Biomarkers for detecting invasive prostate cancer from its indolent form and their treatment

Abstract

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for the detection and treatment of prostate cancer. In a specific embodiment, a method for identifying a patient as having aggressive prostate cancer comprises detecting overexpression of epithelial cell adhesion molecule (EpCAM), H4 clustered histone 5 (H4C5), and tetratricopeptide repeat domain 3 (TTC3) in a urine sample obtained from the patient compared to a control. In an even more specific embodiment, the detecting step includes detecting protein or ribonucleic acid (RNA) levels of EPCAM, H4C5, and TTC3. In another specific embodiment, the method further includes detecting overexpression of messenger ribonucleic acid (mRNA), circular RNA (circRNA), extracellular DNA, and long non-coding RNA (lncRNA) compared to a control. The method can further include detecting metabolites.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 288,157, filed December 10, 2021, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE PRESENT APPLICATION The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for the detection and treatment of prostate cancer.

[0003] Reference to electronic sequence listing The text of the computer readable sequence listing filed herewith, entitled "P17007-02," created on December 12, 2022, and having a file size of 8,614 bytes, is hereby incorporated by reference in its entirety. Summary of the Invention [Problem to be solved by the invention]

[0004] 2. Background of the Invention Prostate cancer (PCa) is the leading cause of cancer death among American men. The National Cancer Institute estimates that there will be 249,000 new cases of prostate cancer in 2021. Approximately 34,000 men will be newly diagnosed with this disease and approximately 34,000 will die from it. This estimated number of diagnoses is a small fraction of the disease-related biopsies performed each year. Prostate-specific antigen (PSA) testing is used to detect this cancer. However, PSA is not a reliable predictor of invasive cancer. Its inability to distinguish from non-invasive cancer forms, its high false positive rate, inconclusive outcomes, and small benefit justify an urgent and unmet need for new and more accurate diagnostic biomarkers for prostate cancer detection and for distinguishing invasive cancer from its indolent forms. [Means for solving the problem]

[0005] Overview of the invention In one embodiment, the present invention provides a method for identifying a patient as having aggressive prostate cancer. In certain embodiments, the present invention is useful for distinguishing between aggressive prostate cancer, indolent prostate cancer, benign prostatic hyperplasia (BPH) and prostatitis (PTT).

[0006] In some embodiments, a method for identifying a patient as having invasive prostate cancer includes detecting overexpression of epithelial cell adhesion molecule (EpCAM), H4 clustered histone 5 (H4C5), and tetratricopeptide repeat domain 3 (TTC3) in a sample obtained from the patient compared to a control.

[0007] In a specific embodiment, the detecting step detects protein levels of EpCAM in a urine sample. In other specific embodiments, the detecting step includes detecting ribonucleic acid (RNA) levels of H4C5 and TTC3. The amplification is carried out using the polymerase chain reaction (PCR).

[0008] In certain embodiments, the method distinguishes between aggressive prostate cancer, indolent prostate cancer, benign prostatic hyperplasia, and prostatitis.

[0009] In an additional embodiment, a method for identifying a patient as having aggressive prostate cancer comprises detecting overexpression of one or more of messenger ribonucleic acid (mRNA), circular RNA (circRNA), extracellular DNA, and long non-coding RNA (lncRNA) compared to a control. Further includes:

[0010] In some embodiments, the mRNA contains one or more of RIDA, H1-4, H4C2 and and H4C3. In certain embodiments, the circRNA comprises one or more of circ842, circ3266, circ1809, circ1979, circ645 and circ1607. In certain embodiments, the lncRNA comprises lnc-CCDC125-13 and / or ZNF667-AS1.

[0011] In some embodiments, the eccDNA comprises one or more of chr22:50276214-50276428; chr20:2236337-2236458; chr6:54059859-54063911; chr16:85975027-85975617; chr3:5565190-5565271; chr10:130300872-130301712; chr11:58900903-59058535 ;chr22:44599233-49967822;chr17:69961543-69961943;chr18:9809075-9809266;chr17:80024303-80024653;chrY:10945178-11295108;c hr7:65038315-65873352;chr1:21669328-93846973;chr6:168914322-168914396;chr6:35786783-35799011;chr6:26305559-28597426;chr 9:34681483-34681981;chr1:207698916-207868701;chr8:57203766-57210492;chr16:20504588-20504731;chr3:67362279-136250669;chr1:248404181-248404245;chr12:1574344-1574491;chr7:66728585-73967789;chr1:55002895-55003057;and chr16:89907819-89908811.

[0012] In certain embodiments, a method for identifying a patient as having invasive prostate cancer includes the steps of: asparagine, aspartate, glycerate, citrate, isocitrate, glutamate, itaconate, malate, meglutol, cisaconitate, isoleucine, leucine, pantothenate, glutamine, nicotinate, threonine, ketoglutarate, alpha-ketoisovaleric acid (KIVA), cysteine, 3P glycerate, xanthine, and detecting one or more metabolites selected from the group consisting of hypoxanthine; Further includes:

[0013] In some embodiments, the method for identifying a patient as having invasive prostate cancer further comprises administering a prostate cancer therapy to the patient identified as having invasive prostate cancer. In certain embodiments, the prostate cancer therapy comprises prostatectomy, radiation therapy, cryotherapy, hormonal therapy, chemotherapy, immunotherapy, and combinations thereof. Further examples of specific treatments are described herein.

[0014] In another aspect, the present invention relates to a method for detecting EPCAM, H4C5 in a sample compared to a control. and administering a prostate cancer therapy to a patient identified as having overexpression of TTC3. In some embodiments, the patient sample has one or more mRNA levels greater than or equal to 100% relative to a control. , further including overexpression of circRNAs, eccDNA and lncRNAs.

[0015] In some embodiments, the mRNA contains one or more of RIDA, H1-4, H4C2 and and H4C3. In certain embodiments, the circRNA comprises one or more of circ842, circ3266, circ1809, circ1979, circ645, and circ1607. In certain embodiments, the lncRNA comprises lnc-CCDC125-13 and / or ZNF667-AS1.

[0016] In some embodiments, the eccDNA comprises one or more of chr22:50276214-50276428; chr20:2236337-2236458; chr6:54059859-54063911; chr16:85975027-85975617; chr3:5565190-5565271; chr10:130300872-130301712; chr11:58900903-59058535 ;chr22:44599233-49967822;chr17:69961543-69961943;chr18:9809075-9809266;chr r17:80024303-80024653;chrY:10945178-11295108;chr7:65038315-65873352;chr1:21669328-93846973;chr6:16891 4322-168914396;chr6:35786783-35799011;chr6:26305559-28597426;chr9:34681483-34681981;chr1:207698916-20 7868701;chr8:57203766-57210492;chr16:20504588-20504731;chr3:67362279-136250669;chr1:248404181-248404245;chr12:1574344-1574491;chr7:66728585-73967789;chr1:55002895-55003057;and chr16:89907819-89908811.

[0017] In another embodiment, the present invention provides a method for detecting overexpression of one or more proteins, mRNAs, circRNAs, eccDNA lncRNAs in a sample obtained from a patient compared to a control. In some embodiments, the protein comprises EPCAM. In certain embodiments, the mRNA comprises one or more of H4C5, TTC3, RIDA, H1-4, H4C2 and H4C3. In certain embodiments, the circRNA comprises one or more of circ842, circ3266, circ1809, circ1979, circ645 and circ1607. In certain embodiments, the lncRNA comprises lnc-CCDC125-13 and / or ZNF667-AS1.

[0018] In some embodiments, the eccDNA comprises one or more of chr22:50276214-50276428; chr20:2236337-2236458; chr6:54059859-54063911; chr16:85975027-85975617; chr3:5565190-5565271; chr10:130300872-130301712; chr11:58900903-59058535 ;chr22:44599233-49967822;chr17:69961543-69961943;chr18:9809075-9809266;chr17:80024303-80024653;chrY:10945178-11295108;c hr7:65038315-65873352;chr1:21669328-93846973;chr6:168914322-168914396;chr6:35786783-35799011;chr6:26305559-28597426;chr 9:34681483-34681981;chr1:207698916-207868701;chr8:57203766-57210492;chr16:20504588-20504731;chr3:67362279-136250669;chr1:248404181-248404245;chr12:1574344-1574491;chr7:66728585-73967789;chr1:55002895-55003057;and chr16:89907819-89908811.

[0019] In some embodiments, the detection step of the methods described herein utilizes a lateral flow device. In specific embodiments, the lateral flow device comprises a dipstick assay.

[0020] In some embodiments, the sample is free flow urine and / or a prostate mask. In some embodiments, the sample comprises blood or serum. In certain embodiments, the detection of H4C5 and TTC3 RNA comprises detection in blood or serum.

[0021] In a specific embodiment, the RNA markers are detected using polymerase chain reaction (PCR). In an even more specific embodiment, the PCR is qPCR. EPCAM, TTC3 and / or Alternatively, in embodiments in which H4C5 is detected via PCR, the primers may comprise SEQ ID NOs: 1-6, respectively.

[0022] In further embodiments, one or more of the following are present relative to a control: Reduced expression may be used in the methods of the invention: COX20, CAPN3, CANX, PBLD, LUC7L3, HMGN2P5, SNURF, PNPO, NUDT4, AK4, RSL1D1, UGDH, TRAPPC5, ZNF181, NPM1, PTMA, VDAC1, HSPD1, HSPE1, NIT2, RBIS, COX6C, ODC1, DDAH1, MRPL51, GATD3B, COA4, ATP5MC1, MAP7, HOMER2, NFIX, CCDC58, and COX5A.

[0023] In some embodiments, one or more of the following are increased compared to the control: Increased (over)expression of the following genes can be used: TTC3, ZNF91, H4C2, EEF1G, TOM1L1, H4C3, ELK4, H1-4, OST4, H4C5, RIDA, MRPS21, NCALD, NDUFB9, RAN, EPCAM, and TMEM2. 63.

