# US 10,870,888 B2 · Methods and systems for analysis of organ transplantation

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- Number: US 10,870,888 B2
- Document ID: US10870888B2
- Status: Granted
- Kind: B2
- Application number: 15/898,513
- Filed: 2018-02-17
- Published: 2018-12-27
- Granted: 2020-12-22
- Earliest claimed: 2013-09-09
- Assignees: The Scripps Research Institute; Northwestern University
- Applicants: The Scripps Research Institute; Northwestern University
- Inventors: Daniel Salomon; Sunil M. Kurian; Steven Head; Michael M. Abecassis; John J. Friedewald; Josh Levitsky
- Web page: https://patentgrep.com/patent/US10870888B2

## CPC

- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G16B 25/00: ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B 25/10: Gene or protein expression profiling; Expression-ratio estimation or normalisation
- G16B 40/00: ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
- G16B 40/20: Supervised data analysis
- G16H 20/40: relating to mechanical, radiation or invasive therapies, e.g. surgery, laser therapy, dialysis or acupuncture
- G16H 50/30: for calculating health indices; for individual health risk assessment
- C12Q 2537/165: Mathematical modelling, e.g. logarithm, ratio
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- G01N 2800/245: Transplantation related diseases, e.g. graft versus host disease
- Y02A 90/10: Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16H: HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed herein are methods of detecting, predicting or monitoring a status or outcome of a transplant in a transplant recipient.

