Biomarkers for the detection of acute rejection in heart transplant

Description

[0001] Transplantation is considered the primary therapy for patients with end-stage vital organ failure. While the availability of immunosuppressants such as cyclosporine and Tacrolimus has improved allograft recipient, e.g., heart transplant recipient, survival and wellbeing, identification of rejection of the allograft as early and as accurately as possible, and effective monitoring and adjusting immunosuppressive medication doses is still of primary importance to the continuing survival of the allograft recipient.

[0002] Rejection of an allograft may be generally described as the result of recipient's immune response to nonself antigens expressed by the donor tissues. Acute rejection may occur within days or weeks of the transplant, while chronic rejection may be a slower process, occurring months or years following the transplant.

[0003] At present, invasive biopsies, such as endomyocardial, liver core, and renal fine-needle aspiration biopsies, are widely regarded as the gold standard for the surveillance and diagnosis of allograft rejections, but are invasive procedures which carry risks of their own (e.g. Mehra MR, et al. Curr.Opin.Cardiol. 2002 Mar; 17(2): 131-136.). Biopsy results may also be subject to reproducibility and interpretation issues due to sampling errors and inter-observer variabilities, despite the availability of international guidelines such as the Banff schema for grading liver allograft rejection (Ormonde et al 1999. Liver Transplantation 5:261-268) or the Revised ISHLT transplantation scale (Stewart et al. 2005. J Heart Lung Transplant, 2005; 24: 1710-20). Although less invasive (imaging) techniques have been developed such as angiography and IVUS for monitoring chronic heart rejection, these alternatives are also susceptible to limitations similar to those associated with biopsies.

[0004] The severity of allograft rejection as determined by biopsy may be graded to provide standardized reference indicia. The International Society for Heart and Lung Transplantation scale (ISHLT) provides a means of grading biopsy samples for heart transplant subjects (Table 1) Table 1: International Society for Heart and Lung Transplantation grading of heart transplant rejection for histopathologic biopsy analysis.Grade Comment 0RNo acute cellular rejection: No evidence of mononuclear inflammation or myocyte damage or necrosis.1RMild, low-grade, acute cellular rejection: Mononuclear cells are present and there may be limited myocyte damage and necrosis.2RModerate, intermediate-grade, acute cellular rejection: Two or more foci of mononuclear cells with associated myocyte damage and necrosis are present. The damage may be found in the same biopsy, or two separate biopsies. Eosinophils may be present.3RSevere, high-grade, acute cellular rejection: Widespread, diffuse myocyte damage and necrosis, and disruption of normal architecture across several biopsies. Edema, interstitial hemorrhage and vasculitis may be present. The infiltrate may be polymorphous.

[0005] Indicators of allograft rejection may include a heightened and localized immune response as indicated by one or more of localized or systemic inflammation, tissue injury, allograft infiltration of immune cells, altered composition and concentration of tissue- and blood- derived proteins, differential oxygenation of allograft tissue, edema, thickening of the endothelium, increased collagen content, altered intramyocardial blood flow, infection, necrosis of the allograft and / or surrounding tissue, and the like.

[0006] Allograft rejection maybe described as 'acute' or 'chronic'. Acute rejection is generally considered to be rejection of a tissue or organ allograft within ~6 months of the subject receiving the allograft. Acute rejection may be characterized by cellular and humoral insults on the donor tissue, leading to rapid graft dysfunction and failure of the tissue or organ. Chronic rejection is generally considered to be reject of a tissue or organ allograft beyond 6 months, and may be several years after receiving the allograft. Chronic rejection may be characterized by progressive tissue remodeling triggered by the alloimmune response may lead to gradual neointimal formation within arteries, contributing to obliterative vasculopathy, parenchymal fibrosis and consequently, failure and loss of the graft. Depending on the nature and severity of the rejection, there may be overlap in the indicators or clinical variables observed in a subject undergoing, or suspected of undergoing, allograft rejection - either chronic or acute.

[0007] Attempts have been made to reduce the number of biopsies per patient, but may be generally unsuccessful, due in part to the difficulty in pinpointing the sites where rejection starts or progresses, and also to the difficulty in assessing tissue without performing the actual biopsy. Noninvasive surveillance techniques have been investigated, and may provide a reasonable negative prediction of allograft rejection, but may be of less practical utility in a clinical setting (Mehra et al., supra).

[0008] The scientific and patent literature is replete with reports of this marker or that being important for identification / diagnosis / prediction / treatment of every medical condition that can be named. Even within the field of allograft rejection, a myriad of markers are recited (frequently singly), and conflicting results may be presented. This conflict in the literature, added to the complexity of the genome (estimates range upwards of 30,000 transcriptional units), the variety of cell types (estimates range upwards of 200), organs and tissues, and expressed proteins or polypeptides (estimates range upwards of 80,000) in the human body, renders the number of possible nucleic acid sequences, genes, proteins or combinations thereof useful for diagnosing acute organ rejection is staggering. Variation between individuals presents additional obstacles, as well as the dynamic range of protein concentration in plasma (ranging from 10"6 to 103 micro g / mL) with many of the proteins of potential interest existing at very low concentrations) and the overwhelming quantities of the few, most abundant plasma proteins (constituting ~ 99 percent of the total protein mass.

[0009] The CARGO study (Cardiac Allograft Rejection Gene Expression Observation) (Deng et al., 2006. Am J. Transplantation 6:150-160) used custom microarray analysis of - 7300 genes and RT-PCR to examine gene expression profile in subjects exhibiting an ISHLT score of 3 A or greater in samples taken 6 months or more post-transplant.

[0010] Immune cells that have a role in recognizing may be useful as indicators of allograft rejection. WO 2005 / 05721 describes methods for distinguishing immunoreactive T-lymphocytes that bind specifically to donor antigen presenting cells, providing a population of T-lymphocytes that are specifically immunoreactive to the donor antigens. Again however, particular markers that may be useful in assessing or diagnosing allograft rejection remain to be determined.

[0011] Traum et al., 2005 (Pediatr. Transplant 9(6):700-711) provides a general overview of transplantation proteomics. Exploration of biomarkers directly in the plasma proteome presents two main challenges - the dynamic range of protein concentrations extends from 10"6 to 103 micro g / mL (Anderson et al. 2004. MoI Cell Proteomics 3:311-326), with many of the proteins of potential interest existing at very low concentrations and the most abundant plasma proteins comprising as much as 99 percent of the total protein mass.

[0012] Maintenance or measurement of B2M serum levels in heart transplant patients was suggested as helpful in managing long-term immunosuppressive therapy (Erez et al., 1998. J Heart Lung Transplant 17:538-541). PCT Publication WO 2009 / 003142 disclose that B2M, along with another protein may be useful as biomarkers for peripheral artery disease.

[0013] Borozdenkova et al. 2004 (J. Proteome Research 3:282-288) discloses that alpha B-crystallin and tropmyosin were elevated in a set of cardiac transplant subjects.

[0014] Ishihara, 2008 (J. Mol Cell Cardiology 45:S33) discloses that ADIPOQ may have a role in cardiac transplantation, and Nakano (Transplant Immunology 2007 17:130-136) suggests that upregulation of ADIPOQ may be necessary for overcoming rejection in liver transplant subjects.

[0015] Antibodies that bind SHBG (PCT Publication WO 2007 / 024715) and FlO (PCT Publication WO 2005 / 020927) are suggested as being useful in preventing graft rejection.

[0016] SERPINFl and CIQ are disclosed as biomarkers associated with an increased risk of a cardiovascular event; the biomarkers maybe detected in a sample of an atherosclerotic plaque from a subject (PCT Publication WO 2009 / 017405); sequences for SERPINFl may also be useful in an assay to select optimal blood vessel graft (US Publication 2006 / 0003338).

[0017] Complement is also known to have a role in rejection of allografts - Csencits et al., 2008 (Am J. Transplantation 8:1622-1630) summarizes past studies on various complement components and observes an accelerated humoral immune response in ClQ- / - mice allograft recipients.

[0018] PCT Publications WO2006 / 083986, WO206 / 122407, US Publications 2008 / 0153092, 2006 / 0141493 and US7235358 disclose methods for using panels of biomarkers (proteomic or genomic) for diagnosing or detecting various disease states ranging from cancer to organ transplantation

[0019] Alakulppi et al, 2007 (Transplantation 83:791-798) discloses the diagnosis of acute renal allograft rejection using RT-PCR for eight markers.

