WO2016044142A1 - Détection et surveillance du cancer de la vessie - Google Patents

Détection et surveillance du cancer de la vessie Download PDF

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Publication number
WO2016044142A1
WO2016044142A1 PCT/US2015/049940 US2015049940W WO2016044142A1 WO 2016044142 A1 WO2016044142 A1 WO 2016044142A1 US 2015049940 W US2015049940 W US 2015049940W WO 2016044142 A1 WO2016044142 A1 WO 2016044142A1
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WIPO (PCT)
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bladder cancer
nucleic acid
patient
mutation
indicator
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PCT/US2015/049940
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English (en)
Inventor
Steve Stone
Michael Perry
Alexander Gutin
Dan Theodorescu
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Myriad Genetics, Inc.
The Regents Of The University Of Colorado
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Publication of WO2016044142A1 publication Critical patent/WO2016044142A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present disclosure generally relates to molecular markers, including methylated markers, for detecting bladder cancer, especially recurrent bladder cancer, and methods of use thereof, including methods of monitoring for the recurrence of bladder cancer.
  • the disclosure also relates to methods of treatment, automated methods of diagnosis, compositions, and kits for detecting the presence of genomic DNA from bladder cancer or tumor cells, including cell-free DNA that can be found in the urine of patients with bladder cancer, and patients with recurrent bladder cancer.
  • Cancer is a major public health problem, accounting for roughly 25% of all deaths in the United States.
  • American Cancer Society FACTS AND FIGURES 2010.
  • treatments have been devised for various cancers, these treatments often vary in severity of side effects. It is useful for clinicians to be able to detect cancers early in their development, particularly through the use of non-invasive methods.
  • Urothelial cell carcinoma (UCC) of the urinary bladder is the 4th most common cancer in the United States, with an estimated 73,510 new cases and 14,880 deaths from bladder cancer in 2012, alone.
  • Each year in the U.S. and EU, bladder cancer is diagnosed in >160,000 men and results in >48,000 deaths. While the five-year survival rate for early-stage bladder cancer is high, over 25% of patients present with advanced disease and around 70%> experience recurrence or progression following treatment.
  • the prognosis for patients with progressive or recurrent invasive bladder cancer is generally poor. Management of recurrent cancers depends on the prior therapy used, site of recurrence, and patient-specific considerations.
  • Urinary cytology and cystoscopy are the current standard-of-care for bladder cancer detection and surveillance. Cystoscopy is the standard method of bladder tumor detection and/or confirmation; however it is an invasive, uncomfortable and costly procedure that results in urinary infection in up to 5% of cases. Almallah, et al., Urology 56:37-39 (2000). Urinary cytology is widely used for detection of bladder cancers, and has the advantage of up to 100% specificity. Unfortunately, urinary cytology is limited by its sensitivity, which is especially poor for low-grade bladder tumors.
  • Bladder cancer-specific mutations were identified by sequencing and analyzing the "exomes" (e.g., coding regions of the genomes) of over 40 human bladder cancer tumors in comparison to reference human genomes. The initially-identified discrepancies were limited to those not found in a database of human single nucleotide polymorphisms. The resulting reduced set of variants were analyzed for the effect that each change was predicted to have on the encoded protein or mRNA transcript.
  • exomes e.g., coding regions of the genomes
  • This gene weighting score was used to identify those genes in which human bladder cancer-specific mutations occurred at a frequency substantially greater than that expected from random chance.
  • the resulting lists of mutations include bladder cancer- specific signature mutations. These mutations and the genes in which these mutations occurred can be used in the various methods, systems, kits, etc. of the present disclosure as diagnostic genes harboring mutations diagnostic of bladder cancer.
  • novel bladder cancer-specific signature mutations thus identified, and other mutations in the diagnostic genes in which they were found, comprise biomarkers that can be used for the detection of bladder cancer.
  • These bladder cancer-specific signature mutations, and the genes in which they occur, are provided herein.
  • Bladder cancer-specific methylation markers are also described by analyzing
  • bladder cancer tumor specimens The percent of methylated cytosines in CpG islands of promoter regions was calculated for 22,670 genes, and gene selection followed a two-step procedure as described herein. Novel bladder cancer-specific methylation markers were thus identified, which comprise indicators that can be used for the detection of bladder cancer. These bladder cancer- specific methylation markers are provided herein.
  • Diagnostic tests based upon the presence of these signature mutations are also provided, as are methods and kits for testing for the presence of bladder cancer, and especially recurrent bladder cancer, in patients. These methods, tests, and kits are suited to detect bladder cancer non-invasively, by testing for the presence of the signature mutations in nucleic acids (e.g., DNA) derived from patient samples (e.g., urine).
  • nucleic acids e.g., DNA
  • patient samples e.g., urine
  • an in vitro method of detecting bladder cancer is provided.
  • the method comprises (1) analyzing nucleic acid (e.g., DNA) in or obtained from a patient biological sample (e.g., urine) to detect the presence or absence of at least one indicator of bladder cancer in said nucleic acid, wherein said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in one or more genes listed in Table 6 or Table 4; and (2)(a) diagnosing a patient in whose sample the presence of said indicator of bladder cancer is detected as having bladder cancer (or as having a high risk of having bladder cancer); or optionally (2)(b) diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having bladder cancer (or as having a low risk of having bladder cancer).
  • said indicator is a signature mutation listed in Table A, B, C, or D.
  • said indicator is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • said indicator is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the presence (or optionally the absence) of a plurality of signature mutations listed in Table A, B, C, or D is detected.
  • the presence (or optionally the absence) of a plurality of missense mutations, nonsense mutations, insertions or deletions, or transcript splicing altering mutations, or any combination thereof, is detected.
  • said bladder cancer is a recurrent bladder cancer.
  • the method further comprises obtaining nucleic acid from a patient biological sample.
  • said obtaining comprises the steps of: (a) isolating cells from a urine sample from said patient; (b) lysing the cells; and (c) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • the nucleic acid analyzed in step (1) is cell-free nucleic acid isolated from a urine sample.
  • the method further comprises the step of obtaining nucleic acid from a patient biological sample, wherein said obtaining comprises isolating cell-free nucleic acid from a urine sample and wherein isolating cell-free nucleic acid from a urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • step (1) comprises detecting the presence or absence of one or more mutations that are the signature mutations listed in Table A, B, C, or D.
  • step (1) comprises detecting the presence or absence of one or more mutations that is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4. In an embodiment, step (1) comprises detecting the presence or absence of one or more mutations that is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the one or more mutations are detected by a technique chosen from allele-specific PCR, mutant-enriched PCR, digital protein truncation test, direct sequencing, massively parallel sequencing, use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • the method further comprises generating a risk profile using the results of steps (1), (2) and (3).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said human patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said one or more genes listed in Table 6 or Table 4 comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 184 (s) genes listed in any of Table 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • an in vitro method of monitoring for recurrent bladder cancer comprises (1) optionally identifying a patient in need of such monitoring; (2) analyzing nucleic acid ⁇ e.g., DNA) in or obtained from a patient biological sample ⁇ e.g., urine) to detect the presence or absence of at least one indicator of bladder cancer in said nucleic acid, wherein said indicator is a mutation ⁇ e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in one or more genes listed in Table 6 or Table 4; and (3)(a) diagnosing a patient in whose sample the presence of said indicator of bladder cancer is detected as having recurrent bladder cancer (or as having a high risk of having recurrent bladder cancer); or optionally (3)(a) diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having recurrent bladder cancer (or
  • said indicator is a signature mutation listed in Table A, B,
  • said indicator is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • said indicator is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the presence (or optionally the absence) of a plurality of signature mutations listed in Table A, B, C, or D is detected.
  • the presence of (or optionally the absence) of a plurality of missense mutations, nonsense mutations, insertions or deletions, or transcript splicing altering mutations, or any combination thereof is detected.
  • said bladder cancer is a recurrent bladder cancer.
  • the method further comprises obtaining nucleic acid from a patient biological sample.
  • said obtaining comprises the steps of: (a) isolating cells from a urine sample from said patient; (b) lysing the cells; and (c) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • the nucleic acid analyzed in step (1) is cell-free nucleic acid isolated from a urine sample.
  • the method further comprises the step of obtaining nucleic acid from a patient biological sample, wherein said obtaining comprises isolating cell-free nucleic acid from a urine sample and wherein isolating cell-free nucleic acid from the urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • step (1) comprises detecting the presence or absence of one or more mutations that are the signature mutations listed in Table A, B, C, or D. In an embodiment, step (1) comprises detecting the presence or absence of one or more mutations that is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • step (1) comprises detecting the presence or absence of one or more mutations that is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the one or more mutations are detected by a technique chosen from allele-specific PCR, mutant-enriched PCR, digital protein truncation test, direct sequencing, massively parallel sequencing, use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • the method further comprises (4) generating a risk profile using the results of steps (1), (2) and (3).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said one or more genes listed in Table 6 or Table 4 comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 184 (s) genes listed in any of Table 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • a kit detects the presence of bladder cancer in a patient, said kit comprising reagents useful, sufficient, or necessary for detecting and/or characterizing at least one indicator of bladder cancer in nucleic acid (e.g., DNA) from a urine sample from a patient, said at least one indicator being chosen from one or more of the signature mutations listed in Table A, B, C, or D, or a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4, or a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4, or combinations thereof.
  • nucleic acid e.g., DNA
  • At least one indicator of bladder cancer to be detected is one or more of the signature mutations listed in Table A, B, C, or D.
  • said at least one indicator of bladder cancer to be detected is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • said at least one indicator of bladder cancer to be detected is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • instructions for using the kit are provided with the kit.
  • an in vitro method of detecting bladder cancer comprises a) obtaining nucleic acid (e.g., DNA) from a biological sample (e.g., urine) from said patient; b) determining the presence of at least one indicator of bladder cancer in said nucleic acid from said sample, wherein said indicator is a mutation (e.g. , a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in one or more genes listed in Table 6 or Table 4; and c) classifying said patient as having bladder cancer, based at least in part on the presence of said indicator of bladder cancer.
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • determining the presence of at least one indicator of bladder cancer in said nucleic acid from said sample wherein said indicator is a mutation (e.g. , a signature mutation listed in Table A, B, C, or D
  • said indicator is a signature mutation listed in Table A, B, C, or D.
  • said indicator is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • said indicator is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • a plurality of signature mutations listed in Table A, B, C, or D are present.
  • a plurality of missense mutations, nonsense mutations, insertions or deletions, or transcript splicing altering mutations, or any combination thereof are present.
  • said bladder cancer is a recurrent bladder cancer.
  • obtaining nucleic acid from a biological sample from said patient comprises the steps of: 1) isolating cells from a urine sample from said patient; 2) lysing the cells; and 3) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • obtaining nucleic acid from a biological sample from said patient comprises isolating cell-free nucleic acid from a urine sample.
  • isolating cell-free nucleic acid from a urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • the determining step comprises detecting one or more mutations that are the signature mutations listed in Table A, B, C, or D.
  • the determining step comprises detecting one or more mutations that is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • the determining step comprises detecting one or more mutations that is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the one or more mutations are detected by a technique chosen from allele-specific PCR, mutant-enriched PCR, digital protein truncation test, direct sequencing, massively parallel sequencing, use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • the method further comprises d) generating a risk profile using the results of steps a), b) and c).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said human patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • an in vitro method of monitoring for recurrent bladder cancer comprises a) optionally identifying a patient in need of such monitoring; b) obtaining nucleic acid ⁇ e.g., DNA) from a biological sample ⁇ e.g., urine) from said patient; c) determining the presence of at least one indicator of bladder cancer in said nucleic acid from said sample, wherein said indicator is a mutation ⁇ e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in one or more genes listed in Table 6 or Table 4; and d) classifying said patient as having recurrent bladder cancer, based at least in part on the presence of said indicator of bladder cancer.
  • nucleic acid ⁇ e.g., DNA
  • a biological sample ⁇ e.g., urine
  • said indicator is a signature mutation listed in Table A, B, C, or D.
  • said indicator is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • said indicator is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • a plurality of signature mutations listed in Table A, B, C, or D are present.
  • a plurality of missense mutations, nonsense mutations, insertions or deletions, or transcript splicing altering mutations, or any combination thereof are present.
  • said bladder cancer is a recurrent bladder cancer.
  • obtaining nucleic acid from a biological sample from said patient comprises the steps of: 1) isolating cells from a urine sample from said patient; 2) lysing the cells; and 3) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • obtaining nucleic acid from a biological sample from said human patient comprises isolating cell-free nucleic acid from a urine sample.