[0024] In certain embodiments, EPCAM protein and H4C5 and TTC RNA may be measured together with one or more other markers described herein. [Brief description of the drawings]

[0025] [Figure 1]Figures 1A-1D. Identification and validation of urine enriched liquid biopsy biomarkers in PCa. Figure 1A: Flowchart for identification of urine enriched liquid biopsy biomarkers in PCa. Figure 1B: Top 50 mRNAs with highest expression in PCa primary and metastases compared to normal from TCGA database. Red and blue indicate up-regulated and down-regulated genes, respectively. Figure 1C: qPCR analysis showing distribution of normalized expression values ​​of 50 mRNAs in pooled PCa and normal urine. Values ​​indicate fold change relative to pooled normal urine. Figure 1D: Differential expression changes of 9 mRNAs in 20 PCa and normal urine were validated by qPCR. N, normal; PCa, prostate cancer; qPCR, quantitative reverse transcription PCR. [Diagram 2] Figure 2A-2C. Urinary EPCAM protein, TTC3 and H4C5 RNA levels in PCa. Figure 2A: Scatter plots representing urinary EPCAM protein concentrations for each normal or PCa patient determined by ELISA. EPCAM protein was detected before and after prostatectomy in each PCa patient. Figure 2B: Scatter plots representing urinary TTC3 RNA concentrations for each normal or PCa patient determined by qPCR. TTC3 RNA was detected before and after prostatectomy in each PCa patient. Figure 2C: Scatter plots representing urinary H4C5 RNA levels for each normal or PCa patient determined by qPCR. H4C5 RNA was detected before and after prostatectomy in each PCa patient. [Diagram 3]Urinary EPCAM protein, TTC3 and H4C5 RNA are potential biomarkers for PCa diagnosis. Receiver Operating Characteristic Curves (ROC) for urinary EPCAM protein, TTC3 and H4C5 RNA levels in patients with PCa versus control urine. Areas under the curve (AUC) are shown for each ROC analysis, 0.99 for EPCAM protein, 0.96 for H4C5 RNA and 0.92 for TTC3 RNA. N, normal; PCa, prostate cancer; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative reverse transcription PCR. [Figure 4] Figures 4A-4D. TTC3 silencing suppresses PCa cell proliferation and invasion in vitro. Figure 4A: Transcript expression of TTC3 in human PCa (PC3, LNCaP) and normal prostate epithelial (HPrEC) cell lines was detected by qPCR. Figure 4B: siRNA-mediated depletion of TTC3 was determined by qPCR. Figure 4C: Effect of TTC3-specific siRNA on PCa cell proliferation by MTS. Figure 4D: Representative images of PC3 and LNCaP invasion of cells treated with TTC3 siRNA on membrane. Data, mean ± SEM. *, p<0.05, **, p<0.01. [Diagram 5] Figures 5A-5C. Expression of FDA approved diagnostic markers urinary prostate cancer antigen 3 (PCA3) and serum prostate specific antigen (PSA) in PCa. Figure 5A: Scatter plots showing urinary PCA3 RNA levels of each normal or PCa patient as determined by qPCR. PCA3 RNA was detected before and after prostatectomy in each PCa patient. Figure 5B: Scatter plots showing urinary SPDEF RNA levels of each normal or PCa patient as determined by qPCR. SPDEF RNA was detected before and after prostatectomy in each PCa patient. Figure 5C: Serum PSA protein was detected before and after prostatectomy in each PCa patient. [Figure 6]Figures 6A-6B. Waterfall plots of urinary biomarker expression in PCa and normal. Figure 6A: Each bar represents the mean value of an individual sample, increasing from left to right. The horizontal black lines on each plot indicate cutoff values ​​of 26.86 pg / ml for EPCAM protein, 69.92 for TTC3 RNA, and 1068.32 for H4C5 RNA. Figure 6B: The horizontal black lines on each plot indicate cutoff values ​​of 5.69 for PCA3 RNA, and 32.76 for SPDEF RNA. The horizontal grey bars indicate the number of patients misdiagnosed as positive or negative at the cut points. N, normal; PCa, prostate cancer; ELISA, enzyme-linked immunosorbent assay; qPCR, quantitative reverse transcription PCR. [Figure 7] Figures 7A-7D. EPCAM silencing suppresses PCa cell proliferation and invasion in vitro. Figure 7A: Transcript expression of EPCAM in human PCa (PC3, LNCaP) and normal prostate epithelial (HPrEC) cell lines was detected by qPCR. Figure 7B: siRNA-mediated depletion of EPCAM was determined by qPCR. Figure 7C: Effect of EPCAM-specific siRNA on PCa cell proliferation by MTS. Figure 7D: Representative images of PC3 and LNCaP invasion of cells treated with EPCAM siRNA on membrane. Data, mean ± SEM. *p<0.05, **, p<0.01. [Figure 8] Figures 8A-8B. Identification of PCa-specific mRNAs in urine. Figure 8A: Principal component analysis (PCA) results depict the separation of normal and tumor samples in RNA-seq data. Figure 8B: A panel of 51 genes separates controls, metastases, and primary tumors. [Figure 9] Figure 9A-9B. qPCR confirmation of selected targets. Figure 9A: Contribution of comparisons 1 and 2. Controls compared to metastases and controls compared to primary tumors in the TCGA dataset. Figure 9B: qPCR validation of candidate genes. [Figure 10]EPCAM, TTC3 and PCA3 gene expression in normal, BPH, prostatitis and PCa urine samples. 20 normal, 11 BPH, 7 prostatitis and 25 PCa urine samples were tested. Both TTC3 and EPCAM clearly separated PCa from the other groups. [Figure 11] Figures 11A-11D. Principal component analysis of the clusters (delta Ct values) of the four groups. Figure 11A: EPCAM+TTC3+PCA. Figure 11B: PCA3+TTC3. Figure 11C: EPCAM+PCA3. Figure 11D: EPCAM+TTC3. EPCAM+TTC3 shows the best separation from the other groups of prostate cancer. [Figure 12] Figures 12A-12B. Prostate cancer-specific circular RNAs in urine. Figure 12A: Volcano plot depicts differentially expressed circRNAs in PCa urine compared to normal. Figure 12B: Highly upregulated circRNAs in PCa urine compared to normal. [Figure 13] Marker significance testing. PCA was unable to distinguish BPH / PTT (prostatitis) v. PCa (non-significant P-values). The most significant comparisons in the data set are highlighted in yellow. [Figure 14] Marker significance testing. The % sensitivity of EPCAM and TTC3 shows the highest sensitivity among the three markers tested. [Figure 15] A novel group of PCa-specific eccDNAs were identified using previously published DNA-seq data. Some of these candidates are being validated in DNA samples from PCa patient samples (ongoing). [Figure 16] Figure 16A-16B. EPCAM ELISA test. A commercial ELISA assay kit for EPCAM was used and a test was developed to measure EPCAM mRNA levels in urine. EPCAM expression in 20 normal (N) and 17 prostate cancer (PCa) patient urine samples. [Figure 17] lnc-RNA expression in PCa urine. PCA decomposition of total RNA. [Figure 18] Volcano plot of lncRNAs only. A total of 53 significant lncRNAs Incipedia annotation. Only two are upregulated in PCa. *(lfc2)>1 and adjPval <0.05. [Figure 19] Figure 19A-19B.DE lncRNAs PCa / Normal. [Figure 20] Figures 20A-20B. PCA (Figure 13A) and heatmap (Figure 13B) plots of 53 lncRNAs. [Figure 21] Figures 21A-21F. Expression of top DE lncRNAs. ENTPD1-AS1 (Figure 14A); LINC01973 (Figure 14B); LINC02312 (Figure 14C); CPB2-AS1 (Figure 14D); lnc-CCDC125-13 (Figure 14E); and ZNF667-AS1 (Figure 14F). [Figure 22] EPCAM ELISA for PCa. Normal 46, pre-PCa 49, post-PCa 24. [Diagram 23] High metabolites in PCa vs. Normal. [Figure 24] Elevated metabolites in PCa vs. BPH. [Diagram 25] Elevated metabolites in PCa versus PTT. [Figure 26] Heatmap of significant metabolites. [Figure 27] Metabolite reduction in PCa versus PTT. [Figure 28] Receiver operating characteristic curve (ROC) analysis of biomarkers. A range of cut-off points are illustrated as black dots. [Figure 29] ROC curve comparison of EPCAM, H4C5 and TTC3. [Diagram 30] EPCAM expression is higher in PCa urine-ELISA assay than in normal urine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description of the Invention

[0027] It is understood that the present invention is not limited to the particular methods, compositions, etc. described herein. It is also understood that the terminology used herein is used for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a" (indefinite article, to be), "an ... The definite articles "and "the" are used in the present specification unless the context clearly indicates otherwise. It should be noted that unless otherwise specified, a reference to a "protein" includes a plural reference. Thus, for example, a reference to a "protein" is a reference to one or more proteins, and and their equivalents known to those of skill in the art, and others.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Specific methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0029] All publications cited herein, including all journal articles, books, manuals, published patent applications, and issued patents, are hereby incorporated by reference. In addition, the meanings of certain terms and phrases employed in this specification, examples, and the appended claims are provided. These definitions are not meant to be limiting in nature, and serve to provide a clearer understanding of certain aspects of the invention.

[0030] In a broad sense, the present invention relates to the testing of one or more classes of biomarker molecules. Classes of molecules are polypeptides / proteins, nucleic acids and polyamino acids, etc. The nucleic acid molecule can be selected from genomic DNA, plasmid DNA, complementary DNA (cDNA), cell-free (e.g., non-encapsulated) DNA (cfDNA) (also called extracellular DNA (eccDNA), circulating tumor DNA (ctDNA), nucleosomal DNA, chromosomal DNA, mitochondrial DNA, and the like. (miDNA), artificial nucleic acid analogs, recombinant nucleic acids, plasmids, viral vectors, and clones. In certain embodiments, the patient sample contains deoxyribonucleic acid (DNA), including chromosomes. The pull includes eccDNA / cfDNA.

[0031] Nucleic acid molecules also include coding and non-coding transcripts, messenger RNA (mRNA), transfer RNA (tRNA), microRNA (mitoRNA), ribosomal RNA (rRNA), circular RNA (cRNA), alternatively spliced ​​mRNAs, long non-coding RNA (lncRNA), small nuclear RNAs (snRNAs), antisense RNA, short hairpins (short hairpins), and ribosomal RNA (rRNA). hairpin RNA (shRNA), or small interfering RNA (siRNA) , ribonucleic acid (RNA). In certain embodiments, the patient sample includes mRNA, circRNA, and / or lncRNA.

[0032] A nucleic acid molecule or fragment thereof may be single-stranded or double-stranded. A sample may contain one or more (hereinafter also referred to as one or more) types of nucleic acid molecule or fragment thereof.

