WO2010136576A1 - Méthode de pronostic/diagnostic in vitro et trousse destinée à évaluer la tolérance dans une transplantation hépatique - Google Patents

Méthode de pronostic/diagnostic in vitro et trousse destinée à évaluer la tolérance dans une transplantation hépatique Download PDF

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WO2010136576A1
WO2010136576A1 PCT/EP2010/057440 EP2010057440W WO2010136576A1 WO 2010136576 A1 WO2010136576 A1 WO 2010136576A1 EP 2010057440 W EP2010057440 W EP 2010057440W WO 2010136576 A1 WO2010136576 A1 WO 2010136576A1
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tolerant
expression profile
graft
liver
genes
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PCT/EP2010/057440
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Alberto Sanchez Fueyo
Juan José LOZANO SALVATELLA
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Tc Land Expression
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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

  • the present invention refers to the field of human medicine, and specifically to the diagnosis of the tolerant state in liver transplant recipients. More precisely, the present invention concerns a method for the in vitro diagnosis of a liver graft tolerant or non- tolerant phenotype, comprising: (a) determining from a liver grafted subject biological sample an expression profile comprising the 25 genes of Table 1, (b) comparing the obtained expression profile with at least one reference expression profile, and (c) determining the graft tolerant or graft non-tolerant 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 a method of treatment of liver transplant recipients.
  • PBMCs peripheral blood mononuclear cells
  • immunosuppressive drugs to prevent graft rejection. These drugs are very effective at preventing graft rejection, but they are also associated with severe side effects, such as nephrotoxicity, an augmented risk of opportunistic infections and tumors, and metabolic complications such as diabetes, hyperlipidemia and arterial hypertension. Due to the side effects of immunosuppressive drugs, the induction of tolerance, defined as a state in which the graft maintains a normal function in the absence of chronic immunosuppression, is one of the main goals of research in transplant immunology. Tolerance induction is possible in a great number of experimental models of transplant in rodents.
  • immunosuppressive drugs While the chronic use of immunosuppressive drugs is currently the only means to ensure long-term survival of transplanted allografts, these drugs are expensive and are associated with severe side effects (nephrotoxicity, tumor and infection development, diabetes, cardiovascular complications, etc.) that lead to substantial morbidity and mortality. Hence, any strategy capable of significantly reducing the use of immunosuppressive drags in transplantation may have a large impact on the health and quality of life of transplant recipients.
  • liver transplanted patients to their liver graft could be diagnosed or prognosed based on the simple analysis of the expression level in blood cells of a set of 25 genes and comparison with the expression level in blood cells of the same 25 genes in reference blood samples of tolerant and non- tolerant patients.
  • the invention thus relates to a method for the in vitro diagnosis of a graft tolerant or non-tolerant phenotype, comprising:
  • the invention also relates to a method for designing a liver transplant recipient immunosuppressive treatment, said method comprising: (a) Determining from a grafted subject biological sample an expression profile comprising or consisting of the 25 genes of following Table 1, (b) Comparing the obtained expression profile with at least one reference expression profile,
  • the invention also relates to a method for weaning the immunosuppressive treatment of a liver transplant recipient, said method comprising: (a) Determining from a grafted subject biological sample an expression profile comprising or consisting of the 25 genes of following Table 1,
  • the invention is also drawn to a method of treatment of a liver grafted subject, comprising: (a) determining from a subject biological sample the presence of a graft tolerant or graft non-tolerant phenotype using a method according to the invention, and (b) adapting the immunosuppressive treatment in function of the result of step (a).
  • Said adaptation of the immunosuppressive treatment may consist in: - a reduction or suppression of said immunosuppressive treatment if the subject has been diagnosed as graft tolerant, or a modification of said immunosuppressive treatment if the subject has been diagnosed as graft non tolerant.
  • a "graft tolerant phenotype" is defined as a state of tolerance of a subject to his graft.