  • isolating cell-free nucleic acid from the urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • the determining step comprises detecting one or more mutations that are the signature mutations listed in Table A, B, C, or D.
  • the determining step comprises detecting one or more mutations that is a missense mutation, nonsense mutation, insertion, or deletion in the coding sequence for the protein encoded by one of the genes listed in Table 6 or Table 4.
  • the determining step comprises detecting one or more mutations that is a mutation that alters the splicing of the transcript of one of the genes listed in Table 6 or Table 4.
  • the one or more mutations are detected by a technique chosen from allele-specific PCR, mutant-enriched PCR, digital protein truncation test, direct sequencing, massively parallel sequencing, use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • the method further comprises d) generating a risk profile using the results of steps a), b) and c).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • an in vitro method of detecting bladder cancer comprises (1) analyzing nucleic acid ⁇ e.g., DNA) in or obtained from a patient biological sample ⁇ e.g., urine) to detect the presence or absence of at least one indicator of bladder cancer in said nucleic acid, wherein said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12; and (2)(a) diagnosing a patient in whose sample the presence of said indicator of bladder cancer is detected as having bladder cancer (or as having a high risk of having bladder cancer); or optionally (2)(b) diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having bladder cancer (or as having a low risk of having bladder cancer).
  • nucleic acid ⁇ e.g., DNA
  • a patient biological sample ⁇ e.g., urine
  • said bladder cancer is a recurrent bladder cancer.
  • the method further comprises obtaining nucleic acid from a patient biological sample, wherein said obtaining comprises the steps of: (a) isolating cells from a urine sample from said patient; (b) lysing the cells; and (c) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • the nucleic acid analyzed in step (1) is cell-free nucleic acid isolated from a urine sample.
  • the method further comprises the step of obtaining nucleic acid from a patient biological sample, wherein said obtaining comprises isolating cell-free nucleic acid from a urine sample and wherein isolating cell-free nucleic acid from a urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • methylation levels are detected by determining the percent of methylated cytosines in CpG islands of promoter regions of the genes listed in Table 12.
  • the method further comprises (4) generating a risk profile using the results of steps (1), (2) and (3).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said human patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80%) at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said method comprises detecting the presence or absence of at least one second indicator of bladder cancer, wherein said second indicator is a signature mutation listed in Table A, B, C, or D.
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • an in vitro method of monitoring for recurrent bladder cancer comprises (1) optionally identifying a patient in need of such monitoring; (2) analyzing nucleic acid (e.g., DNA) in or obtained from a patient biological sample (e.g., urine) to detect the presence or absence of at least one indicator of bladder cancer in said nucleic acid, wherein said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12; and (3)(a) diagnosing a patient in whose sample the presence of said indicator of bladder cancer is detected as having recurrent bladder cancer (or as having a high risk of having recurrent bladder cancer); or optionally (3)(a) diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having recurrent bladder cancer (or as having a low risk of having recurrent bladder cancer).
  • nucleic acid e.g., DNA
  • a patient biological sample e.g., urine
  • said method comprises detecting the presence or absence of at least one second indicator of bladder cancer, wherein said second indicator is a signature mutation listed in Table A, B, C, or D.
  • said bladder cancer is a recurrent bladder cancer.
  • the method further comprises obtaining nucleic acid from a patient biological sample wherein said obtaining comprises the steps of: (a) isolating cells from a urine sample from said patient; (b) lysing the cells; and (c) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • the nucleic acid analyzed in step (1) is cell- free nucleic acid isolated from a urine sample.
  • the method further comprises the step of obtaining nucleic acid from a patient biological sample, wherein said obtaining comprises isolating cell-free nucleic acid from a urine sample and wherein isolating cell-free nucleic acid from the urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • the method further comprises (4) generating a risk profile using the results of steps (1), (2) and (3).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • a kit is provided.
  • the kit is for detecting the presence of bladder cancer in a patient, said kit comprising reagents useful, sufficient, or necessary for detecting and/or characterizing at least one indicator of bladder cancer in nucleic acid (e.g., DNA) from a urine sample from a patient, wherein said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12.
  • the kit comprises reagents useful, sufficient, or necessary for detecting and/or characterizing at least one second indicator of bladder cancer, wherein said second indicator is a signature mutation listed in Table A, B, C, or D.
  • the kit comprises instructions for using the kit.
  • an in vitro method of detecting bladder cancer comprises a) obtaining nucleic acid ⁇ e.g., DNA) from a biological sample ⁇ e.g., urine) from said patient; b) determining the presence of at least one indicator of bladder cancer in said nucleic acid from said sample, wherein said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12; and c) classifying said patient as having bladder cancer, based at least in part on the presence of said indicator of bladder cancer.
  • said method comprises detecting the presence or absence of at least one second indicator of bladder cancer, wherein said second indicator is a signature mutation listed in Table A, B, C, or D.
  • said bladder cancer is a recurrent bladder cancer.
  • the method comprises obtaining nucleic acid from a biological sample from said patient comprises the steps of: 1) isolating cells from a urine sample from said patient; 2) lysing the cells; and 3) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • obtaining nucleic acid from a biological sample from said patient comprises isolating cell-free nucleic acid from a urine sample.
  • isolating cell-free nucleic acid from a urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • the method further comprises d) generating a risk profile using the results of steps a), b) and c).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said human patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80%) at a specificity of at least 90%>, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • an in vitro method of monitoring for recurrent bladder cancer comprises a) optionally identifying a patient in need of such monitoring; b) obtaining nucleic acid ⁇ e.g., DNA) from a biological sample ⁇ e.g., urine) from said patient; c) determining the presence of at least one indicator of bladder cancer in said nucleic acid from said sample, wherein said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12; and d) classifying said patient as having recurrent bladder cancer, based at least in part on the presence of said indicator of bladder cancer.
  • nucleic acid ⁇ e.g., DNA
  • a biological sample ⁇ e.g., urine
  • said method comprises detecting the presence or absence of at least one second indicator of bladder cancer, wherein said second indicator is a signature mutation listed in Table A, B, C, or D.
  • said bladder cancer is a recurrent bladder cancer.
  • obtaining nucleic acid from a biological sample from said patient comprises the steps of: 1) isolating cells from a urine sample from said patient; 2) lysing the cells; and 3) extracting the nucleic acid from the cell lysate.
  • the isolating step comprises isolating the cells by centrifugation or filtration of the urine sample.
  • the lysing step comprises treating the isolated cells with a detergent or surfactant to provide a lysate.
  • the extracting step comprises treating the cell lysate with a nucleic acid extraction reagent and concentrating the extracted nucleic acid by precipitation or by binding it to a cationic matrix.
  • obtaining nucleic acid from a biological sample from said human patient comprises isolating cell-free nucleic acid from a urine sample.
  • isolating cell-free nucleic acid from the urine sample comprises passing the urine sample through a cationic matrix that binds the cell-free nucleic acid or concentrating the cell-free nucleic acid by ultrafiltration of the urine sample.
  • the method further comprises d) generating a risk profile using the results of steps a), b) and c).
  • said bladder cancer is selected from the group consisting of premalignant and malignant bladder cancer.
  • said method permits detection of bladder cancer in said patient with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70% at a specificity of at least 95%.
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • Figure 1 illustrates Spearman rank correlations of bladder tumors, and normal bladder tissues, in terms of the methylation levels of 22,670 genes.
  • Figure 2 illustrates Spearman rank correlations of 31 methylated genes that best discriminate tumor from normal bladder tissue, after adjusting for tumor versus normal tissue status.
  • the phrase “indicator of bladder cancer,” refers to a mutation in a diagnostic gene of the disclosure, as detected by the methods disclosed herein below. As such, the phrase “indicator of bladder cancer,” can refer to one of the mutations specifically listed in the Tables provided herewith. Additionally, the phrase “indicator of bladder cancer,” can refer to other activating or deactivating (depending on the gene) mutations in the diagnostic genes of the disclosure besides those listed in the Tables, or a mutation predicted to either activate or deactivate such gene.
  • the phrase "indicator of bladder cancer,” can refer to missense mutations that alter the sequence of amino acids in the protein encoded by a signature gene of the disclosure by converting the original codon encoding a first amino acid to a mutant codon encoding a second amino acid that is different from the first amino acid.
  • the phrase "indicator of bladder cancer,” can refer to mutations that result in truncations of the protein encoded by a diagnostic gene of the disclosure.
  • Such "truncation inducing" mutations include "nonsense mutations" where a single base change converts an amino acid encoding codon to a stop codon.
  • truncation inducing mutations include "framesfiift mutations," which are insertions or deletions of one, two, or more (typically a multiple of one or two but not three) nucleotides, that either alter the normal or native reading frame of the codons that make up the coding sequence of the mRNA transcript of a diagnostic gene of the disclosure, or result in insertions or deletions of one or more amino acids in the protein encoded by a diagnostic gene of the disclosure.
  • These insertion or deletion mutations can result in truncations of the encoded protein since altered reading frames can contain stop codons that will be encountered by the translational machinery of the cell in advance of the native stop codon.
  • insertions or deletions that result in altered reading frames can result in a different sequence of amino acids being added to the carboxyl-terminus of a protein as a result of the translational machinery translating in a different reading frame before encountering a stop codon in this new frame.
  • the phrase "indicator of bladder cancer,” can refer to mutations that adversely alter the splicing of exons, or the removal of introns, from the transcript transcribed from a diagnostic gene of the disclosure.
  • Such mutations that alter splicing the splicing of transcripts can occur at or near one of the so-called “splice junctions" that are found at the boundaries of the encoded exons and the introns that separate them.
  • Such mutations can cause alterations in the amino acid sequence in the protein encoded by a diagnostic gene of the disclosure.
  • such mutations result in truncations of the encoded protein, because stop codons can occur in multiple reading frames.
  • the phrase "indicator of bladder cancer” can refer to diagnostic genes of the disclosure that has increased or decreased levels of methylation (e.g., hypermethylation or hypomethylation).
  • hypomethylation refers to an increase in methylation of cytosine and adenosine residues in the diagnostic genes as described herein.
  • a sample or DNA in or extracted from a sample is hypermethylated if 20% or more of tested CpG sites are detected to be methylated.
  • hypomethylation refers to a decrease in methylation of cytosine and adenosine residues in the diagnostic genes as described herein.
  • a sample or DNA in or extracted from a sample is hypomethylated if fewer than 20% of tested CpG sites are detected to be methylated.
  • CpG site or “methylation site” refers to regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. Cytosines in CpG dinucleotides are capable of being methylated by DNA
  • CpG islands methyltransferases to form 5-methylcytosine. Regions of the DNA which have a higher concentration of CpG sites are known as CpG islands.
  • a “methylation state” refers to the presence or absence of one or more methylated nucleotide bases or the ratio of methylated cytosine to unmethylated cytosine for a methylation site in a nucleic acid target region (e.g., the promoter region of a gene listed in Table 12). Said ratio may also be referred to as "relative methylation.” For example, a nucleic acid target region containing at least one methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid target region is methylated). A nucleic acid target region that does not contain any methylated nucleotides is considered unmethylated.
  • the methylation state of a nucleotide locus in a nucleic acid target region refers to the presence or absence of a methylated nucleotide at a particular locus in the nucleic acid target region.
  • the methylation state of a cytosine at the 7th nucleotide in a nucleic acid target region is methylated when the nucleotide present at the 7th nucleotide in the nucleic acid target region is 5-methylcytosine.
  • the methylation state of a cytosine at the 7th nucleotide in a nucleic acid target region is unmethylated when the nucleotide present at the 7th nucleotide in the nucleic acid target region is cytosine (and not 5-methylcytosine).
  • the ratio of methylated cytosine to unmethylated cytosine for a methylation site or sites can provide a methylation state of a nucleic acid target region.
  • methylation detection methods refer to the methods, conditions and instrumentation for analysis of DNA methylation.
  • detection methods include, but are not limited to, multiplexed hME assays, fluorescence-based real-time PCR, methylation-sensitive single nucleotide primer extension, methylated CpG island amplification, methylation-specific PCR, restriction landmark genomic scanning, methylation-sensitive- representational difference analysis (MS-RDA), methylation-specific AP-PCR (MS-AP-PCR) methyl-CpG binding domain column/segregation of partly melted molecules (MBD/SPM), bisulphite sequencing direct, or combined bisulfite restriction analysis (COBRA).