[0033] A nucleic acid molecule or fragment thereof can contain any number of nucleotides. For example, a single stranded nucleic acid molecule or fragment thereof can contain at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, ...10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least 10, at least at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, At least 300, at least 350, at least 400, or more nucleotides In the illustrative example of a double stranded nucleic acid molecule or fragment thereof, the nucleic acid molecule or fragment thereof may comprise at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least at least 110, at least 120, at least 130, at least 140, at least 150, at least At least 160, at least 170, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 350, At least 400 or more base pairs (bp), e.g., including nucleotide pairs In some cases, double-stranded nucleic acid molecule or its fragment can comprise between 100 and 200bp, for example, between 120 and 180bp.For example, sample can comprise cfDNA molecule comprised between 120 and 180bp.

[0034] Classes of biomarker molecules can also include polyamino acids, including polyamino acids, peptides, or proteins. As used herein, the term polyamino acid refers to a polymer in which the monomers are amino acid residues joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer may be used. can also be used, with the L-isomer being preferred. In one example, the analyte is an autoantibody.

[0035] Further examples of classes of molecules (or analytes) include metabolites such as, for example, sugars, lipids, amino acids, fatty acids, phenolic compounds, or alkaloids. In one embodiment, the analyte is a carbohydrate. In another embodiment, the analyte is a carbohydrate antigen. In even more specific embodiments, the carbohydrate antigen is attached to an O-glycan. In certain embodiments, the analyte is a monosaccharide, a disaccharide, a trisaccharide, or a tetrasaccharide.

[0036] In specific embodiments, biomarker classes include RNA (mRNA, circRNA, lncRNA), eccDNA, proteins, metabolites, and combinations of the foregoing.

[0037] A sample containing one or more analytes / classes of biomarkers can be processed to provide or purify a particular analyte or collection thereof. A sample containing analytes can be processed to separate one type of analyte (e.g., protein or eccDNA / cfDNA) from other types of analytes (e.g., mRNA, circRNA, lncRNA). In another embodiment, the sample is divided into aliquots for analysis of a different analyte in each aliquot from the sample. In yet another embodiment, one or more nuclei are separated. A sample containing nucleic acid molecules or fragments thereof of different sizes (e.g., lengths) will tend to produce higher molecular weight and / or longer nucleic acid molecules or fragments thereof, or more highly fragmented nucleic acid molecules. The present invention provides a method for removing lower molecular weight and / or shorter nucleic acid molecules or fragments thereof. The material can be treated to remove

[0038] Sample processing can include one or more processes such as, for example, centrifugation, filtration, selective precipitation, tagging, barcoding, and partitioning. For example, cellular DNA can be separated from cfDNA by selective polyethylene glycol and bead-based precipitation processes such as centrifugation or filtration processes.

[0039] As further described herein, assays useful for detecting the biomarkers of the invention include, but are not limited to, whole genome sequencing (WGS), whole genome bisulfite sequencing, Sequencing (WGSB), small RNA sequencing, quantitative immunoassay, enzyme-linked immunosorbent assay Standard method (ELISA), proximity extension assay (PEA), protein Microarray, mass spectrometry, low-coverage whole genome sequencing lcWGS), cf-protein immunoquantitation ELISAs, SIMOA; and cf-miRNA sequencing, as well as cell type or cell table derived from any of the above assays. Includes current mix ratio.

[0040] Analyses for biomarker detection may include, but are not limited to, the following: linear discriminant analysis (LDA); partial least squares (PLS); random forest; principal component analysis (PCA); k-nearest neighbor (KNN); support with radial basis function kernel Support Vector Machines (SVM) (SVMRadial); SVM with a linear basis function kernel (SVMLinear); SVM with a polynomial basis function kernel (SVMPoly); with classifiers trained and constructed according to one or more of decision trees, multilayer perceptrons, mixtures of experts, sparse factor analysis, hierarchical decomposition, and combinations of linear algebraic routines and statistics. This can be executed.

[0041] I. Definition

[0042] As used herein, a "subject," "patient," or "individual" is a human. A subject may be suffering from, or have previously been diagnosed with, a condition, disease, or disorder for which treatment is sought (e.g., prostate cancer), or one or more complications associated with the condition, disease, or disorder. have been diagnosed or identified, and optionally have been treated for, that condition, disease, or disorder, or for one or more complications related to that condition, disease, or disorder; Alternatively, the subject may have previously been diagnosed with a condition, disease, or disorder, or one or more complications associated with the condition, disease, or disorder. For example, a subject may exhibit one or more risk factors for a condition, disease, or disorder, or one or more complications associated with the condition, disease, or disorder, or may not exhibit risk factors. A "subject" for which treatment is "required" for a particular condition, disease, or disorder may be a subject suspected of having the condition, disease, or disorder, diagnosed with the condition, disease, or disorder, already treated or being treated for the condition, disease, or disorder, or not being treated for the condition, disease, or disorder, or for the risk of developing the condition, disease, or disorder.

[0043] In some embodiments, the subject is selected from the group consisting of a subject suspected of having a disease, a subject having a disease, a subject diagnosed with a disease, a subject receiving treatment for a disease, a subject being treated for a disease, and a subject at risk of developing a disease.

[0044] In some embodiments, the subject is a subject suspected of having prostate cancer, a subject with prostate cancer, a subject diagnosed with prostate cancer, a subject with non-aggressive prostate cancer, a subject suspected of having aggressive prostate cancer, a subject undergoing treatment for prostate cancer, a subject with benign prostate hyperplasia, a subject with prostatitis, The subject is selected from the group consisting of a subject undergoing treatment for prostate cancer, a subject undergoing treatment for prostate cancer, and a subject at risk for developing prostate cancer.

[0045] By "at risk" it is intended to mean at increased risk compared to normal subjects or a control group, e.g., a patient population. Thus, subjects carrying a particular marker can be identified as being at increased risk compared to a particular condition, disease or disorder and in need of further testing. "Increased risk" or "risk" refers to a condition that is at increased risk for a particular condition, disease or disorder. By "increased risk" is meant, for example, any statistically significant increase in the probability that a subject has a disorder. The risk can be increased by at least 10%, at least 20%, and even at least 50% relative to a control group to which the comparison is made. In certain embodiments, the subject can be at risk for developing aggressive prostate cancer.

[0046] "Sample" is used herein in its broadest sense. As used herein, the term "biological sample" refers to a sample taken or isolated from a biological organism. Samples or biological samples include blood, serum, plasma, tears, aqueous humor, etc. and bodily fluids, including vitreous humor, spinal fluid; soluble fractions of cell or tissue preparations, or the medium in which cells have been grown; or membranes isolated or extracted from cells or tissues; polypeptides, or peptides in solution or bound to a matrix; cells; tissues, tissue prints, fingerprints, skin or hair; fragments and derivatives thereof. Non-limiting examples of samples or biological samples include cheek swabs; mucus; whole blood, blood, serum; plasma; urine; saliva, semen; lymph; fecal extracts; sputum; other bodily fluids or biofluids; cell samples. and tissue samples, etc. The term also includes mixtures of the above samples or biological samples. The term "sample" also includes unprocessed or preprocessed (or pre-processed) biological samples. In some embodiments, a sample or biological sample is one or more samples from a subject. The subject sample or biological sample may include cells of the subject. The subject sample or biological sample typically includes blood, plasma, and serum, as well as derivatives of blood products. In some embodiments, the sample is a biological sample. In some embodiments, the sample is blood. In some embodiments, the sample is plasma. In some embodiments, the sample is blood, plasma, serum, or urine. In certain embodiments, the sample is a serum sample. In certain embodiments, the sample is a urine sample.

[0047] The term "body fluids" or "body fluids" refers to liquids originating from within an organism. Body fluids include amniotic fluid, aqueous humor, vitreous humor, bile, blood (e.g., serum), breast milk, cerebrospinal fluid, earwax, chyle, chyme, endolymph and perilymph, exudate, and feces. , female ejaculate, gastric acid, gastric juices, lymph, mucus (e.g., nasal drainage and phlegm), pericardial fluid, peritoneal fluid ), pleural effusion, pus, mucous membrane secretions (rheum), saliva, sebum (skin oil), serous fluid, semen, sputum. These include sputum, synovial fluid, sweat, tears, urine, vaginal secretions, and vomit. Extracellular fluids include intravascular fluid (plasma), interstitial fluid, lymphatic fluid, and transcellular fluid. "Biological sample" also includes mixtures of the above bodily fluids. A "biological sample" may be an untreated or pretreated (or pre-processed) biological sample. In certain embodiments, bodily fluid refers to urine.

[0048] Sample collection procedures and devices known in the art are suitable for use with various embodiments of the present invention. Examples of sample collection procedures and devices include, but are not limited to, phlebotomy tubes (e.g., vacutainer blood / specimen collection devices for collection and / or storage of blood / specimens), dried blood spots, Microvette CB300 Capillary Collection Device (Sarstedt), HemaXis Blood Collection Device (Microfluidics, Hemaxis), Volumetric Absorptive Microsampling. (e.g., the CE-IVD Mitra microsampling device (Neoteryx) for accurate dried blood sampling) Things, HemaSpot TM -HF blood collection device (HemaSpot TM -HF Blood Collection Device( TMis a trademark of the U.S. and other countries; tissue sample collection device; standard collection / storage device Collection and / or storage of samples (e.g., blood, plasma, serum, urine, etc.) includes dried blood spot sampling devices; collection / storage devices for storage; In some embodiments, Volumetric Absorptive Microsampling (VAMS) is used. 1M ) Samples can be stored and mailed, and The assay can be performed remotely.

[0049] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, -carboxyglutamate, and O-phosphoserine. Amino acid analogs include, but are not limited to, amino acids that are not naturally occurring or synthetic. Compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine, Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but do not resemble naturally occurring Amino acids that retain the same basic chemical structure as the amino acid they mimic. An amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids, as used herein, are referred to by their commonly known three letter symbols. or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides are also referred to by their commonly accepted one-letter codes. It can be done.

