one non-rejecting reference expression profiles.
  • the diagnosis (or prognostic) may thus be performed using one rejecting reference expression profile and one non-rejecting reference expression profile.
  • said diagnosis is carried out using several rejecting reference expression profiles and several non-rejecting reference expression profiles.
  • the comparison of a tested subject expression profile with said reference expression profiles can be done using statistical models or machine learning methods which aim is to predict a clinical response (eg: 0 if Not-rejecting, 1 if rejecting) based on a combination of the explanatory variables (the genes).
  • Statistical models such as logistic regression and fisher linear discriminant analysis are particularly relevant to predict outcome.
  • Other discriminating algorithms include kNN (k nearest neighbour), decision trees, SVM (support vector machine), NN (neural networks) and forest.
  • the PLS regression, MIPP, sparse linear discrimination and PAM predictive analysis of microarrays are particularly relevant to give prediction in the case of pangenomic analyses with small reference samples.
  • the expression profile may be determined by any technology known by a man skilled in the art.
  • each gene expression level may be measured at the genomic and/or nucleic and/or proteic level.
  • the expression profile is determined by measuring the amount of nucleic acid transcripts of each gene.
  • the expression profile is determined by measuring the amount of each gene corresponding protein.
  • the amount of nucleic acid transcripts can be measured by any technology known by a man skilled in the art.
  • the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well- know in the art.
  • mRNA messenger RNA
  • cDNA retrotranscribed complementary DNA
  • the amount of nucleic acid transcripts may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative PCR, and hybridization with a labelled probe.
  • the expression profile is determined using quantitative PCR.
  • Quantitative, or real-time, PCR is a well known and easily available technology for those skilled in the art and does not need a precise description.
  • the determination of the expression profile using quantitative PCR may be performed as follows. Briefly, the real-time PCR reactions are carried out using the TaqMan Universal PCR Master Mix (Applied Biosystems). 6 ⁇ l cDNA is added to a 9 ⁇ l PCR mixture containing 7.5 ⁇ l TaqMan Universal PCR Master Mix, 0.75 ⁇ l of a 2OX mixture of probe and primers and 0.75 ⁇ l water. The reaction consisted of one initiating step of 2 min at 50 deg.
  • the reaction and data acquisition can be performed using the ABI PRISM 7900 Sequence Detection System (Applied Biosystems).
  • the number of template transcript molecules in a sample is determined by recording the amplification cycle in the exponential phase (cycle threshold or C T ), at which time the fluorescence signal can be detected above background fluorescence.
  • cycle threshold or C T cycle threshold
  • the expression profile is determined by the use of a nucleic microarray.
  • a nucleic microarray consists of different nucleic acid probes that are attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead.
  • a microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose.
  • Probes can be nucleic acids such as cDNAs ("cDNA microarray”) or oligonucleotides (“oligonucleotide microarray”), and the oligonucleotides may be about 25 to about 60 base pairs or less in length.
  • a target nucleic sample is labelled, contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The presence of labelled hybridized complexes is then detected.
  • Many variants of the microarray hybridization technology are available to the man skilled in the art.
  • the nucleic acid microarray is an oligonucleotide microarray comprising or consisting of 45 oligonucleotides specific for the 45 genes of Table 1, or comprising or consisting of 29 oligonucleotides specific for the 29 genes of Table 2, or comprising or consisting of 15 oligonucleotides specific for the 15 genes of Table 3, or comprising or consisting of 14 oligonucleotides specific for the 14 genes of Table 4.
  • the oligonucleotides are about 50 bases in length. It is acknowledged that the nucleic acid microarray, or oligonucleotide microarray of the invention encompass the microarrays specific for an Equivalent Expression Profile as defined above.
  • Suitable oligonucleotides specific for any gene of Table 1, 2, 3 or 4 may be designed, based on the genomic sequence of each gene (see Genbank accession numbers), using any method of microarray oligonucleotide design known in the art.
  • any available software developed for the design of microarray oligonucleotides may be used, such as, for instance, the OligoArray software (available at bSS ⁇ llh ⁇ IJy .
  • the expression profile is determined by the use of proteic microarrays.
  • antibodies, aptamers, or aff ⁇ bodies microarrays are mainly used, most of the time antibodies microarrays (Hall et al, 2007).