  • MS-RDA methylation-sensitive- representational difference analysis
  • MS-AP-PCR methylation-specific AP-PCR
  • MBD/SPM partly melted molecules
  • COBRA combined bisulfite restriction analysis
  • Examples of different instruments for the analysis of DNA methylation include, but are not limited to, mass spectrometers, nucleic acid sequencers (e.g., capillary sequencer), gel electrophoresis, fluorescence detectors (e.g., charge-coupled devices), thermal cyclers in conjunction with methylation-specific PCR, and HPLC.
  • the phrase "deleterious mutation” as used herein, refers to a mutation that results in altered structure, expression or activity of the protein encoded by one of the diagnostic genes of the disclosure such that the mutation promotes the development or progression of cancer.
  • This may include a deactivating mutation (e.g., ARID 1 A C132T) that results in, e.g., truncation of the encoded protein, an altered amino acid sequence of the encoded protein that yields a protein with no or attenuated activity, alterations in the splicing of the transcript of one of the diagnostic genes of the disclosure, reduction in the in vivo stability of the encoded transcript that yields lower expression of the protein, etc.
  • a deactivating mutation e.g., ARID 1 A C132T
  • This may also include an activating mutation (e.g., TERT promoter mutations C228T and/or C250T) that results in, e.g., an altered promoter that yields increased (e.g., constitutive) expression of the encoded protein, an altered amino acid sequence of the encoded protein that yields a protein with enhanced (e.g., constitutive) activity, increase in the in vivo stability of the encoded transcript that yields higher expression of the protein, etc.
  • an activating mutation e.g., TERT promoter mutations C228T and/or C250T
  • the phrases “signature mutation,” “tumor-specific mutation,” or “tumor-specific signature mutation” specifically refer to a mutation in a diagnostic gene of the disclosure, as detected by the methods disclosed herein below. As such, the phrases “signature mutation,” “tumor-specific mutation,” or “tumor-specific signature mutation” refers to one of the mutations specifically identified in the Tables provided herewith.
  • biomarker when used in the context of describing mutations or methylation status in the diagnostic genes of the present disclosure, is synonymous with the phrase “indicator of bladder cancer,” and refers not only to one of the mutations specifically identified in the Tables provided herewith, but also to other mutations (e.g., nonsense, frameshift, large rearrangement, missense mutations) or methylation statuses in the diagnostic genes of the disclosure besides those mutations identified in the Tables provided herewith.
  • diagnosis gene refers to one of the genes identified by the methods disclosed herein as containing or “harboring” a "signature mutation,” “tumor-specific mutation,” “tumor-specific signature mutation,” “hypermethylation,” or “hypomethylation.”
  • diagnostic genes are those genes identified in Tables 1-11, and 12 provided herewith, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z as defined below.
  • the term "diagnostic test panel” means a predetermined group of diagnostic genes to be used in the methods, systems or kits of the disclosure (e.g., for the detection of bladder cancer or for the monitoring of bladder cancer recurrence).
  • blade cancer refers to all forms of malignancy of urinary bladder tissues, but particularly includes malignancies arising from the epithelial lining (e.g. , the urothelium) of the urinary bladder.
  • bladder cancer The most common type of bladder cancer, which recapitulates the normal histology of the urothelium, is known as transitional cell carcinoma, or urothelial cell carcinoma. Bladder cancers are often staged according to the following criteria:
  • Stage I Cancer at this stage occurs in the bladder's inner lining but has not invaded the muscular bladder wall. This stage is commonly referred to as Tl .
  • Stage II At this stage, cancer has invaded the bladder wall but is still confined to the bladder. This stage is commonly referred to as T2.
  • Stage III At this stage the cancer cells have spread through the bladder wall to surrounding tissue, and may also have spread to the prostate in men or the uterus or vagina in women. This stage is commonly referred to as T3.
  • Stage IV Cancer cells, by this stage may have spread to the lymph nodes and other organs, such as the lungs, bones or liver. This stage is commonly referred to as T4.
  • recurrent bladder cancer refers to those forms of bladder cancer that have reoccurred following an intervention (initial or subsequent), including surgical interventions, intended to remove any existing bladder cancer.
  • the surgical intervention utilized for early stage bladder cancers is transurethral resection, in which superficial tumors (those which have not entered the surrounding muscle layer) are removed by electrocautery. The removed material can then be used for pathological examination and subsequent staging of the cancer.
  • Recurrent bladder cancers may also arise after any combination of treatment by surgical intervention, chemotherapy, immunotherapy, and radiation treatment, with the latter methods typically being used in stage II, III and IV bladder cancers.
  • Recurrent bladder cancers may also arise after surgical removal of all or part of the urinary bladder (e.g., cystectomy), particularly with metastatic forms of bladder cancer.
  • the term "gene length,” when used in reference to a diagnostic gene as disclosed herein, refers to the length, in basepairs, of that portion of the diagnostic gene over which the gene was evaluated for the presence of bladder cancer-specific signature mutations according to the methods described in the Examples below.
  • the "gene length" for a particular diagnostic gene includes the length of the exons of that diagnostic gene, plus 100 base pairs from each (e.g., 5' and 3') end of any intervening sequences (e.g., introns) within that diagnostic gene, plus 100 base pairs of the untranslated regions of the exons abutting the 5'-most and 3'-most ends of the coding region.
  • sample is an amount of tissue or bodily fluid taken from a subject, such as a mammal, or any biomolecule derived therefrom.
  • Biomolecules derived from a tissue or fluid include molecules present in such tissue or fluid and extracted therefrom as well as artificial counterparts synthesized based on such endogenous biomolecules.
  • Non- limiting examples of artificial counterparts include PCR products using endogenous nucleic acids as templates (e.g., cDNA synthesized from mRNA, PCR amplification of genomic DNA, etc.).
  • bodily fluids include urine, blood, plasma, serum, semen, perspiration, tears, mucus, and tissue lystates.
  • sample or biological sample further refers to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or component parts, or a fraction or portion thereof.
  • sample or biological sample can refer to non-cellular biological material, such as blood or urine. In an exemplary embodiment, the sample is urine.
  • nucleic acid refers to deoxyribunucleotides, e.g., DNA, or ribonucleotides, e.g., RNA, and polymers thereof in either single- or double-stranded form.
  • the term encompasses a nucleic acid containing known nucleotide analogs or modified backbone residues or linkages, which can be synthetic, naturally occurring, or nonnaturally occurring, which can have similar binding properties as the reference nucleic acids, and which can be metabolized in a manner similar to the reference nucleotides.
  • predicting prognosis will often be used herein to refer to either or both.
  • a “poor prognosis” will generally refer to an increased likelihood of recurrence, metastatic progression, or both.
  • subject refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, etc.
  • patient refers to a human subject.
  • the genomic sequence of the coding regions (e.g., exomes) of bladder cancer tumors can obtained and analyzed as compared to a reference human genome. Discrepancies between the bladder cancer tumor exomes and a reference human genome can be identified. Such discrepancies can be assessed for reproducibility between sequence reads. The discrepancies that represent real differences between the tumor exome and the reference genome can be classified as variants. Such variants can be assessed for novelty by comparing the variants against a database of known single nucleotide polymorphisms. Variants found in the database can be excluded from further analysis. These resulting subsets of variants can be further analyzed for their effect on the encoded protein and mR A transcript encoding the protein.
  • Variants causing a truncation of the encoded protein through the introduction of stop codons (e.g., nonsense mutations) and frameshift mutations can be included in further analyses, as can be missense mutations if at least one nonsense mutation or frameshift mutation had been mapped to the same gene. Variations that resulted in the change of the codon that was initially present in the protein for a synonymous codon (e.g., synonymous mutations) can be excluded.
  • EXAMPLE A a list of genes harboring at least one "suspected deleterious" mutation was prepared, and the genes in the list were subjected to a weighting procedure (as described in EXAMPLE A), which can include, but is not limited to:
  • Weight (for a given gene) ((# of Unique Variants A 2) / (# of Variants A 2)) * (Number of samples affected by all variants) / (Square root Length of the gene) to place a greater value on genes bearing unique variants as compared to those genes bearing variants that occurred in multiple samples, to place a greater value on genes that were affected by a greater number of unique variants, and to give a lower value to longer genes, which would be expected to have a greater number of variations due to random chance.
  • the top 40 genes so identified in Example A were then evaluated based on their known functions to produce a list of 10 diagnostic genes harboring tumor-specific mutations.
  • GENE LIST A which includes TP53, NUP188, MUC16, CCDC168, KDM6A, SPTAN1, MLL2, ERBB3, ARID 1 A, and RBI.
  • the tumor-specific signature mutations that were observed in these diagnostic genes ⁇ e.g., MUTATION PANEL A) are disclosed in Example A, and further information about the diagnostic genes is provided in Table 1.
  • EXAMPLE B demonstrates how additional efforts can be taken to further refine the list of diagnostic genes and tumor- specific signature mutations. Following sequencing and the identification of discrepancies between tumor genomes and the reference human genome, and following removal of polymorphisms known from the database single nucleotide polymorphisms, remaining variant calls can be also compared to a dataset of polymorphisms identified in coding regions of genomic sequences. For example, EXAMPLE B identified polymorphisms in 105 unrelated human blood samples. In EXAMPLE B, if a variant call was shared by more than 2 of the 105 blood samples, it was excluded from further consideration.
  • Weight (for a given gene) (((p*l) A n) / n!) * e A -(p*l)
  • n number of unique variants.
  • GENE LIST B includes TP53, NUP188, XIRP2, PLCG2, KDM6A, CCDC168, KIAA1671, KPRP, OR5L2, SPTAN1, ERBB3, SRRM2, ARID 1 A, FOXM1, MUC16, ISG20L2, ZC3H7A, MYBPC2, AHNAK2, HSPBAPl, SYNEl, ZNF208, PLDl, SMC2, OR8I2, BTN2A2, MLL2, JMJDIC, SLC35G6, VCAN, VPS13D, VCX3B, ZNF705G, RBBP8, IGSF6, DOCK9, C9orfl 74, NPC1L1, PCDHGA9, ACTB, DNHD1, LYST, SCAF11, ZNF846, LOC100133128, DNAH17, DYNC1H1, ANK3, KIAA0100, STAG2, FLG, ZNF623, DCHS1, CARD6, KIF13A, HEATR1,
  • HEATR1 55127 14505 5 5 5 7.69E-09
  • EXAMPLE C demonstrates how additional efforts can be taken to further refine the list of diagnostic genes and tumor-specific signature mutations.
  • EXAMPLE C the same starting data and refined procedure as in EXAMPLE B was used, with one exception: Variants predicted to adversely affect the splicing of the encoded transcript can be also included for further analyses.
  • genes bearing splice-site altering mutations in addition to genes bearing missense mutations, nonsense mutations, insertions or deletions can be subjected to the same weighting procedure as described for EXAMPLE B.
  • genes with a weighted score of less than 10 "7 (1 x E-7) can be retained as diagnostic genes.
  • GENE LIST C which substantially overlaps with GENE LIST B, includes TP53, NUP188, XIRP2, PLCG2, FOXM1, KDM6A, ARID 1 A, CCDC168, KIAA1671, KPRP, MUC16, OR5L2, SPTAN1, ERBB3, SRRM2, SNRNP200, ISG20L2, ZC3H7A, MYBPC2, AHNAK2, HSPBAP1, SYNE1, ZNF208, PLD1, SMC2, OR8I2, STAG2, BTN2A2, MLL2, JMJD1C, SLC35G6, VCAN, VPS13D, VCX3B, ZNF705G, RBBP8, IGSF6, DOCK9, C9orfl 74, NPCILI, PCDHGA9, ACTB, DNHDl, LYST, SCAFll, ZNF846, NF1, CA
  • VPS13D 55187 30041 7 7 5 l .l lE-10
  • HEATR1 55127 14505 5 5 5 7.69E-09
  • GENE LIST X comprises AHNAK2, AKAP13, BTN2A2, CARD 6, CCL20, CLCA4, COL12A1, COL21A1, CPSF2, DCHSl, DNAH17, DNAJCIO, DOCK9, DYNCIHI, FAM193A, FLG, GLDC, HMCN1, HSPBAP1, IGSF6, ISG20L2, ITGA8, JMJD1C, KDM6A, KIAA0100, KIAA1671, KPRP, LYST, MFHASl, MLL2, MYBPC2, NCKAPIL, NPCILI, NPHP3, NUP188, ODZ3, PCLO, PDE4A, PLCG2, PLXNA2, PSD4, RBBP8, SAMD4A, SCN9A, SMC2, SNRNP200, SPAG17, STAG2, SYNEl, TNP2, UGGTl, UGT1A3, VCX3B, VPS13D, WDR6,
  • EXAMPLE D demonstrates how additional efforts can be taken to further refine the list of diagnostic genes and tumor-specific signature mutations.