## Claims

### Claim 1

A method of detecting gene expression products and treating acute rejection in a transplant recipient on an immunosuppressive drug, the method comprising:
(a) obtaining a blood sample, wherein the blood sample comprises mRNA from the transplant recipient on an immunosuppressive drug or DNA complements of mRNA from the transplant recipient on an immunosuppressive drug;
(b) performing a microarray assay or sequencing assay to determine an expression level of the mRNA from the transplant recipient on an immunosuppressive drug or DNA complements of mRNA from the transplant recipient on an immunosuppressive drug;
(c) detecting or predicting acute rejection in the transplant recipient by applying a trained algorithm to the expression level determined in step (b), wherein the trained algorithm comprises one or more classifiers, wherein the trained algorithm is trained on 25 or more genes selected from the group consisting of SMARCB1, STP6AP2, PLEKHA3, TP53, PRKAG2, ND2, YWHAE, SSR1, CCDC91, PRKAG2, ADPGK, CHFR, PRNP, CDYL, SPTLC1, CHMP1B, KBTBD2, MST4, CDC42EP3, UBXN4, PGGT1B, CCR2, ZNF426, SP1, MED18, FLI1, LUC7L2, CD46, CASKIN1, UGP2, ARNTL, PLEKHF2, ZNF117, ITGB1, MAP3K1, FBXW7, SRGAP2B, ZNF136, ARPC5, FBXL18, TMED2, YWHAZ, SNAP23, TMEM30A, CD46, INSIG1, SRSF2, API5, FLI1, SLC35E1, PAPOLA, MLLT10, SLC35B4, ARL2BP, CNOT8, SYPL1, BRWD3, RBBP4, GLCCI1, MBP, RAB8B, YTHDF3, TOR1AIP1, ATF1, TMEM230, LRCH3, ARID2, CNOT8, ZBTB20, PSMD10, RHOP, ATF71P, LOC100131541, STYX, CN1H4, EMC1, MACF1, NOP14-AS1, HHEX, DHX36, DAPP1, WTAP, PSEN1, MAP3K7, PPP2R5E, FAM108B1, TAB2, FAR1, METTL21A, FYTTD1, TMED2, TROVE2, LNPEP, CAB39, RC3H2, SLBP, SNAP23, VWA9, MGAT2, OCIAD1, SH2D1B, PEX7, CD4, BZW1, RNF125, GNL3L, TRMT10B, RHEB, TMF1, STAG3L1, WSB1, PDE4B, ARID2, ATXN3, CUL4B, PRMT2, CTSS, ANAPC2, GLUD1, SGPP1, FAM178A, 2-Sep, KCTD5, ZEB1, RAC1, UVSSA, RBM4, ALKBH7, 214405_PM_at_EST1, SIVA1, 214482_PM_at_EST2, CHD2, POLR2C, 1556865_PM_at_EST3, GTF2H4, RNF25, DICER1, C9orf86, C5orf24, KIDINS220, ADPGK, HNRNPA0, 236237_PM_at_EST4, BAX, MBNL1, RAB5A, POLR2G, CELF2, 1561909_PM_at_EST5, FNBP1, FOXO3 /// FOXO3B, 233303_PM_at_EST6, 244219_PM_at_EST7, R3HCC1, DLST, CALM3, U2AF1, ANTXR2, LILRA4, TLE4, SMARCC1, BET1L, VAMP2, MRPS18A, KLF7, 242726_PM_at_EST8, USP34, APPL2, 240991_PM_at_EST9, NCR3, TXN1P, SDHAF1, HECTD1, C6orf62, 243751_PM_at_EST10, ATAD2B, MLL3, ATF6, Clorf128, SERPINF1, PAFAH1B1, C2ORF7, FOXO3 /// FOXO3B, CHMP4A, ANXA11, TAF10, DLEU2, 15622_PM_at_EST11, SEPT9, RBM5, MGEA5, TRAF4, LOC100132707, OTUD4, KLHL12, MED1, 