[0020] A review by Fildes et al 2008 (Transplant Immunology 19:1-11) discusses the role of cell types in immune processes following lung transplantation, and discloses that AICL (CLEC2B) interaction with NK cell proteins may have a role in acute and chronic rejection

[0021] Integration of multiple platforms (proteomics, genomics) has been suggested for diagnosis and monitoring of various cancers, however discordance between protein and mRNA expression is identified in the field (Chen et al., 2002.Mol Cell Proteomicsl:304-313; Nishizuka et al., 2003 Cancer Research 63:5243-5250). Previous studies have reported low correlations between genomic and proteomic data (Gygi SP et al. 1999. Mol Cell Biol.\9?12Q-\ 730; Huber et al., 2004 Mol Cell Proteomics 3:43-55). Hollander et al. 2010 discusses whole blood biomarkers of acute cardiac allograft rejection. Cohen Freue et al. 2013 discusses computational biomarker pipeline from discovery to clinical implementation, including plasma proteomic biomarkers for cardiac transplantation. Kasamatsu et al. 2005 discusses the identification of candidate genes associated with salivary adenoid cystic carcinomas using combined comparative genomic hybridization and oligonucleotide microarray analyses. Thach et al.2005 discusses the surveillance of transcriptomes in basic military trainees with normal, febrile respiratory illness, and convalescent phenotypes. Lin et al. 2009 discusses whole blood genomic biomarkers of acute cardiac allograft rejection.BRIEF SUMMARY OF THE INVENTION

[0022] In a first aspect, the invention provides a method of determining the acute rejection status of a heart transplant in a subject using a biomarker panel comprising nucleic acid markers, the method comprising the steps of: a. determining the nucleic acid expression profile of nucleic acid markers in a biological sample from the subject, wherein the nucleic acid markers comprise or consist of CD177, CPA3, HEBP1, ORM1, VNN 1 and CNTNAP3, b. comparing the nucleic acid expression profile to a control profile, c. determining whether expression of the nucleic acid markers is increased or decreased relative to the control profile, wherein the increased or decreased expression of the nucleic acid markers is indicative of the acute rejection status of the subject.

[0023] The disclosure further provides a kit for determining the acute rejection status of heart transplant in a patient, comprising a plurality of detection reagents for detecting the nucleic acid expression of nucleic acid markers, wherein the nucleic acid markers comprise or consists of CD177, CNTNAP3, CPA3, HEBP1, ORM1 and VNN1.

[0024] In some embodiments, the nucleic acid expression profile is determined by PCR, HTG EdgeSeq or NanoString nCounter.

[0025] In some embodiments, the biomarker panel has an AUC of at least 0.6, and / or a sensitivity of at least 80%, and / or a specificity of at least 15%, and / or a positive predictive value (PPV) of at least 4%, and / or a negative predictive value (NPV) of at least 98%, in predicting the status of acute rejection of heart transplant. In some embodiments, the assays using the biomarker panel described herein show comparable performance (e.g., NPV and PPV) to commercially available tests, such as the AlloMap assay by CareDx. In some embodiments, the assays described herein can be used in the first 2 months post-transplant where commercial tests have not demonstrated utility. For example, in some embodiments, assays using the biomarkers described herein achieve a PPV of 5% and a NPV of 98-100% for samples obtained during the first 2 months after the heart transplant.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows a range of biological processes in which the nucleic acid markers disclosed herein participate. Figure 2 shows a range of biological processes in which the proteomic markers disclosed herein participate. DETAILED DESCRIPTION OF THE INVENTION

[0027] In the description that follows, a number of terms are used extensively, the following definitions are provided to facilitate understanding of various aspects of the invention. Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to," and the word "comprises" has a corresponding meaning.

[0028] The present disclosure provides nucleic acidexpression profiles related to the assessment, prediction or diagnosis of allograft rejection in a subject, as defined in the claims. The specific combination of the altered expression levels (increased or decreased relative to a control) of specific sets of genomic markers comprise a novel combination useful for assessment, prediction or diagnosis of allograft rejection in a subject.

[0029] An allograft is an organ or tissue transplanted between two genetically different subjects of the same species. The subject receiving the allograft is the 'recipient', while the subject providing the allograft is the 'donor'. A tissue or organ allograft may alternately be referred to as a 'transplant', a 'graft', an 'allograft', a 'donor tissue' or 'donor organ', or similar terms. A transplant between two subjects of different species is a xenograft.

[0030] Subjects may present with a variety of symptoms or clinical variables well-known in the literature, however none of these of itself is a predictive or diagnostic of allograft rejection. A myriad of clinical variables may be used in assessing a subject having, or suspected of having, allograft rejection, in addition to biopsy of the allograft. The information gleaned from these clinical variables is then used by a clinician, physician, veterinarian or other practitioner in a clinical field in attempts to determine if rejection is occurring, and how rapidly it progresses, to allow for modification of the immunosuppressive drug therapy of the subject. Examples of clinical variables are described in Table 2. Table 2. Clinical variables for possible use in assessment of allograft rejection.Clinical Variable Name Renal / Heart / Liver / All Variable Explanation Primary DiagnosisAllDiagnosis leading to transplantSecondary DiagnosisAllDiagnosis leading to transplant"Transplant Procedure - Living related, Living unrelated, or cadaveric"Blood TypeAllBlood TypeBlood RhAllBlood RhHeight (cm)AllHeight (cm)Weight (kg)AllWeight (kg)BMIAllCalculation: Weight / (Height)2Liver AscitesAllHLA A1AllHLA A2AllHLA B1AllHLA B2AllHLA DR1AllHLA DR2AllCMVAllViral StatusCMV DateAllDate of viral statusHIVAllViral StatusHBVAllViral StatusHBV DateAllDate of viral statusHbsAbAllViral StatusHbcAb (Total)AllViral StatusHBvDNAAllViral StatusHCVAllViral StatusHCV GenotypeAllHepatitis C genotypeHCV Genotype SubAll"Hepatitis C genotype, subtype"EBVAllViral StatusZosterAllViral StatusDialysis Start DateAllDialysis Start DateDialysis TypeAllDialysis TypeCytoxicity Current LevelAllCytoxicity Current DateAllCytoxicity Peak LevelAllCytoxicity Peak DateAllFlush SolnAllType of Flush Solution used at transplantCold Time 1AllCold Time 2AllRe-Warm Time 1AllRe-Warm Time 2AllHTLV 1AllHTLV 2AllHCV RNAAll24hr UrineAll24 Hour urine outputSystolic Blood PressureAllBlood Pressure readingDiastolic Blood PressureAllBlood Pressure reading24 Hr UrineAll24 hour urineSodiumAllBlood testPotassiumAllBlood testChlorideAllBlood testTotal CO2AllBlood testAlbuminAllBlood testProteinAllBlood testCalciumAllBlood testInorganic PhosphateAllBlood testMagnesiumAllBlood testUric AcidAllBlood testGlucoseAllBlood testHemoglobin A1CAllBlood testCPKAllBlood testParathyroid HormoneAllBlood testHomocysteineAllBlood testUrine ProteinAllUrine testCreatinineAllBlood testBUNAllBlood testHemaglobinAllBlood testPlatelet CountAllBlood testWBC CountAllBlood testProthrombin TimeAllBlood testPartial Thromboplastin TimeAllBlood testINRAllBlood testGamma GTAllBlood testASTAllBlood testAlkaline PhosphataseAllBlood testAmylaseAllBlood testTotal BilirubinAllBlood testDirect BilirubinAllBlood testLDHAllBlood testALTAllBlood testTriglyceridesAllBlood testCholesterolAllBlood testHDL CholesterolAllBlood testLDL CholesterolAllBlood testFEV1AllLung function testFVCAllLung function testTotal FerritinAllBlood testTIBCAllBlood testTransferrin SaturatedAllBlood testFerritinAllBlood testAngiographyHeartHeart function testIntravascular ultrasoundHeartHeart function testDobutamine Stress EchocardiographyHeartHeart function testCyclosporine WBAllImmunosuppressive levelsCyclosporine 2 hrAllImmunosuppressive levelsTacrolimus WBAllImmunosuppressive levelsSirolimus WBAllImmunosuppressive total daily doseSolumedrolAllImmunosuppressive total daily dosePrednisoneAllImmunosuppressive total daily dosePrednisone ALTAllImmunosuppressive total daily doseTacrolimusAllImmunosuppressive total daily doseCyclosporineAllImmunosuppressive total daily doseImuranAllImmunosuppressive total daily doseMycophonelate MofetilAllImmunosuppressive total daily doseSirolimusAllImmunosuppressive total daily doseOKT3AllImmunosuppressive total daily doseATGAllImmunosuppressive total daily doseALGAllImmunosuppressive total daily doseBasiliximabAllImmunosuppressive total daily doseDaclizumabAllImmunosuppressive total daily doseGanciclovirAllAnti-viral total daily doseLamivudineAllAnti-viral total daily doseRibovironAllAnti-viral total daily doseInterferonAllAnti-viral total daily doseHepatisis C Virus RNAAlltest for presence of HCV values ()CMV AntigenemiaAllAntiviral and VirusValganciclovirAllAnti-viral total daily doseNeutrophil NumberAllBlood testC PeptideAllBlood testPeg InterferonAllAnti-viral total daily doseGFRAllGlomerular Filtration RateComplication EventsAllComplication TypeBiopsy ScoresRenalBorderline, 1A, 1B, 2A, 2B, 3, HyperacuteBiopsy ScoresLiverPortal inflammation, Bile duct inflammation damage, Venous endothelial inflammation each scored from 1 to 3Donor Blood TypeAllDonor Blood TypeDonor Blood RhAllDonor RhDonor HLA A1AllDonor HLA A1Donor HLA A2AllDonor HLA A2Donor HLA B 1AllDonor HLA B 1Donor HLA B2AllDonor HLA B2Donor HLA DR1AllDonor HLA DR1Donor HLA DR2AllDonor HLA DR2Donor CMVAllDonor CMVDonor HIVAllDonor HIVDonor HBVAllDonor HBVDonor HbsAbAllDonor HbsAbDonor HbcAb (total)AllDonor HbcAb (total)Donor HbdnaAllDonor HbdnaDonor HCVAllDonor HCVDonor EBVAllDonor EBV