  • EXAMPLE D demonstrates how genomic sequences of coding regions (e.g., exomes) of 53 human bladder cancer tumors can be obtained and analyzed. Of these samples, 45 were the same as those used in EXAMPLES A-C, and 8 were new. Discrepancies between the bladder cancer tumor exomes and a reference human genome can be identified. Such discrepancies can be assessed for reproducibility between sequence reads. The discrepancies that represented real differences between the tumor exome and the reference genome can be classified as variants.
  • variants can be assessed for novelty by comparing the variants against a database of known single nucleotide polymorphisms. Variants found in the database can be excluded from further analysis. These resulting subsets of variants can be further analyzed for their effect on the encoded protein and mRNA transcript encoding the protein. Variants causing a truncation of the encoded protein through the introduction of stop codons (e.g., nonsense mutations), insertion or deletion mutations that result in a shift in the reading frame (e.g., frameshift mutations), and intronic mutations that potentially alter transcript splicing were categorized as "class 1" variants, having a higher chance in causing a change in function.
  • stop codons e.g., nonsense mutations
  • insertion or deletion mutations that result in a shift in the reading frame
  • intronic mutations that potentially alter transcript splicing were categorized as "class 1" variants, having a higher chance in causing a change in function.
  • Variants that resulted in missense mutations, insertions or deletions that maintained the reading frame, and intronic mutations that likely would not alter transcript splicing can be categorized as "class 0" variants, having a lower chance of causing a functional change. Unlike with the previous Examples, variants of both classes (class 1 and class 0) can be included in the gene weighting calculations. Additionally, as described below, the weighting protocol for EXAMPLE D differed from the weighting protocol for the previous Examples.
  • the four additional genes used in EXAMPLE D are FIBROBLAST GROWTH FACTOR RECEPTOR 3 (FGFR3; (GRCh37): 4:1, 795,038 - 1,810,598; OMIM 134934); PHOSPHATIDYL-INOSITOL 3-KINASE, CATALYTIC, ALPHA (PIK3CA; (GRCh37): 3:178,866,310 - 178,952,499; OMIM: 171834); V-KI-RAS2 KIRSTEN RAT SARCOMA VIRAL ONCOGENE HOMOLOG (KRAS; (GRCh37): 12:25,358,179 - 25,403,869; OMIM: 190070); and TELOMERASE REVERSE TRANSCRIPTASE (TERT; (GRCh37): 5:1,253,281 - 1,295,177; OMIM: 187270).
  • the first three of these additional genes i.e., FGFR3, PIK3CA
  • ETS E-twenty-six
  • each variant (n) is deweighted by the number of samples in which it appears, so that if a variant is unique to 1 sample, it is weighted as 1. If a variant appears in 2 samples, it would be 1 ⁇ 2, etc.
  • GENE LIST D includes TP53, MLL2, ARID 1 A, KDM6A, PCLO, ClOorfll, ZFHX4, PCNXL2, XIRP2, FOXM1, ODZ3, DNAH17, FLG, PLEC, RPILI, LOCI 00130830, OBSCN, NLRP13, AGRN, SPTAN1, PCDHGA2, KPRP, RBBP8, PCDHGA9, OR2T4, AHNAK2, MUC16, RNF111, COL6A1, PCDH8, NACAD, UNC93B1, WDR6, ZRANB3, SRRM2, TMEM175, AKAP13, INPP5D, KIF7, CHD8, NEB, ZSCAN5D, CCDC40, RBI, CAMTA2, KIAA1683, HSPBAP1, GYG2, VPS13D, GLIS2, SUV420
  • GENE LIST Z includes: ABCA5, ABCB5, ACTB, AGRN, AHNAK2, AKAP13, ANK3, APOB, ARIDIA, ATP2C2, AZUl, BTN2A2, ClOorftl, C16orf96, C2orfl6, C7orfi8, C9orfl 74, CACNA2D3, CAMTA2, CARD6, CCDC168, CCDC40, CCL20, CDH13, CDRT15L2, CEP95, CHD6, CHD8, CLCA4, COL12A1, COL21A1, COL6A1, CPSF1, CPSF2, CRTAC1, DCHS1, DNAH17, DNAJC10, DNHD1, DOCK9, DST, DYNC1H1, ERBB2, ERBB3, FAM193A, FAM75D1, FAN1, FAT1, FGFR
  • An exemplary panel can include TERT (promoter), TP53, MLL2, KDM6A,
  • Target regions for designing sequencing probes for TERT promoters of this exemplary panel can be, for example, TERT_p01 (chr5 1294920-1295648), TERT_p02 (chr5 1294584-1295334), TERT_p03 (chr5 1297264-1298000), TERT_p04 (chr5 1291797-1292266), and TERT_p05 (chr5 1291877-1292695).
  • KRAS hot spot regions can include, for example: codons 12 and 13 (accession NP_004976.2; mRNA position starting at c.34 and c.37 of NM_004985.4) contained within chrl2:25,398,277-25,398,288; codon 61 (accession NP_004976.2; mRNA position starting at c.181 of NM_004985.4) contained within chrl2:25,380,269-25,380,283; and codon 146 (accession NP 004976.2; mRNA position starting at c.436 of accession NM 004985.4) contained within chrl2:25, 378,554-25,378, 568.
  • KRAS amino acid modifications can include, for example, G12V, G12D, G12C, G12A, G12S, G12R, G13D, G13C, Q61L, Q61K, Q61H, Q61R, Q61P, Q61E, A146T, A146V, and A146P.
  • HRAS hot spot regions can include, for example: codons 12 and 13 (accession NP 001123914.1; mRNA position starting at c.34 and c.37 of accession NM_001130442.1) contained within chrl 1 :534,281-534,292; and codon 61 (accession NP 001123914.1; mRNA position starting at c.181 of accession NM 001130442.1) that is contained within chrl 1 :533,867-533,881.
  • HRAS amino acid modifications can include, for example, G12V, G12D, G12S, G12D, G12A, G13R, G13V, G13D, G13C, G13S, Q61L, Q61R, Q61K, and Q61H.
  • PIK3CA hot spot regions can include, for example: codons 542 and 545 (accession NP 006209.2; mRNA position starting at c.1624 and c.1633) contained within chr3: 178,936,076-178,936,099; and codon 1047 (accession NP 006209.2; mRNA c.3139 of accession NM 006218.2) contained within chr3: 178,952,078-178,952,092.
  • PIK3CA amino acid modifications can include E542K, E545K, E545A, E545G, H1047R, and H1047L.
  • the "gene length" for a particular diagnostic gene includes the length, in basepairs, of the exons of that diagnostic gene, plus 100 base pairs from each (e.g., 5 ' and 3 ') end of any intervening sequences (e.g., introns) within that diagnostic gene, plus 100 base pairs of the untranslated regions of exons abutting the 5 '-most and 3 '-most ends of the coding region.
  • This length was used in the gene weighting calculations of the studies to detect the diagnostic genes of the disclosure, as described in detail below.
  • the mutations identified in the diagnostic genes in Table 6 by the methods disclosed herein, and potentially other mutations in these same genes that cause truncations of the encoded protein, alterations in the amino acid sequence of the encoded protein, or alterations in the splicing of the encoded transcript, have been identified as biomarkers to be used in detecting bladder cancer, or in monitoring for recurrent bladder cancer. Diagnostic tests based upon these biomarkers are disclosed, as are methods of testing and monitoring, and kits for testing for the presence to bladder cancer, and especially recurrent bladder cancer. These methods, tests, and kits are designed to detect bladder cancer, in some embodiments non- invasively by testing for the presence or level of such biomarkers in the diagnostic genes of the disclosure, for example using DNA isolated from urine samples. IDENTIFICATION OF TUMOR-SPECIFIC METHYLATED GENES
  • the methylation status of a target nucleic acid may be determined using a number of different methods.
  • a non- limiting example of determining methylation status is bisulfite sequencing, which can be used to identify methylations of CpGs of a target nucleic acid molecule.
  • a nucleic acid target molecule may be treated with a reagent that modifies the nucleic acid target molecule as a function of its methylation state by adding a reagent such as bisulfite to a solution containing the nucleic acid target region.
  • any unmethylated nucleotide, such as any unmethylated C nucleotide, present in the nucleic acid target molecule can be chemically modified, such as deaminated; however, if the nucleic acid target molecule contains no unmethylated selected nucleotide, such as no
  • nucleic acid target molecule treated with such a reagent may not be chemically modified.
  • Grunau, C., et al. provide several different bisulfite treatment methods in "Bisulfite genomic sequencing: systematic investigation of critical experimental parameters" Nucleic Acids Res, 29, E65-65 (2001). The converted nucleic acid molecule is amplified and sequenced.
  • PCR reverse transcriptase-PCR
  • RT-qPCR reverse transcription quantitative real-time PCR
  • SDA strand displacement amplification
  • 3SR self-sustained sequence replication
  • Applicable PCR amplification techniques are described in Current Protocols in Molecular Biology, Ausubel et al. (Eds.), John Wiley and Sons, NY (1989), and Innis et al. (PCR Protocols, A Guide to Methods and
  • PCR a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length.
  • annealing temperatures may vary between about 32°C and 48°C depending on primer length.
  • high stringency PCR amplification a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).
  • DNA methylation of a nucleic acid target region can also be obtained by
  • MALDI-TOF MS analysis of base-specific cleavage products derived from amplified nucleic acid target molecules In general, a PCR amplification product is generated from bisulfite treated DNA, which is transcribed in vitro into a single stranded RNA molecule and subsequently cleaved base-specifically by an endoribonuclease. The conversion of cytosine to uracil during bisulfite treatment generates different base specific cleavage patterns that can be readily analyzed by MALDI-TOF MS. These spectral analyses may be used to determine the ratio of methylated versus non-methylated nucleotide at each methylation site of the nucleic acid target region.
  • the methods of the present invention are particularly useful for quantitative methylation analysis.
  • methylation state of one or a plurality of CpG sites and/or islands within a DNA sequence involve, among other techniques, DNA sequencing of bisulfite-treated DNA (Frommer et al., Proc. Natl. Acad. Sci. USA 89: 1827-1831, (1992)), PCR (for sequence-specific amplification), Southern blot analysis, use of methylation-sensitive restriction enzymes, etc.
  • restriction enzyme digestion of PCR products amplified from converted DNA can be used, e.g., the method described by Sadri & Hornsby (Nucl. Acids Res. 24:5058-5059, (1996)), or COBRA (Combined Bisulfite Restriction Analysis) (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, (1997)).
  • COBRA analysis is a quantitative methylation assay useful for determining DNA methylation levels at specific gene loci in small amounts of genomic DNA (Xiong & Laird, Nucleic Acids Res. 25:2532-2534, 1997).
  • restriction enzyme digestion is used to reveal methylation-dependent sequence differences in PCR products of sodium bisulfite-treated DNA. Methylation-dependent sequence differences are first introduced into the genomic DNA by standard bisulfite treatment according to the procedure described by Frommer et al. (Proc. Natl. Acad. Sci. USA 89:1827-1831 (1992)). PCR amplification of the bisulfite converted DNA is then performed using primers specific for the interested CpG islands, followed by restriction endonuclease digestion, gel electrophoresis, and detection using specific, labeled hybridization probes.
  • Methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNA methylation levels.
  • this technique can be reliably applied to DNA obtained from microdissected paraffin-embedded tissue samples.
  • MCA Molarity of methylation sites
  • Another method for analyzing methylation sites is a primer extension assay, including an optimized PCR amplification reaction that produces amplified targets for subsequent primer extension genotyping analysis using mass spectrometry.
  • the assay can also be done in multiplex. This method (particularly as it relates to genotyping single nucleotide polymorphisms) is described in detail in PCT publication WO05012578A1 and US publication US20050079521A1.
  • the assay can be adopted to detect bisulfite introduced methylation dependent C to T sequence changes.
  • tumor specimens and/or normal samples can be formalin-fixed paraffin-embedded (FFPE) or fresh frozen.
  • FFPE formalin-fixed paraffin-embedded
  • the percent of methylated cytosines in CpG islands of promoter regions can be calculated for any number of genes interest (e.g., the genes of Table 12 or a subset thereof, 22,670 total genes, etc.). This percent of methylated CpG islands can be used as a score to distinguish bladder cancer from normal tissue.
  • Genes can be considered hypermethylated when ⁇ 20% or more of test CpG sites (or islands) are methylated. Genes can be considered not to be hypermethylated (or hypomethylated) when fewer than 20% of tested CpG sites (or islands) are methylated. Genes to be tested in a particular sample can be selected based on having significant discriminatory power based on results of Wilcoxon signed-ranked tests.