[0050] As used herein, the term "peptide" refers to a peptide that is a single amino acid residue adjacent to the carboxyl group. At least two amino acid residues joined by amide bonds In some embodiments, a peptide typically refers to a polymer of amino acid residues ranging in length from 2 to about 30, or to about 40, or to about 50, or to about 60, or to about 70 residues. In certain embodiments, a peptide The peptides range in length from about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 60, 50, 45, 40, 45, 30, 25, 20, or 15 residues. In certain embodiments, the peptides range in length from about 8, 9, 10, 11, or 12 residues to about 15, 20, or 25 residues. In some embodiments, the peptides range in length from 2 to about 12 residues, or from 2 to about 20 residues, or from 2 to about 30 residues. up to about 40 residues, or from 2 to about 50 residues, or from 2 to about 60 residues In certain embodiments, the peptides may range in length from 2 to about 70 residues. The amino acid residues contained herein are "L-form" amino acid residues, although it is recognized that in various embodiments, "D" amino acids can be incorporated into the peptides. Similar to naturally occurring amino acid polymers, one or more amino acid residues may be present in the corresponding In addition, the term includes amino acid polymers that are artificial chemical analogues of naturally occurring amino acids. This applies to amino acids conjugated with aryl groups (e.g., substituted with aryl esters, hydroxylates, and the like) (see, e.g., Spatola et al., (1983) Chem. Biochem. Amino Acids and Proteins 7:267-357), where the amide is replaced with a saturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542, U.S. Pat. No. 5,311,621, and U.S. Pat. No. 5,311,621). It is incorporated herein by reference, and Kaltenbronn et al., (1990) pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOM Science Publishers. Publishers, The Netherlands, and others of the same type. reference)).

[0051] A protein is a large molecule made up of one or more long chains of amino acids. Proteins refer to any of a class of nitrogenous organic compounds that contain and are an essential part of all living organisms. Proteins may contain various modifications to the amino acid structure, such as disulfide bond formation, phosphorylation, and glycosylation. A linear chain of amino acid residues may be called a "polypeptide," and a protein consists of at least one polypeptide. Short polypeptides, e.g., less than 20-30 residues, are sometimes referred to as polypeptides. , called "peptides."

[0052] "Antibody" refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds to and recognizes an epitope (e.g., an antigen). Recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, as well as the myriad immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases, including, for example, Fab' and F(ab)'2 fragments. As used herein, "antibody" refers to a polypeptide ligand that specifically binds to and recognizes an epitope (e.g., an antigen). Recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, as well as the myriad immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases, including, for example, Fab' and F(ab)'2 fragments. The term "antibody" includes either antibody fragments produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal, monoclonal, chimeric, humanized, or single chain antibodies. The "Fc" portion of an antibody comprises one or more heavy chain constant region domains of an immunoglobulin heavy chain. The term "heavy chain variable region" refers to that portion that contains the CH1, CH2 and CH3 domains, but does not include the heavy chain variable region.

[0053] The phrases "specifically (or selectively) bind" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refer to a binding reaction that defines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a specified antibody binds to a particular protein at least twice background and to other proteins present in a sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select an antibody specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays can be used to select an antibody specifically immunoreactive with a protein. (See, for example, Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immune reactivity.) )reference).

[0054] As used herein, the term "threshold" refers to a magnitude or strength that must be exceeded to occur or be considered relevant for a certain reaction, event, outcome, or condition. The association can depend on the association, for example, it can refer to a positive, reactive, or statistically significant association.

[0055] By "binding assay" is meant a biochemical assay in which a biomarker is detected by binding to an agent, such as an antibody, through which the detection process occurs. The detection process may involve fluorescent or radioactive labels, and the like. The assay may involve immobilization of the biomarker, or may be performed in solution.

[0056] An "immunoassay" is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). Immunoassays use a method to isolate, target, and / or quantify an antigen. , characterized by the use of the specific binding properties of a particular antibody. Non-limiting examples of immunoassays include ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, SISCAPA (stable isotopically coupled assay), and the like. These include antibody capture with anti-peptide antibodies and Western blots.

[0057] "Diagnosis" means identifying the presence or nature of a pathological condition, disease, or disorder, and includes identifying patients at risk for developing a particular condition, disease, or disorder. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the proportion of diseased individuals who test positive (proportion of "true positives"). Diseased individuals not detected by the assay are "false negatives." Subjects who do not get the disease and test negative by the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, The "false positive" rate is then defined as the proportion of those without the disease that test positive. While a particular diagnostic test may not provide a definitive diagnosis of a condition, disease, or disorder, it suffices if the diagnostic test provides a positive indication that aids in diagnosis.

[0058] The terms "statistically significant" or "significantly" refer to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is in fact true. The determination is often made using the p-value.

[0059] As used herein, the term "sensitivity" refers to the ability of a method to detect or identify the presence of a disease in a subject. For example, when used in reference to any of the various methods described herein that can detect the presence of cancer (e.g., invasive prostate cancer) in a subject, high sensitivity means that the method correctly identifies the presence of invasive prostate cancer in a subject a large percentage of the time. For example, a method described herein that correctly detects invasive prostate cancer in a subject 95% of the time the method is performed is said to have a sensitivity of 95%. In certain embodiments, a method described herein that can detect invasive prostate cancer (or distinguish between invasive and non-invasive prostate cancer) in a subject provides a sensitivity of at least 70% (e.g., about 70%, about 72%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or about 100%). In certain embodiments, two or more classes of biomarkers (e.g., nucleic acid biomarkers and / or protein biomarkers) are included. The methods provided herein include detecting the presence of one or more members of provides greater sensitivity than methods that involve detecting the presence of one or more members of only one class of biomarkers.

[0060] As used herein, the term "specificity" refers to the ability of a method to detect the presence of a disease in a subject (e.g., the specificity of a method can be used to distinguish true positives compared to true negatives in a subject and / or to distinguish truly occurring sequence variants from sequencing artifacts or other densities). High specificity can be described as the ability of the method to distinguish from adjacent related sequences. For example, when used in connection with any of the various methods described herein that can detect the presence of cancer (e.g., invasive prostate cancer) in a subject, high specificity means that the method will correctly identify the absence of cancer in the subject a large percentage of the time (e.g., the method will not falsely identify the presence of cancer in the subject a large percentage of the time). In some embodiments, detecting the absence of cancer (normal, BPH or other non-invasive cancer) in a subject can be used to detect the absence of cancer in a subject. The methods described herein provide a specificity of at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more). A method with high specificity (e.g., compared to other methods) produces minimal or no false positive results. False positive results can come from any source. In some embodiments, the methods provided herein provide a method for detecting two or more classes of variances. detecting the presence of one or more members of a biomarker (e.g., a nucleic acid biomarker, and / or protein biomarkers), one or more of only one class of biomarkers This provides greater specificity than methods that involve detecting the presence of members of the above.

[0061] The terms "detection," "detecting," and the like, may be used in the context of detecting a biomarker, detecting a peptide, detecting a protein, or detecting a condition, disease, or disorder (e.g., when a positive assay result is obtained). In the latter context, when mere detection is indicative of a diagnosis, "detecting" and "diagnosing" are considered synonymous. The term is also used synonymously with the term "measuring."

[0062] The terms "marker" or "biomarker" are used interchangeably herein, and in the context of the present invention, refer to a protein or peptide (e.g., a prostate cancer or prostate cancer-related protein or peptide described herein) that is differentially present in samples taken from patients with a particular disease or disorder compared to a control value, which may consist, for example, of the mean or median value in comparable samples taken from control subjects (e.g., persons with a negative diagnosis, normal or healthy subjects). Biomarkers may be determined, for example, as specific peptides or proteins that can be detected by antibodies or mass spectrometry. In some applications, for example, mass spectrometry or other profiles of various antibodies may be used to determine the various biomarkers, and the differences between the individual biomarkers and / or partial or complete profiles may be used to diagnose a diagnosis. In some embodiments, the biomarkers may be detected by antibodies, mass spectrometry, or a combination thereof. In certain embodiments, the markers or biomarkers include RNA (e.g., circulating RNA (circRNA), IncRNA, mRNA), DNA (e.g., extracellular DNA (eccDNA) (also known as cell-free DNA or cfDNA), peptides / proteins, and / or metabolites. In certain embodiments, the markers or biomarkers include RNA (e.g., circulating RNA (circRNA), IncRNA, mRNA), DNA (e.g., extracellular DNA (eccDNA) (also known as cell-free DNA or cfDNA)), peptides / proteins, and / or metabolites. - is measured in urine.

[0063] A "test amount" of a marker refers to the amount of marker present in a sample being tested. A test amount can be either an absolute amount (e.g., g / mL) or a relative amount (e.g., relative intensity of signals).

[0064] A "diagnostic amount" of a marker refers to the amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or disorder. A diagnostic amount can be either an absolute amount (e.g., μg / ml) or a relative amount (e.g., relative intensity of signals).

[0065] A "control amount" of a marker can be any amount or range of amounts that is compared to a test amount of the marker. For example, a control amount of a marker can be the amount of the marker in a person who does not suffer from the disease or disorder being diagnosed, and the control amount can be either an absolute amount (e.g., μg / ml) or a relative amount (e.g., the relative intensity of the signals).

[0066] The term "differentially present" or "altered in level" refers to the difference in the amount and / or frequency of a marker present in a sample taken from a patient having a particular disease or disorder compared to a control subject. The marker may be present at elevated or decreased levels in samples from patients with a disease or disorder compared to a threshold value (e.g., determined from a sample from a control subject). Alternatively, the marker may be detected at a higher or lower frequency in a patient sample compared to a control subject sample. The marker may be differentially present in terms of amount, frequency, or both, as well as the ratio of differences between two or more specific modified amino acid residues and / or the proteins themselves. In one embodiment, an increase in the ratio of the modification to the unmodified proteins and peptides herein is diagnostic of any one or more of the diseases described herein. In certain embodiments, the marker may be present at a higher or lower frequency in a patient sample compared to a control subject sample. The subjects included control subjects with or without non-invasive prostate cancer. It can be differentially present in patients with invasive prostate cancer compared to those with PCa. Differentially present can refer to PCa versus normal, BPH, and / or PTT.

[0067] A marker, compound, composition or substance is differentially present in a sample if the amount of the marker, compound, composition or substance in a sample (a patient with invasive prostate cancer) is statistically significantly different from the amount of the marker, compound, composition or substance in another sample (a patient with non-invasive cancer or no cancer) or from a control value (e.g., an indicator or value representing non-invasive cancer or the absence of cancer). For example, a marker is differentially present in a sample if it is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 400%, at least about 400%, at least about 400%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500 at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000%; and is differentially present if it is present in less than one sample (e.g., a control) or it is detectable in one sample and not the other. a marker is differentially present if it is present at least about 50%, 1000% greater or less than it is presence in the other sample (e.g., control), or if it is detectable in one sample and not detectable in the other.