  • the antibodies, aptamers, or aff ⁇ bodies are attached to various supports using various attachment methods, using a contact or non-contact spotter (Hall et al, 2007).
  • suitable supports include glass and silicon microscope slides, nitrocellulose, microwells (for instance made of a silicon elastomer) (Hall et al, 2007).
  • a coating is generally added. Examples of coatings for random attachment (i.e.
  • resulting in a random orientation of attached proteins to the support include aldehyde- and epoxy-derivatized coatings for random attachment through amines, and nitrocellulose, gel pads or poly-L-lysine coatings (Hall et al, 2007).
  • coatings for non random attachment include nickel coating fro use with His6-tag proteins, and streptavidin coating for use with biotinylated proteins (Hall et al, 2007).
  • two main technologies are used: 1) direct labelling, single capture assays and 2) dual-antibody sandwich immunoassays (Kingsmore, 2006, see notably Figure 1).
  • proteins contains in one or more samples are labelled with distinct labels (generally fluorescent or radioisotope labels), hybridized to the microarray, and labelled hybridized proteins are directly detected (Kingsmore, 2006, see notably Figure Ia).
  • label generally fluorescent or radioisotope labels
  • dual-antibody sandwich immunoassays the sample is hybridized to the microarray, and a secondary tagged antibody is added.
  • a third labelled (generally fluorescent or radioisotope label) antibody specific for the tag of the secondary antibody is then used for detection (Kingsmore, 2006, see notably Figure Ib). Further details concerning antibodies microarrays may be found in Haab, 2005 and Eckel-Passow et al, 2005.
  • said method may further comprise determining from a non invasive biological sample of the subject at least one additional parameter useful for the diagnosis.
  • additional parameter useful for the diagnosis are parameters that cannot be used alone for a diagnosis but that have been described as displaying significantly different values between grafted subjects in chronic antibody mediated rejection and non-rejecting subjects and may thus also be used to refine and/or confirm the diagnosis according to the above described method according to the invention. They may notably be selected from: standard biological and histological parameters specific for said subject grafted organ type, phenotypic analyses of peripheral blood cells, and qualitative and/or quantitative analysis of peripheral blood cells immune repertoire.
  • standard biological parameters specific for said subject grafted organ type means biological parameters that are usually used by clinicians to monitor the stability of grafted subjects status and to detect chronic antibody mediated rejection.
  • These standard biological parameters specific for said subject grafted organ usually comprise serum or plasma concentrations of particular proteins as well as the presence of anti-HLA antibodies, which vary depending on the grafted organ type.
  • these standard biological parameters specific for said subject grafted organ type are, for each organ type, well known of those skilled in the art.
  • standard biological parameters specific for kidney include serum or plasma urea and creatinine concentrations.
  • the serum creatinine concentration is usually comprised between 40 to 80 ⁇ mol/L for a woman and 60 to 100 ⁇ mol/L for a man, and the serum urea concentration between 4 to 7 mmol/L.
  • a serum creatinine concentration superior to 80 ⁇ mol/L for a woman and to 100 ⁇ mol/L for a man, or a serum urea concentration superior to 7 mmol/L may confirm a chronic rejection status.
  • the phenotypic analyses of peripheral blood mononuclear cells may comprise various types of phenotypic analysis.
  • they may comprise: - measuring the percentage of CD4 + CD25 + T cells in peripheral blood lymphocytes, which may be performed by any technology known in the art, in particular by flow cytometry using labelled antibodies specific for the CD4 and CD25 molecules.
  • the percentage of CD4 + CD25 + T cells in peripheral blood lymphocytes of a subject undergoing chronic rejection is significantly lower (p ⁇ 0.05) from that of a healthy volunteer.
  • determining the cytokine expression profile of T cells which may be performed using any technology known in the art, including quantitative PCR and flow cytometry analysis.
  • the oligoclonal V ⁇ families of a grafted subject undergoing chronic rejection express increased levels compared to a healthy volunteer of THl or TH2 effector molecules, including interleukin 2 (IL-2), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 13 (IL- 13), transforming growth factor beta (TGF- ⁇ ), interferon gamma (IFN- ⁇ ) and perform, whereas oligoclonal V ⁇ families of a grafted subject with stable graft function do not express increased levels of those effector molecules compared to a healthy volunteer.
