# US 11,306,360 B2 · Assessment of cellular signaling pathway activity using linear combination(s) of target gene expressions

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- Number: US 11,306,360 B2
- Document ID: US11306360B2
- Status: Granted
- Kind: B2
- Application number: 14/652,805
- Filed: 2013-12-18
- Published: 2015-12-03
- Granted: 2022-04-19
- Earliest claimed: 2012-12-26
- Assignees: Koninklijke Philips N.V.
- Applicants: Koninklijke Philips N.V.
- Inventors: Hendrik Jan Van Ooijen; Wilhelmus Franciscus Johannes Verhaegh; Paul Arnold Van De Wiel
- Web page: https://patentgrep.com/patent/US11306360B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- G06N 5/048: Fuzzy inferencing
- G16B 5/00: ICT specially adapted for modelling or simulations in systems biology, e.g. gene-regulatory networks, protein interaction networks or metabolic networks
- 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
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/118: Prognosis of disease development
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- 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
- G06N: COMPUTING ARRANGEMENTS BASED ON SPECIFIC COMPUTATIONAL MODELS
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

The present application mainly relates to specific methods for inferring activity of a cellular signaling pathway in tissue and/or cells of a medical subject based at least on expression levels of one or more target gene(s) of the cellular signaling pathway measured in an extracted sample of the tissue and/or cells of the medical subject, an apparatus comprising a digital compressor configured to perform such methods and a non-transitory storage medium storing instructions that are executable by a digital processing device to perform such methods.

## Claims

### Claim 1

A method for correcting abnormal activity of a cellular signaling pathway in a subject diagnosed with cancer, wherein the cellular signaling pathway comprises a Wnt pathway, an ER pathway, an AR pathway, or an HH pathway, the method comprising:
obtaining, by using at least one of Polymerase Chain Reaction (PCR), a microarray technique, and RNA-sequencing, expression levels of three or more target genes of the cellular signaling pathway derived from a sample extracted from the subject;
determining, by a digital processing device using a mathematical model of the cellular signaling pathway, activity of the cellular signaling pathway in a tissue and/or cells of the subject based at least on the expression levels of the three or more target genes of the cellular signaling pathway measured in an extracted sample of the tissue and/or cells of the subject, wherein the determining comprises:
determining a level of a transcription factor (TF) element in the extracted sample of the tissue and/or cells of the subject, the TF element controlling transcription of the three or more target genes of the cellular signaling pathway, the determining being based at least in part on evaluating the mathematical model relating expression levels of the three or more target genes of the cellular signaling pathway to the level of the TF element, the model being based at least in part on one or more linear combination(s) of expression levels of the three or more target genes; and
determining the activity of the cellular signaling pathway in the tissue and/or cells of the subject based on the determined level of the TF element in the extracted sample of the tissue and/or cells of the subject; and
determining that the cellular signaling pathway is operating abnormally in the subject based on the determined activity of the cellular signaling pathway, wherein the determined abnormal operation of the cellular signaling pathway is overactive operation of the cellular signaling pathway; and
selecting, based on the determined abnormal operation of the cellular signaling pathway, a specific treatment configured to remedy the determined abnormal operation of the cellular signaling pathway, wherein the specific treatment is a signaling pathway inhibitor configured to inhibit the operation of the cellular signaling pathway; and
administering, in response to the determined abnormal operation of the cellular signaling pathway and the selection of the specific treatment configured to remedy the determined abnormal operation of the cellular signaling pathway, the signaling pathway inhibitor;
wherein the signaling pathway inhibitor is a Wnt pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the Wnt pathway, wherein the signaling pathway inhibitor is an ER pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the ER pathway, wherein the signaling pathway inhibitor is an AR pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the AR pathway, and wherein the signaling pathway inhibitor is an HH pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the HH pathway.

### Claim 2 (depends on 1)

The method of claim 1, wherein for each of the three or more target genes one or more expression level(s) measured in the extracted sample of the tissue and/or cells of the subject is provided, and wherein the one or more linear combination(s) comprises a linear combination of all expression levels of the one or more expression level(s) provided for the three or more target genes.

### Claim 3 (depends on 1)

The method of claim 1, wherein for each of the three or more target genes one or more expression level(s) measured in the extracted sample of the tissue and/or cells of the subject is provided, and wherein the one or more linear combination(s) comprise a linear combination including for each of the three or more target genes a weighted term, each weighted term being based on only one expression level of the one or more expression level(s) provided for the respective target gene.