[0031] Clinical variables (optionally accompanied by biopsy), while currently the only practical tools available to a clinician in mainstream medical practice, are not always able to cleanly differentiate between an AR (an "acute rejector"; ISHLT grade 2 R or higher) and an NR (a "mild or non-rejector"; ISHLT grade 0R or 1R) subject. While the extreme left and right subjects are correctly classified as AR or NR, the bulk of the subjects are represented in the middle range and their status is unclear. This does not negate the value of the clinical variables in the assessment of allograft rejection, but instead indicates their limitation when used in the absence of other methods.

[0032] The multifactorial nature of allograft rejection prediction, diagnosis and assessment is considered in the art to exclude the possibility of a single biomarker that meets even one of the needs of prediction, diagnosis or assessment of allograft rejection. Strategies involving a plurality of markers may take into account this multifactorial nature. Alternately, a plurality of markers may be assessed in combination with clinical variables that are less invasive (e.g. a biopsy not required) to tailor the prediction, diagnosis and / or assessment of allograft rejection in a subject.

[0033] Regardless of the methods used for prediction, diagnosis and assessment of allograft rejection, earlier is better - from the viewpoint of preserving organ or tissue function and preventing more systemic detrimental effects. There is no 'cure' for allograft rejection, only maintenance of the subject at a suitably immunosuppressed state, or in some cases, replacement of the organ if rejection has progressed too rapidly or is too severe to correct with immunosuppressive drug intervention therapy.

[0034] Applying a plurality of mathematical and / or statistical analytical methods to a nucleic acid expression dataset may indicate varying subsets of significant markers, leading to uncertainty as to which method is 'best' or 'more accurate'. Regardless of the mathematics, the underlying biology is the same in a dataset. By applying a plurality of mathematical and / or statistical methods to a microarray dataset and assessing the statistically significant subsets of each for common markers, uncertainty may be reduced, and clinically relevant core group of markers may be identified.

[0035] "Markers", "biological markers" or "biomarkers" may be used interchangeably and refer generally to detectable (and in some cases quantifiable) molecules or compounds in a biological sample. A marker may be down-regulated (decreased), up-regulated (increased) or effectively unchanged in a subject following transplantation of an allograft. Markers may include nucleic acids (DNA or RNA), a gene, or a transcript, or a portion or fragment of a transcript in reference to 'genomic' markers (alternately referred to as "nucleic acid markers"). In some usages, these terms may reference the level or quantity of a nucleic acid or polynucleotide (in absolute terms or relative to another sample or standard value) or the ratio between the levels of two polynucleotidesin a subject's biological sample. The level may be expressed as a concentration, for example micrograms per milliliter; as a colorimetric intensity, for example 0.0 being transparent and 1.0 being opaque at a particular wavelength of light, with the experimental sample ranked accordingly and receiving a numerical score based on transmission or absorption of light at a particular wavelength; or as relevant for other means for quantifying a marker, such as are known in the art. In some examples, a ratio may be expressed as a unitless value. A "marker" may also reference to a ratio, or a net value following subtraction of a baseline value. A marker may also be represented as a 'fold-change', with or without an indicator of directionality (increase or decrease / up or down). The increase or decrease in expression of a marker may also be referred to as 'down-regulation' or 'up-regulation', or similar indicators of an increase or decrease in response to a stimulus, physiological event, or condition of the subject. A marker may be present in a first biological sample, and absent in a second biological sample; alternately the marker may be present in both, with a statistically significant difference between the two. Expression of the presence, absence or relative levels of a marker in a biological sample may be dependent on the nature of the assay used to quantify or assess the marker, and the manner of such expression will be familiar to those skilled in the art.

[0036] A marker may be described as being differentially expressed when the level of expression in a subject who is rejecting an allograft is significantly different from that of a subject or sample taken from a non-rejecting subject. A differentially expressed marker may be overexpressed or underexpressed as compared to the expression level of a normal or control sample.

[0037] A "profile" is a set of one or more markers and their presence, absence, relative level or abundance (relative to one or more controls). A genomic or nucleic acid profile a dataset of the presence, absence, relative level or abundance of expressed nucleic acids (e.g. transcripts, mRNA, EST or the like). A profile may alternately be referred to as an expression profile.

[0038] The increase or decrease, or quantification of the markers in the biological sample may be determined by any of several methods known in the art for measuring the presence and / or relative abundance of a gene product or transcript, or a nucleic acid molecule comprising a particular sequence or the like. The level of the markers may be determined as an absolute value, or relative to a baseline value, and the level of the subject's markers compared to a cutoff index (e.g. a non-rejection cutoff index). Alternately the relative abundance of the marker may be determined relative to a control. The control may be a clinically normal subject (e.g. one who has not received an allograft) or may be an allograft recipient that has not previously demonstrated rejection.

[0039] In some embodiments, the control may be an autologous control, for example a sample or profile obtained from the subject before undergoing allograft transplantation. In some embodiments, the profile obtained at one time point (before, after or before and after transplantation) may be compared to one or more than one profiles obtained previously from the same subject. By repeatedly sampling the same biological sample from the same subject over time, a composite profile, illustrating marker level or expression over time may be provided. Sequential samples can also be obtained from the subject and a profile obtained for each, to allow the course of increase or decrease in one or more markers to be followed over time. For example, an initial sample or samples may be taken before the transplantation, with subsequent samples being taken weekly, biweekly, monthly, bimonthly or at another suitable, regular interval and compared with profiles from samples taken previously. Samples may also be taken before, during and after administration of a course of a drug, for example an immunosuppressive drug.

[0040] Techniques, methods, tools, algorithms, reagents and other necessary aspects of assays that may be employed to detect and / or quantify a particular marker or set of markers are varied. Of significance is not so much the particular method used to detect the marker or set of markers, but what markers to detect. As is reflected in the literature, tremendous variation is possible. Once the marker or set of markers to be detected or quantified is identified, any of several techniques may be well suited, with the provision of appropriate reagents. One of skill in the art, when provided with the set of markers to be identified, will be capable of selecting the appropriate assay (for example, a PCR based or a microarray based assay for nucleic acid markers, an ELISA, protein or antibody microarray or similar immunologic assay, or in some examples, use of an MRM, iTRAQ, iCAT or SELDI proteomic mass spectrometric based method) for performing the methods disclosed herein.