216112_PM_at_EST12, ACSL4, ATXN7L3b, DCTN3, MGEA4, MS4A6A, CCDC69, H2AFY, TMEM165, INO80D, PSEN1, C15orf17, RBM27, GZMM, ATP6, ZDHHC1, TLE4, SDHC, HNRNPUL1, SF3A2, 236545_PM_at_EST13, CDH23, LOC100506866, C4orf48, EMD, NEK1, HNRNPM, 241240_PM_at_EST15, LIMD2, THOC2, 243046_PM_at_EST16, BAT2L2, ZCCHC6, 228723_PM_at_EST17, 242695_PM_at_EST18, PLEC, U1MC1, LRRFIP1, 1556055_PM_at_EST19, AFFX-, M27830_5_at_EST20, GRB10, CD84, KLHDC3, BAX, NRD1, RASA2, 1559589_PM_a_at_EST21, MTPAP, ZFP36L2, AGPAT1, KIDINS220, ISOC2, KLF7, MED27, TLR4, ABHD14B, 244349_PM_at_EST22, 244418_PM_at_EST23, MLXIP, PPID, MRPL43, ND3 /// SH3KBP1, TNRC6B, KLHL28, SLC25A37, NELF, FARS2, ZCCHC6, TRIM25, TAF15, LTB, 221995_PM_s_at_EST24, IFNGR1, 228826_PM_at_EST25, C7orf16, NEDD9, NDUFA7, ZC3H11A, DNAJA4, ERICH1, RALB, MPZL1, EZR, RBKS, TMOD1 /// TSTD2, LOC729683, PTK2B, APBB11P, UBXN4, SETD5, 243032_PM_at_EST26, 216380_PM_x_at_EST27, TRAPPC4, CKB, CANX, 1558624_PM_at_EST28, SIVA1, 240652_PM_at_EST29, HOXA1, C11orf49, SLC11A1, C9orf86, IVNS1ABP, PDXP /// SH3BP1, CHTF8, FKBP5, FGFR1OP2, SECISBP2L, ARF1, B4GALT1, PHF19, C16orf5, C11orf59, PIP4K2A, GCLC, EMILIN2, NGLY1, ANKHD1 /// ANKHD1-EIF4EBP3, DLEU2, PBLS, RALGAPA2, 230733_PM_at_EST30, 1557804_PM_at_EST31, PKN2, and GGNBP2, wherein the trained algorithm is capable of distinguishing between acute rejection and acute dysfunction with no rejection, and wherein the trained algorithm has a negative predictive value of greater than 70%; and
(d) administering a higher dosage of the immunosuppressive drug or administering a new immunosuppressive drug in order to treat or prevent the acute rejection detected or predicted in the transplant recipient in step (c).

### Claim 2 (depends on 1)

The method of claim 1, wherein the trained algorithm comprises a linear classifier.

### Claim 3 (depends on 2)

The method of claim 2, wherein the linear classifier comprises one or more of linear discriminant analysis, Fisher's linear discriminant, Nave Bayes classifier, Logistic regression, Perceptron, Support vector machine (SVM) or a combination thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the trained algorithm comprises a Diagonal Linear Discriminant Analysis (DLDA) algorithm.

### Claim 5 (depends on 1)

The method of claim 1, wherein the trained algorithm comprises a Nearest Centroid algorithm.

### Claim 6 (depends on 1)

The method of claim 1, wherein the trained algorithm comprises a Random Forest algorithm or statistical bootstrapping.