  • All analyses can be conducted using R version 3.0.2.
  • Heatmaps can be generated, and clustering can be performed using the R heatmap library. Clustering can use the complete method, with clustering distance determined by Spearman rank correlation.
  • a Wilcoxon signed-rank test can be conducted to determine whether methylation levels are significantly higher in tumors than in normal samples.
  • P-values from Wilxocon tests can be adjusted for multiple testing according to the Bonferroni correction method. Adjusted p-values can be considered to indicate significant results at the one-sided 5% level.
  • Gene selection can occur in two steps.
  • the median percent methylation can be calculated for both tumors and normal tissue.
  • some subset of genes e.g., 4,641 genes
  • a Wilcoxon signed-ranked test can be conducted. After correcting for multiple testing, the genes showing significantly higher methylation in tumors than in normal tissues at the 55 significance level (e.g., the genes in Table 12) can be selected for use in the methods and kits described herein.
  • the genes showing significantly higher methylation can include GCM2, NKX2-6, NPY, WRAP73, PDX1, T, VSX1, HIST1H4F, CARTPT, EN1, SIX6, OTX20S1, TALI, ALX1, SLFN12L, NKX2-3, RAX, OLIG2, GABRA ⁇ FEZF2, CA2, CNTNAP5, SHOXY, OTX2, TCF24, NPAS4, GAD2, FOXF1, LOC386758, PCDHGA2, and MAFB.
  • Figure 2 shows an example of Spearman correlations of the top performing genes (e.g., the genes in Table 12), which can be adjusted for tumor versus normal tissue status.
  • the present disclosure provides methods, systems, and kits for detecting bladder cancer (e.g., diagnosing bladder cancer, monitoring for bladder cancer recurrence, etc.) using 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180 or more genes in any of GENE LISTS A, B, C, D, and/or Z.
  • bladder cancer e.g., diagnosing bladder cancer, monitoring for bladder cancer recurrence, etc.
  • the diagnostic genes of the disclosure are provided in four different overlapping lists, GENE LISTS A, B, C, and D, and one comprehensive list, GENE LIST Z, and a 61 -member subset of that GENE LIST C, GENE LIST X.
  • the 184 individual diagnostic genes of GENE LIST Z are further described in Table 6, above.
  • the genes with respect to diagnostic methylation are provided in Table 12.
  • bladder cancer-specific signature mutations in the diagnostic genes of Table 6 enables diagnostic (including monitoring, e.g., recurrence monitoring) and prognostic methods, systems, and kits.
  • the diagnostic genes identified in Table 6, not only provide the particular bladder cancer-specific mutations of MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, and MUTATION PANEL D, but will also harbor additional mutations (e.g., "biomarkers") that can be used to detect bladder cancer.
  • the present disclosure provides methods, systems, and kits using particular bladder cancer-specific mutations identified in the diagnostic genes in Table 6 - mutations that comprise the bladder cancer-specific signature mutations of the disclosure.
  • These mutations as identified by the procedures described in EXAMPLES A, B, C and D, are referred to as MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, and MUTATION PANEL D respectively, and are described in detail in Tables A, B, C, and D, respectively.
  • all of the mutations provided in Tables A, B, C, and D comprise indicators of bladder cancer that can be used in the methods of detecting bladder cancer, as further disclosed herein.
  • the mutations described in Tables A, B, C, and D are collectively referred to as bladder cancer-specific signature mutations of the present disclosure.
  • the present disclosure provides methods, systems, and kits using particular bladder cancer-specific genes in Table 12— having altered methylation patterns in bladder cancer.
  • the present disclosure provides methods, systems, and kits using indicators of bladder cancer that in this aspect are mutations which result in truncation of the protein encoded by one of the diagnostic genes listed in Tables 1-11, alteration of the amino acid sequence of the encoded protein, or alteration of the splicing of the transcript of one of the diagnostic genes listed in Tables 1-11, but that are not present in MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D.
  • diagnostic test panels of mutations comprising a predetermined subset of diagnostic genes are envisioned. Such diagnostic test panels can be used for the detection of bladder cancer, or for the monitoring of bladder cancer recurrence, by determining the presence or absence of a limited, and predetermined set of bladder cancer-specific signature mutations of the disclosure.
  • such diagnostic test panels of mutations can comprise a single mutation from MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D for each one of the 184 diagnostic genes listed in Table 6.
  • such diagnostic test panels of mutations can comprise the full set of disclosed bladder cancer-specific signature mutations for each single diagnostic gene, but for only a subset of the diagnostic genes listed in Table 6, such as the subset provided in Table 4.
  • a diagnostic test panel can comprise a subset of the bladder cancer-specific signature mutations as disclosed in MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D, for a subset of diagnostic genes listed in Table 6, such as the subset provided in Table 4.
  • diagnostic methods of the disclosure may comprise determining whether a patient sample has one or more mutations in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the genes listed in Tables 1- 11 or in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, or GENE LIST Z.
  • diagnostic test panels may comprise all cancer-specific signature mutations known for any single diagnostic gene listed in Table 6, with the diagnostic panel in these embodiments comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the diagnostic genes listed in Tables 1-11 or in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, or GENE LIST Z.
  • diagnostic test panels may comprise a predetermined set of 10, 20, 40, 60, 80, 100, 200, 400, 600, or 800 of the cancer-specific signature mutations disclosed in MUTATION PANEL A, MUTATION PANEL B, and MUTATION PANEL C.
  • a diagnostic test panel may comprise only those bladder cancer-specific signature mutations with high predictive value that are found most frequently in bladder cancer.
  • the panel may comprise TERT (promoter), TP53, MLL2, KDM6A, FGFR3, ARID 1 A, STAG2, ERBB2, ERBB3, RBI, KRAS (hot spots), HRAS (hot spots), and PIK3CA (hot spots).
  • the present disclosure also provides methods of detecting bladder cancer (whether for initial diagnosis or monitoring for recurrent bladder cancer) that utilize the disclosed indicators of bladder cancer, including the bladder cancer-specific signature mutations of the disclosure or methylated genes of the disclosure, as identified in the diagnostic genes of the disclosure, to classify patients as having bladder cancer or having recurrent bladder cancer.
  • the present disclosure provides an in vitro method of detecting bladder cancer in patients, comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • said indicator is a mutation (e.g., a signature mutation listed in Tables A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 184 gene(s) listed in Table 6 or Table 4; and
  • this method further includes a step (2)(b), which comprises the optional step of diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having bladder cancer (or as having a low risk of having bladder cancer).
  • this method further comprises a step of generating a risk profile for the patient using the results of steps (1) and (2)
  • the present disclosure provides an in vitro method of monitoring for recurrent bladder cancer in a patient, comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or a combination thereof) in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 184 gene(s) listed in Table 6 or Table 4; and
  • this method further includes a step (3)(b), which comprises diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having recurrent bladder cancer (or as having a low risk of having recurrent bladder cancer).
  • this method further comprises a step of generating a risk profile for the patient using the results of steps (1), (2), and (3).
  • the determining step may rely exclusively on detecting the presence of one or more of the bladder cancer-specific signature mutations of the disclosure, as disclosed in MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D, or some combination thereof.
  • the determining step may comprise detecting the presence of one or more mutations in one or more of the diagnostic genes in Table 6 or Table 4, e.g., mutations that result in a truncation of the encoded protein, and alteration in the amino acid sequence of the encoded protein, or altered splicing of the transcript of a diagnostic gene of the disclosure, wherein the mutation, or mutations are not listed in MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D.
  • the determining step may comprise some combination of detecting the presence of one or more of the bladder cancer-specific signature mutations of the disclosure, and detecting the presence of one or more mutations in one or more of the diagnostic genes in Table 6 or Table 4 that are not listed in MUTATION PANEL A, MUTATION PANEL B, MUTATION PANEL C, or MUTATION PANEL D (in some embodiments such other mutation(s) resulting in a truncation of the encoded protein, and alteration in the amino acid sequence of the encoded protein, or altered splicing of the transcript of a diagnostic gene of the disclosure).
  • the determining step may comprise detecting the presence of one or more of the bladder cancer-specific signature mutations in a particular diagnostic test panel, as described above.
  • the individual predictive power of each gene (or mutations therein) may be used to rank them in importance. Such rankings may further be used to weight the various genes in a diagnostic panel of the disclosure.
  • the inventors have determined that the diagnostic genes of the disclosure can be ranked in various ways as shown in Tables 1-11 according to the predictive power of each individual gene.
  • the plurality of diagnostic genes analyzed for the presence of an indicator of bladder cancer comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 65, 70, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, or 184 gene(s) genes listed in any of Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the genes listed in Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Tables 1-11.
  • the plurality of diagnostic genes comprises at least some number of genes listed in Table 6 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more) and this plurality of diagnostic genes comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Tables 1-11.
  • the present disclosure provides an in vitro method of detecting bladder cancer in patients, comprising:
  • nucleic acid e.g., DNA
  • a patient biological sample e.g., urine
  • said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12
  • this method further includes a step (3)(b), which comprises diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having recurrent bladder cancer (or as having a low risk of having recurrent bladder cancer).
  • this method further comprises a step of generating a risk profile for the patient using the results of steps (1), (2), and (3).
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • the present disclosure provides an in vitro method of detecting bladder cancer in patients, comprising:
  • nucleic acid e.g., DNA
  • patient biological sample e.g., urine
  • indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12;
  • this method further includes a step (3)(b), which comprises diagnosing a patient in whose sample the absence of any indicator of bladder cancer is detected as not having recurrent bladder cancer (or as having a low risk of having recurrent bladder cancer).
  • this method further comprises a step of generating a risk profile for the patient using the results of steps (1), (2), and (3).
  • said one or more indicator of bladder cancer is one or more genes listed in Table 12 that 1) comprises any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12, or 2) comprises any of the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 genes listed in Table 12.
  • Biological samples can be obtained from a subject ⁇ e.g., a human patient) using any known device or method so long as the analytes to be measured by the methods of detecting are not rendered undetectable by the detection step.
  • devices or methods suitable for taking bodily fluid from a mammal include urine sample cups, urethral catheters, swabs, hypodermic needles, thin needle biopsies, hollow needle biopsies, punch biopsies, metabolic cages, and aspiration.
  • the test sample may be diluted to reduce the concentration of the sample analytes prior to analysis.
  • the degree of dilution may depend on a variety of factors including but not limited to the type of assay used to measure the analytes, the reagents utilized in the assay, and the type of bodily fluid contained in the test sample.
  • Non-limiting examples of suitable diluents include deionized water, distilled water, saline solution, Ringer's solution, phosphate buffered saline solution, TRIS-buffered saline solution, standard saline citrate, and HEPES -buffered saline.
  • the biological sample is an amount of bodily fluid obtained from a subject, such as a mammal.
  • Bodily fluids can include urine, blood, plasma, serum, semen, perspiration, tears, mucus, and tissue lystates.
  • the sample is urine.
  • nucleic acid e.g., DNA, RNA
  • a biological sample e.g., urine sample
  • an indicator of bladder cancer can be DNA from cells (e.g., nucleated cells), cell fragments or microvesicles present in the urine, or cell-free DNA.
  • the cells may be isolated by sedimentation (e.g., through centrifugation), subsequently lysed, and the DNA extracted from the lysate. If the DNA that is obtained from a urine sample and used to determine the presence of an indicator of bladder cancer is cell-free DNA, it may be isolated by, e.g., ultrafiltration of the urine, or passage of the urine through a cationic matrix that binds the negatively-charged DNA.
  • determining the presence of indicators of bladder cancer can be accomplished by adapting suitable techniques used in the art, of which there are many with which those skilled in the art would be familiar, to the methods disclosed herein. Determining generally refers to the detection of the presence (or absence or level or structure) of a target nucleic acid molecule, which can include a target nucleic acid molecule having a polymorphism or indicator of bladder cancer of interest.
  • a nucleic acid molecule is "detected" as used herein where the level of nucleic acid measured (e.g., by quantitative PCR), or the level of detectable signal provided by the detectable label (including the level of nucleic acid having a certain structure or sequence, e.g., an indicator of bladder cancer) is at all above the background level, thus allowing for a qualitative (e.g., present or absent) or quantative (e.g., amount) detection of the analyte.
  • detection can occur by a process wherein the signal generated by a directly or indirectly labeled probe nucleic acid molecule (capable of hybridizing to a target in a sample) is measure or observed. Detection of the probe nucleic acid is directly indicative of the presence, and thus the detection of, an indicator of bladder cancer, such as a sequence encoding a marker gene.