  • a “state of tolerance” means that this subject (referred to as a "graft tolerant subject”) does not reject his graft m the absence of an immunosuppressive treatment with a well functioning graft
  • a "graft non- tolerant phenotype” refers to the absence in said subject of a state of tolerance, meaning that said subject (referred to as a "graft non-tolerant subject”) would, at the time of the diagnosis, reject its graft if the immunosuppressive treatment was withdrawn
  • the population of graft tolerant subjects only includes subjects m a state of tolerance to their graft
  • the population of graft non-tolerant subjects thus includes all other subjects and is composed of a variety of different states' patients already suffering from obvious chronic rejection, patients at the early non symptomatic stage of chronic rejection, but also stable patients, who cannot at this time be considered as tolerant but who may later develop a graft tolerant phenotype.
  • the mechanisms of tolerance are complex and still not elucidated, and the cellular and molecular processes of tolerance induction may require a prolonged laps of time.
  • the population of graft tolerant subjects only includes subjects who have already reached a stable state of tolerance to their graft
  • the population of graft non-tolerant subjects is heterogeneous and includes all other subjects, i.e. both subjects in the process of developing chrome rejection and subjects in the process of developing tolerance.
  • the present invention possesses two major interests
  • 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. Such a sample must allow for the determination of an expression profile comprising or consisting of the 25 genes of Table 1.
  • Preferred biological samples for the determination of an expression profile include samples such as 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).
  • PBMC peripheral blood mononuclear cells
  • the biological sample may be a purified V51TCR+ T cells population obtained from a blood sample.
  • V52TCR+ T cells largely predominate in peripheral blood (>80%), while V61TCR+ T cells are the major subtype in tissues such as intestine, liver and spleen.
  • peripheral blood V81TCR+ T cells expand and typically outnumber V ⁇ 2TCR+ T cells.
  • V61TCR+ T cells are the only ⁇ TCR+ T cell subset clearly influencing tolerance-related transcriptional signatures, and that t peripheral blood V ⁇ lTCR+ T cells from tolerant liver recipients exhibit unique expression and cell surface traits that distinguish them from those present on either non-tolerant recipients or non-transplanted healthy individuals. V61TCR+ T cells have been reported to exert immunoregulatory functions in a variety of non- transplantation experimental and clinical settings.
  • expression profile is meant a group of at least 25 values corresponding to the expression levels of the 25 genes of Table 1, with optionally further other values corresponding to the expression levels of other genes.
  • the expression profile consists of a maximum of 500, 400, 300, 200, preferably 100, 90, 80, 75, more preferably 70, 65, 60, even more preferably 55, 50, 45, 40, 35, 30, 29, 28, 27, 26 or even 25 genes, 25 of which are the genes of Table 1.
  • the expression profile consists of the 25 genes of Table 1, since this expression profile has been demonstrated to be particularly relevant for assessing liver graft tolerance/non- tolerance.
  • the list of 25 genes of Table 1 has been determined as the best expression profile to assess graft tolerance/non-tolerance, the omission of a restricted number of genes from Table 1 , for example the omission of 1 or 2 genes from the list of 25 genes of Table 1, still permits to assess graft tolerance, although with less reliability.
  • liver graft tolerant subjects as defined above (ToI) and liver graft non- tolerant subjects are listed in the following Table 1.
  • 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 tolerant subject ("tolerant reference expression profile"), and/or from a biological sample from a graft non-tolerant subject ("non-tolerant reference expression profile").
  • At least one reference expression profile is a tolerant reference expression profile.
  • at least one reference expression profile may be a non- tolerant reference expression profile.
  • the determination of the presence or absence of a graft tolerant phenotype is carried out by comparison with at least one tolerant and at least one non-tolerant reference expression profiles.
  • the diagnosis (or prognostic) may thus be performed using one tolerant reference expression profile and one non-tolerant reference expression profile.
  • said diagnosis is carried out using several tolerant reference expression profiles and several non-tolerant reference expression profiles.
  • the comparison of a tested subject expression profile with said reference expression profiles can be done using the PLS regression (Partial Least Square) which aim is to extract components, which are linear combinations of the explanatory variables (the genes), in order to model the variable response (eg: 0 if Not-TOL, 1 if TOL).