### Claim 4 (depends on 1)

The method of claim 1, wherein for each of the three or more target genes one or more expression level(s) measured in the extracted sample of the tissue and/or cells of the subject is provided, wherein the one or more linear combination(s) comprises for each of the three or more target genes a first linear combination of all expression levels of the one or more expression level(s) provided for the respective target gene, and wherein the model is further based at least in part on a further linear combination including for each of the three or more target genes a weighted term, each weighted term being based on the first linear combination for the respective target gene.

### Claim 5 (depends on 1)

The method of claim 1, wherein the determining comprises:
determining activity of a Wnt pathway in the tissue and/or cells of the subject based at least on expression levels of three or more target genes of the Wnt pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: KIAA1199, AXIN2, RNF43, TBX3, TDGF1, SOX9, ASCL2, IL8, SP5, ZNRF3, KLF6, CCND1, DEFA6 and FZD7.

### Claim 6 (depends on 1)

The method of claim 1, wherein the determining comprises:
determining activity of an ER pathway in the tissue and/or cells of the subject based at least on expression levels of three or more target genes of the ER pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: CDH26, SGK3, PGR, GREB1, CA12, XBPI, CELSR2, WISP2, DSCAM, ERBB2, CTSD, TFF1 and NRIP1.

### Claim 7 (depends on 1)

The method of claim 1, wherein the determining comprises: determining activity of an HH pathway in the tissue and/or cells of the subject based at least on expression levels of three or more target genes of the HH pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: GLI1, PTCH1, PTCH2, IGFBP6, SPP1, CCND2, FST, FOXL1, CFLAR, TSC22D1, RAB34, S100A9, S100A7, MYCN, FOXM1, GLI3, TCEA2, FYN and CTSL1.

### Claim 8 (depends on 1)

The method of claim 1, wherein the determining comprises:
determining activity of an AR pathway in the tissue and/or cells of the subject based at least on expression levels of three or more target genes of the AR pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: KLK2, PMEPA1, TMPRSS2, NKX3 1, ABCC4, KLK3, FKBPS, ELL2, UGT2B15, DHCR24, PPAP2A, NDRG1, LRIG1, CREB3L4, LCP1, GUCY1A3, AR and EAF2.

### Claim 9 (depends on 5)

The method of claim 5, wherein the determining is further based on expression levels of at least one target gene of the Wnt pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: NKD1, OAT, FAT1, LEF1, GLUL, REG1B, TCF7L2, COL18A1, BMP7, SLC1A2, ADRA2C, PPARG, DKK1, HNF1A and LECT2.

### Claim 10 (depends on 6)

The method of claim 6, wherein the determining is further based on expression levels of at least one target gene of the ER pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: AP1B1, ATPSJ, COL18A1, COX7A2L, EBAG9, ESR1, HSPB1, IGFBP4, KRT19, MYC, NDUFV3, PISD, PRDM15, PTMA, RARA, SOD1 and TRIM25.

### Claim 11 (depends on 7)

The method of claim 7, wherein the determining is further based on expression levels of at least one target gene of the HE pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: BCL2, FOXA2, FOXF1, H19, HHIP, IL1R2, JAG2, JUP, MIF, MYLK, NKX2.2, NKX2.8, PITRM1 and TOM1.

### Claim 12 (depends on 8)

The method of claim 8, wherein the determining is further based on expression levels of at least one target gene of the AR pathway measured in the extracted sample of the tissue and/or cells of the subject selected from the group comprising: APP, NTS, PLAU, CDKN1A, DRG1, FGF8, IGF1, PRKACB, PTPN1, SGK1 and TACC2.

### Claim 13 (depends on 1)

The method of claim 1, further comprising:
prescribing a drug for the subject configured to remedy the determined abnormal operation of the cellular signaling pathway.

### Claim 14 (depends on 1)

The method of claim 1, wherein the method is used in at least one of the following activities:
diagnosis based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
prognosis based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
drug prescription based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
prediction of drug efficacy based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
prediction of adverse effects based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
monitoring of drug efficacy;
drug development;
assay development;
pathway research;
cancer staging;
enrollment of the subject in a clinical trial based on the determined activity of the cellular signaling pathway in the tissue and/or cells of the subject;
selection of subsequent test to be performed, and
selection of companion diagnostics tests.