  • IL-2 interleukin 2
  • IL-8 interleukin 8
  • IL-10 interleukin 10
  • IL- 13 interleukin 13
  • TGF- ⁇ transforming growth factor beta
  • IFN- ⁇ interferon gamma
  • the analysis of peripheral blood cells immune repertoire consists advantageously in the qualitative and quantitative analysis of the T cell repertoire (2), such as the T cell repertoire oligoclonality and the level of TCR transcripts or genes.
  • the T cell repertoire oligoclonality may be determined by any technology enabling to quantify the alteration of a subject T cell repertoire diversity compared to a control repertoire.
  • said alteration of a subject T cell repertoire diversity compared to a control repertoire is determined by quantifying the alteration of T cell receptors (TCR) complementary determining region 3 (CDR3) size distributions.
  • TCR T cell receptors
  • CDR3 complementary determining region 3
  • TCR CDR3 size distribution displays a Gaussian form, which may be altered in the presence of clonal expansions due to immune response, or when the T cell repertoire diversity is limited and reaches oligoclonality.
  • the level of TCR expression at the genomic, transcriptomic or proteic level is preferably determined independently for each V ⁇ family by any technology known in the art.
  • the level of TCR transcripts of a particular V ⁇ family may be determined by calculating the ratio between these V ⁇ transcripts and the transcripts of a control housekeeping gene, such as the HPRT gene.
  • a significant percentage of V ⁇ families display an increase in their transcript numbers compared to patients with stable kidney graft function or to healthy individuals.
  • Such additional parameters may be used to confirm the diagnosis obtained using the expression profile comprising or consisting of the 45 genes from Table 1 or subsets of these 45 genes such as those described in Tables 2, 3 and 4, or Equivalent Expression Profiles.
  • the invention further concerns a kit for the in vitro diagnosis of a graft rejection or graft non-rejection phenotype, comprising at least one reagent for the determination of an expression profile comprising, or consisting of, the 45 genes from Table 1 or subsets of these 45 genes such as those described in Tables 2, 3 and 4, or Equivalent Expression Profiles thereof.
  • a reagent for the determination of an expression profile is meant a reagent which specifically allows for the determination of said expression profile, i.e. a reagent specifically intended for the specific determination of the expression level of the genes comprised in the expression profile, either on the transcription (RNA) of the translation (proteic) levels.
  • kits for the in vitro diagnosis of a graft rejection or graft non-rejection phenotype may further comprise instructions for determination of the presence or absence of a graft rejection phenotype.
  • kit for the in vitro diagnosis of a graft rejection phenotype may also further comprise at least one reagent for the determining of at least one additional parameter useful for the diagnosis such as standard biological parameters specific for said subject grafted organ type (notably the presence of anti-HLA antibodies), phenotypic analyses of peripheral blood cells, and quantitative and/or qualitative analysis of peripheral blood cells immune repertoire (such as the T cell repertoire oligoclonality and the level of TCR transcripts).
  • the reagent(s) for the determination of an expression profile comprising, or consisting of, the 45 genes from Table 1 or subsets of these 45 genes such as those described in Tables 2, 3 and 4 or Equivalent Expression Profiles thereof, preferably include specific amplification primers and/or probes for the specific quantitative amplification of transcripts of genes of Table 1, and/or a nucleic microarray for the detection of genes of Table 1.
  • the determination of the expression profile may thus be performed using quantitative PCR and/or a nucleic microarray, preferably an oligonucleotide microarray.
  • the instructions for the determination of the presence or absence of a graft rejection phenotype preferably include at least one reference expression profile, or at least one reference sample for obtaining a reference expression profile.
  • at least one reference expression profile is a graft rejection expression profile.
  • at least one reference expression profile may be a graft non- rejection expression profile.
  • the determination of the level of graft rejection is carried out by comparison with both graft rejection and graft non-rejection expression profiles as described above.
  • the invention is also directed to a nucleic acid microarray comprising or consisting of nucleic acids specific for the 45 genes from Table 1, or subsets of these 45 genes such as those described in Tables 2, 3 and 4 or Equivalent Expression Profiles thereof.