  • a target nucleic acid molecule e.g., an indicator of bladder cancer
  • the detecting step may involve detection by a technique chosen from allele-specific polymerase chain reactions (PCR), mutant-enriched PCR, digital protein truncation tests, DNA or RNA sequencing (including, e.g., direct sequencing, massively parallel sequencing), use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • Quantitative amplification methods such as, but not limited to, TaqMan® (a commercially available quantitative PCR system) can also be used to "detect” an indicator of bladder cancer according to the disclosure.
  • TaqMan® a commercially available quantitative PCR system
  • Methods and techniques for "detecting" fluorescent, radioactive, and other chemical labels may be found in Ausubel et al. (1995, Short protocols in Molecular biology, 3 rd Ed. John Wiley and Sons, Inc.).
  • a nucleic acid can be "indirectly detected" wherein a moiety is attached to a probe nucleic acid that will hybridize with the target, wherein the moiety comprises, for example, an enzyme activity, allowing detection of the target in the presence of an appropriate substrate, or a specific antigen or other marker allowing detection by addition of an antibody or other specific indicator.
  • Nucleic acid molecules having for example, indicators of bladder cancer can be detected and/or isolated by specific hybridization under particular stringency conditions.
  • Stringency conditions for hybridization is a term of art that refers to incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid.
  • the first nucleic acid can be perfectly complementary to the second, or the first and second can share some degree of complementarity less than perfect ⁇ e.g., 70%, 75%, 85%, 95%).
  • certain high stringency conditions can be used that distinguish perfectly complementary nucleic acids from those of less complementarity.
  • 0.2xSSC, 0.1 xSSC temperature ⁇ e.g., room temperature, 42°C, 68°C
  • concentration of destabilizing agents such as formamide or denaturing agents such as SDS
  • factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • the detection of bladder cancer, or monitoring for recurrent bladder cancer can be accomplished with a sensitivity of at least 85% at a specificity of at least 85%, with a sensitivity of at least 80% at a specificity of at least 90%, with a sensitivity of at least 75% at a specificity of at least 95%, or with a sensitivity of at least 70%> at a specificity of at least 95%.
  • the present disclosure provides methods that classify or diagnosing a subject ⁇ e.g., a human patient) as having bladder cancer or having recurrent bladder cancer.
  • classifying a cancer and “cancer classification” refer to determining one or more clinically-relevant features of a cancer and/or determining a particular prognosis of a patient having said cancer.
  • classifying a cancer includes, but is not limited to: (i) diagnosis of bladder cancer; (ii) evaluating risk or likelihood of recurrence; (iii) evaluating metastatic potential, potential to metastasize to specific organs, risk of recurrence, and/or course of the tumor; (iv) evaluating tumor stage; (v) determining patient prognosis in the absence of treatment of the cancer; (vi) determining prognosis of patient response ⁇ e.g., tumor shrinkage or progression-free survival) to treatment ⁇ e.g., surgery to excise tumor, adjuvant therapy, including immunotherapy, targeted therapy, or conventional chemotherapy, etc.); (vii) diagnosis of actual patient response to current and/or past treatment; (viiii) determining a preferred course of treatment for the patient; (ix) prognosis for patient relapse after treatment (either treatment in general or some particular treatment); (x) prognosis of patient life expectancy ⁇ e.g., prognosis for overall survival), etc.
  • risk profile generally means the likelihood of a subject as described herein having or developing bladder cancer. This involves correlating a particular assay or analysis result o routput to some likelihood ⁇ e.g., increased, not increased, decreased, etc.) of some clinical feature ⁇ e.g., increased risk ⁇ e.g., increased hereditary risk) of cancer), or additionally or alternatively concluding or communicating such clinical feature based at least in part on such particular assay or analysis result, such correlating, concluding or communicating may comprise assigning a risk or likelihood of the clinical feature occurring based at least in part on the particular assay or analysis result. In some embodiments, such risk is a percentage probability of the event or outcome occurring.
  • the patient is assigned to a risk group (e.g., low risk, intermediate risk, high risk, etc.).
  • low risk is any percentage probability below 5%, 10%, 15%, 20%, 25%, 30%), 35%o, 40%), 45%, or 50%>.
  • intermediate risk is any percentage probability above 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% and below 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
  • high risk is any percentage probability above 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • the terms “increased,” “normal,” and “decreased” when referring to risk preferably mean that the subject has that risk level when compared with the risk associated with an individual having a different nucleic acid in one or more genes from Tables 1-11, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z, or a hypermethylated or hypomethylated gene listed in Table 12.
  • results of the methods of the present disclosure can be combined with, or interpreted in light of, the results from other diagnostic procedures involving other bladder cancer biomarkers.
  • Descriptions of other bladder cancer biomarkers and their use have been provided in a recent review article by Parker and Spiess. See, Parker, J. & Spiess, P.E.; TheScientificWorldJournal 11 : 1103-1112 (2011).
  • FISH fluorescence in situ hybridization
  • results of the methods of the present disclosure can also be combined with, or interpreted in light of, the results from immunoflurometric assays designed to test the levels of expression of proteins of the minichromosome maintenance (Mem) family, and specifically Mcm5, as described by Kelly et al. (PLoS ONE 7(7)e40305: l-8(2012).
  • the results of the methods of the present disclosure can also be combined with, or interpreted in light of, the assays designed to detect epigenetic changes in the FOXEl, GATA4, TWISTI, NID2, CCNAl genes, as described in U.S. Patent Application Publication No. 2012/0027870.
  • results of the methods of the present disclosure can also be combined with, or interpreted in light of, the multiparametric assays described in U.S. Patent Application Publication No. 2012/0244536.
  • the present disclosure provides methods that include treatment based on the classification of bladder cancer as described herein.
  • the levels of one or more analyte biomarkers or the levels of a specific panel of analyte biomarkers in a sample are compared to a reference standard ("reference standard” or “reference level”) in order to direct treatment decisions.
  • the reference standard used for any embodiment disclosed herein may comprise average, mean, or median levels of the one or more analyte biomarkers or the levels of the specific panel of analyte biomarkers in a control population.
  • the reference standard may additionally comprise cutoff values or any other statistical attribute of the control population, such as a standard deviation from the mean levels of the one or more analyte biomarkers or the levels of the specific panel of analyte biomarkers.
  • the control population may comprise healthy individuals or individuals with bladder cancer.
  • individuals with levels of one or more analyte biomarkers or levels of a specific panel of analyte biomarkers greater than the reference levels would be more likely to have bladder cancer. Therefore, an individual presenting with levels of the one or more analyte biomarkers or levels of the specific panel of analyte biomarkers greater than the reference standard would be a candidate for more aggressive therapy. On the other hand, an individual presenting with levels of the one or more analyte biomarkers or levels of the specific panel of analyte biomarkers less than or equal to the reference standard would be less likely to have bladder cancer and therefore be a candidate for less aggressive therapy.
  • individuals with levels of one or more analyte biomarkers or levels of a specific panel of analyte biomarkers less than the reference levels would be more likely to have bladder cancer. Therefore, an individual presenting with levels of the one or more analyte biomarkers or levels of the specific panel of analyte biomarkers less than the reference standard would be a candidate for more aggressive therapy. On the other hand, an individual presenting with levels of the one or more analyte biomarkers or levels of the specific panel of analyte biomarkers greater than or equal to the reference standard would be less likely to have bladder cancer and therefore be a candidate for less aggressive therapy.
  • a patient is treated more or less aggressively than a reference therapy.
  • a reference therapy is any therapy that is the standard of care for bladder cancer.
  • the standard of care can vary temporally and geographically, and a skilled person can easily determine the appropriate standard of care by consulting the relevant medical literature.
  • treatment will be either 1) more aggressive, or 2) less aggressive than a standard therapy.
  • a more aggressive therapy than the standard therapy comprises beginning treatment earlier than in the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises administering additional treatments than in the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises treating on an accelerated schedule compared to the standard therapy. In some embodiments, a more aggressive therapy than the standard therapy comprises administering additional treatments not called for in the standard therapy.
  • a less aggressive therapy than the standard therapy comprises delaying treatment relative to the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering less treatment than in the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering treatment on a decelerated schedule compared to the standard therapy. In some embodiments, a less aggressive therapy than the standard therapy comprises administering no treatment.
  • Chemotherapy and surgery are non-limiting examples of treatment options.
  • active treatment in bladder cancer is well-understood by those skilled in the art and, as used herein, has the conventional meaning in the art. Generally speaking, active treatment in bladder cancer can include anything other than “watchful waiting.” Active treatment currently applied in the art of bladder cancer treatment can include, e.g., radiotherapy, transurethral resection, cystectomy, hormonal therapy, chemotherapy, immunotherapy, etc. Active treatment can include a drug regimen, which can include, but is not limited to, Adriamycin, Cisplatin, Doxorubicin Hydrochloride, and Platinol. Each treatment option has with it certain risks as well as side-effects of varying severity.
  • the practitioner adjusts the therapy based on a comparison between a reference level and the levels of one or more analyte biomarkers or the levels of a specific panel of analyte biomarkers in a sample from a patient. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different combination of drugs. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting drug dosage.
  • the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug combination and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and dose schedule. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting dose schedule and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting dose schedule. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, and adjusting length of therapy.
  • the practitioner adjusts the therapy by selecting and administering a different drug, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy. In one embodiment, the practitioner adjusts the therapy by selecting and administering a different drug, adjusting drug dosage, adjusting dose schedule, and adjusting length of therapy.
  • treatment comprises a less aggressive therapy than a reference therapy.
  • a less aggressive therapy comprises not administering drugs and taking a "watchful waiting" approach.
  • Watchful-waiting also sometimes called “active surveillance,” also has its conventional meaning in the art. This generally means observation and regular monitoring without treatment of the underlying disease. Watching- waiting can also be suggested when the risks of surgery, radiation therapy, hormonal therapy, or chemotherapy, for example, outweighs the possible benefits. Other treatments can be started if symptoms develop, or if there are signs that the cancer growth is accelerating.
  • a less aggressive therapy comprises delaying treatment.
  • a less aggressive therapy comprises selecting and administering less potent drugs. In one embodiment a less aggressive therapy comprises decreasing the frequency treatment. In one embodiment a less aggressive therapy comprises shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decreasing drug dosage. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and decelerating dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage and decelerating dose schedule. In one embodiment, less aggressive therapy comprises decreasing drug dosage and shortening length of therapy. In one embodiment, less aggressive therapy comprises decelerating dose schedule and shortening length of therapy.
  • less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and decelerating dose schedule. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decelerating dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises decreasing drug dosage, decelerating dose schedule, and shortening length of therapy. In one embodiment, less aggressive therapy comprises selecting and administering less potent drugs, decreasing drug dosage, decelerating dose schedule, and shortening length of therapy. In some embodiments, a less aggressive therapy comprises administering only non-drug-based therapies.
  • treatment comprises a more aggressive therapy than a reference therapy.
  • a more aggressive therapy comprises increased length of therapy.
  • a more aggressive therapy comprises increased frequency of the dose schedule.
  • more aggressive therapy comprises selecting and administering more potent drugs and increasing drug dosage.
  • more aggressive therapy comprises selecting and administering more potent drugs and
  • more aggressive therapy comprises selecting and administering more potent drugs and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage and accelerating dose schedule. In one embodiment, more aggressive therapy comprises increasing drug dosage and increasing length of therapy. In one embodiment, more aggressive therapy comprises accelerating dose schedule and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and accelerating dose schedule. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, and increasing length of therapy. In one embodiment, more aggressive therapy comprises selecting and administering more potent drugs, accelerating dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, accelerating dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, accelerating dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, accelerating dose schedule, and increasing length of therapy. In one embodiment, more aggressive therapy comprises increasing drug dosage, accelerating dose schedule, and increasing length of therapy.
  • more aggressive therapy comprises selecting and administering more potent drugs, increasing drug dosage, accelerating dose schedule, and increasing length of therapy.
  • a more aggressive therapy comprises administering a combination of drug- based and non-drug-based therapies.
  • the present disclosure provides methods of treating a subject (e.g., a human cancer patient) that includes an in vitro method generally comprising detecting an indicator of bladder cancer in a patient sample; diagnosing a patient in whose sample an indicator of bladder cancer is detected as having bladder cancer; and recommending, prescribing, or administering a treatment for a patient diagnosed as having bladder cancer.