[0068] Alternatively, or in addition, a marker, compound, composition or substance is differentially present between samples if the frequency of detection of the marker, etc., is statistically significantly higher or lower in samples from patients suffering from a particular disease or disorder than in control samples or control values ​​obtained from controls, such as subjects with non-invasive prostate cancer, benign lesions and the like, or otherwise healthy individuals. For example, a biomarker may be present when it is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 40 ...500%, at least about 500%, at least about 500%, at least about 500%, at least about or at least about 80%, at least about 90%, or at least about 100% more frequently than other sets of samples (e.g., patients with aggressive prostate cancer). A differential expression is present between two sets of samples if it is observed at a lower frequency in a set of samples (e.g., patients with non-invasive prostate cancer or without cancer). Regardless of the exact value, it is expected that a skilled practitioner will be able to determine a cut-off point that represents a statistically significant difference, etc., for determining whether a marker is differentially present.

[0069] The term "one or more (also referred to as "one or more")" refers to a combination of various biomarkers. , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40...N, where N is the total number of biomarker proteins in certain embodiments. The term also includes at least 1, at least 2, at least 3, The biomarkers herein include at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 15, 16, 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40...N, and are used interchangeably. The phrase "one or more" of the biomarkers, and in particular, each column of the biomarker panel In the embodiments recited, language such as "at least 1, at least 2, at least 3" may be used. It is understood to include.

[0070] A "detectable moiety" or "label" refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include: 32 P , 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin-streptavidin, digoxigenin, antisera, or monoclonal antibodies. Detectable moieties include haptens and proteins that are available, or nucleic acid molecules with sequences complementary to the target. Detectable moieties often generate measurable signals, such as radioactive, chromogenic, or fluorescent signals, that can be used to quantify the amount of bound detectable moiety in a sample. Quantification of the signal is accomplished, for example, by scintillation counting, densitometry, flow cytometry, or direct analysis of intact proteins or peptides by mass spectrometry. In some embodiments, the detectable moiety is a stable isotope. In some embodiments, the stable isotope is 15 N,13 C. 18 O and 2 H.

[0071] As used herein, the terms "treat," "treatment," "treating," or "amelioration," when used in reference to a disease, disorder, or medical condition, refer to both therapeutic treatment and prophylactic or preventative measures, where the objective is to reverse, alleviate, ameliorate, inhibit, reduce, slow or halt the progression or severity of the symptoms, condition, disease, or disorder. The term "treating" refers to an alleviation or amelioration of at least one deleterious effect or side effects of a condition, disease, or disorder. Treatment generally involves reducing or alleviating one or more symptoms or clinical signs. A treatment is "effective" if the progression of a disease, disorder, or medical condition is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least delay of the development or worsening of symptoms that would be expected in the absence of treatment. "Treatment" can also mean pursuing or obtaining a beneficial result, or reducing the likelihood that an individual will develop a condition, disease, or disorder, if the treatment is ultimately unsuccessful. Those for whom treatment is sought include those already having a condition, disease, or disorder, as well as those prone to having a condition, disease, or disorder, or those for whom a condition, disease, or disorder is to be prevented.

[0072] Non-limiting examples of treatments or therapeutic procedures include pharmacological or biological therapy and / or includes interventional surgical procedures.

[0073] The term "prophylactic treatment" means maintaining or improving the health or non-diseased state of a healthy or disease-free subject. The term "prophylactic treatment" or "health monitoring" also means preventing or slowing the appearance of symptoms associated with a condition, disease, or disorder. The term "prophylactic treatment" also means preventing or slowing a subject from acquiring a condition, disease, or disorder.

[0074] As used herein, the term "administering" refers to placing an agent or treatment as disclosed herein into a subject by a method or route that results in at least partial localization of the agent or treatment at a desired site. "Route of administration" may refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, by inhalation, oral, anal, intraanal, perianal, transmucosal, transdermal, parenteral, enteral, topical, or local. "Parenteral" refers to intratumoral, intracranial, intraventricular, intrathecal, intrathecal, intraperitoneal ... Epidural, intrathecal, intraorbital, injection, intracapsular, intraarticular, intracardiac, intradermal, intramuscular intraperitoneal, intrapulmonary, intraspinal, intravertebral, intrastemal, intrathecal, intrauterine, intravascular, intravenous, intraarterial, arachnoid Generally by injection, including intrathecal, subcapsular, subcutaneous, transmucosal, or transtracheal Via the parenteral route, the composition may be in the form of a solution or suspension for infusion or injection, or may be a lyophilized powder. Through the topical route, the pharmaceutical composition can be in the form of a tablet, gel capsule, dragee, syrup, suspension, solution, powder, granule, emulsion, microsphere or nanosphere, or lipid or polymer vesicle, allowing for controlled release. Through the topical route, the pharmaceutical composition can be in the form of an aerosol, lotion, cream, gel, ointment, suspension, solution or emulsion. According to the present invention, "administering" can be administering to oneself. For example, as disclosed herein, the subject's consumption of a composition is considered to be "administering".

[0075] II. Detection / measurement of nucleic acid markers

[0076] Nucleic acids can be sequenced using sequencing methods, such as next generation sequencing, high throughput sequencing, massively parallel sequencing, sequencing-by-synthesis, paired-end sequencing, Single Molecule Sequencing, Nanopore Sequencing, Pyrosequencing, Semiconductor Sequencing, Sequencing by Ligation, Sequencing by Hybridization, RNA-Seq, Digital Gene Expression, Single Molecular Sequencing by Synthesis (SMSS), Clonal Single Molecular Array (Solexa) The sequences may be determined using such methods as shotgun sequencing, Maxim-Gilbert sequencing, primer walking, and Sanger sequencing.

[0077] Sequencing methods include targeted sequencing, whole genome sequencing (WGS), low-pass sequencing, and The sequencing method may include sequencing, bisulfite sequencing, whole genome bisulfite sequencing (WGBS), or a combination thereof. The sequencing method may include preparation of a suitable library. The sequencing method may include amplification of nucleic acids (e.g., targeted or non-targeted amplification such as PCR). or by universal amplification).

[0078] Sequencing reads can be obtained from a variety of sources, including whole genome sequencing, whole exome sequencing, targeted sequencing, next generation sequencing, pyrosequencing, sequencing-by-synthesis, ion semiconductor sequencing, tag-based next generation sequencing semiconductor sequencing, single molecule sequencing, nanopore sequencing, sequencing-by-ligation, sequencing-by-hybridization, digital gene expression (DGE), massively parallel sequencing, sequencing using clonal single molecule arrays (Solexa / Illumina), PacBio, and sequencing by oligonucleotide ligation and detection (SOLiD). Included.

[0079] In some embodiments, sequencing can include, for example, tagging a barcode, unique molecular identifier (UMI), or another tag to a nucleic acid molecule or fragment thereof. In particular, modifications of the nucleic acid molecule or fragments thereof may be included. Ligating the barcode to one end of a nucleic acid molecule or fragment thereof can facilitate analysis of the nucleic acid molecule or fragment thereof after sequencing. In some embodiments, the barcode is a unique barcode (i.e., a UMI). In specific embodiments, the barcode is a unique barcode (i.e., a UMI). The code is non-unique, and the barcode sequence can be used in conjunction with endogenous sequence information, such as the start and stop sequences of the target nucleic acid (e.g., the target nucleic acid is flanked by the barcode and the barcode sequence in conjunction with sequences at the beginning and end of the target nucleic acid to generate a uniquely tagged molecule).

[0080] Sequencing reads are de-multiplexed, de-deduplicated, and (e.g. using unique molecular identifiers, UMIs), adapter trimming, quality filtering The sequences can be processed using methods such as filtering, GC correction, amplification bias correction, batch effect correction, depth normalization, removal of sex chromosomes, and removal of low quality genomic bins.

[0081] In various embodiments, the sequencing reads can be aligned to a reference nucleic acid sequence.In one example, the reference nucleic acid sequence is a human reference genome.For example, the human reference genome can be hg19, hg38, GrCH38, GrCH37, NA12878 or GM12878.

[0082] III. Protein Marker Detection / Measurement

[0083] In a specific embodiment, the proteins of the present invention are detected and / or assayed by immunoassay. Immunoassays require a biospecific capture reagent / binding agent, such as an antibody, to capture the biomarker. Many antibodies are commercially available. Antibodies can also be produced by methods well known in the art, e.g., by immunizing animals with the biomarkers. Biomarkers can be isolated from samples based on their binding properties. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well-known in the art. Biospecific capture reagents useful in immunoassays can also include lectins. In other embodiments, the biospecific capture reagents bind to a particular biomarker and dissimilar forms thereof.

[0084] The present invention contemplates traditional immunoassays, such as ELISA or fluorescence-based immunoassays. These include sandwich immunoassays, including turbidimetry, immunoblots, and Western blots (WB), as well as other enzyme immunoassays. Turbidimetry is an assay performed in the liquid phase, where the antibody is in solution. Binding of the antigen to the antibody results in a change in absorbance that is measured. In SELDI-based immunoassays, a biospecific capture A reagent is attached to the surface of an MS probe, such as a preactivated ProteinChip array, etc. Biomarkers are then specifically captured onto the biochip through the reagent, and the captured biomarkers are detected by mass spectrometry.

[0085] In certain embodiments, the expression levels of protein biomarkers used herein are quantified by immunoassays, such as enzyme-linked immunoassay (ELISA) techniques. In a specific embodiment, the expression level of the biomarker is determined by contacting the biological sample with an antibody or antigen-binding fragment thereof that selectively binds to the biomarker; and detecting binding of the antibody, or antigen-binding fragment thereof, to the biomarker. In certain embodiments, the binding agent employed in the disclosed methods and compositions is labeled with a detectable moiety. In other embodiments, a binding agent and a detection agent are used, where the detection agent is labeled with a detectable moiety. For ease of reference, the term antibody is used in describing the binding agent or capture molecule. However, it is understood that reference to an antibody in the context of describing an exemplary binding agent in the methods of the invention also includes reference to other binding agents, including, but not limited to, lectins.

