Methods of Detecting Bladder Cancer

Abstract

Compositions and methods for detecting bladder cancer are provided. In some embodiments, methods of monitoring recurrence of bladder cancer are provided. In some embodiments, the methods comprise detecting a set of markers consisting of CRH, IGF2, KRT20, and ANXA10.

1 . A method for detecting a set of bladder cancer markers in a sample from the subject comprising:
 (a) obtaining a urine sample or bladder washing sample from the subject;   (b) contacting RNA from the sample with a set of primers, wherein the set of primers consists of
 i. a first and second primer for detecting corticotrophin releasing hormone (CRH), 
 ii. a first and second primer for detecting insulin-like growth factor 2 (IGF2), 
 iii. a first and second primer for detecting keratin 20 (KRT20), and 
 iv. a first and second primer for detecting annexin 10 (ANXA10); and 
   (c) detecting hybridization between the RNA and the set of primers.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises detecting an endogenous control and/or exogenous control. 
     
     
         3 . The method of  claim 2 , wherein the endogenous control is selected from ABL, GUSB, GAPDH, TUBB, and UPK1. 
     
     
         4 . (canceled) 
     
     
         5 . The method of  claim 2 , wherein the exogenous control is an RNA. 
     
     
         6 . The method of  claim 1 , wherein the detecting comprises RT-PCR. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 2 , wherein the method comprises comparing a Ct value or a ΔCt value to a threshold Ct value or ΔCt value, wherein ΔCt is the Ct value for the control minus the Ct value for the marker. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the detecting comprises a RT-PCR reaction that takes less than 2 hours from an initial denaturation step through a final extension step. 
     
     
         11 .- 14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the first and second primer for detecting CRH comprise:
 a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 19 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 20, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or   b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 35 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 36, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.   
     
     
         16 . The method of  claim 1 , wherein the first and second primer for detecting IGF2 comprise:
 a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 16 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 17, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or   b) a first primer comprising at least 12 contiguous nucleotides of at least 12 contiguous nucleotides of SEQ ID NO: 32 and a second primer comprising SEQ ID NO: 33, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.   
     
     
         17 . The method of  claim 1 , wherein the first and second primer for detecting KRT20 comprise:
 a) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 13 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 14, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or   b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 29 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 30, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.   
     
     
         18 . The method of  claim 1 , wherein the first and second primer for detecting ANXA10 comprise:
 a) comprises a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 26 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 27, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or   b) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 38 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 39, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long; or   c) a first primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 48 and a second primer comprising at least 12 contiguous nucleotides of SEQ ID NO: 39, wherein each primer is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long.   
     
     
         19 .- 22 . (canceled) 
     
     
         23 . The method of  claim 1 , wherein the method further comprises forming a set of bladder cancer marker amplicons, wherein the set of bladder cancer marker amplicons consists of a CRH amplicon, an IGF2 amplicon, a KRT20 amplicon, and an ANXA10 amplicon, and contacting the bladder cancer marker amplicons with a set of bladder cancer marker probes, wherein the set of bladder cancer marker probes consists of a first probe for detecting the CRH amplicon, a second probe for detecting the IGF2 amplicon, a third probe for detecting the KRT20 amplicon, and a fourth probe for detecting the ANXA10 amplicon. 
     
     
         24 . The method of  claim 23 , wherein the first probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 21 or SEQ ID NO: 37, wherein the first probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long. 
     
     
         25 . The method of  claim 23 , wherein the second probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 34 or at least 12 SEQ ID NO: 18, wherein the second probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long. 
     
     
         26 . The method of  claim 23 , wherein the third probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 15 or SEQ ID NO: 31, wherein the third probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long. 
     
     
         27 . The method of  claim 23 , wherein the fourth probe comprises at least 12 contiguous nucleotides of SEQ ID NO: 28 or SEQ ID NO: 40, wherein the fourth probe is less than 50, less than 45, less than 40, less than 35, or less than 30 nucleotides long. 
     
     
         28 . The method of  claim 23 , wherein each bladder cancer marker probe comprises a dye, and wherein each dye is detectably different from the other three dyes. 
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 23 , wherein the method further comprises forming an endogenous control amplicon, and contacting the endogenous control amplicon with an endogenous control probe, and/or forming an exogenous control amplicon, and contacting the exogenous control amplicon with an exogenous control probe, wherein each probe comprises a dye, and wherein each dye is detectably different from the other dyes. 
     
     
         31 .- 41 . (canceled) 
     
     
         42 . A composition comprising a set of bladder cancer marker primer pairs, wherein the set of bladder cancer marker primer pairs consists of a first primer pair for detecting CRH, a second primer pair for detecting IGF2, a third primer pair for detecting KRT20, and a fourth primer pair for detecting ANXA10. 
     
     
         43 .- 61 . (canceled) 
     
     
         62 . A kit comprising the composition of  claim 42 .