# US 10,501,809 B2 · Methods for detecting gene dysregulation by intragenic differential expression

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- Number: US 10,501,809 B2
- Document ID: US10501809B2
- Status: Granted
- Kind: B2
- Application number: 16/153,108
- Filed: 2018-10-05
- Published: 2019-03-28
- Granted: 2019-12-10
- Earliest claimed: 2009-05-26
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments LLC
- Inventors: Heather R. Sanders; Maher Albitar; Aurelia Meloni-Ehrig
- Web page: https://patentgrep.com/patent/US10501809B2

## CPC

- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6858: Allele-specific amplification
- 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 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Described herein are methods, compositions and kits directed to the detection of gene dysregulations such as those arising from gene fusions and/or chromosomal abnormalities, e.g., translocations, insertions, inversions and deletions. Samples containing dysregulated gene(s) of interest may show independent expression patterns for the 5′ and 3′ regions of the gene. The methods, compositions and kits are useful for detecting mutations that cause the differential expression of a 5′ portion of a target gene relative to the 3′ region of the target gene.

## Claims

### Claim 1

A method for distinguishing prostate cancer from benign prostate hyperplasia in a subject suspected of having prostate cancer comprising:
(a) amplifying a 5′ region of a TMPRSS2 gene transcript, in a biological sample from the subject with one or more 5′ target primer pairs which are complementary to the 5′ region of the TMPRSS2 gene;
(b) amplifying a 3′ region of the TMPRSS2 gene transcript, if present, in the biological sample with one or more 3′ target primer pairs which are complementary to the 3′ region of the TMPRSS2 gene;
(c) detecting the amounts of amplification products produced by the one or more 5′ target primer pairs and the one or more 3′ target primer pairs; and
(d) diagnosing the subject as having prostate cancer when the relative expression level of the 5′ region to the 3′ region of the TMPRSS2 gene is above a cutoff level, and diagnosing the subject as having benign prostate hyperplasia when the relative expression level of the 5′ region to the 3′ region of the TMPRSS2 gene is below the cutoff level.

### Claim 2 (depends on 1)

The method of claim 1, wherein the amplifying is performed by real time RT-PCR and the relative expression is determined as an IDE Score using a formula selected from the group consisting of
(Ct3′-target gene)−(Ct5′-target gene) IDE Score=2, and
(3′ Target)/(5′ Target)
wherein
the target gene is TMPRSS2;
Ct denotes threshold cycle,
3′ Target denotes the expression level of the 3′ region of the TMPRSS2 gene, and
5′ Target denotes the expression level of the 5′ region of the target TMPRSS2 gene.

### Claim 3 (depends on 2)

The method of claim 2, further comprising determining an IDE score for one or more additional target genes selected from the group consisting of ERG, ETV1, ETV4, and ETV5.

### Claim 4 (depends on 1)

The method of claim 1, wherein the amounts of the amplification products produced by steps (a) and (b) are each normalized to the amount of an endogenous control gene transcript.

### Claim 5 (depends on 1)

The method of claim 1, further comprising amplifying a region of an endogenous control gene transcript present in the biological sample with a primer pair complementary to the endogenous control gene and detecting the amplification product of the region of the endogenous control gene.

### Claim 6 (depends on 5)

The method of claim 5, wherein the endogenous control gene is ABL.

### Claim 7 (depends on 1)

The method of claim 1, wherein the biological sample is selected from the group consisting of tissue, whole blood, isolated blood cells, serum, and urine.

### Claim 8 (depends on 1)

The method of claim 1, wherein the 5′ target primer pair that is complementary to a 5′ region of a TMPRSS2 gene transcript comprises a first primer comprising the sequence of SEQ ID NO: 1 and/or a second primer comprising the sequence of SEQ ID NO: 2.

### Claim 9 (depends on 1)

The method of claim 1, wherein the 3′ target primer pair that is complementary to a 3′ region of a TMPRSS2 gene transcript comprises a first primer comprising the sequence of SEQ ID NO: 5 and/or a second primer comprising the sequence of SEQ ID NO: 6.

### Claim 10 (depends on 1)

The method of claim 1, wherein detecting the amounts of amplification products produced by the one or more 5′ target primer pairs and the one or more 3′ target primer pairs comprises contacting the amplification products with an oligonucleotide probe that is complementary to an amplification product generated using the 5′ target primer pair or 3′ target primer pair.

### Claim 11 (depends on 1)

The method of claim 1, wherein the oligonucleotide probe that is complementary to an amplification product generated using the 5′ target primer pair or 3′ target primer pair is labeled with a donor fluorophore and a quenching moiety.

### Claim 12 (depends on 10)

The method of claim 10, wherein the oligonucleotide probe that is complementary to an amplification product generated using the 5′ target primer pair comprises the sequence of SEQ ID NO: 3.

### Claim 13 (depends on 10)

The method of claim 10, wherein the oligonucleotide probe that is complementary to an amplification product generated using the 3′ target primer pair comprises the sequence of SEQ ID NO: 6.