[0086] For example, the level of a biomarker in a sample can be assayed by contacting the biological sample with an antibody or antigen-binding fragment thereof that selectively binds to a target protein (called a capture molecule or antibody or binder), and detecting binding of the antibody or antigen-binding fragment thereof to the protein. Detection can be accomplished by detecting a second antibody to bind to the capture antibody complexed with the target biomarker. The target biomarker can be a whole protein or a modified or altered form thereof. Kits for the detection of proteins as described herein include pre-coated strips / plates, biotinylated secondary antibodies, Includes running standards, controls, buffer, streptavidin-horseradish peroxidase (HRP), tetramethylbenzidine (TMB), stop reagent, and standards. The test may contain detailed instructions for performing the test.

[0087] The present disclosure also provides a method for detecting a protein in a sample obtained from a subject, where the expression level of the protein in the biological sample is determined simultaneously. For example, in one embodiment, the method comprises: (a) detecting a biological sample obtained from a subject; and (b) contacting the one or more biomarker proteins with a plurality of binding agents, each of which selectively binds to the one or more biomarker proteins for a period of time sufficient to form a binding agent-biomarker complex; and (b) detecting binding of the binding agents to the one or more biomarker proteins. In a further embodiment, the detecting thereby determines a level of expression of the biomarker in the biological sample; and the method further comprises (c) detecting binding of the binding agents to the one or more biomarker proteins in the biological sample. The expression levels of one or more biomarker proteins in the sample are compared to a predetermined threshold value. wherein a level of expression of at least one of the biomarker proteins above or below a predetermined threshold indicates, for example, that the subject has prostate cancer. the severity of prostate cancer and / or the likelihood of responding to prostate cancer therapy. It is pointed out that such embodiments can aid in distinguishing whether a subject has PCa versus normal, BPH and / or PTT. Examples of binding agents that can be usefully employed in such methods include, but are not limited to, antibodies or antigen-binding fragments thereof, aptamers, lectins, and the like.

[0088] Although antibodies are useful for their extensive characterization, any other suitable agent (e.g., peptides, aptamers, or small organic molecules) that specifically binds to the biomarkers of the invention is optionally used in place of the antibodies in the above immunoassays. For example, aptamers that specifically bind one or more of the biomarkers and / or their degradation products may be used. Aptamers are nucleic acid-based molecules that bind to specific ligands. Methods for generating aptamers with specific binding specificities are known, as detailed in U.S. Patent Nos. 5,475,096; 5,670,637; 5,696,249; 5,270,163; 5,707,796; 5,595,877; 5,660,985; 5,567,588; 5,683,867; 5,637,459; and 6,011,020.

[0089] In a specific embodiment, the assay performed on the biological sample comprises reacting the biological sample with one or more capture agents (e.g., an antibody or a combination thereof) to form a biomarker:capture agent complex. The complex can then be detected and / or quantified. Elephants are identified as having invasive prostate cancer based on a comparison of detected / quantified / measured levels of biomarkers compared to one or more reference controls as described herein. It is possible.

[0090] One method involves the use of a protein biomarker, such as an antibody that specifically binds to a protein biomarker of interest. Such a first, or capture, binding agent is immobilized on a suitable solid phase substrate or carrier. The test biological sample is then contacted with the capture antibody and incubated for a desired period of time. After washing to remove unbound material, a second, detection, antibody that binds to a different, non-overlapping, epitope on the biomarker (or the bound capture antibody) is introduced. Then, it is used to detect the binding of polypeptide biomarkers compared to the capture antibody.Detection antibody is preferably conjugated to a detectable moiety, either directly or indirectly.The examples of detectable moieties that can be employed in such a method include, but are not limited to, chemical fluorescent and luminescent agents; fluorophores, such as fluorescein, rhodamine and eosin; radioisotopes; colorimetric agents; and enzyme substrate labels, such as biotin.

[0091] In an even more specific embodiment, a biotinylated lectin that specifically binds to the biomarker can be added to a patient sample, and a streptavidin-labeled fluorescent marker that binds the biotinylated lectin bound to the biomarker is then added and the biomarker is detected.

[0092] In another embodiment, the assay is a competitive binding assay, in which a labeled protein biomarker is used in place of a labeled detection antibody, and the labeled biomarker and any unlabeled biomarker present in the test sample compete for binding to the capture antibody. The amount of biomarker bound to the capture antibody can be determined based on the detected proportion of labeled biomarker.

[0093] The solid phase substrate or carrier that can be effectively used in such assays is well known to those skilled in the art, and includes, for example, 96-well microtiter plate, glass, paper, and microporous membranes that are composed of, for example, nitrocellulose, nylon, polyvinylidene fluoride, polyester, cellulose acetate, mixed cellulose esters, and polycarbonate.Suitable microporous membranes include, for example, those described in U.S. Patent Application Publication No. US2010 / 0093557A1.Methods for automating immunoassays are well known in the art, and include, for example, those described in U.S. Patent Nos. 5,885,530, 4,985,785, 6,159,750, and 5,358,691.

[0094] The presence of several different protein biomarkers in a "0001" test sample can be detected simultaneously using a multiplex assay, such as a multiplex ELISA. The assay offers the advantages of high throughput, requiring only small amounts of sample, and the ability to detect different proteins across the dynamic range of concentrations of the board.

[0095] In certain embodiments, such methods employ arrays, in which multiple binding agents (e.g., capture antibodies) specific for multiple biomarkers are immobilized on a substrate, such as a membrane, with each capture agent located at a specific, predefined, location on the substrate. Methods for performing assays employing such arrays include, for example, those described in U.S. Patent Application Publication Nos. US2010 / 0093557A1 and US2010 / 0190656A1, the disclosures of which are specifically incorporated herein by reference.

[0096] For example, multiplex arrays in several different formats based on the use of flow cytometry, chemiluminescence, or electrochemiluminescence technologies can be used. Flow cytometry multiplex arrays, also known as bead-based multiplex arrays, include the Cytometric Bead Array (CBA) system from BD Biosciences (Bedford, Mass.) and the Multi-Array (Multiplexer) from Luminex Corp (Austin, Tex). Analyte Profiling (xMAP (R) ( (R) The technology includes ELISA (registered trademark in the U.S. and other countries), all of which employ bead sets that can be distinguished by flow cytometry. Each bead set is coated with a specific capture antibody. Fluorescent or streptavidin-labeled detection antibodies bind to the specific capture antibody-biomarker complexes that form on the bead set. Various biomarkers can be recognized and measured by their differences in the bead sets, and color or fluorescent emissions are detected using flow cytometry analysis.

[0097] An alternative format is available from Quansys Biosciences. The multiplex ELISA from Sigma (Logan, Utah) coats multiple specific capture antibodies on multiple spots (one antibody per spot) in the same well on a 96-well microtiter plate. The present invention uses chemiluminescence technology to detect a variety of biomarkers in vivo.

[0098] "0002" In some embodiments, the biomarkers of the present invention may be detected using an electrochemiluminescence assay developed by Meso Scale Discovery (Gaithersburg, MD). Electrochemiluminescence detection uses labels that emit light when electrochemically stimulated. Because the stimulation mechanism (electricity) is decoupled from the signal (light), background signal is minimal. The labels are stable, non-radioactive, and offer a convenient choice of coupling chemistry. They emit light at .about.620 nm, eliminating the problem of color quenching. See U.S. Patent Nos. 7,497,997; 7,491,540; 7,288,410; 7,036,946; 7,052,861; 6,977,722; 6,919,173; 6,673,533; 6,413,783; 6,362,011; 6,319,670; 6,207,369; 6,140,045; 6,090,545; and 5,866,434. Published Applications No. 2009 / 0170121; No. 2009 / 006339; No. 2009 / 0065357; No. 2006 / 0172340; No. 2006 / 0019319; No. 2005 / 0142033; No. 2005 / 0052646; No. 2004 / 0022677; No. 2003 / 0124572; No. 2003 / 0113713; No. 2003 / 0003460; No. 2002 / 0137234; No. 2002 / 0086335; and No. 2001 / 0021534.

[0099] Proteins in the present invention can be detected by other suitable methods. Detection paradigms that can be employed for this purpose include optical methods, electrochemical methods (voltammetry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Optical methods Exemplary methods are microscopy, both confocal and non-confocal, as well as fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index detection (e.g. surface plasmon resonance, ellipsometry, resonant mirror method, grating coupler waveguide method or interferometry).

[0100] In certain embodiments, the protein biomarker proteins of the present invention can be captured and concentrated using nanoparticles. (R) (Nanotrap) Technology (Ceres Nanosciences, Inc. The Nanotrap platform can be used to capture and concentrate nanoparticles using a fluorophore such as fluorophore ... Enabling biomarker enrichment, by removal of high abundance analytes, and preventing degradation to highly labile analytes in an innovative, one-step collection workflow Multiple analytes isolated from a single sample are concentrated and eluted in a small volume, increasing the concentration by up to 100-fold depending on the starting sample volume. These techniques can effectively amplify DNA up to 1000 kDa (Shafagati, 2014; Shafagati, 2013; Longo et al., 2009), providing significant improvements in downstream analytical sensitivity.

[0101] Additionally, samples can also be analyzed using biochips. Biochips often include a solid substrate and have a typically planar surface to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Often, the surface of the biochip includes multiple addressable locations, each of which has a capture reagent bound thereto. Protein biochips are biochips adapted for the capture of polypeptides. Many protein biochips have been described in the art. These include, for example, the protein biochips available from Ciphergen Biosystems, Inc. (Fremont, Calif.) (Nashville, MD), Invitrogen Corp. (Carlsbad, CA), Affymetrix, Inc. (Fremon, CA), ), Zyomyx (Hayward, CA), R&D Systems, Inc. Indo-D Systems (Minneapolis, MN), Biacore ( Biacore (Uppsala, Sweden) and Procognia Protein produced by Locognia (Berkshire, UK) Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent No. 6,536,749; U.S. Patent No. 6,329,209; U.S. Patent No. 6,225,047; U.S. Patent No. 5,242,828; PCT International Publication No. WO 00 / 56934; and PCT International Publication No. WO 03 / 048768.

[0102] In a specific embodiment, the invention includes a microarray chip. More specifically, the chip includes a small wafer carrying a collection of surface-bound binding agents in a regular pattern, with each binding agent occupying a specific location on the chip. The set of binding agents may be one or more of the binding agents described herein. The binding agents specifically bind to each of the biomarkers above. In certain embodiments, a few microliters of serum or plasma are dropped onto the chip array. Protein biomarkers present in the tested specimen bind to the binding agents that are specifically recognized by them. The subtypes and amounts of binding marks are detected and quantified, for example, using fluorescently labeled secondary subtype-specific antibodies. In certain embodiments, an optical reader is used for bound biomarker detection and quantification. Thus, the system can include a chip array and an optical reader. In other embodiments, a chip is provided.