### Claim 7 (depends on 1)

The method of claim 1, wherein the trained algorithm comprises a Prediction Analysis of Microarrays (PAM) algorithm.

### Claim 8 (depends on 1)

The method of claim 1, wherein the trained algorithm is not validated by a cohort-based analysis of an entire cohort.

### Claim 9 (depends on 1)

The method of claim 1, wherein the determining the expression level in step (b) comprises determining the expression level of 25 or more gene expression products with different sequences.

### Claim 10 (depends on 9)

The method of claim 9, wherein the 25 or more gene expression products correspond to less than 200 genes listed selected from the group consisting of SMARCB1, STP6AP2, PLEKHA3, TP53, PRKAG2, ND2, YWHAE, SSR1, CCDC91, PRKAG2, ADPGK, CHFR, PRNP, CDYL, SPTLC1, CHMP1B, KBTBD2, MST4, CDC42EP3, UBXN4, PGGT1B, CCR2, ZNF426, SP1, MED18, FLI1, LUC7L2, CD46, CASKIN1, UGP2, ARNTL, PLEKHF2, ZNF117, ITGB1, MAP3K1, FBXW7, SRGAP2B, ZNF136, ARPC5, FBXL18, TMED2, YWHAZ, SNAP23, TMEM30A, CD46, INSIG1, SRSF2, API5, FLI1, SLC35E1, PAPOLA, MLLT10, SLC35B4, ARL2BP, CNOT8, SYPL1, BRWD3, RBBP4, GLCCI1, MBP, RAB8B, YTHDF3, TOR1AIP1, ATF1, TMEM230, LRCH3, ARID2, CNOT8, ZBTB20, PSMD10, RHOP, ATF71P, LOC100131541, STYX, CN1H4, EMC1, MACF1, NOP14-AS1, HHEX, DHX36, DAPP1, WTAP, PSEN1, MAP3K7, PPP2R5E, FAM108B1, TAB2, FAR1, METTL21A, FYTTD1, TMED2, TROVE2, LNPEP, CAB39, RC3H2, SLBP, SNAP23, VWA9, MGAT2, OCIAD1, SH2D1B, PEX7, CD4, BZW1, RNF125, GNL3L, TRMT10B, RHEB, TMF1, STAG3L1, WSB1, PDE4B, ARID2, ATXN3, CUL4B, PRMT2, CTSS, ANAPC2, GLUD1, SGPP1, FAM178A, 2-Sep, KCTD5, ZEB1, RAC1, UVSSA, RBM4, ALKBH7, 214405_PM_at_EST1, SIVA1, 214482_PM_at_EST2, CHD2, POLR2C, 1556865_PM_at_EST3, GTF2H4, RNF25, DICER1, C9orf86, C5orf24, KIDINS220, ADPGK, HNRNPA0, 236237_PM_at_EST4, BAX, MBNL1, RAB5A, POLR2G, CELF2, 1561909_PM_at_EST5, FNBP1, FOXO3 /// FOXO3B, 233303_PM_at_EST6, 244219_PM_at_EST7, R3HCC1, DLST, CALM3, U2AF1, ANTXR2, LILRA4, TLE4, SMARCC1, BET1L, VAMP2, MRPS18A, KLF7, 242726_PM_at_EST8, USP34, APPL2, 240991_PM_at_EST9, NCR3, TXN1P, SDHAF1, HECTD1, C6orf62, 243751_PM_at_EST10, ATAD2B, MLL3, ATF6, Clorf128, SERPINF1, PAFAH1B1, C2ORF7, FOXO3 /// FOXO3B, CHMP4A, ANXA11, TAF10, DLEU2, 15622_PM_at_EST11, SEPT9, RBM5, MGEA5, TRAF4, LOC100132707, OTUD4, KLHL12, MED1, 216112_PM_at_EST12, ACSL4, ATXN7L3b, DCTN3, MGEA4, MS4A6A, CCDC69, H2AFY, TMEM165, INO80D, PSEN1, C15orf17, RBM27, GZMM, ATP6, ZDHHC1, TLE4, SDHC, HNRNPUL1, SF3A2, 236545_PM_at_EST13, CDH23, LOC100506866, C4orf48, EMD, NEK1, HNRNPM, 241240_PM_at_EST15, LIMD2, THOC2, 243046_PM_at_EST16, BAT2L2, ZCCHC6, 228723_PM_at_EST17, 242695_PM_at_EST18, PLEC, U1MC1, LRRFIP1, 1556055_PM_at_EST19, AFFX-, M27830_5_at_EST20, GRB10, CD84, KLHDC3, BAX, NRD1, RASA2, 1559589_PM_a_at_EST21, MTPAP, ZFP36L2, AGPAT1, KIDINS220, ISOC2, KLF7, MED27, TLR4, ABHD14B, 244349_PM_at_EST22, 244418_PM_at_EST23, MLXIP, PPID, MRPL43, ND3 /// SH3KBP1, TNRC6B, KLHL28, SLC25A37, NELF, FARS2, ZCCHC6, TRIM25, TAF15, LTB, 221995_PM_s_at_EST24, IFNGR1, 228826_PM_at_EST25, C7orf16, NEDD9, NDUFA7, ZC3H11A, DNAJA4, ERICH1, RALB, MPZL1, EZR, RBKS, TMOD1 /// TSTD2, LOC729683, PTK2B, APBB11P, UBXN4, SETD5, 243032_PM_at_EST26, 216380_PM_x_at_EST27, TRAPPC4, CKB, CANX, 1558624_PM_at_EST28, SIVA1, 240652_PM_at_EST29, HOXA1, C11orf49, SLC11A1, C9orf86, IVNS1ABP, PDXP /// SH3BP1, CHTF8, FKBP5, FGFR1OP2, SECISBP2L, ARF1, B4GALT1, PHF19, C16orf5, C11orf59, PIP4K2A, GCLC, EMILIN2, NGLY1, ANKHD1 /// ANKHD1-EIF4EBP3, DLEU2, PBLS, RALGAPA2, 230733_PM_at_EST30, 1557804_PM_at_EST31, PKN2, and GGNBP2.