[0041] The present disclosure provides nucleic acid expression profiles related to the assessment, prediction or diagnosis of allograft rejection in a subject.

[0042] For example, detection or determination, and in some cases quantification, of a nucleic acid may be accomplished by any one of a number methods or assays employing recombinant DNA technologies known in the art, including but not limited to, as sequence-specific hybridization, polymerase chain reaction (PCR), RT-PCR, microarrays and the like. Such assays may include sequence-specific hybridization, primer extension, or invasive cleavage. Furthermore, there are numerous methods for analyzing / detecting the products of each type of reaction (for example, fluorescence, luminescence, mass measurement, electrophoresis, etc.). Furthermore, reactions can occur in solution or on a solid support such as a glass slide, a chip, a bead, or the like.

[0043] Methods of designing and selecting probes for use in microarrays or biochips, or for selecting or designing primers for use in PCR-based assays are known in the art. Once the marker or markers are identified and the sequence of the nucleic acid determined by, for example, querying a database comprising such sequences, or by having an appropriate sequence provided (for example, a sequence listing as provided herein), one of skill in the art will be able to use such information to select appropriate probes or primers and perform the selected assay.

[0044] Standard reference works setting forth the general principles of recombinant DNA technologies known to those of skill in the art include, for example: Ausubel et al, Current Protocols In Molecular Biology, John Wiley and Sons, New York (1998 and Supplements to 2001); Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, New York (1989); Kaufman et al, Eds., Handbook Of Molecular And Cellular Methods In Biology And Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991).

[0045] A subject's rejection status may be described as an "acute rejector" (ISHLT grade 2R of higher; AR) or as a "non-rejector" (ISHLT grade 0R or 1R; NR) and is determined by comparison of the concentration of the markers to that of a non-rejector cutoff index. A "non-rejector cutoff index" is a numerical value or score, beyond or outside of which a subject is categorized as having an AR rejection status. The non- rejector cutoff index maybe alternately referred to as a 'control value', a 'control index', or simply as a 'control'. A non-rejector cutoff-index maybe the concentration of individual markers in a control subject population and considered separately for each marker measured; alternately the non-rejector cutoff index may be a combination of the concentration of the markers, and compared to a combination of the concentration of the markers in the subject's sample provided for diagnosing. The control subject population may be a normal or healthy control population, or may be an allograft recipient population that has not, or is not, rejecting the allograft. The control maybe a single subject, and for some embodiments, maybe an autologous control. A control, or pool of controls, may be constant e.g. represented by a static value, or may be cumulative, in that the sample population used to obtain it may change from site to site, or over time and incorporate additional data points. For example, a central data repository, such as a centralized healthcare information system, may receive and store data obtained at various sites (hospitals, clinical laboratories or the like) and provide this cumulative data set for use with the methods of the invention at a single hospital, community clinic, for access by an end user (i.e. an individual medical practitioner, medical clinic or center, or the like).

[0046] The non-rejector cutoff index may be alternately referred to as a 'control value', a 'control index' or simply as a 'control'. In some embodiments the cutoff index may be further characterized as being a genomic cutoff index (for genomic expression profiling of subjects), or the like.

[0047] A "biological sample" refers generally to body fluid or tissue or organ sample from a subject. For example, the biological sample may a body fluid such as blood, plasma, lymph fluid, serum, urine or saliva. A tissue or organ sample, such as a non-liquid tissue sample maybe digested, extracted or otherwise rendered to a liquid form - examples of such tissues or organs include cultured cells, blood cells, skin, liver, heart, kidney, pancreas, islets of Langerhans, bone marrow, blood, blood vessels, heart valve, lung, intestine, bowel, spleen, bladder, penis, face, hand, bone, muscle, fat, cornea or the like. A plurality of biological samples may be collected at any one time. A biological sample or samples may be taken from a subject at any time, including before allograft transplantation, at the time of translation or at anytime following transplantation. A biological sample may comprise nucleic acid, such as deoxyribonucleic acid or ribonucleic acid, or a combination thereof, in either single or double- stranded form. When an organ is removed from a donor, the spleen of the donor or a part of it may be kept as a biological sample from which to obtain donor T-cells. When an organ is removed from a living donor, a blood sample may be taken, from which donor T-cells may be obtained. Alloreactive T-cells may be isolated by exploiting their specific interaction with antigens (including the MHC complexes) of the allograft. Methods to enable specific isolation of alloreactive T-cells are described in, for example PCT Publication WO 2005 / 05721.

[0048] A lymphocyte is nucleated or 'white' blood cell (leukocyte) of lymphoid stem cell origin. Lymphocytes include T-cells, B-cells natural killer cells and the like, and other immune regulatory cells. A "T-cell" is a class of lymphocyte responsible for cell-mediated immunity, and for stimulating B-cells. A stimulated B-cell produces antibodies for specific antigens. Both B- cells and T-cells function to recognize non-self antigens in a subject. Non-self antigens include those of viruses, bacteria and other infectious agents as well as allografts.

[0049] The term "subject" or "patient" generally refers to mammals and other animals including humans and other primates, companion animals, zoo, and farm animals, including, but not limited to, cats, dogs, rodents, rats, mice, hamsters, rabbits, horses, cows, sheep, pigs, goats, poultry, etc. A subject includes one who is to be tested, or has been tested for prediction, assessment or diagnosis of allograft rejection. The subject may have been previously assessed or diagnosed using other methods, such as those described herein or those in current clinical practice, or maybe selected as part of a general population (a control subject).

[0050] A fold-change of a marker in a subject, relative to a control maybe at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 or more, or any amount therebetween. The fold change may represent a decrease, or an increase, compared to the control value.

[0051] One or more than one includes 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more.

[0052] "Down-regulation" or 'down-regulated may be used interchangeably and refer to a decrease in the level of a marker, such as a gene, nucleic acid, transcript, protein or polypeptide. "Up-regulation" or "up-regulated" may be used interchangeably and refer to an increase in the level of a marker, such as a gene, nucleic acid or transcript.

[0053] For the purpose of this invention, a patient has treatable acute rejection status to heart transplant if he or she had response that fits into the "2R" or "3R" category according to the International Society for heart and Lung transplantation standard (Table 1). A patient has non rejection status if the response to heart transplant fit into the "0R" category and moderate rejection status if the response fits into the "1R" category according to the above standard.

[0054] Once a subject is identified as an acute rejector, or at risk for becoming an acute rejector by any method (genomic, proteomic, or a combination thereof), therapeutic measures may be implemented to alter the subject's immune response to the allograft. The subject may undergo additional monitoring of clinical values more frequently, or using more sensitive monitoring methods. Additionally the subject may be administered immunosuppressive medicaments to decrease or increase the subject's immune response. Even though a subject's immune response needs to be suppressed to prevent rejection of the allograft, a suitable level of immune function is also needed to protect against opportunistic infection. Various medicaments that maybe administered to a subject are known; see for example, Goodman and Gilman's The Pharmacological Basis of Therapeutics 11th edition. Ch 52, pp 1405-1431 and references therein; LL Brunton, JS Lazo, KL Parker editors. Standard reference works setting forth the general principles of medical physiology and pharmacology known to those of skill in the art include: Fauci et al., Eds., Harrison's Principles Of Internal Medicine, 14th Ed., McGraw-Hill Companies, Inc. (1998). Other preventative and therapeutic strategies are reviewed in the medical literature- see, for example Kobashigawa et al. 2006. Nature Clinical Practice. Cardiovascular Medicine 3:203-21.GENOMIC NUCLEIC ACID EXPRESSION PROFILING

[0055] A method of diagnosing acute allograft rejection in a subject comprises 1) determining the expression profile of nucleic acid markers in a biological sample from the subject, wherein the nucleic acid markers comprise or consist of the nucleic acid markers listed in Table 7; 2) comparing the expression profile of the nucleic acid markers to a non-rejector profile; and 3) determining whether the expression level of the nucleic acid markers is up-regulated or down- regulated relative to the control profile, wherein up-regulation or down-regulation of the nucleic acid markers is indicative of the rejection status.

[0056] Therefore, the disclosure also provides for a method of predicting, assessing or diagnosing allograft rejection in a subject comprising 1) measuring the increase or decrease of the nucleic acid markers listed in Table 7 and 2) determining the 'rejection status' of the subject, wherein the determination of 'rejection status' of the subject is based on comparison of the subject's nucleic acid marker expression profile to a control nucleic acid marker expression profile.