  • Said nucleic acid microarray may comprise additional nucleic acids specific for genes other than the 45 genes from Table 1, but preferably consists of a maximum of 500, 400, 300, 200 preferably 100, 90, 80, 70 more preferably 60, 50, or even 45 distinct nucleic acids, 45 of which are specific for the 45 genes of Table 1.
  • the nucleic acid microarray may include even less distinct nucleic acids.
  • said microarray consists of nucleic acids specific for the 45 genes of Table 1 or subsets of these 45 genes such as those described in Tables 2, 3 and 4.
  • said nucleic acid microarray is an oligonucleotide microarray comprising or consisting of oligonucleotides specific for the 45 genes from Table 1 or subsets of these 45 genes such as those described in Tables 2, 3 and 4, or for Equivalent Expression Profiles thereof.
  • Figure 1 Scheme of a first network of interacting genes.
  • Figure 2. Scheme of a second network of interacting genes.
  • Shaded shapes represent the molecules that are part of the 45 list.
  • the types of line represent the type of interaction: - plain arrows: means the first molecule directly acts on the second one (such as but not limited to activation) dotted arrows: means the first molecule indirectly acts on the second one A line ending with a short segment indicates inhibition A simple line represents interaction (such a but not limited to protein-protein interaction)
  • Peripheral blood samples from 19 patients with ABMR and 124 relevant controls were identified and analyzed by microarrays (HG-Ul 33 Plus2) on an Affymetrix platform.
  • the group of patients with ABMR was composed both of a group at lyr post transplant in patients with no renal dysfunction (protocol biopsies), and another group at a median of 5 years post transplant with renal failure (biopsies for cause).
  • pangenomic nature of the microarrays made it possible to probe the samples for the expression of virtually all genes of the genome without any a priori as to which genes may be of interest.
  • the next step was to transfer the gene signature from the microarray platform to a quantitative PCR platform.
  • the primers and probes corresponding to the 135 genes previously identified were first validated in terms of efficacy and specificity, leading to the removal of 17% of the genes.
  • 60 came from the statistical analysis of the gene expression data obtained by microarray (22 from an optimal multigene discrimination ⁇ Predictive Analysis of Microarray (PAM) package -like), 38 individually expressed genes) and 52 came from the Intelligent Data Mining step.
  • the 112 gene candidates were analyzed by qPCR in the same samples used for the microarray analysis.
  • the group of 45 genes according to the invention was able to correctly classify 77% of the samples analyzed. This yielded sensitivity and negative predictive value for rejection of 79% and 96%, respectively. The AUC is 83%.
  • EXAMPLE 3 Identification of subsets of genes able to classify the patients with a good accuracy through an analysis of the pathways
  • IPKB Ingg ⁇ uity_Pa11iw ⁇ y ⁇ jCnowledge ⁇ ase
  • IPA Ingenuity Pathway Analysis
  • the group of 14 genes according to the invention was able to correctly classify respectively 76,8% of the samples analyzed. This yielded sensitivity and negative predictive value for rejection of 68% and 94%, respectively.

Abstract

La présente invention concerne le domaine de la médecine humaine, et spécifiquement le diagnostic de l'état de rejet chez les receveurs d'une transplantation rénale. Plus précisément, la présente invention concerne une méthode permettant le diagnostic in vitro d'un phénotype de rejet ou de non-rejet de greffon rénal consistant à : (a) déterminer à partir d'un échantillon biologique d'un patient greffé d'un rein un profil d'expression comprenant les 45 gènes du tableau 1 ou les sous-groupes de cette liste de gènes décrits dans les tableaux 2, 3 et 4, (b) comparer le profil d'expression obtenu avec au moins un profil d'expression de référence, et (c) déterminer le phénotype de rejet de greffe ou de non-rejet de greffe à partir de ladite comparaison. La présente invention concerne également des kits et des micropuces d'acide nucléique pour mettre en œuvre ladite méthode. La présente invention concerne également des méthodes de traitement du receveur d'une transplantation rénale.
PCT/EP2010/058126 2009-06-10 2010-06-10 Méthode de diagnostic/pronostic in vitro et kit d'évaluation du rejet chronique médié par anticorps dans la transplantation rénale WO2010142751A1 (fr)

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