### Claim 11 (depends on 1)

The method of claim 1, wherein the blood sample is a peripheral blood sample.

### Claim 12 (depends on 1)

The method of claim 1, wherein the blood sample is a whole blood sample.

### Claim 13 (depends on 1)

The method of claim 1, wherein the blood sample is not derived from tissue from a biopsy of a transplanted organ of the transplant recipient on an immunosuppressive drug.

### Claim 14 (depends on 1)

The method of claim 1, wherein the performing a microarray assay or sequencing assay in step (b) comprises performing an RNA sequencing assay on the mRNA from the transplant recipient on an immunosuppressive drug.

### Claim 15 (depends on 1)

The method of claim 1, wherein the performing a microarray assay or sequencing assay comprises performing a DNA sequencing assay on the DNA complements of mRNA from the transplant recipient on an immunosuppressive drug.

### Claim 16 (depends on 1)

The method of claim 1, wherein the determining the expression level in step (b) comprises determining the expression level of 25 or more genes selected from the group consisting of SMARCB1, STP6AP2, PLEKHA3, TP53, PRKAG2, ND2, YWHAE, SSR1, CCDC91, PRKAG2, ADPGK, CHFR, PRNP, CDYL, SPTLC1, CHMP1B, KBTBD2, MST4, CDC42EP3, UBXN4, PGGT1B, CCR2, ZNF426, SP1, MED18, FLI1, LUC7L2, CD46, CASKIN1, UGP2, ARNTL, PLEKHF2, ZNF117, ITGB1, MAP3K1, FBXW7, SRGAP2B, ZNF136, ARPC5, FBXL18, TMED2, YWHAZ, SNAP23, TMEM30A, CD46, INSIG1, SRSF2, API5, FLI1, SLC35E1, PAPOLA, MLLT10, SLC35B4, ARL2BP, CNOT8, SYPL1, BRWD3, RBBP4, GLCCI1, MBP, RAB8B, YTHDF3, TOR1AIP1, ATF1, TMEM230, LRCH3, ARID2, CNOT8, ZBTB20, PSMD10, RHOP, ATF71P, LOC100131541, STYX, CN1H4, EMC1, MACF1, NOP14-AS1, HHEX, DHX36, DAPP1, WTAP, PSEN1, MAP3K7, PPP2R5E, FAM108B1, TAB2, FAR1, METTL21A, FYTTD1, TMED2, TROVE2, LNPEP, CAB39, RC3H2, SLBP, SNAP23, VWA9, MGAT2, OCIAD1, SH2D1B, PEX7, CD4, BZW1, RNF125, GNL3L, TRMT10B, RHEB, TMF1, STAG3L1, WSB1, PDE4B, ARID2, ATXN3, CUL4B, PRMT2, CTSS, ANAPC2, GLUD1, SGPP1, FAM178A, 2-Sep, KCTD5, ZEB1, RAC1, UVSSA, RBM4, ALKBH7, 214405_PM_at_EST1, SIVA1, 214482_PM_at_EST2, CHD2, POLR2C, 1556865_PM_at_EST3, GTF2H4, RNF25, DICER1, C9orf86, C5orf24, KIDINS220, ADPGK, HNRNPA0, 236237_PM_at_EST4, BAX, MBNL1, RAB5A, POLR2G, CELF2, 1561909_PM_at_EST5, FNBP1, FOXO3 /// FOXO3B, 233303_PM_at_EST6, 244219_PM_at_EST7, R3HCC1, DLST, CALM3, U2AF1, ANTXR2, LILRA4, TLE4, SMARCC1, BET1L, VAMP2, MRPS18A, KLF7, 242726_PM_at_EST8, USP34, APPL2, 240991_PM_at_EST9, NCR3, TXN1P, SDHAF1, HECTD1, C6orf62, 243751_PM_at_EST10, ATAD2B, MLL3, ATF6, Clorf128, SERPINF1, PAFAH1B1, C2ORF7, FOXO3 /// FOXO3B, CHMP4A, ANXA11, TAF10, DLEU2, 15622_PM_at_EST11, SEPT9, RBM5, MGEA5, TRAF4, LOC100132707, OTUD4, KLHL12, MED1, 216112_PM_at_EST12, ACSL4, ATXN7L3b, DCTN3, MGEA4, MS4A6A, CCDC69, H2AFY, TMEM165, INO80D, PSEN1, C15orf17, RBM27, GZMM, ATP6, ZDHHC1, TLE4, SDHC, HNRNPUL1, SF3A2, 236545_PM_at_EST13, CDH23, LOC100506866, C4orf48, EMD, NEK1, HNRNPM, 241240_PM_at_EST15, LIMD2, THOC2, 243046_PM_at_EST16, BAT2L2, ZCCHC6, 228723_PM_at_EST17, 242695_PM_at_EST18, PLEC, U1MC1, LRRFIP1, 1556055_PM_at_EST19, AFFX-, M27830_5_at_EST20, GRB10, CD84, KLHDC3, BAX, NRD1, RASA2, 1559589_PM_a_at_EST21, MTPAP, ZFP36L2, AGPAT1, KIDINS220, ISOC2, KLF7, MED27, TLR4, ABHD14B, 244349_PM_at_EST22, 244418_PM_at_EST23, MLXIP, PPID, MRPL43, ND3 /// SH3KBP1, TNRC6B, KLHL28, SLC25A37, NELF, FARS2, ZCCHC6, TRIM25, TAF15, LTB, 221995_PM_s_at_EST24, IFNGR1, 228826_PM_at_EST25, C7orf16, NEDD9, NDUFA7, ZC3H11A, DNAJA4, ERICH1, RALB, MPZL1, EZR, RBKS, TMOD1 /// TSTD2, LOC729683, PTK2B, APBB11P, UBXN4, SETD5, 243032_PM_at_EST26, 216380_PM_x_at_EST27, TRAPPC4, CKB, CANX, 1558624_PM_at_EST28, SIVA1, 240652_PM_at_EST29, HOXA1, C11orf49, SLC11A1, C9orf86, IVNS1ABP, PDXP /// SH3BP1, CHTF8, FKBP5, FGFR1OP2, SECISBP2L, ARF1, B4GALT1, PHF19, C16orf5, C11orf59, PIP4K2A, GCLC, EMILIN2, NGLY1, ANKHD1 /// ANKHD1-EIF4EBP3, DLEU2, PBLS, RALGAPA2, 230733_PM_at_EST30, 1557804_PM_at_EST31, PKN2, and GGNBP2.

### Claim 17 (depends on 1)

The method of claim 1, wherein the method has a sensitivity of at least about 80%.

### Claim 18 (depends on 1)

The method of claim 1, wherein the method has a specificity of at least about 80%.

### Claim 19 (depends on 1)

The method of claim 1, wherein the transplant recipient on an immunosuppressive drug has a serum creatinine level of at least 1.5 mg/dL.