[0057] The phrase "gene expression data", "gene expression profile" "nucleic acid expression profile" or "marker expression profile" as used herein refers to information regarding the relative or absolute level of expression of a gene or set of genes in a biological sample. The level of expression of a gene may be determined based on the level of a nucleic acid such as RNA including mRNA, transcribed from or encoded by the gene.

[0058] A "polynucleotide", "oligonucleotide", "nucleic acid" or "nucleotide polymer" as used herein may include synthetic or mixed polymers of nucleic acids, including RNA, DNA or both RNA and DNA, both sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e. g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), and modified linkages (e.g., alpha anomeric polynucleotides, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.

[0059] An oligonucleotide includes variable length nucleic acids, which may be useful as probes, primers and in the manufacture of microarrays (arrays) for the detection and / or amplification of specific nucleic acids. Oligonucleotides may comprise DNA, RNA, PNA or other polynucleotide moieties as described in, for example, US 5,948,902. Such DNA, RNA or oligonucleotide strands may be synthesized by the sequential addition (5 '-3' or 3'-5') of activated monomers to a growing chain which may be linked to an insoluble support. Numerous methods are known in the art for synthesizing oligonucleotides for subsequent individual use or as a part of the insoluble support, for example in arrays (BERNFIELD MR. and ROTTMAN FM. J. Biol. Chem. (1967) 242(18):4134-43; SULSTON J. et al. PNAS (1968) 60(2):409-415; GILLAM S. et al. Nucleic Acid Res.(1975) 2(5):613-624; BONORA GM. et al. Nucleic Acid Res.(1990) 18(11):3155-9; LASHKARI DA. et al. PNAS (1995) 92(17):7912-5; MCGALL G. et al. PNAS (1996) 93(24):13555-60; ALBERT TJ. et al. Nucleic Acid Res.(2003) 31(7):e35; GAO X. et al. Biopolymers (2004) 73(5):579-96; and MOORCROFT MJ. et al. Nucleic Acid Res.(2005) 33(8):e75). In general, oligonucleotides are synthesized through the stepwise addition of activated and protected monomers under a variety of conditions depending on the method being used. Subsequently, specific protecting groups may be removed to allow for further elongation and subsequently and once synthesis is complete all the protecting groups may be removed and the oligonucleotides removed from their solid supports for purification of the complete chains if so desired.

[0060] A "gene" is an ordered sequence of nucleotides located in a particular position on a particular chromosome that encodes a specific functional product and may include untranslated and untranscribed sequences in proximity to the coding regions (5' and 3' to the coding sequence). Such non-coding sequences may contain regulatory sequences needed for transcription and translation of the sequence or splicing of introns, for example, or may as yet to have any function attributed to them beyond the occurrence of the mutation of interest. A gene may also include one or more promoters, enhancers, transcription factor binding sites, termination signals or other regulatory elements. A gene may be generally referred to as 'nucleic acid'.

[0061] The term "microarray," "array," or "chip" refers to a plurality of defined nucleic acid probes coupled to the surface of a substrate in defined locations. The substrate may be preferably solid. Microarrays, their methods of manufacture, use and analysis have been generally described in the art in, for example, U.S. Patent Nos. 5,143,854 (Pirrung), 5,424,186 (Fodor), 5,445,934 (Fodor), 5,677,195 (Winkler), 5,744,305 (Fodor), 5,800,992 (Fodor), 6,040,193 (Winkler), and Fodor et al. 1991. Science, 251 -.161-111.

[0062] "Hybridization" includes a reaction in which one or more polynucleotides and / or oligonucleotides interact in an ordered manner (sequence-specific) to form a complex that is stabilized by hydrogen bonding - also referred to as 'Watson-Crick' base pairing. Variant base- pairing may also occur through non-canonical hydrogen bonding includes Hoogsteen base pairing. Under some thermodynamic, ionic or pH conditions, triple helices may occur, particularly with ribonucleic acids. These and other variant hydrogen bonding or base-pairing are known in the art, and may be found in, for example, Lehninger - Principles of Biochemistry, 3rd edition (Nelson and Cox, eds. Worth Publishers, New York.).

[0063] Hybridization reactions can be performed under conditions of different "stringency". The stringency of a hybridization reaction includes the difficulty with which any two nucleic acid molecules will hybridize to one another. Stringency may be increased, for example, by increasing the temperature at which hybridization occurs, by decreasing the ionic concentration at which hybridization occurs, or a combination thereof. Under stringent conditions, nucleic acid molecules at least 60 percent, 65 percent, 70 percent, 75 percent or more identical to each other remain hybridized to each other, whereas molecules with low percent identity cannot remain hybridized. An example of stringent hybridization conditions are hybridization in 6x sodium chloride / sodium citrate (SSC) at about 44-45 degrees centigrade, followed by one or more washes in 0.2xSSC, 0.1 percent SDS at 5ODegrees centigrade 55Degrees centigrade 60Degrees centigrade 65 degrees centigrade, or at a temperature therebetween.

[0064] Hybridization between two nucleic acids may occur in an antiparallel configuration - this is referred to as 'annealing', and the paired nucleic acids are described as complementary. A double-stranded polynucleotide may be "complementary", if hybridization can occur between one of the strands of the first polynucleotide and the second. The degree of which one polynucleotide is complementary with another is referred to as homology, and is quantifiable in terms of the proportion of bases in opposing strands that are expected to hydrogen bond with each other, according to generally accepted base-pairing rules.

[0065] In general, sequence-specific hybridization involves a hybridization probe, which is capable of specifically hybridizing to a defined sequence. Such probes may be designed to differentiate between sequences varying in only one or a few nucleotides, thus providing a high degree of specificity. A strategy which couples detection and sequence discrimination is the use of a "molecular beacon", whereby the hybridization probe (molecular beacon) has 3' and 5' reporter and quencher molecules and 3' and 5' sequences which are complementary such that absent an adequate binding target for the intervening sequence the probe will form a hairpin loop. The hairpin loop keeps the reporter and quencher in close proximity resulting in quenching of the fluorophor (reporter) which reduces fluorescence emissions. However, when the molecular beacon hybridizes to the target the fluorophor and the quencher are sufficiently separated to allow fluorescence to be emitted from the fluorophor.

[0066] Probes used in hybridization may include double-stranded DNA, single-stranded DNA and RNA oligonucleotides, and peptide nucleic acids. Hybridization conditions and methods for identifying markers that hybridize to a specific probe are described in the art - see, for example, Brown, T. "Hybridization Analysis of DNA Blots" in Current Protocols in Molecular Biology. FM Ausubel et al, editors. Wiley and Sons, 2003. doi: 10.1002 / 0471142727.mb0210s21. Suitable hybridization probes for use in accordance with the invention include oligonucleotides, polynucleotides or modified nucleic acids from about 10 to about 400 nucleotides, alternatively from about 20 to about 200 nucleotides, or from about 30 to about 100 nucleotides in length.

[0067] Specific sequences may be identified by hybridization with a primer or a probe, and this hybridization subsequently detected.

[0068] A "primer" includes a short polynucleotide, generally with a free 3'-OH group that binds to a target or "template" present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or "set of primers" consisting of "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are well known in the art, and are taught, for example, in Beverly, SM. Enzymatic Amplification of RNA by PCR (RT-PCR) in Current Protocols in Molecular Biology. FM Ausubel et al, editors. Wiley and Sons, 2003. doi: 10.1002 / 0471142727.mb 1505s56. Synthesis of the replicate copies may include incorporation of a nucleotide having a label or tag, for example, a fluorescent molecule, biotin, or a radioactive molecule. The replicate copies may subsequently be detected via these tags, using conventional methods.

[0069] A primer may also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses (see, e.g., Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0070] A "probe set" (or 'primer set') as used herein refers to a group of oligonucleotides that may be used to detect one or more expressed nucleic acids, or expressed genes. Detection may be, for example, through amplification as in PCR and RT-PCR, or through hybridization, as on a microarray, or through selective destruction and protection, as in assays based on the selective enzymatic degradation of single or double stranded nucleic acids. Probes in a probe set may be labeled with one or more fluorescent, radioactive or other detectable moieties (including enzymes). Probes may be any size so long as the probe is sufficiently large to selectively detect the desired gene - generally a size range from about 15 to about 25, or to about 30 nucleotides is of sufficient size. A probe set maybe in solution, e.g. for use in multiplex PCR. Alternately, a probe set may be adhered to a solid surface, as in an array or microarray.

