# US 12,595,518 B1 · Methods for assaying enriched nucleic acids

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- Number: US 12,595,518 B1
- Document ID: US12595518B1
- Status: Granted
- Kind: B1
- Application number: 18/905,647
- Filed: 2024-10-03
- Published: 2026-04-09
- Granted: 2026-04-07
- Earliest claimed: 2008-05-30
- Assignees: British Columbia Cancer Agency Branch; The University of North Carolina at Chapel Hill; University of Utah Research Foundation; Washington University
- Applicants: The University of North Carolina at Chapel Hill; Washington University; University of Utah Research Foundation; British Columbia Cancer Agency Branch
- Inventors: Charles M. Perou; Joel S. Parker; James Stephen Marron; Andrew Nobel; Philip S. Bernard; Matthew J. Ellis; Elaine Mardis; Torsten O. Nielsen; Maggie Chon U. Cheang
- Web page: https://patentgrep.com/patent/US12595518B1

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6869: Methods for sequencing
- 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 40/20: Supervised data analysis
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q 2600/158: Expression markers
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

In an aspect, a method comprises providing a biological sample of a subject, and assaying nucleic acid molecules derived from RNA expression products of the biological sample to determine an RNA expression level of a plurality of genes, wherein the assaying comprises use of probes having sequences complementary to the nucleic acid molecules derived from the RNA expression products, to enrich the nucleic acid molecules for the plurality of genes.

## Claims

### Claim 1

A method comprising:
(a) providing a biological sample of a subject, and
(b) assaying nucleic acid molecules derived from RNA expression products of said biological sample to determine RNA expression levels corresponding to genes of said subject wherein said assaying comprises use of capture probes having sequences complementary to said nucleic acid molecules derived from said RNA expression products;
(c) generating a gene expression profile comprising RNA expression levels for a plurality of genes consisting of (i) 40 to 50 genes selected from ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDCA1, CDH3, CENPF, CEP55, CXXC5, EGFR, ERBB2, ESR1, EXO1, FGFR4, FOXA1, FOXC1, GPR160, GRB7, HSPC150 (UBE2T), KIF2C, KNTC2, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MYBL2, MYC, NAT1, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, and UBE2C and (ii) a set of housekeeping genes;
(d) comparing said gene expression profile to each of a set of centroids, wherein said set of centroids correspond to a plurality of breast cancer intrinsic subtypes; and
(e) assigning said biological sample to a breast cancer intrinsic subtype of said plurality of breast cancer intrinsic subtypes corresponding to a centroid among said set of centroids based at least upon a distance of said gene expression profile to said centroid.

### Claim 2 (depends on 1)

The method of claim 1, wherein said subject has been diagnosed with cancer.

### Claim 3 (depends on 2)

The method of claim 2, wherein said cancer is breast cancer.

### Claim 4 (depends on 3)

The method of claim 3, wherein said biological sample is a breast tumor tissue sample.

### Claim 5 (depends on 4)

The method of claim 4, wherein said breast tumor tissue sample is a formalin-fixed paraffin-embedded (FFPE) breast tumor tissue sample.

### Claim 6 (depends on 1)

The method of claim 1, wherein said subject is undergoing a breast cancer therapy.

### Claim 7 (depends on 6)

The method of claim 6, wherein said breast cancer therapy is an endocrine therapy.

### Claim 8 (depends on 7)

The method of claim 7, wherein said endocrine therapy is a neoadjuvant endocrine therapy.

### Claim 9 (depends on 6)

The method of claim 6, wherein said breast cancer therapy is a chemotherapy.

### Claim 10 (depends on 1)

The method of claim 1, wherein said plurality of genes consists of
(i) 42 to 50 genes selected from ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDCA1, CDH3, CENPF, CEP55, CXXC5, EGFR, ERBB2, ESR1, EXO1, FGFR4, FOXA1, FOXC1, GPR160, GRB7, HSPC150 (UBE2T), KIF2C, KNTC2, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MYBL2, MYC, NAT1, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, and UBE2C and
(ii) a set of housekeeping genes.