  • the disclosure provides a method of treating bladder cancer patients comprising:
  • nucleic acid e.g., DNA
  • a biological patient sample e.g. , urine
  • said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or combination thereof) in one or more genes listed in Table 6 or Table 4;
  • step (3) recommending, prescribing, or administering a treatment regimen a treatment for a patient diagnosed as having bladder cancer in step (2).
  • the present disclosure provides methods of treating a subject (e.g., a human cancer patient) that includes an in vitro method generally comprising detecting an indicator of bladder cancer in a patient sample; diagnosing a patient in whose sample an indicator of bladder cancer is detected as having bladder cancer; and recommending, prescribing, or administering a treatment for a patient diagnosed as having bladder cancer.
  • a subject e.g., a human cancer patient
  • an in vitro method generally comprising detecting an indicator of bladder cancer in a patient sample; diagnosing a patient in whose sample an indicator of bladder cancer is detected as having bladder cancer; and recommending, prescribing, or administering a treatment for a patient diagnosed as having bladder cancer.
  • the disclosure provides a method of treating bladder cancer patients comprising:
  • nucleic acid e.g., DNA
  • a biological patient sample e.g. , urine
  • said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12;
  • the present disclosure provides methods of treating a subject (e.g., a human cancer patient) that includes an in vitro method generally comprising detecting bladder cancer in a patient according to the present disclosure, classifying the patient as having bladder cancer, and recommending, prescribing, or administering a treatment for the cancer patient based on the classification.
  • a subject e.g., a human cancer patient
  • an in vitro method generally comprising detecting bladder cancer in a patient according to the present disclosure, classifying the patient as having bladder cancer, and recommending, prescribing, or administering a treatment for the cancer patient based on the classification.
  • the disclosure provides a method of treating a cancer patient comprising:
  • nucleic acid e.g., DNA
  • a biological patient sample e.g. , urine
  • said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or combination thereof) in one or more genes listed in Table 6 or Table 4;
  • step (3) (a) recommending, prescribing, or administering an aggressive treatment regimen for a patient diagnosed as having bladder cancer in step (2)(a); or
  • step (3)(b) recommending, prescribing, or administering a passive treatment regimen (e.g., watchful waiting or active surveillance) for a patient diagnosed as not having bladder cancer in step (3)(a).
  • a passive treatment regimen e.g., watchful waiting or active surveillance
  • the present disclosure provides methods of treating a subject
  • the disclosure provides a method of treating a cancer patient comprising:
  • nucleic acid e.g., DNA
  • a biological patient sample e.g. , urine
  • said indicator is a hypermethylated (or optionally hypomethylated) gene listed in Table 12;
  • step (3) (a) recommending, prescribing, or administering an aggressive treatment regimen for a patient diagnosed as having bladder cancer in step (2)(a); or
  • step (3)(b) recommending, prescribing, or administering a passive treatment regimen (e.g., watchful waiting or active surveillance) for a patient diagnosed as not having bladder cancer in step (3)(a).
  • a passive treatment regimen e.g., watchful waiting or active surveillance
  • the present disclosure provides methods of treating a subject (e.g., a human cancer patient) that includes an in vitro method of monitoring for recurrent bladder cancer in a patient, classifying the patient as having recurrent bladder cancer, and recommending, prescribing, or administering a treatment for the cancer patient based on the classification.
  • a subject e.g., a human cancer patient
  • the disclosure provides a method of treating bladder cancer patients comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or combination thereof) in one or more genes listed in Table 6 or Table 4;
  • step (3)(a) recommending, prescribing, or administering an aggressive treatment regimen for a patient diagnosed as having recurrent bladder cancer in step (3)(a);
  • the present disclosure provides methods of treating a subject
  • the disclosure provides a method of treating bladder cancer patients comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • step (3)(a) recommending, prescribing, or administering an aggressive treatment regimen for a patient diagnosed as having recurrent bladder cancer in step (3)(a);
  • step (3)(b) recommending, prescribing, or administering a passive treatment regimen (e.g., watchful waiting or active surveillance) for a patient diagnosed as not having recurrent bladder cancer in step (3)(b).
  • a passive treatment regimen e.g., watchful waiting or active surveillance
  • the present disclosure provides methods of treating a subject
  • the disclosure provides a method of treating a cancer patient comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • said indicator is a mutation (e.g., a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, splicing mutation, or large rearrangement, or combination thereof) in one or more genes listed in Table 6 or Table 4;
  • diagnosing said patient as having recurrent bladder cancer, based at least in part on the presence of said indictor of bladder cancer; and
  • step (3) recommending, prescribing, or administering a treatment regimen.
  • the present disclosure provides methods of treating a subject (e.g., a human cancer patient) that includes an in vitro method of monitoring for recurrent bladder cancer in a patient, classifying the patient as having recurrent bladder cancer, and recommending, prescribing, or administering a treatment for the cancer patient based on the classification.
  • a subject e.g., a human cancer patient
  • the disclosure provides a method of treating a cancer patient comprising:
  • nucleic acid e.g., DNA
  • a biological sample e.g., urine
  • step (3) recommending, prescribing, or administering a treatment regimen.
  • compositions for use in the above methods include, but are not limited to, nucleic acid probes hybridizing to a set of bladder cancer diagnostic genes (or to any nucleic acids encoded thereby or complementary thereto); nucleic acid primers and primer pairs suitable for amplifying (e.g., by PCR) all or a portion of a set of bladder cancer diagnostic genes or any nucleic acids encoded thereby; antibodies binding immunologically to a polypeptide encoded by a set of bladder cancer diagnostic genes; probe sets comprising a plurality of said nucleic acid probes, nucleic acid primers, antibodies, and/or polypeptides; microarrays comprising any of these; etc.
  • the disclosure provides computer methods, systems, software and/or modules for use in the above methods.
  • the disclosure provides a set of probes comprising isolated (or synthetic) oligonucleotides capable of selectively hybridizing to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 or more genes from Tables 1-12, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z.
  • probe and "oligonucleotide” (also “oligo"), when used in the context of nucleic acids, interchangeably refer to a relatively short nucleic acid fragment.
  • the disclosure also provides primers useful in the methods of the disclosure.
  • “Primers” are oligonucleotides capable, under the right conditions and with the right companion reagents, of selectively priming the biochemical synthesis of (e.g., amplifying) a target nucleic acid (e.g., a target gene or portion thereof).
  • target nucleic acid e.g., a target gene or portion thereof.
  • the probe can generally be of any suitable size/length. In some embodiments the probe has a length from about 8 to 200, 15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length. They can be labeled with detectable markers with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Research 14:6115-6128 (1986); Nguyen et al., Biotechniques 13: 116-123 (1992); Rigby et al., J. Mol. Bio.
  • probes may be modified in any suitable manner for various molecular biological applications.
  • General techniques for producing such oligonucleotide probes are conventional in the art and, based on the present disclosure, can be adapted and applied to the present disclosure to produce compositions of the disclosure.
  • Probes according to the disclosure can be used in the hybridization / amplification / detection techniques discussed above.
  • some embodiments of the disclosure comprise probe sets suitable for use in a microarray in detecting, amplifying and/or quantitating a plurality of bladder cancer diagnostic genes.
  • the probe sets have a certain proportion of their probes directed to bladder cancer diagnostic genes - e.g., a probe set comprising at least 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), 96%o, 97%), 98%), 99%, or 100% probes specific for bladder cancer diagnostic genes according to the present disclosure.
  • the probe set comprises probes directed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 or more genes from Tables 1-12, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z.
  • Such probe sets can be incorporated into high-density arrays comprising 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1 ,000,000 or more different probes.
  • the probe sets comprise primers (e.g., primer pairs) for amplifying nucleic acids comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 or more genes from Tables 1-12, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z.
  • primers e.g., primer pairs
  • the present disclosure provides the use of such compositions.
  • the disclosure provides the use of a plurality of oligonucleotide probes for detecting bladder cancer in a patient sample, wherein said plurality of probes comprises at least one probe selectively hybridizing to each of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 or more genes from Tables 1-12, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z.
  • the disclosure also provides the use of an oligonucleotide probe set for detecting bladder cancer in a patient sample, wherein said probe set comprises at least one probe per gene directed to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,
  • the disclosure also provides the use of a plurality of oligonucleotide primers for detecting bladder cancer in a patient sample, wherein said plurality of primers comprises at least one primer selectively hybridizing to each of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 or more genes from Tables 1-12, and in GENE LIST A, GENE LIST B, GENE LIST C, GENE LIST D, GENE LIST X, and GENE LIST Z.
  • the probe sets comprise primers (e.g., primer pairs) for amplifying nucleic acids comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,
  • kits for conducting the methods of the present disclosure that utilize the disclosed indicators of bladder cancer, including, but not limited to, the bladder cancer-specific signature mutations of the disclosure, as identified in the diagnostic genes of the disclosure, to classify patients as having bladder cancer or having recurrent bladder cancer.
  • kits may comprise reagents useful, sufficient, or necessary for detecting and/or characterizing at least one indicator of bladder cancer in nucleic acid (e.g., DNA) from a biological sample (e.g., urine sample) from a patient (e.g., a human patient), said at least one indicator being chosen from one or more mutation (e.g. , a signature mutation listed in Table A, B, C, or D, or another missense mutation, nonsense mutation, frameshift mutation, a splicing mutation, or large rearrangement, or a combination thereof) in a gene listed in Table 6.
  • Such kits may also comprise instructions for using the kit, and preferably instructions on using the kit to practice a diagnostic method of the present disclosure using samples (e.g., human samples), in some embodiments urine samples.
  • kits may optionally comprise additional reagents and devices required for obtaining nucleic acid (e.g. , DNA) from a biological (e.g. , urine) sample, including centrifuge tubes for isolating cells from a sample, filtration or ultrafiltration devices for isolating cells or cell-free nucleic acid from a sample, reagents for lysing cells isolated from a sample, reagents for extracting nucleic acid from cell lysates, cationic matrices or media, including matrices and media installed in cationic spin-tubes or spin-columns, for binding cell-free nucleic acid or binding nucleic acid extracted from cell lysates, and any reagent necessary for elution of bound nucleic acid from such cationic matrices or media, and storage of such eluted nucleic acid.
  • nucleic acid e.g. , DNA
  • a biological sample e.g. , urine
  • kits may also optionally comprise reagents in dry or liquid form that are intended to be added directly to samples (e.g. , urine samples) to inhibit degradation of nucleic acid (e.g., DNA or RNA), including such reagents as ethylenediaminetetraacetic acid (EDTA) or other preservatives, protease inhibitors such as proteinase K, nuclease inhibitors, antimicrobial agents such as sodium azide, buffers, salts, etc.
  • reagents ethylenediaminetetraacetic acid (EDTA) or other preservatives
  • protease inhibitors such as proteinase K
  • nuclease inhibitors such as sodium azide, buffers, salts, etc.
  • the reagents useful, sufficient, or necessary for detecting and/or characterizing at least one indicator of bladder cancer in nucleic acid (e.g., DNA) from a biological (e.g., urine) sample can comprise any reagent know in the art for use in any technique designed to detection mutations and alterations in nucleic acids.
  • the reagents included can be reagents useful for specific detection of mutations by allele-specific polymerase chain reactions (PCR), mutant-enriched PCR, digital protein truncation tests, DNA or RNA sequencing (e.g., direct sequencing, massively parallel sequencing), use of molecular beacon probes or primers, BEAMing digital PCR, or allele-specific hybridization.
  • oligonucleotide primers suitable for the amplification of a target nucleic acid sequence DNA polymerases such as thermostable DNA polymerases to be used in amplification reactions and other types of nucleic acid template-directed synthetic reactions, other nucleic acid modifying enzymes such as RNase A and RNase H, nucleotides (including deoxyribonucleotides and modified nucleotides, such as fluorescently-labeled dideoxynucleotides for terminating amplification reactions), buffers, and other additives required for amplification of target sequences from isolated nucleic acid, and for sequencing such amplified nucleic acid, or isolated nucleic acid.
  • DNA polymerases such as thermostable DNA polymerases to be used in amplification reactions and other types of nucleic acid template-directed synthetic reactions
  • other nucleic acid modifying enzymes such as RNase A and RNase H
  • nucleotides including deoxyribonucleotides and modified nucleotides
  • probes such as fluorescently-labeled probes, designed to detect specific nucleotide sequences in isolated and/or amplified nucleic acid.
  • probes such as fluorescently-labeled probes
  • gene chips that comprise a microarray of oligonucleotides that can have a nucleotide sequence containing one of the signature mutations listed in the accompanying tables in context with surrounding genomic nucleotide sequence, or the complement thereof.