[0071] In the invention, a probe set for detection of nucleic acids markers, wherein the nucleic acid markers comprise or consist of CD177, CPA3, HEBP1, ORM1, VNN 1 and CNTNAP3 is provided. Such a probe set may be useful for determining the rejection status of a subject. The probe set may comprise one or more pairs of primers for specific amplification (e.g. PCR or RT- PCR) of nucleic acid sequences. In another embodiment of the invention, the probe set is part of a microarray.

[0072] It will be appreciated that numerous other methods for sequence discrimination and detection are known in the art and some of which are described in further detail below. It will also be appreciated that reactions such as arrayed primer extension mini sequencing, tag microarrays and sequence-specific extension could be performed on a microarray. One such array based genotyping platform is the microsphere based tag-it high throughput array (BORTOLINS. et al. 2004 Clinical Chemistry 50: 2028-36). This method amplifies genomic DNA by PCR followed by sequence-specific primer extension with universally tagged primers. The products are then sorted on a Tag-It array and detected using the Luminex xMAP system.

[0073] It will be appreciated by a person of skill in the art that any numerical designations of nucleotides or amino acids within a sequence are relative to the specific sequence. Also, the same positions may be assigned different numerical designations depending on the way in which the sequence is numbered and the sequence chosen. Furthermore, sequence variations such as insertions or deletions, may change the relative position and subsequently the numerical designations of particular nucleotides or amino acids at or around a mutational site.

[0074] Selection and / or design of probes, primers or probe sets for specific detection of expression of any gene of interest is within the ability of one of skill in the relevant art, when provided with one or more nucleic acid sequences of the gene of interest. Further, any of several probes, primers or probe sets, or a plurality of probes, primers or probe sets may be used to detect a gene of interest, for example, an array may include multiple probes for a single gene transcript - the aspects of the invention as described herein are not limited to any specific probes exemplified.

[0075] Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program (for DNA or RNA sequences, or fragments or portions thereof) or an amino acid sequence comparison program (for protein, polypeptide or peptide sequences, or fragments or portions thereof), such as that provided within DNASIS (for example, but not limited to, using the following parameters: GAP penalty 5, #of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith and Waterman (1981, Adv. Appl. Math. 2:482), Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), Pearson and Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA, and BLAST), or by manual alignment and visual inspection.

[0076] If a nucleic acid or gene, polypeptide or sequence of interest is identified and a portion or fragment of the sequence (or sequence of the gene polypeptide or the like) is provided, other sequences that are similar, or substantially similar may be identified using the programs exemplified above. For example, when constructing a microarray or probe sequences, the sequence and location are known, such that if a microarray experiment identifies a 'hit' (the probe at a particular location hybridizes with one or more nucleic acids in a sample, the sequence of the probe will be known (either by the manufacturer or producer of the microarray, or from a database provided by the manufacturer - for example the NetAffx databases of Affymetrix, the manufacturer of the Human Genome U133 Plus 2.0 Array). If the identity of the sequence source is not provided, it may be determined by using the sequence of the probe in a sequence-based search of one or more databases. For peptide or peptide fragments identified by proteomics assays, for example iTRAQ, the sequence of the peptide or fragment may be used to query databases of amino acid sequences as described above. Examples of such a database include those maintained by the National Centre for Biotechnology Information, or those maintained by the European Bioinformatics Institute.

[0077] A protein or polypeptide, nucleic acid or fragment or portion thereof may be considered to be specifically identified when its sequence may be differentiated from others found in the same phylogenetic Species, Genus, Family or Order. Such differentiation may be identified by comparison of sequences. Comparisons of a sequence or sequences may be done using a BLAST algorithm (Altschul et al. 1009. J. MoI Biol 215:403-410). A BLAST search allows for comparison of a query sequence with a specific sequence or group of sequences, or with a larger library or database (e.g. GenBank or GenPept) of sequences, and identify not only sequences that exhibit 100 percent identity, but also those with lesser degrees of identity. For example, regarding a protein with multiple isoforms (either resulting from, for example, separate genes or variant splicing of the nucleic acid transcript from the gene, or post translational processing), an isoform may be specifically identified when it is differentiated from other isoforms from the same or a different species, by specific detection of a structure, sequence or motif that is present on one isoform and is absent, or not detectable on one or more other isoforms.

[0078] Access to the methods of the invention may be provided to an end user by, for example, a clinical laboratory or other testing facility performing the individual marker tests - the biological samples are provided to the facility where the individual tests and analyses are performed and the predictive method applied; alternately, a medical practitioner may receive the marker values from a clinical laboratory and use a local implementation or an internet-based implementation to access the predictive methods of the invention.

[0079] Determination of statistical parameters such as multiples of the median, standard error, standard deviation and the like, as well as other statistical analyses as described herein are known and within the skill of one versed in the relevant art. Use of a particular coefficient, value or index is exemplary only and is not intended to constrain the limits of the various aspects of the invention as disclosed herein.

[0080] Interpretation of the large body of gene expression data obtained from, for example, microarray experiments, or complex RT-PCR experiments may be a formidable task, but is greatly facilitated through use of algorithms and statistical tools designed to organize the data in a way that highlights systematic features. Visualization tools are also of value to represent differential expression by, for example, varying intensity and hue of colour (Eisen et al. 1998. Proc Natl Acad Sci 95:14863-14868). The algorithm and statistical tools available have increased in sophistication with the increase in complexity of arrays and the resulting datasets, and with the increase in processing speed, computer memory, and the relative decrease in cost of these.

[0081] Mathematical and statistical analysis of nucleic acid profiles may accomplish several things - identification of groups of genes that demonstrate coordinate regulation in a pathway or a domain of a biological system, identification of similarities and differences between two or more biological samples, identification of features of a gene expression profile that differentiate between specific events or processes in a subject, or the like. This may include assessing the efficacy of a therapeutic regimen or a change in a therapeutic regimen, monitoring or detecting the development of a particular pathology, differentiating between two otherwise clinically similar (or almost identical) pathologies, or the like.

[0082] Clustering methods are known and have been applied to microarray datasets, for example, hierarchical clustering, self-organizing maps, k-means or deterministic annealing. (Eisen et al, 1998 Proc Natl Acad Sci USA 95:14863- 14868; Tamayo, P., et al. 1999. Proc Natl Acad Sci USA 96:2907-2912; Tavazoie, S., et al. 1999. Nat Genet 22:281-285; Alon, U., et al. 1999. Proc Natl Acad Sci USA 96:6745-6750). Such methods may be useful to identify groups of genes in a gene expression profile that demonstrate coordinate regulation, and also useful for the identification of novel genes of otherwise unknown function that are likely to participate in the same pathway or system as the others demonstrating coordinate regulation.

[0083] The pattern of nucleic acid in a biological sample may also provide a distinctive and accessible molecular picture of its functional state and identity (DeRisi 1997; Cho 1998; Chu 1998; Holstege 1998; Spellman 1998). Two different samples that have related gene expression patterns are therefore likely to be biologically and functionally similar to one another, conversely two samples that demonstrate significant differences may not only be differentiated by the complex expression pattern displayed, but may indicate a diagnostic subset of gene products or transcripts that are indicative of a specific pathological state or other physiological condition, such as allograft rejection.GENOMIC EXPRESSION PROFILING MARKERS ("GENOMIC MARKERS")

[0084] The methods of the present invention use a core group of markers to determine the acute rejection status of a heart transplant in a subject, which comprises or consists of CD177, CPA3, HEBP1, ORM1, VNN 1 and CNTNAP3.

[0085] The sensitivity of the assay to determine the acute rejection status of a heart transplant in a subject using panels of nucleic acid markers described herein may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. The specificity of the assay using the panels of nucleic acid markers may be at least 10%, at least 15%, at least 22%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 74%, at least 80%, at least 90%, at least 91%, or at least 95%. The PPV of the assay using the panels of the nucleic acid markers may be at least 2%, at least 4%, at least 5%, at least 7%, at least 14%, or at least 32%.