  • the PLS regression is particularly relevant to give prediction in the case of small reference samples.
  • the comparison may also be performed using PAM (predictive analysis of microarrays) statistical method.
  • a non supervised PAM 3 classes statistical analysis is thus performed. Briefly, tolerant reference expression profiles, non-tolerant (preferably chronic rejection) reference expression profiles, and the expression profile of the tested subject are subjected to a clustering analysis using non supervised PAM 3 classes statistical analysis.
  • a cross validation (CV) probability may be calculated (CV to i), which represents the probability that the tested subject is tolerant.
  • another cross validation probability may be calculated (CV no n-toi), which represents the probability that the tested subject is non-tolerant. The diagnosis is then performed based on the CV tO ⁇ and/or CV non-to i probabilities.
  • a subject is diagnosed as a tolerant subject if the CV to i probability is of at least 0.5, at least 0.6, at least 0.7, at least 0.75, at least 0.80, at least 0.85, more preferably at least 0.90, at least 0.95, at least 0.97, at least 0.98, at least 0.99, or even 1.00, and the CV non - to i probability is of at most 0.5, at most 0.4, at most 0.3, at most 0.25, at most 0.20, at most 0.15, at most 0.10, at most 0.05, at most 0.03, at most 0.02, at most 0.01, or even 0.00. Otherwise, said subject is diagnosed as a graft non-tolerant subject.
  • 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. From the mRNA or cDNA sample, 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. C, followed by 10 min at 95 deg. C, and 40 cycles of amplification including 15 sec at 95 deg. C and 1 min at 60 deg. C.
  • 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 starting number of template transcript molecules is inversely related to C T -
  • 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.
  • cDNA microarray cDNA microarray
  • oligonucleotide microarray oligonucleotides
  • the oligonucleotides may be about 25 to about 60 base pairs or less in length.
  • the nucleic microarray is an oligonucleotide microarray comprising, or consisting of 25 oligonucleotides specific for the 25 genes of Table 1.
  • the oligonucleotides are about 50 bases in length.
  • Suitable microarray oligonucleotides specific for any gene of Table 1 may be designed, based on the genomic sequence of each gene (see Table 1 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 http://berry.engin.umich.edu/oliRoarray/), the GoArrays software (available at http://www.isima.fr/bioinfo/goaiTays/), the Array Designer software (available at http://www.premierbiosoft.com/dnamicroarrav/index.html), the Primer3 software (available at http://frodo.wi.mit.edu/primer3/primer3 code.html), or the Promide software (available at http://oligos.molgen.mpg.de/).
  • the OligoArray software available at http://berry.engin.umich.edu/oliRoarray/
  • the GoArrays software available at http://www.isima.fr/bioinfo/goaiTays/
  • the Array Designer software available at http://www.premierbiosoft.com
  • the expression profile is determined by the use of proteic microarrays.
  • antibodies, aptamers, or affibodies microarrays are mainly used, most of the time antibodies microarrays (Hall et al, 2007).
  • the antibodies, aptamers, or affibodies 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 for 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 contained 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 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 tolerant grafted subjects and subjects in chronic rejection 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 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 graft rejection.
  • GTT gamma glutamyl transpeptidase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • LDH lactate dehydrogenase
  • bilirubin total or conjugated
  • 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.
  • the phenotypic analyses of peripheral blood cells may comprise various types of phenotypic analysis. In particular 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 Preferably, the percentage of CD4 + CD25 + T cells in peripheral blood lymphocytes of a tolerant subject is not statistically different from that of a healthy volunteer, whereas it is significantly lower (p ⁇ 0.05) in a non-tolerant grafted subject.
  • - 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 non-tolerant grafted subject 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-IO), interleukin 13 (IL- 13), transforming growth factor beta (TGF- ⁇ ), interferon gamma (IFN- ⁇ ) and perforin, whereas oligoclonal V ⁇ families of a tolerant grafted subject do not express increased levels of those effector molecules compared to a healthy volunteer.