### Claim 20 (depends on 1)

The method of claim 1, wherein the transplant recipient on an immunosuppressive drug has a serum creatinine level of at least 3 mg/dL.

### Claim 21 (depends on 1)

The method of claim 1, wherein the transplant recipient is a kidney transplant recipient.

### Claim 22 (depends on 1)

The method of claim 1, wherein the trained algorithm is further capable of distinguishing normal transplant functioning from acute rejection and from acute dysfunction with no rejection.

### Claim 23 (depends on 1)

The method of claim 1, wherein the method comprises administering an increased dose of the immunosuppressive drug to the transplant recipient in order to treat or prevent the acute rejection detected or predicted in the transplant recipient in step (c).

### Claim 24 (depends on 1)

The method of claim 1, wherein the method comprises administering a new immunosuppressive drug to the transplant recipient in order to treat or prevent the acute rejection detected or predicted in the transplant recipient in step (c).

### Claim 25 (depends on 1)

The method of claim 1, wherein the immunosuppressive drug or new immunosuppressive drug is selected from the group consisting of a calcineurin inhibitor, an mTOR inhibitor, an anti-proliferative, a corticosteroid, and an anti-T-cell antibody.

### Claim 26 (depends on 1)

The method of claim 1, wherein the immunosuppressive drug or new immunosuppressive drug is selected from the group consisting of cyclosporine, tacrolimus, azathioprine, mycophenolic acid, prednisolone, hydrocortisone, basiliximab, daclizumab, Orthoclone, anti-thymocyte globulin, and anti-lymphocyte globulin.

### Claim 27 (depends on 1)

The method of claim 1, wherein the detecting or predicting comprises diagnosing the acute rejection in the transplant recipient.

### Claim 28 (depends on 1)

The method of claim 1, wherein the detecting or predicting comprises detecting the acute rejection in the transplant recipient.

### Claim 29 (depends on 1)

The method of claim 1, wherein the detecting or predicting comprises predicting a risk of acute rejection in the transplant recipient.