### Claim 15 (depends on 1)

The method of claim 1, further comprising:
determining activity of a Wnt pathway in tissue and/or cells of a subject based at least on expression levels of two, three or more target genes of a set of target genes of the Wnt pathway measured in an extracted sample of the tissue and/or cells of the subject,
and/or
determining activity of an ER pathway in tissue and/or cells of a subject based at least on expression levels of two, three or more target genes of a set of target genes of the ER pathway measured in an extracted sample of the tissue and/or cells of the subject,
and/or
determining activity of a HH pathway in tissue and/or cells of a subject based at least on expression levels of two, three or more target genes of a set of target genes of the HH pathway measured in an extracted sample of the tissue and/or cells of the subject,
and/or
determining activity of an AR pathway in tissue and/or cells of a subject based at least on expression levels of two, three or more target genes of a set of target genes of the AR pathway measured in an extracted sample of the tissue and/or cells of the subject.

### Claim 16 (depends on 15)

The method of claim 15, wherein
the set of target genes of the Wnt pathway includes at least nine target genes selected from the group comprising: KIAA1199, AXIN2, RNF43, TBX3, TDGF1, SOX9, ASCL2, IL8, SP5, ZNRF3, KLF6, CCND1, DEFA6 and FZD7,
and/or
the set of target genes of the ER pathway includes at least nine target genes selected from the group comprising: CDH26, SGK3, PGR, GREB1, CA12, XBP1, CELSR2, WISP2, DSCAM, ERBB2, CTSD, TFF1 and NRIP1,
and/or
the set of target genes of the HH pathway includes at least nine target genes selected from the group comprising: GLI1, PTCH1, PTCH2, IGFBP6, SPP1, CCND2, FST, FOXL1, CFLAR, TSC22D1, RAB34, S100A9, S100A7, MYCN, FOXMl, GLI3, TCEA2, FYN and CTSL1,
and/or
the set of target genes of the AR pathway includes at least nine target genes selected from the group comprising: KLK2, PMEPA1, TMPRSS2, NKX3 1, ABCC4, KLK3, FKBPS, ELL2, UGT2B15, DHCR24, PPAP2A, NDRG1, LRIG1, CREB3L4, LCP1, GUCY1A3, AR and EAF2.

### Claim 17 (depends on 16)

The method of claim 16, wherein
The set of target genes of the Wnt pathway further includes at least one target gene selected from the group comprising: NKD1, OAT, FAT1, LEF1, GLUL, REG1B, TCF7L2, COL18A1, BMP7, SLC1A2, ADRA2C, PPARG, DKK1, HNF1A, and LECT2,
and/or
the set of target genes of the ER pathway further includes at least one target gene selected from the group comprising: AP1B1, ATPSJ, COL18A1, COX7A2L, EBAG9, ESR1, HSPB1, IGFBP4, KRT19, MYC, NDUFV3, PISD, PRDM15, PTMA, RARA, SOD1 and TRIM25,
and/or
the set of target genes of the HH pathway further includes at least one target gene selected from the group comprising: BCL2, FOXA2, FOXF1, H19, HHIP, IL1R2, JAG2, JUP, MIF, MYLK, NKX2.2, NKX2.8, PITRM1 and TOM1,
and/or
the set of target genes of the AR pathway further includes at least one target gene selected from the group comprising: APP, NTS, PLAU, CDKN1A, DRG1, FGF8, IGF1, PRKACB, PTPN1, SGK1 and TACC2.

### Claim 18 (depends on 1)

The method of claim 1, wherein the obtaining of the expression levels of the three or more target genes includes measuring the expression levels of the three or more target genes.

### Claim 19 (depends on 1)

The method of claim 1,
wherein the target genes are mRNA direct target genes,
wherein the method further comprises calibrating the mathematical model, and
wherein the method further comprises:
constructing a network between the expression levels of the three or more mRNA direct target genes and the activity of the cellular signaling pathway, and
training the network by:
determining the level of the TF element in the extracted sample of the tissue and/or cells of the subject, the TF element controlling transcription of the three or more mRNA direct target genes of the cellular signaling pathway;
determining the activity of the cellular signaling pathway in the tissue and/or cells of the subject based on the determined level of the TF element in the extracted sample of the tissue and/or cells of the subject; and
measuring the nodes representing corresponding probe set nodes of the corresponding three or more mRNA direct target genes.