  • IL-2 interleukin 2
  • IL-8 interleukin 8
  • IL-IO interleukin 10
  • IL- 13 interleukin 13
  • TGF- ⁇ transforming growth factor beta
  • IFN- ⁇ interferon gamma
  • the proportion of peripheral blood V51TCR+ T cells and V ⁇ l/V ⁇ 2 T cell ratio in peripheral blood cells of tolerant subjects is higher than the proportion of peripheral blood V51TCR+ T cells and V ⁇ l/V ⁇ 2 T cell ratio in peripheral blood cells of non-tolerant subjects.
  • the tolerant subjects exhibit an increased number of CD4+CD25+Foxp3+, ⁇ TCR+ and 51TCR+ T cells compared with the non-tolerant subjects.
  • 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
  • the level of TCR expression at the genomic, transcriptional or protein 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 a normal healthy subject.
  • a graft tolerant subject displays a T cell repertoire with a significantly higher oligoclonality than a normal healthy subject.
  • Such additional parameters may be used to confirm the diagnosis obtained using the expression profile comprising or consisting of the 25 genes from Table 1.
  • said subject is a liver transplanted subject.
  • a "liver transplanted subject” is a subject that was grafted with a non syngeneic, including allogenic or even xenogenic, liver. Said liver transplanted subject may further have been grafted with another organ of the same donor providing the liver.
  • the invention further concerns a kit for the in vitro diagnosis of a graft tolerant or graft non-tolerant phenotype, comprising at least one reagent for the determination of an expression profile comprising, or consisting of, the 25 genes from Table 1.
  • 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 This definition excludes generic reagents useful for the determination of the expression level of any gene, such as taq polymerase or an amplification buffer, although such reagents may also be included in a kit according to the invention.
  • kit for the in vitro diagnosis of a graft tolerant or graft non-tolerant phenotype may further comprise instructions for determination of the presence or absence of a graft tolerant phenotype.
  • kit for the in vitro diagnosis of a graft tolerant 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, phenotypic analyses of peripheral blood cells (notably the percentage of CD4 + CD25 + T cells in peripheral blood lymphocytes, the percentage and relative composition of ⁇ T cells, and the cytokine expression profile of T 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 25 genes from Table 1, 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 tolerant 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 tolerant expression profile.
  • at least one reference expression profile may be a graft non- tolerant expression profile. More preferably, the determination of the level of graft tolerance is carried out by comparison with both graft tolerant and graft non-tolerant expression profiles as described above.
  • the invention is also directed to a nucleic acid microarray comprising or consisting of nucleic acids specific for the 25 genes from Table 1.
  • Said nucleic acid microarray may comprise additional nucleic acids specific for genes other than the 25 genes from Table 1, but preferably consists of a maximum of 500, 400, 300, 200 preferably 100, 90, 80, 70 more preferably 60, 50, 40, even more preferably 30, even 25 distinct nucleic acids, 25 of which are specific for the 25 genes of Table 1.
  • said microarray consists of the 25 genes of Table 1.
  • said nucleic acid microarray is an oligonucleotide microarray comprising or consisting of oligonucleotides specific for the 25 genes from Table 1.
  • FIG. 1 Scheme of the prospective immunosuppression weaning study.
  • Peripheral blood samples from stable liver recipients under maintenance immunosuppression are collected before (timepoint 1) these drugs are gradually discontinued during a period of about 6 months.
  • Patients are followed-up for 12 months after complete discontinuation of all immunosuppressive drugs (timepoint 2), at which time a second blood sample is taken.
  • timepoint 2 those who have not rejected their graft are considered as tolerant
  • Blood samples at timepoint 1 are used to obtain an expression profile of the 25 genes signature according to the invention using Affymetrix microarray and PAM analysis.
  • the inventors conducted a prospective immunosuppression weaning study in which peripheral blood samples from stable liver recipients under maintenance immunosuppression are collected before these drugs are gradually discontinued during a period of about 6 months. Patients are followed-up for 12 months after complete discontinuation of all immunosuppressive drugs, and those who maintain a stable liver graft function during this period of time are considered as tolerant (see Figure 1).