### Claim 30

A method of detecting gene expression products and treating acute rejection in a transplant recipient on an immunosuppressive drug, the method comprising:
(a) obtaining a blood sample, wherein the blood sample comprises mRNA from the transplant recipient or DNA complements of mRNA from the transplant recipient;
(b) performing a microarray assay or sequencing assay to determine an expression level of mRNA from the transplant recipient or DNA complements of mRNA from the transplant recipient; and
(c) detecting or predicting acute rejection in the transplant recipient by applying a trained algorithm to the expression level determined in step (b), wherein the trained algorithm is trained on 25 or more genes selected from the group consisting of SMARCB1, STP6AP2, PLEKHA3, TP53, PRKAG2, ND2, YWHAE, SSR1, CCDC91, PRKAG2, ADPGK, CHFR, PRNP, CDYL, SPTLC1, CHMP1B, KBTBD2, MST4, CDC42EP3, UBXN4, PGGT1B, CCR2, ZNF426, SP1, MED18, FLI1, LUC7L2, CD46, CASKIN1, UGP2, ARNTL, PLEKHF2, ZNF117, ITGB1, MAP3K1, FBXW7, SRGAP2B, ZNF136, ARPC5, FBXL18, TMED2, YWHAZ, SNAP23, TMEM30A, CD46, INSIG1, SRSF2, API5, FLI1, SLC35E1, PAPOLA, MLLT10, SLC35B4, ARL2BP, CNOT8, SYPL1, BRWD3, RBBP4, GLCCI1, MBP, RAB8B, YTHDF3, TOR1AIP1, ATF1, TMEM230, LRCH3, ARID2, CNOT8, ZBTB20, PSMD10, RHOP, ATF71P, LOC100131541, STYX, CN1H4, EMC1, MACF1, NOP14-AS1, HHEX, DHX36, DAPP1, WTAP, PSEN1, MAP3K7, PPP2R5E, FAM108B1, TAB2, FAR1, METTL21A, FYTTD1, TMED2, TROVE2, LNPEP, CAB39, RC3H2, SLBP, SNAP23, VWA9, MGAT2, OCIAD1, SH2D1B, PEX7, CD4, BZW1, RNF125, GNL3L, TRMT10B, RHEB, TMF1, STAG3L1, WSB1, PDE4B, ARID2, ATXN3, CUL4B, PRMT2, CTSS, ANAPC2, GLUD1, SGPP1, FAM178A, 2-Sep, KCTD5, ZEB1, RAC1, UVSSA, RBM4, ALKBH7, 214405_PM_at_EST1, SIVA1, 214482_PM_at_EST2, CHD2, POLR2C, 1556865_PM_at_EST3, GTF2H4, RNF25, DICER1, C9orf86, C5orf24, KIDINS220, ADPGK, HNRNPA0, 236237_PM_at_EST4, BAX, MBNL1, RAB5A, POLR2G, CELF2, 1561909_PM_at_EST5, FNBP1, FOXO3 /// FOXO3B, 233303_PM_at_EST6, 244219_PM_at_EST7, R3HCC1, DLST, CALM3, U2AF1, ANTXR2, LILRA4, TLE4, SMARCC1, BET1L, VAMP2, MRPS18A, KLF7, 242726_PM_at_EST8, USP34, APPL2, 240991_PM_at_EST9, NCR3, TXN1P, SDHAF1, HECTD1, C6orf62, 243751_PM_at_EST10, ATAD2B, MLL3, ATF6, Clorf128, SERPINF1, PAFAH1B1, C2ORF7, FOXO3 /// FOXO3B, CHMP4A, ANXA11, TAF10, DLEU2, 15622_PM_at_EST11, SEPT9, RBM5, MGEA5, TRAF4, LOC100132707, OTUD4, KLHL12, MED1, 216112_PM_at_EST12, ACSL4, ATXN7L3b, DCTN3, MGEA4, MS4A6A, CCDC69, H2AFY, TMEM165, INO80D, PSEN1, C15orf17, RBM27, GZMM, ATP6, ZDHHC1, TLE4, SDHC, HNRNPUL1, SF3A2, 236545_PM_at_EST13, CDH23, LOC100506866, C4orf48, EMD, NEK1, HNRNPM, 241240_PM_at_EST15, LIMD2, THOC2, 243046_PM_at_EST16, BAT2L2, ZCCHC6, 228723_PM_at_EST17, 242695_PM_at_EST18, PLEC, U1MC1, LRRFIP1, 1556055_PM_at_EST19, AFFX-, M27830_5_at_EST20, GRB10, CD84, KLHDC3, BAX, NRD1, RASA2, 1559589_PM_a_at_EST21, MTPAP, ZFP36L2, AGPAT1, KIDINS220, ISOC2, KLF7, MED27, TLR4, ABHD14B, 244349_PM_at_EST22, 244418_PM_at_EST23, MLXIP, PPID, MRPL43, ND3 /// SH3KBP1, TNRC6B, KLHL28, SLC25A37, NELF, FARS2, ZCCHC6, TRIM25, TAF15, LTB, 221995_PM_s_at_EST24, IFNGR1, 228826_PM_at_EST25, C7orf16, NEDD9, NDUFA7, ZC3H11A, DNAJA4, ERICH1, RALB, MPZL1, EZR, RBKS, TMOD1 /// TSTD2, LOC729683, PTK2B, APBB11P, UBXN4, SETD5, 243032_PM_at_EST26, 216380_PM_x_at_EST27, TRAPPC4, CKB, CANX, 1558624_PM_at_EST28, SIVA1, 240652_PM_at_EST29, HOXA1, C11orf49, SLC11A1, C9orf86, IVNS1ABP, PDXP /// SH3BP1, CHTF8, FKBP5, FGFR1OP2, SECISBP2L, ARF1, B4GALT1, PHF19, C16orf5, C11orf59, PIP4K2A, GCLC, EMILIN2, NGLY1, ANKHD1 /// ANKHD1-EIF4EBP3, DLEU2, PBLS, RALGAPA2, 230733_PM_at_EST30, 1557804_PM_at_EST31, PKN2, and GGNBP2, wherein the trained algorithm is a three-way classifier capable of distinguishing between at least three conditions, and wherein one of the at least three conditions is acute rejection, wherein one of the at least three conditions is acute dysfunction with no rejection, and wherein the trained algorithm has a negative predictive value of greater than 70%; and
(d) administering a higher dosage of the immunosuppressive drug or administering a new immunosuppressive drug in order to treat the acute rejection detected or predicted in the transplant recipient in step (c).

### Claim 31 (depends on 30)

The method of claim 30, wherein a frozen robust multichip average (fRMA) algorithm is used to produce normalized expression level data in (b).