### Claim 11 (depends on 1)

The method of claim 1, wherein said plurality of genes consists of (i) 44 to 50 genes selected from ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDCA1, CDH3, CENPF, CEP55, CXXC5, EGFR, ERBB2, ESR1, EXO1, FGFR4, FOXA1, FOXC1, GPR160, GRB7, HSPC150 (UBE2T), KIF2C, KNTC2, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MYBL2, MYC, NAT1, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, and UBE2C and (ii) a set of housekeeping genes.

### Claim 12 (depends on 1)

The method of claim 1, wherein said plurality of genes consists of (i) 46 to 50 genes selected from ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDCA1, CDH3, CENPF, CEP55, CXXC5, EGFR, ERBB2, ESR1, EXO1, FGFR4, FOXA1, FOXC1, GPR160, GRB7, HSPC150 (UBE2T), KIF2C, KNTC2, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MYBL2, MYC, NAT1, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, and UBE2C and (ii) a set of housekeeping genes.

### Claim 13 (depends on 1)

The method of claim 1, wherein said plurality of genes consists of (i) 48 to 50 genes selected from ACTR3B, ANLN, BAG1, BCL2, BIRC5, BLVRA, CCNB1, CCNE1, CDC20, CDC6, CDCA1, CDH3, CENPF, CEP55, CXXC5, EGFR, ERBB2, ESR1, EXO1, FGFR4, FOXA1, FOXC1, GPR160, GRB7, HSPC150 (UBE2T), KIF2C, KNTC2, KRT14, KRT17, KRT5, MAPT, MDM2, MELK, MIA, MKI67, MLPH, MMP11, MYBL2, MYC, NAT1, ORC6L, PGR, PHGDH, PTTG1, RRM2, SFRP1, SLC39A6, TMEM45B, TYMS, and UBE2C and (ii) a set of housekeeping genes.

### Claim 14 (depends on 1)

The method of claim 1, further comprising:
wherein said set of centroids is constructed at least in part by:
(1) detecting RNA expression levels of said plurality of genes in a plurality of training breast cancer samples that have been classified according to breast cancer intrinsic subtype, thereby generating a training gene expression profile, wherein each of the Luminal A (LumA), Luminal B (LumB), Basal-like (Basal), and HER2-enriched (HER2) breast cancer intrinsic subtypes is represented in the plurality of training breast cancer samples, and
(2) constructing centroids for each of said breast cancer intrinsic subtypes in said plurality of training breast cancer samples, at least in part by comparing, using a nearest centroid algorithm, said training gene expression profile of said training breast cancer samples to reference gene expression data.

### Claim 15 (depends on 1)

The method of claim 1, further comprising predicting a response or a non-response to neoadjuvant therapy in the subject based at least in part on said assigning in (e), wherein said breast cancer intrinsic subtype assigned in (e) is indicative of said predicted response or non-response to said neoadjuvant therapy.

### Claim 16 (depends on 15)

The method of claim 15, wherein said neoadjuvant therapy is neoadjuvant endocrine therapy, and wherein said biological sample is obtained from said subject after initiation of said neoadjuvant endocrine therapy.

### Claim 17 (depends on 15)

The method of claim 15, further comprising predicting a response to said neoadjuvant therapy in said subject.

### Claim 18 (depends on 17)

The method of claim 17, further comprising administering said neoadjuvant therapy to said subject responsive to said predicting.

### Claim 19 (depends on 1)

The method of claim 1, wherein said RNA expression levels of said plurality of genes are normalized prior to said comparing in (d).

### Claim 20 (depends on 19)

The method of claim 19, wherein said normalizing comprises normalization to said set of housekeeping genes.

### Claim 21 (depends on 1)

The method of claim 1, wherein said set of housekeeping genes comprise a gene selected from the group consisting of MRPL19, PSMC4, SF3A1, PUM1, ACTB, GAPD, GUSB, RPLPO, and TFRC.

### Claim 22 (depends on 1)

The method of claim 1, wherein the subject is node-negative.

### Claim 23 (depends on 1)

The method of claim 1, wherein the subject is node-positive.

### Claim 24 (depends on 1)

The method of claim 1, further comprising determining a likelihood of breast cancer recurrence of the subject.