Methods for detecting gene dysregulation by intragenic differential expression

Patent metadata

Field	Value
Publication number	US-11021758-B2
Application number	US-201916707661-A
Country	US
Kind code	B2
Filing date	Dec 9, 2019
Priority date	May 26, 2009
Publication date	Jun 1, 2021
Grant date	Jun 1, 2021

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.

First claim

What is claimed is:

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 an ETS Related Gene (ERG) 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 ERG gene;
(b) amplifying a 3′ region of the ERG 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 ERG 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 3′ region to the 5′ region of the ERG gene is above a cutoff level, and diagnosing the subject as having benign prostate hyperplasia when the relative expression level of the 3′ region to the 5′ region of the ERG gene is below the cutoff level.

2. 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
(Ct 3′-target gene )−(Ct 5′-target gene ) IDE Score=2, and

(3′ Target)/(5′ Target)

wherein
the target gene is ERG;
Ct denotes threshold cycle,
3′ Target denotes the expression level of the 3′ region of the ERG gene, and
5′ Target denotes the expression level of the 5′ region of the ERG gene.

3. The method of claim 2 , further comprising determining an IDE score for TMPRSS2.

4. 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.

5. 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.

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

7. 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.

8. The method of claim 1 , wherein the 5′ target primer pair that is complementary to a 5′ region of the ERG gene transcript comprises a first primer comprising the sequence of SEQ ID NO: 7 and/or a second primer comprising the sequence of SEQ ID NO: 8.

9. The method of claim 1 , wherein the 3′ target primer pair that is complementary to a 3′ region of the ERG gene transcript comprises a first primer comprising the sequence of SEQ ID NO: 10 and/or a second primer comprising the sequence of SEQ ID NO: 11.

10. The method of claim 1 , wherein detecting the amounts of amplification products produced by the 5′ target primer pair and the 3′ target primer pair 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.

11. The method of claim 10 , 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.

12. 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: 9.

13. 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: 12.

14. The method of claim 3 , wherein a 5′ target primer pair that is complementary to a 5′ region of TMPRSS2 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.

15. The method of claim 3 , wherein the 3′ target primer pair that is complementary to a 3′ region of TMPRSS2 comprises a first primer comprising the sequence of SEQ ID NO: 4 and/or a second primer comprising the sequence of SEQ ID NO: 5.

16. The method of claim 14 , further comprising detecting an amount of amplification products produced by the 5′ target primer pair by contacting the amplification products with an oligonucleotide probe that is complementary to an amplification product generated using the 5′ target primer pair.

17. The method of claim 16 , wherein the oligonucleotide probe is labeled with a donor fluorophore and a quenching moiety.

18. The method of claim 16 , wherein the oligonucleotide probe comprises the sequence of SEQ ID NO: 3.

19. The method of claim 15 , further comprising detecting an amount of amplification products produced by the 3′ target primer pair by contacting the amplification products with an oligonucleotide probe that is complementary to an amplification product generated using the 3′ target primer pair.

20. The method of claim 19 , wherein the oligonucleotide probe is labeled with a donor fluorophore and a quenching moiety.

21. The method of claim 19 , wherein the oligonucleotide probe comprises the sequence of SEQ ID NO: 6.

Assignees

• Quest Diagnostics Invest Llc

Inventors

• Sanders Heather R
• Albitar Maher
• Meloni-Ehrig Aurelia

Classifications

• G16B25/10

  Gene or protein expression profiling; Expression-ratio estimation or normalisation · CPC title

• C12Q1/6809

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q1/6858

  Allele-specific amplification · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title