  • Pre-weaning peripheral blood samples were taken from each patient and employed to conduct gene expression profiling analysis using Affymetrix Human Genome U133 Plus 2.0 microarrays (see Figure 1).
  • the nearest shrunken centroid classifier implemented in the Predictive Analysis of Microarray (PAM) package was used with the expression levels of the 25 genes of Table 1 to classify patients as either tolerant or non-tolerant on the basis of the pre- weaning peripheral blood sample.
  • the group of 25 genes according to the invention was able to correctly classify
  • the 25genes signature according to the invention may be useful for diagnosing or prognosing tolerance in liver transplant recipients. Indeed, while not all tolerant patients are predicted using this signature, the test is very specific, so that there is only a very low risk that a patient predicted as tolerant actually rejects its graft after immunosuppression weaning.
  • the inventors next investigated the stability over time of the expression measurements of this group of 25 genes by comparing microarray experiments conducted before weaning was started and 18 months afterwards, in a group of 11 Non- tolerant and 9 Tolerant recipients. Note that while all patients were receiving immunosuppressive drugs at timepoint 1, at timepoint 2 only non-tolerant recipients were on drugs. The differences in expression of the 25 genes taken together between timepoint 1 and timepoint2 were then, computed. Results show that for most patients, there were only minor changes in expression.
  • Table 2 shows that the correlation between timepoint 1 and timepoint2 is significant for almost all of the 25 genes analyzed (see Table 2).

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Abstract

La présente invention concerne le domaine de la médecine humaine, et plus précisément le diagnostic de l'état de tolérance chez des patients ayant reçu une transplantation hépatique. Plus précisément encore, la présente invention concerne une méthode de diagnostic in vitro d'un phénotype de tolérance ou d'intolérance au greffon hépatique, consistant à : (a) déterminer à partir d'un échantillon biologique d'un sujet ayant reçu un greffon hépatique un profil d'expression comprenant les 25 gènes du tableau 1, (b) comparer le profil d'expression obtenu à au moins un profil d'expression de référence, et (c) déterminer le phénotype de tolérance ou d'intolérance au greffon d'après ladite comparaison. La présente invention concerne également des trousses et des puces à ADN permettant de mettre en œuvre ladite méthode. La présente invention concerne également une méthode de traitement d'un patient ayant reçu une transplantation hépatique.
PCT/EP2010/057440 2009-05-29 2010-05-28 Méthode de pronostic/diagnostic in vitro et trousse destinée à évaluer la tolérance dans une transplantation hépatique WO2010136576A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2016075232A1 (fr) * 2014-11-12 2016-05-19 Institut National De La Sante Et De La Recherche Medicale Signature génique associée à une tolérance à une allogreffe rénale
WO2018015551A1 (fr) * 2016-07-22 2018-01-25 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés permettant de distinguer un sujet tolérant
WO2019217990A1 (fr) * 2018-05-15 2019-11-21 The Council Of The Queensland Institute Of Medical Research Modulation de réponses immunitaires

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Publication number Priority date Publication date Assignee Title
WO2016075232A1 (fr) * 2014-11-12 2016-05-19 Institut National De La Sante Et De La Recherche Medicale Signature génique associée à une tolérance à une allogreffe rénale
WO2018015551A1 (fr) * 2016-07-22 2018-01-25 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés permettant de distinguer un sujet tolérant
JP2019527062A (ja) * 2016-07-22 2019-09-26 インセルム（インスティチュート ナショナル デ ラ サンテ エ デ ラ リシェルシェ メディカル） 寛容な対象を識別するための方法
US11479817B2 (en) 2016-07-22 2022-10-25 INSERM (Institut National de la Santé et de la Recherche Médicale Methods for discriminating a tolerant subject
WO2019217990A1 (fr) * 2018-05-15 2019-11-21 The Council Of The Queensland Institute Of Medical Research Modulation de réponses immunitaires

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