### Claim 20

A method for correcting abnormal activity of a cellular signaling pathway in a subject diagnosed with cancer, wherein the cellular signaling pathway comprises a Wnt pathway, an ER pathway, an AR pathway, or an HH pathway, the method comprising:
obtaining, by using at least one of Polymerase Chain Reaction (PCR), a microarray technique, and RNA-sequencing, expression levels of three or more target genes of the cellular signaling pathway derived from a sample extracted from the subject;
determining, by a digital processing device using a mathematical model of the cellular signaling pathway, activity of the cellular signaling pathway in a tissue and/or cells of the subject based at least on the expression levels of the three or more target genes of the cellular signaling pathway measured in an extracted sample of the tissue and/or cells of the subject, wherein the determining comprises/:
determining a level of a transcription factor (TF) element in the extracted sample of the tissue and/or cells of the subject, the TF element controlling transcription of the three or more target genes of the cellular signaling pathway, the determining being based at least in part on evaluating the mathematical model relating expression levels of the three or more target genes of the cellular signaling pathway to the level of the TF element, the model being based at least in part on one or more linear combination(s) of expression levels of the three or more target genes; and
determining the activity of the cellular signaling pathway in the tissue and/or cells of the subject based on the determined level of the TF element in the extracted sample of the tissue and/or cells of the subject; and
determining that the cellular signaling pathway is operating abnormally in the subject based on the determined activity of the cellular signaling pathway, wherein the determined abnormal operation of the cellular signaling pathway is inactive or underactive operation of the cellular signaling pathway; and
selecting, based on the determined abnormal operation of the cellular signaling pathway, a specific treatment configured to remedy the determined abnormal operation of the cellular signaling pathway, wherein the specific treatment is a signaling pathway upregulator configured to upregulate the operation of the cellular signaling pathway; and
administering, in response to the determined abnormal operation of the cellular signaling pathway and the selection of the specific treatment configured to remedy the determined abnormal operation of the cellular signaling pathway, the signaling pathway upregulator;
wherein the signaling pathway upregulator is a Wnt pathway upregulator when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the Wnt pathway, wherein the signaling pathway upregulator is an ER pathway upregulator when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the ER pathway, wherein the signaling pathway upregulator is an AR pathway upregulator when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the AR pathway, and wherein the signaling pathway upregulator is an HH pathway upregulator when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the HH pathway.

### Claim 21

A method for treating a subject diagnosed with cancer, comprising:
selecting, based on a determined abnormal operation of a cellular signaling pathway, a specific treatment configured to remedy the determined abnormal operation of the cellular signaling pathway, wherein the specific treatment is a signaling pathway inhibitor configured to inhibit the operation of the cellular signaling pathway, and wherein the cellular signaling pathway comprises a Wnt pathway, an ER pathway, an AR pathway, or an HH pathway, wherein the abnormal operation of a cellular signaling pathway is determined by:
obtaining, by using at least one of Polymerase Chain Reaction (PCR), a microarray technique, and RNA-sequencing, expression levels of three or more target genes of the cellular signaling pathway derived from a sample extracted from the subject;
determining, by a digital processing device using a mathematical model of the cellular signaling pathway, activity of the cellular signaling pathway in a tissue and/or cells of the subject based at least on the expression levels of the three or more target genes of the cellular signaling pathway measured in an extracted sample of the tissue and/or cells of the subject, wherein the determining comprises:
determining a level of a transcription factor (TF) element in the extracted sample of the tissue and/or cells of the subject, the TF element controlling transcription of the three or more target genes of the cellular signaling pathway, the determining being based at least in part on evaluating the mathematical model relating expression levels of the three or more target genes of the cellular signaling pathway to the level of the TF element, the model being based at least in part on one or more linear combination(s) of expression levels of the three or more target genes; and
determining the activity of the cellular signaling pathway in the tissue and/or cells of the subject based on the determined level of the TF element in the extracted sample of the tissue and/or cells of the subject; and
determining that the cellular signaling pathway is operating abnormally in the subject based on the determined activity of the cellular signaling pathway, wherein the determined abnormal operation of the cellular signaling pathway is overactive operation of the cellular signaling pathway; and
administering the signaling pathway inhibitor;
wherein the signaling pathway inhibitor is a Wnt pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the Wnt pathway, wherein the signaling pathway inhibitor is an ER pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the ER pathway, wherein the signaling pathway inhibitor is an AR pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the AR pathway, and wherein the signaling pathway inhibitor is an HH pathway inhibitor when the determined abnormal operation of the cellular signaling pathway is abnormal operation of the HH pathway.