[0086] The disclosure also provides for a kit for determining the acute rejection status of heart transplant in a patient, comprising a plurality of detection reagents for detecting the nucleic acid expression of nucleic acid markers, wherein the nucleic acid markers comprise or consists of CD177, CNTNAP3, CPA3, HEBP1, ORM1 and VNN1, along with instructions for the use of such reagents and methods for analyzing the resulting data. The kit may be used alone for determining the acute rejection status of heart transplant in a patient, or it may be used in conjunction with other methods for determining clinical variables, or other assays that may be deemed appropriate. The kit may include, for example, one or more labelled oligonucleotides capable of selectively hybridizing to the marker. The kit may further include, for example, one or more oligonucleotides operable to amplify a region of the marker (e.g. by PCR). Instructions or other information useful to combine the kit results with those of other assays to provide a non-rejection cutoff index for the prediction or diagnosis of a subject's rejection status may also be provided.RNA EXTRACTION AND MICROARRAY ANALYSIS

[0087] Subjects were enrolled according to Biomarkers in Transplantation standard operating procedures. Subjects waiting for a cardiac transplant at the St. Paul's Hospital, Vancouver, BC were approached by the research coordinators and consented subjects were enrolled in the study. All heart transplants are overseen by the British Columbia Transplant (BCT) and all subjects receive standard immunosuppressive therapy consisting of cyclosporine, prednisone and mycophenolate mofetil. Cyclosporine was replaced by tacrolimus for women and by sirolimus in cases of renal impairment. Blood samples from consented subjects were taken before transplant (baseline) and collected in PAXGene tubes, placed in an ice bath for delivery, frozen at -20°C for one day and then stored at -80°C until RNA extraction for nucleic acid marker analysis.

[0088] RNA extraction was performed on thawed samples using the PAXgene ™< Blood RNA Kit [Cat #762134] to isolate total RNA. Between 4 and 10 micro g of RNA was routinely isolated from 2.5 ml whole blood and the RNA quality confirmed using the Agilent BioAnalyzer. Samples with 1.5 micro g of RNA, an RIN number >5, and A240 / A280 > 1.9 were packaged on dry ice and shipped by Federal Express to the Microarray Core (MAC) Laboratory, Children's Hospital, Los Angeles, CA for Affymetrix microarray analysis. The microarray analysis was performed by a single technician at the CAP / CLIA accredited MAC laboratory. Nascent RNA was used for double stranded cDNA synthesis. The cDNA was then labeled with biotin, fragmented, mixed with hybridization cocktail and hybridized onto GeneChip Human Genome U133 Plus 2.0 Arrays. The arrays were scanned with the Affymetrix System in batches of 48 with an internal RNA control made from pooled normal whole blood. Microarrays were checked for quality issues using Affymetrix version 1.16.0 and affyPLM version 1.14.0 BioConductor packages (Bolstad, B., Low Level Analysis of High-density Oligonucleotide Array Data: Background, Normalization and Summarization. 2004, University of California, Berkeley; Irizarry et al. 2003. Biostatistics 4(2): 249-64). The arrays with lower quality were repeated with a different RNA aliquot from the same time point. The Affymetrix ™< NetAffx ™< Annotation database Update Release 25 (March 2008) was used for identification and analysis of microarray results.

[0089] The expression profile of nucleic acid markers can also be confirmed by RT-PCR or NanoString nCounter technology. The expression of these markers can also be detected and validated using more clinically-amenable technologies, e.g., the HTG Molecular qNPA (quantitative nuclease protection assay) platform. The HTG Edge System is a desired platform for clinical assay development because it is fully-automated, which greatly simplifies laboratory workflow, requires small sample input and minimal hands-on time. One or more housekeeping genes can be used in these assay platforms, for example, ACTB, ANT, B2M, OAZ1, RPL11, or SDHA.STATISTICAL ANALYSIS

[0090] Applying a plurality of mathematical and / or statistical analytical methods to a microarray dataset may indicate varying subsets of significant markers, leading to uncertainty as to which method is 'best' or 'more accurate'. Regardless of the mathematics, the underlying biology is the same in a dataset. By applying a plurality of mathematical and / or statistical methods to a microarray dataset or the mass spectrometry dataset and assessing the statistically significant subsets of each for common markers to all, the uncertainty is reduced, and clinically relevant core group of markers is identified.

[0091] Exemplar statistical models that can be used include a robust moderated t-test (eBayes - Smyth GK) for the evaluation of differential protein expression levels, and linear models and empirical Bayes methods for assessing differential expression in microarray experiments. StatAppl Genet MoI Biol. 2004;3:Article3 (Berkeley Electronic Press).

[0092] Classification methods such as elaticnet, random forest, Linear Discriminant Analysis (LDA), regression, and others were applied to identify a subset of the markers to be included in the mRNA panel.

[0093] Various parameters are employed to evaluate the performance of panels of biomarkers used in determining acute rejection status in patients. AUC, "area under the curve", which is examined within the scope of ROC (receiver-operator characteristic) analysis and which is a measure of the quality of the individual parameter (biomarker) or a combination of biomarkers, based on the cases examined. Thus, the sensitivity on the ordinate is plotted against specificity on the abscissa in the diagram. Specificity is defined as the number of actually negative samples divided by the sum of the numbers of the actually negative and false positive samples. A specificity of 1 means that a test recognizes all acute rejectors as acute rejectors, i.e., no non-rejector is identified as being an acute rejector. This says nothing about how reliably the test recognizes acute rejectors. Sensitivity is defined as the number of actually acute rejectors divided by the sum of the numbers of the actually acute rejecters and the number of non rejectors that has been false diagnosed as acute rejectors. A sensitivity of 1 means that the test recognizes all acute rejectors. This says nothing about how reliably the test recognizes non-rejectors. Thus, an AUC value of 1 means that all samples have been assigned correctly (specificity and sensitivity of 1), an AUC value of 0.5 means that the samples have been assigned with guesswork probability and the parameter thus has no significance.

[0094] In a preferred embodiment the panel of biomarkers employed to determine the acute rejection status in patients has an AUC value that is greater than 0.6, preferably greater than 0.7. In another preferred embodiment the sensitivity of the panels is equal to or greater than 91%, and the specificity of the panels is equal to or greater than 15%.

[0095] "Positive predictive value" or "PPV" is calculated by TP / (TP+FP) or the true positive fraction of all positive test results. It is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. "Negative predictive value" or "NPV" is calculated by TN / (TN + FN) or the true negative fraction of all negative test results. It also is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested. In one preferred embodiment of the invention, the PPV of the panels of markers used to determine the acute rejection status in patients is equal to or greater than 4% and the NPV of the panels is equal to or greater than 98%.

[0096] The present invention is described by reference to the following Examples. Standard techniques well known in the art or the techniques specifically described below were utilized.EXAMPLESEXAMPLE 1. NUCLEIC ACID AND PROTEIN MARKERS FOR DIAGNOSING TRANSPLANT REJECTION.

[0097] The development of the biomarker panel in determining the acute rejection status of a patient involves three phases: a biomarker discovery phase, a biomarker replication phase, and an assay migration and validation phase. In the biomarker discovery phase: 65 heart transplant patients were recruited from a single site (Vancouver, Canada). Nucleic acid expression of over 36,000 nucleic acid markers were analyzed using Affymetrix microarrays, HTG EdgeSeq, and NanoString nCounter technology. Over 200 proteomic markers in plasma were analyzed using mass spectrometry and ELISA. Panels of nucleic acid markers or proteomic markers with an area under the receiver operating characteristics curve (AUC) above 0.8 were moved to the biomarker replication phase.

[0098] In the biomarker replication phase: 84 heart transplant patients were recruited from eight enrolling sites across Canada. Nucleic acid expression and proteomic expression were performed on the markers identified in the discovery phase with the same technologies. Over 99% negative predictive value (NPV) was achieved for panels of nucleic acid markers and panels of proteomic panels. The best performing panels were selected for development in the assay migration and validation phase.

[0099] In the assay migration and validation phase, panels of markers identified in previous phases were migrated into clinically-amenable technologies, e.g., the HTG Molecular qNPA (quantitative nuclease protection assay) platform for detection of nucleic acid expression. The HTG Edge System is a desired platform for clinical assay development because it is fully-automated, which greatly simplifies laboratory workflow; and it requires small sample input and minimal hands-on time. Over 100 patients (and 350 samples) were collected through the 8 pan-Canadian sites for testing in this stage, in which 40 mRNA markers (Table 3) were tested. See Table 3. In the initial testing on the multiplex HTG study, a panel of 10 mRNA markers (Table 5) was identified and its performance in determining the acute rejection status is discussed in Example 2. The mRNA markers identified herein participate in a range of biological processes: cellular and humoral immune responses, acute phase inflammatory pathways, proliferation, chemotaxis, development, cell adhesion, apoptosis, signal transduction, cell cycle, and reproduction. See Figure 1.