  • kits of the disclosure can be reagents necessary and sufficient to serve as positive and negative controls for the method being used to detect the signature mutations of the present disclosure.
  • the kit may be designed to include only those reagents useful, sufficient, or necessary for detecting and/or characterizing a subset of the signature mutations listed in Table A, B, C, or D, such as, for example only those mutations that comprise a particular diagnostic test panel. Consequently, different types of kits, each type designed to test for mutants that comprise a particular diagnostic test panel, are envisioned.
  • kits may designed to only detect signature mutations in a particular diagnostic gene, or a particular set of diagnostic genes, selected from all diagnostic genes identified in Table 6.
  • kits may be designed to detect indicators of bladder cancer in (e.g., the coding sequence of) only those diagnostic genes in GENE LIST X, as identified in Table 4.
  • a system for diagnosing, monitoring, or determining bladder cancer in a subject includes a database to store a plurality of bladder cancer database entries, and a processing device that includes the modules of a bladder cancer determining application.
  • the modules are executable by the processing device, and include an analyte input module, a comparison module, and an analysis module.
  • the analyte input module receives three or more sample analyte concentrations that include the biomarker analytes.
  • the sample analyte concentrations are entered as input by a user of the application.
  • the sample analyte concentrations are transmitted directly to the analyte input module by the sensor device used to measure the sample analyte concentration via a data cable, infrared signal, wireless connection or other methods of data transmission known in the art.
  • the comparison module compares each sample analyte concentration to an entry of a bladder cancer database.
  • Each entry of the bladder cancer database includes a list of minimum diagnostic concentrations reflective of a bladder cancer.
  • the entries of the bladder cancer database may further contain additional minimum diagnostic concentrations to further define diagnostic criteria including but not limited to minimum diagnostic concentrations for additional types of bodily fluids, additional types of subjects, and severities of a particular cancer.
  • the analysis module determines a bladder cancer by combining the bladder cancers identified by the comparison module for all of the sample analyte concentrations.
  • the bladder cancer has the most minimum diagnostic concentration that is less than the corresponding sample analyte concentrations.
  • the bladder cancer has the most minimum diagnostic concentrations that are all less than the corresponding sample analyte concentrations.
  • the analysis module combines the sample analyte concentrations algebraically to calculate a combined sample analyte concentration that is compared to a combined minimum diagnostic concentration calculated from the corresponding minimum diagnostic criteria using the same algebraic operations. Other combinations of sample analyte concentrations from within the same test sample, or combinations of sample analyte concentrations from two or more different test samples containing two or more different bodily fluids may be used to determine a bladder cancer.
  • the system includes one or more processors and volatile and/or nonvolatile memory and can be embodied by or in one or more distributed or integrated components or systems.
  • the system may include computer readable media (CRM) on which one or more algorithms, software, modules, data, and/or firmware is loaded and/or operates and/or which operates on the one or more processors to implement the systems and methods identified herein.
  • CRM computer readable media
  • the computer readable media may include volatile media, nonvolatile media, removable media, non-removable media, and/or other media or mediums that can be accessed by a general purpose or special purpose computing device.
  • computer readable media may include computer storage media and communication media, including but not limited to computer readable media.
  • Computer storage media further may include volatile, nonvolatile, removable, and/or non-removable media implemented in a method or technology for storage of information, such as computer readable instructions, data structures, program modules, and/or other data.
  • Communication media may, for example, embody computer readable instructions, data structures, program modules, algorithms, and/or other data, including but not limited to as or in a modulated data signal.
  • the communication media may be embodied in a carrier wave or other transport mechanism and may include an information delivery method.
  • the communication media may include wired and wireless connections and technologies and may be used to transmit and/or receive wired or wireless communications. Combinations and/or sub-combinations of the above and systems, components, modules, and methods and processes described herein may be made.
  • Illumina Tru-seq Capture Protocol (Illumina, Inc.; San Diego, CA)
  • isolated tumor genomic DNA was sequenced using a massively parallel sequencing procedure employing an Illumina HiSeq2000 Sequencing Apparatus (Illumina, Inc.; San Diego, CA).
  • the resulting reads were aligned against HG19, and all discrepancies were cataloged. Discrepancies that occurred in 20% or greater of the reads were classified as variants.
  • the variant calls were then compared with the dbSNP database; any variants that were present within dbSNP were excluded from further analysis. Variants were then classified in 3 categories: synonymous, missense, and suspected deleterious. Synonymous variants have nucleic but no amino acid changes, and were ignored.
  • Missense variant result in single amino acid changes, and may be detrimental to gene function.
  • Suspected deleterious variants are stops (e.g., nonsense mutations), or insertions or deletions that result in a frameshift; these both lead to truncations of the gene.
  • Genes having at least one frameshift or nonsense mutation were considered for further analysis.
  • the individual variants considered during further analysis of the candidate diagnostic genes are shown in Table 7 as belonging to "Variant Class 1 ,” which, in this study, comprised stop mutations, frameshift mutations and missense mutations.
  • Variants excluded from these analyses are shown in Table 7 as belonging to "Variant Class 0," which comprised synonymous mutations and splice site mutations.
  • Genes were then weighted based on their missense/suspected deleterious variants with the following formula:
  • Weight ((# of Unique Variants A 2) / (# of Variants A 2)) * (Number of samples affected by all variants) / (Square root Length of the gene)
  • the "gene length" for a particular diagnostic gene includes the length, in basepairs, of the exons of that diagnostic gene, plus 100 base pairs from each ⁇ e.g., 5' and 3') end of any intervening sequences ⁇ e.g., introns) within that diagnostic gene, plus 100 base pairs of the untranslated regions abutting the 5 '-most and 3 '-most ends of the coding region. This length was used in the gene weighting calculations of the studies to detect the diagnostic genes of the disclosure, as described above.
  • MUTATION PANEL A The mutations identified in the genes in GENE LIST A are referred to herein as MUTATION PANEL A, and are specifically identified in Table 7.
  • the tumor sample sequence dataset used for this analysis was the dataset generated in Example A, except that sequencing reads from only 45 samples were included as it was determined that 3 samples were inadvertently run twice, and those duplicate reads were removed.
  • 48 fresh-frozen human bladder tumor samples were processed through the Illumina Tru-seq Capture Protocol (Illumina, Inc.; San Diego, CA), and isolated tumor genomic DNA was sequenced using a massively parallel sequencing procedure employing an Illumina HiSeq2000 Sequencing Apparatus (Illumina, Inc.; San Diego, CA)
  • the reads from 45 unique samples were analyzed as follows. The resulting reads were first aligned against HG19, and all discrepancies were cataloged.
  • variant calls that occurred in 20% or greater of the reads were classified as variants.
  • the variant calls were then compared with the dbSNP database; any variants that were present within dbSNP were excluded from further analysis.
  • the remaining variant calls were then compared to the genomic sequences obtained from 106 unrelated human blood samples; if a variant call existed in more than 2 blood samples it was excluded.
  • the purpose of comparing variant calls to the genomic sequences obtained from the 106 unrelated blood samples was two-fold: to supplement dbSNP in germline variant removal, and to remove any process-specific artifacts. The latter proved most relevant, as most of the variants removed following the comparison existed in all the blood genomic sequences and all the tumor genomic sequences.
  • Weight (for a given gene) (((p*l) A n) / n!) * e A -(p*l)
  • n number of unique variants
  • GENE LIST B TP53, NUP188, XIRP2, PLCG2, KDM6A, CCDC168,
  • ARID 1 A 8289 12201 7 11 9 2.18E-13
  • HEATR1 55127 14505 5 5 5 7.69E-09
  • the "gene length" for a particular diagnostic gene includes the length, in basepairs, of the exons of that diagnostic gene, plus 100 base pairs from each ⁇ e.g., 5' and 3') end of any intervening sequences ⁇ e.g., introns) within that diagnostic gene, plus 100 base pairs of the untranslated regions abutting the 5 '-most and 3 '-most ends of the coding region. This length was used in the gene weighting calculations of the studies to detect the diagnostic genes of the disclosure, as described above.
  • the tumor sample sequence dataset used for this analysis was the dataset generated in Example A, except that sequencing reads from only 45 samples were included as it was determined that 3 samples were inadvertently run twice, and those duplicate reads were removed.
  • 48 fresh-frozen human bladder tumor samples were processed through the Illumina Tru-seq Capture Protocol (Illumina, Inc.; San Diego, CA), and isolated tumor genomic DNA was sequenced using a massively parallel sequencing procedure employing an Illumina HiSeq2000 Sequencing Apparatus (Illumina, Inc.; San Diego, CA)
  • the reads from 45 unique samples were analyzed as follows. The resulting reads were first aligned against HG19, and all discrepancies were cataloged.
  • variant calls that occurred in 20% or greater of the reads were classified as variants.
  • the variant calls were then compared with the dbSNP database; any variants that were present within dbSNP were excluded from further analysis.
  • the remaining variant calls were then compared to the genomic sequences obtained from 106 unrelated human blood samples; if a variant call existed in more than 2 blood samples it was excluded.
  • the purpose of comparing variant calls to the genomic sequences obtained from the 106 unrelated blood samples was two-fold: to supplement dbSNP in germline variant removal, and to remove any process-specific artifacts. The latter proved most relevant, as most of the variants removed following the comparison existed in all the blood genomic sequences and all the tumor genomic sequences.
  • variants were then classified in 3 categories: synonymous, missense, and suspected deleterious.
  • Synonymous variants have nucleic but no amino acid changes, and were ignored. Missense variant result in single amino acid changes, and may be detrimental to gene function.
  • Suspected deleterious variants are stops (nonsense) or insertions and deletions that result in a frameshift, which both lead to truncations of the gene, as well as mutations that likely adversely affect the correct splicing of exons of the encoded gene product.
  • the individual variants considered during further analysis of the candidate diagnostic genes in this study are shown in Table 9 as belonging to "Variant Class 1," which comprised stop mutations, frameshift mutations, missense mutations and splice site mutations.
  • Variants excluded from these analyses are shown in Table 9 as belonging to "Variant Class 0," which comprised only synonymous mutations. Genes were then weighted based upon their missense/suspected deleterious variants with the following formula, based on the Poisson distribution:
  • Weight (for a given gene) (((p*l) A n) / n!) * e A -(p*l)
  • n number of unique variants
  • GENE LIST C TP53, NUP188, XIRP2, PLCG2, FOXM1, KDM6A,
  • ARID 1 A CCDC168, KIAA1671, KPRP, MUC16, OR5L2, SPTAN1, ERBB3, SRRM2, SNRNP200, ISG20L2, ZC3H7A, MYBPC2, AHNAK2, HSPBAP1, SYNE1, ZNF208, PLD1, SMC2, OR8I2, STAG2, BTN2A2, MLL2, JMJD1C, SLC35G6, VCAN, VPS13D, VCX3B, ZNF705G, RBBP8, IGSF6, DOCK9, C9orfl 74, NPC1L1, PCDHGA9, ACTB, DNHD1, LYST, SCAF11, ZNF846, NF1, CACNA2D3, LAPTM4B, LOC100133128, PCLO, DNAH17, DYNC1H1, ANK3, KIAA0100, FLG, ABCB5, POLR3C, ZNF623, DCHS1, CARD6, KIF13A, HEATR1, WDR6,

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Abstract

La présente invention concerne des marqueurs moléculaires pour détecter le cancer de la vessie, notamment un cancer de la vessie récidivant, et des procédés d'utilisation de ceux-ci, comprenant des procédés de surveillance pour la récidive du cancer de la vessie.
PCT/US2015/049940 2014-09-15 2015-09-14 Détection et surveillance du cancer de la vessie WO2016044142A1 (fr)

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CN111868264A (zh) * 2018-03-19 2020-10-30 学校法人庆应义塾 尿路上皮癌的风险的判定方法
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CN113621704B (zh) * 2021-07-22 2023-08-29 武汉艾米森生命科技有限公司 肝癌的检测和诊断的试剂及试剂盒
CN113652490A (zh) * 2021-09-27 2021-11-16 广州凯普医药科技有限公司 一种用于膀胱癌早期筛查和/或预后监测的引物探针组合及试剂盒
CN113652490B (zh) * 2021-09-27 2022-07-22 广州凯普医药科技有限公司 一种用于膀胱癌早期筛查和/或预后监测的引物探针组合及试剂盒
CN116555432A (zh) * 2023-07-05 2023-08-08 广州凯普医药科技有限公司 一种膀胱癌快速检测试剂盒
CN116555432B (zh) * 2023-07-05 2023-09-05 广州凯普医药科技有限公司 一种膀胱癌快速检测试剂盒

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