[0103] IV. Metabolite detection / measurement

[0104] Metabolites useful in the present invention include, but are not limited to, asparagine, aspartate, glycerate, citrate, isocitrate, glutamate, itaconate, malate, meglutol, cis-aconitate, isoleucine, leucine, pantothenate, glutamine, nicotinate, and threonine. Compositions for detecting / measuring metabolites and methods are known in the art. Examples include those available from Metabolon, Inc. (Morrisville, NC) (e.g., U.S. Pat. Nos. 10,890,592; 11,181,530; 11,061,005; 10,965,183; 10,573,406; and 10,267,777); Abcam plc (Cambridge, UK) (e.g., Asparagine Assay Kit (Fluorometric), Glutamine Assay Kit (Colorimetric), Aspartate Assay Kit, and Citrate Assay Kit). Promega Corporation (Madison, WI) (e.g., Glutamate-Glo assay kits); TM Assay (Glutamate-Glo TM Asse B), and Glutamine / Glutamate-Glo (R) Assay (Glutamine / Glutamate-Glo (R) and Sigma-Aldrich, Inc. (St. Louis, MO). Truist, MO (examples include the Glutatmate Assay Kit), Citrate Assay Kit, and Isocitrate Assay Kit. See also Malate Assay Kit.

[0105] In other embodiments, the metabolic biomarkers of the present invention may be detected by mass spectrometry, a method that uses a mass spectrometer to detect gas phase ions. Examples of mass spectrometers include time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, Orbitrap, hybrid or combinations of the above, and the like.

[0106] In certain embodiments, metabolites are detected using selected reaction monitoring (SRM) mass spectrometry techniques. Selected reaction monitoring (SRM) is a non-scanning mass spectrometry technique performed on triple quadrupole-like instruments, in which collision-induced dissociation is used as a means to increase selectivity. SRM experiments In the experiment, the two mass analyzers were used as static mass filters to filter out selected precursor ions. Selected precursor ions and fragment ions are monitored. A particular pair of mass-over-charge (m / z) values ​​that is related to an on is called a "transition." In contrast to conventional MS-based proteomics, in SRM analysis mass spectra are recorded. Instead, the detector acts as a counting device for ions that match a selected transition, thereby returning a distribution of intensities over time. Multiple SRM transitions can be measured within the same experiment on the chromatographic time scale by rapidly toggling between different precursor / fragment pairs (sometimes called multiple reaction monitoring, MRM). Typically, a triple quadrupole instrument cycles through a series of transitions and records the signal of each transition as a function of elution time. This method allows for additional selectivity by monitoring the chromatographic coelution of multiple transitions for a given analyte. The terms SRM / MRM are also sometimes used to describe experiments performed on mass spectrometers other than triple quadrupoles. They are also used (e.g. in trapping instruments) where a narrow mass range is scanned in MS2 mode during fragmentation of a particular precursor ion, and flags specific to the precursor of interest are generated. The term SRM and MRM or also SRM / MRM is used interchangeably in this application, since they both refer to the same principle of operation of a mass spectrometer. As a matter of clarity, the term MRM will be used throughout the text, but The term includes both SRM and MRM, as well as any similar techniques, such as highly selective reaction monitoring, hSRM, LC-SRM, or any other SRM / MRM-like or SRM / MRM-mimicking approaches implemented in any type of mass spectrometry and / or where The tides can be fragmented by any other method, such as CAD (collision-activated dissociation, also known as CID or collision-induced dissociation), HCD (higher energy CID), ECD (electron capture Fragmentation is performed using techniques such as photodissociation (PD), photodissociation (PD), or electron transfer dissociation (ETD). It will be turned into a token.

[0107] In another specific embodiment, the mass spectrometry method is a matrix-assisted laser desorption / ionization flight. In another embodiment, the method comprises MALDI-TOF tandem mass spectrometry (MALDI-TOF MS / MS). In yet another embodiment, the mass spectrometry is performed by a method according to the art. As can be envisaged by one of ordinary skill in the art, MALDI-TOF can be combined with other suitable method(s). For example, MALDI-TOF can be combined with trypsin digestion and tandem digestion as described herein. It can be used in conjunction with simultaneous mass spectrometry.

[0108] In an alternative embodiment, the mass spectrometry technique may be Surface Enhanced Laser Desorption and Ionization, or "SELDI," as described, for example, in U.S. Pat. Nos. 6,225,047 and 5,719,060. Briefly, SELDI refers to a method of desorption / ionization gas phase ion spectrometry (e.g., mass spectrometry) in which an analyte (here, one or more of the biomarkers) is are captured on the surface of a SELDI mass spectrometry probe. There are several versions of SELDI that can be utilized, including but not limited to Affinity Capture Mass Spectrometry (also called Surface-Enhanced Affinity Capture (SEAC)) and Surface-Enhanced Neat Desorption (SEND), which involves the use of a probe that contains an energy absorbing molecule chemically bonded to the probe surface (the SEND probe). Another SELDI method is shown in Table Surface-Enhanced Photolabile Attachment and Release ) (SEPAR), which is a surface-attached molecule capable of covalently binding an analyte. These involve the use of a probe having an analyte and then releasing the analyte through breaking of a photosensitive bond on the moiety after exposure to light, e.g., laser light (see U.S. Pat. No. 5,719,060). SEPAR and other forms of SELDI can be used in accordance with the present invention as biomarkers or is easily adapted to detect a panel of biomarkers.

[0109] In another mass spectrometry method, the biomarkers can first be captured on a chromatography resin that has chromatographic properties that bind to the biomarkers. , cation exchange resin, such as CM Ceramic HyperD F resin, etc. Such methods could capture the biomarkers on a cation exchanger such as PBS, wash the resin, elute the biomarkers, and detect by MALDI. This could be preceded by fractionating the sample on an anion exchange resin before applying it to the exchange resin. In another alternative, one could fractionate on an anion exchange resin and then perform the fractionation by MALDI. In yet another method, one could capture the biomarker on an immunochromatographic resin containing antibodies to bind the biomarker, wash the resin to remove unbound material, elute the biomarker from the resin, and detect the eluted biomarker by MALDI or by SELDI.

[0110] V. Point-of-Care Assays for Detecting Target Proteins / Nucleic Acids

[0111] The above assay types are amenable to developing point-of-care (POC) devices. A number of different devices can be used to make the system self-contained, with the output being readable by the user. This property is particularly useful when collection of the sample to be tested does not require medical intervention (examples are urine, saliva, or sputum). One device that makes this possible is the Lateral Flow Device (LFD). These devices use a multi-layer structure that contains both absorbent and non-absorbent components to form the solid phase. Capture and / or recognition reagents (antigens or antibodies) are pre-applied to specific areas within the assembled instrument. A reagent is used, and the analyte is allowed to flow through the system to come into contact with the reagent. Often, for self-contained purposes, the reagent components are added in a dried state so that fluid from the sample rehydrates and activates them. Conventional ELISA techniques, the antibody It can be used to detect the analyte in the original antibody complex. In some embodiments, the system can be designed to provide a colorimetric readout for visual estimation of a binary response ("yes" or "no"), or it can be configured to be quantitative.

[0112] Many of the embodiments contemplated herein for lateral flow devices are described with respect to detecting proteins (e.g., EPCAM), and lateral flow devices include, but are not limited to, H4C5 and TTC3 (as well as other nuclear RNAs described herein, including mRNAs, circRNAs, eccDNAs, and lncRNAs). The method can be used to detect nucleic acids, including nucleic acid biomarkers. U.S. Patent No. 9,121,849 (Rapid Pathogen Screening, Inc.) ); U.S. Patent Application Publication No. 20090305290 (Rapid Pathogen Screening, Inc.); See International Patent Application Publication No. WO 2004 / 092342 (Applera Corporation). Metabolites or small molecules can also be detected using lateral flow technology. U.S. Patent No. 8,399,261 (Inbios International, Inc.) ) (International Patent Application WO 2017 / 075649) and Nuntawong et al., 76 J. NAT. MED. (Journal of Natural Medicines) 521-45 (2022). Thus, it is contemplated herein that proteins, nucleic acids and small molecules (e.g., metabolites) can be detected via lateral flow. In certain embodiments, lateral flow assays are used to detect EpCAM, and qPCR is used to detect H4C5 and TTC3. Used for.

[0113] In certain embodiments, the presently disclosed methods can use lateral flow devices or dipstick assays, including immunochromatographic strip tests that rely on direct (double antibody sandwich) reactions. Without wishing to be bound by theory, this direct reaction scheme can be used when sampling for a larger analyte that may have multiple antigenic sites. For example, a combination of different antibodies can be used, where different antibodies are included on the capture (detection) line, the control line, and, for example, gold particles, e.g., gold microparticles, gold nanoparticles, etc. It can be conjugated to a particle or a fluorescent dye and included in the mobile phase of the assay.

[0114] The term "dipstick assay" as used herein refers to any assay that uses a dipstick to contact a sample solution with the dipstick to move the sample solution to the capture zone of the dipstick by capillary action, thereby allowing the target antigen in the sample solution to be captured and detected in the capture zone. To test for the presence of an analyte, the contact end of the dipstick is contacted with a test solution. If the analyte is present in the test solution, the analyte moves to the capture zone of the dipstick by capillary action where it is captured by the capture antibody. The presence of the analyte in the capture zone of the dipstick is detected by an additional anti-analyte antibody (detection antibody) labeled with, for example, colloidal gold.

[0115] These stick tests have several advantages: they are easy and inexpensive to perform, do not require specialist equipment, and results are obtained quickly and can be read visually. Thus, these tests are particularly suitable for use in physicians' offices, at home, in remote locations, and in countries where specialist equipment may not be available. They can be used, for example, to detect PCa.

[0116] To carry out the method of the first aspect of the invention, the targeting agent and the label are simply added to a test solution. and then contacting the test solution with the contact end of the chromatography strip. Such a method is easier to carry out than the method disclosed in WO 00 / 25135, which requires two separate wicking steps. Thus, the results are It can be obtained more quickly and also the sensitivity of analyte detection is more sensitive.