[0100] Six proteomic markers (Table 4), originally identified by MS technologies, were also confirmed by immunoassays (ELISAs) to be suitable as markers for determining the acute rejection status. The performance of the protein panel comprising these six proteomic markers is described in Example 2. These proteomic markers participate in a range of biological processes, including cell adhesion, transport, blood coagulation, and inflammation. See Figure 2. These proteomic markers, along with housekeeping genes, will be migrated onto a multiplexed, immuno-based microfluidics point-of-care platform for further testing and validation. Table 3. 40 nucleic acid markers.SymbolGene NameCD177CD177 moleculeCPA3carboxypeptidase A3 (mast cell)HEBP 1heme binding protein 1ORM 1orosomucoid 1VNN1vanin 1CNTNAP3contactin associated protein-like 3ADD2adducin 2 (beta)AKAP12A kinase (PRKA) anchor protein 12APOBEC3Capolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3CCTSEcathepsin EKELKell blood groUp, metallo-endopeptidaseLTBlymphotoxin-beta isoform aRNF5ring finger protein 5UBL7ubiquitin-like 7 (bone marrow stromal cell-derived)HCLS 1hematopoietic cell-specific Lyn substrate 1PSTPIP1proline-serine-threonine phosphatase interacting protein 1ENO1BH3 interacting domain death agonistIL16Interleukin 16 (lymphocyte chemoattractant factor)LOC284454Hypothetical protein LOC284454RAB37RAB37, member RAS oncogene familyTIMP1TIMP metallopeptidase inhibitor 1C3complement component 3CD44CD44 molecule (Indian blood group)FCER1GFc fragment of IgE, high affinity I, receptor for; gamma polypeptideIFIT2interferon-induced protein with tetratricopeptide repeats 2IKZF 1IKAROS family zinc finger 1 (Ikaros)IL4Rinterleukin 4 receptorLILRA5leukocyte immunoglobulin-like receptor, subfamily A (with TM domain), member 5NFKB1nuclear factor of kappa light polypeptide gene enhancer in B-cells 1SERPING1serpin peptidase inhibitor, clade G (C1 inhibitor), member 1SOCS3suppressor of cytokine signaling 3STAT5Bsignal transducer and activator of transcription 5BBTKBruton agammaglobulinemia tyrosine kinaseCD99CD99 moleculeCTNNB1catenin (cadherin-associated protein), beta 1, 88kDaIRF7interferon regulatory factor 7LTBRLymphotoxin Beta ReceptorNFKBIZnuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zetaSTAT5Asignal transducer and activator of transcription 5ASYKspleen tyrosine kinaseSymbolProtein NameFXIIIFactor XIIIFVFactor VCFIComplement factor IADPAdiponectinPLTPPhospholipid transfer proteinPROCREndothelial protein C receptor Table 4. Proteomics markers. FXIIIFactor XIII FVFactor VCFIComplement factor IADPAdiponectinPLTPPhospholipid transfer proteinPROCREndothelial protein C receptor EXAMPLE 2. INITIAL BIOMARKER PERFORMANCE ON HTG mRNA ASSAY AND ELISA.

[0101] 37 banked samples were used in the initial assay migration and validation phase study. 14 of them were previously diagnosed with acute rejection status (AR), and 23 with no rejection status (NR). The panel of 10 nucleic acid markers in Table 5 was assayed using the multiplex HTG mRNA assay and the panel of six proteomic markers in Table 4 were assayed using ELISA kits. Table 5. 10 nucleic acid markers.HEBP 1Heme binding protein 1ORM 1Orosomucoid 1IL4RInterleukin 4 receptorCD44CD44 molecule (Indian blood group)SERPING1serpin peptidase inhibitor, clade G (C1 inhibitor), member 1FCER1GFc fragment of IgE, high affinity I, receptor for; gamma polypeptideC3complement component 3NFKB1nuclear factor of kappa light polypeptide gene enhancer in B-cells 1LTBRLymphotoxin Beta ReceptorBTKBruton agammaglobulinemia tyrosine kinase

[0102] The results show that the assay, which employs a panel comprising the 10 nucleic acid markers to determine the acute rejection status in a patient, had a sensitivity of 100%, a specificity of 74%. This indicates that by using only 10 mRNA measurements on the HTG assay, those samples from patients without acute rejection, i.e. non rejectors (NR) and moderate rejectors (MR), can be identified 100% of the time; and samples from patients who had acute rejection, i.e. acute rejectors, can be identified 74% of the time. The assay using the panel showed a positive predictive value (PPV) of 14%, a negative predictive value (NPV) of 100%, and an AUC of 0.85. The panel comprising the six proteomic markers had a sensitivity of 100% and a specificity of 48%. The PPV for the panel was 7%, and NPV was 100%. The AUC for the panel was 0.62. The result also shows that the a biomarker panel combining the 6 proteomic markers and the 10 nucleic acid markers through computational methods improved the specificity of the HTG assay using the 10 nucleic acid markers alone, from 74% to 91%. See Table 6. Table 6. Performance of the diagnostic biomarker panels.PanelsensitivitySpecificityPPVNPVAUC10 mRNAs100%74%14%100%0.856 proteins100%48%7%100%0.62combined100%91%32%100%0.91 EXAMPLE 3. INITIAL BIOMARKER PERFORMANCE ON NANO STRING NCOUNTER.

[0103] A panel consisting of the 6 nucleic acid markers in Table 7 was tested in two different cohorts using the NanoString nCounter technology. The first is the recalibration cohort, in which the 6 nucleic acid marker panel was tested on samples from 38 subjects. 15 subjects had acute rejection and 23 had no rejection or moderate rejection to heart transplant. The second is the replication cohort, in which the panel of the 6 nucleic acid makers was tested on samples from 126 subjects, of which 22 had acute rejection and 104 had no rejection or moderate rejection.

[0104] The results (Table 8) show that the assay used in the recalibration cohort had a sensitivity of 100%, a specificity of 22%, a PPV of 5%, and a NPV of 100%. The assay used in the replication cohort had a sensitivity of 91%, a specificity of 15%, a PPV of 4%, and a NPV of 98%. Table 7. 6 nucleic acid markers.Gene SymbolGene nameHEBP1Heme binding protein 1CD 177CD 177 moleculeCPA3Carboxypeptidase A3 (Mast Cell)VNN1Vanin 1ORM1Orosomucoid 1CNTNAP3Contactin associated protein-like 3 Table 8. The performance of the 6 nucleic acid markers. PanelCohortSensitivitySpecificityPPVNPV6 mRNAsRecalibration100%22%5%100%Replication91%15%4%98%

Claims

1. A method of determining the acute rejection status of a heart transplant in a subject using a biomarker panel comprising nucleic acid markers, the method comprising the steps of: a. determining the nucleic acid expression profile of nucleic acid markers in a biological sample from the subject, wherein the nucleic acid markers comprise or consist of CD177, CPA3, HEBP1, ORM1, VNN 1 and CNTNAP3, b. comparing the nucleic acid expression profile to a control profile, c. determining whether expression of the nucleic acid markers is increased or decreased relative to the control profile, wherein the increased or decreased expression of the nucleic acid markers is indicative of the acute rejection status of the subject.

2. The method of claim 1, wherein the step (a) further comprises determining the nucleic acid expression profile of housekeeping genes, wherein the housekeeping genes are selected from a group consisting of ACTB, ANT, B2M, OAZ1, RPL11, and SDHA.

3. The method of claim 2, wherein the housekeeping genes are ACTB, B2M, and OAZ 1.

4. The method of any one of claims 1 to 3, wherein step (b) comprises comparing a concentration of the nucleic acid markers to a non-rejector cutoff index.

5. The method of any one of claims 1 to 4, wherein the biomarker panel has a sensitivity of 90%, a specificity of at least 15%, a positive predictive value (PPV) of at least 2%, and a negative predictive value (NPV) of at least 95%, in determining the acute rejection status of heart transplant of the subject.

6. The method of any one of claims 1 to 5, wherein the biological sample is blood.