[0117] The term "chromatographic strip" is used herein to mean any porous strip of material capable of transporting a solution by capillary action. Chromatographic strips can be capable of bibulous or non-bibulous lateral flow, but are preferably bibulous lateral flow. By the term "non-bibulous lateral flow" it is meant that all of the dissolved or dispersed components of the liquid flow at substantially equal rates, and in contrast to the preferential retention of one or more components that would occur in "bibulous lateral flow". Lateral flow refers to the flow of a liquid that is carried laterally through a membrane in a relatively unimpeded manner. Materials capable of bibulous lateral flow include paper, nitrocellulose, and nylon. A preferred example is nitrocellulose.

[0118] The label may be attached to the targeting agent by premixing the targeting agent with the label before the targeting agent is added to (or otherwise contacted with) the test solution. However, in some circumstances, it is preferable that the targeting agent and the label are not premixed, since such premixing can cause the targeting agent and the label to precipitate. Thus, the targeting agent and the label may be added to (or contacted with) the test solution separately. The targeting agent and the label can be added to (or contacted with) the test solution substantially simultaneously or in any order.

[0119] The test solution may be pre-incubated with the targeting agent and the label before the test solution contacts the contact end of the chromatography strip to ensure complex formation. The optimal time of pre-incubation depends on the ratio of the reagents and the flow rate of the chromatography strip. In some cases, too long pre-incubation can reduce the detection signal obtained and even lead to false positive detection signals. Thus, it may be necessary to optimize the pre-incubation time for the specific conditions used.

[0120] It may be desirable to pre-incubate the targeting agent with the test solution prior to conjugating the label to the targeting agent, so as to allow the targeting agent to bind to the analyte in the test solution under optimal binding conditions.

[0121] As used herein, the term "lateral flow" refers to the flow of liquid along the plane of a substrate or carrier, e.g., a lateral flow membrane. Generally, a lateral flow device includes a strip (or multiple strips in fluid communication) of material capable of transporting a solution by capillary action, where different regions or zones in the strip(s) contain assay reagents, either diffusively or non-diffusively bound to the substrate, that generate a detectable signal when a solution is transported to or moves through such zones. Typically, such an assay includes an application zone adapted to receive a liquid sample, a reagent zone laterally spaced from and in fluid communication with the application zone, and a detection zone laterally spaced from and in fluid communication with the reagent zone. The reagent zone is mobile in the liquid, and in the sample, e.g., can include a compound capable of interacting with the analyte to form an analyte-reagent complex, and / or with a molecule bound in the detection zone. The detection zone may be immobilized on the strip and contain analytes and / or reagents and / or binding molecules capable of interacting with analyte-reagent complexes to generate a detectable signal. Such assays can be used to detect analytes in a sample directly (sandwich assays) or through competitive binding. Examples of lateral flow devices are described in U.S. Pat. No. 6,194,220 to Malick et al., U.S. Pat. No. 5,998,221 to Malick et al., U.S. Pat. No. 5,798,273 to Shuler et al., and U.S. Pat. No. 5,798,273 to Rosenstein et al. No. RE38,430 to Senstein et al.

[0122] In some embodiments, the presently disclosed methods can be used with assays including sandwich lateral flow or dipstick assays. In a sandwich assay, a liquid sample, which may or may not contain an analyte of interest, is applied to an application zone and allowed to pass into the reagent zone by capillary action. The term "analyte" as used herein refers to a target protein, including but not limited to EPCAM, H4C5 and / or TTC3. In certain embodiments, the presence or absence of the analyte is qualitatively determined in the sample. In other embodiments, a quantitative determination of the amount or concentration of the analyte is determined in the sample. In other embodiments, the H4C5 and / or TTC3 nucleic acid, e.g. In a specific embodiment, the target analyte is EPCAM protein. In some embodiments, the target analyte includes H4C5 and / or TTC3 RNA. The target analyte may be any protein, nucleus, or the like of any of the biomarkers described herein. It can be an acid or a metabolic product.

[0123] If present, the analyte is reacted with the target in the reagent zone to form an analyte-reagent complex. The analyte interacts with the identified reagent, and the analyte-reagent complex is transported by capillary action to the detection zone. The labeled reagent migrates through the detection zone. The analyte-reagent complex is captured in the detection zone by interaction with binding molecules specific for the analyte and / or reagent. Unbound sample can pass by capillary action through the detection zone to a control zone or absorbent pad that is laterally apposed to and in fluid communication with the detection zone. The labeled reagent can then be detected in the detection zone by appropriate means.

[0124] Generally, and without limitation, a lateral flow device includes a sample pad. The sample pad comprises a membrane surface, also referred to herein as a "sample application zone," adapted to receive a liquid sample. Standard cellulose sample pads have been shown to facilitate the absorption and flow of biological samples, including, but not limited to, urine. The sample pad comprises the portion of the lateral flow device that is in direct contact with the liquid sample, i.e., it receives the sample to be tested for an analyte of interest. The sample pad can be part of the lateral flow membrane or separate from it. Thus, the liquid sample can migrate from the sample pad through lateral or capillary flow toward the portion of the lateral flow membrane that contains the detection zone. The sample pad is in fluid communication with the lateral flow membrane that contains the analyte detection zone. This fluid communication can arise through top-to-bottom, or be a top-to-bottom overlap, or be an end-to-end fluid connection between the sample pad and the lateral flow membrane. In certain embodiments, the sample pad comprises a porous material, such as, and without limitation, paper.

[0125] Typically, the sample pad is disposed adjacent to and in fluid communication with the conjugate pad. The conjugate pad is configured to receive one or more samples of interest. It contains a labeled reagent that has specificity for the analyte. In some embodiments, the conjugate pad contains a non-absorbent, synthetic material (e.g., polyester) to ensure release of its contents. The detection conjugate is dried into place on the conjugate pad and is released only when a liquid sample is applied to the sample pad. The detection conjugate can be applied to the pad by dipping or spraying.

[0126] In certain embodiments, the detection conjugate comprises an antibody that specifically binds to EPCAM. In this embodiment, the detection conjugate is an antibody that specifically binds to H4C5 and / or TTC3 In some embodiments, the antibody is a monoclonal antibody.

[0127] Antibodies, e.g., monoclonal antibodies (MAbs), fluorescent dyes or gold particles, e.g., colloids With gold, one can conjugate to something like gold microspheres or gold nanoparticles, such as about 40 nm gold nanoparticles. For example, using streptavidin-coated microspheres, one can biotinylate the conjugated MAb to take advantage of the strong affinity that biotin has for streptavidin. An alternative includes Protein A coated microspheres which bind to the Fc region of IgGs.

[0128] In certain embodiments, the conjugate pad is adjacent to and in fluid communication with the lateral flow membrane. Capillary action causes antibody-antigen complexes to form and promote lateral flow. The fluid mixture is drawn through the conjugate pad, which enters the membrane, onto the sample pad. Lateral flow is a function of the properties of the lateral flow membrane. Lateral flow membranes are typically very thin and hydrophilic enough to be wetted, thereby allowing unhindered lateral flow and mixing of reactants and analytes at essentially the same rate.

[0129] Lateral flow membranes can include any substrate capable of providing liquid flow, including, but not limited to, substrates such as nitrocellulose, polyester or nitrocellulose blends with cellulose, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymers, plastic, glass, nylon, and the like. Lateral flow membranes can be porous. Typically, the pores of a lateral flow membrane are large enough that particles, e.g., microparticles containing reagents capable of forming complexes with analytes, flow through the entire membrane. Lateral flow membranes can generally have pore sizes ranging from about 3 μm to about 100 μm, and in some embodiments, have pore sizes ranging from about 10 μm to about 50 μm. The pore size affects the capillary flow rate and the overall performance of the device.

[0130] The use of nitrocellulose for the primary membrane has the following advantages: low cost, capillary flow, High affinity for protein binding and handling Nitrocellulose has high protein binding. Another alternative is cellulose acetate, which has low protein binding. Size dictating surface area is a function of membrane capacity (the amount of sieving material that can pass through a membrane per unit time). These variables determine the volume of the pipe that will produce lateral flow. Since the flow rate controls the rate at which the membrane is spun, they can affect the sensitivity and specificity of the assay. The flow rate also varies with the viscosity of the sample. Several different sizes and polymers are available for use as microspheres, which are displaced down the membrane upon introduction of a fluidic sample. Optimal flow rates are generally achieved using spheres that are no larger than 1 / 10 the pore size of the membrane.

[0131] Those skilled in the art will recognize other materials that allow for liquid flow. Lateral flow membranes, in some embodiments, may include one or more substrates in fluid communication with the membrane. For example, the conjugate pad can be on the same substrate or on a separate substrate (i.e., pad) within the lateral flow membrane or in fluid communication with the lateral flow membrane. In some embodiments, the nitrocellulose membrane can include a very thin Mylar sheet coated with a nitrocellulose layer. do.

[0132] The lateral flow membrane further comprises at least one indicator or detection zone. The terms "indicator zone" and "detection zone" are used interchangeably herein and refer to a portion of a carrier or porous membrane that contains an immobilized binding reagent. As used herein, the term "binding reagent" refers to any molecule or molecule bound to a particle where the molecule recognizes or binds an analyte of interest. The binding reagent can form a binding complex with an analyte-labeled reagent complex. The binding reagent is immobilized in the detection zone and is not affected by the lateral flow of the liquid sample due to its immobilization on the membrane. When the binding reagent binds to the analyte-labeled reagent complex, it prevents the analyte-labeled reagent complex from continuing through the flow of the liquid sample. In some embodiments, the binding reagent specifically binds to EPCAM. In another embodiment, the binding reagent further comprises an antibody that specifically binds to TTC3.

[0133] Thus, during the actual reaction between the analyte and the reagent, the first member becomes the indicator zone. The second member binds to the second member at the junction, and the resulting binding complex binds to a specific antigen. Detection can be accomplished by a variety of labels and / or markers, e.g., enzymes (with appropriate substrates) Any of a variety of reagents may be used, including alkaline phosphatase or horseradish peroxidase with ion exchangers (e.g., ion exchangers with ion exchangers), radioisotopes, liposomes or latex beads impregnated with fluorescent tags, polymeric dyes or colored particles, and the like. Thus, results can be interpreted by any direct or indirect reaction. Colloidal gold particles, which impart a purple or red coloration, are currently most commonly used.

[0134] Capture and immobilization of the assay reagent (complementary member of the binding pair) in the indicator zone can be achieved by covalent binding or, more usually, by adsorption, such as by drying. Such capture can also be indirect, for example by binding of latex beads coated with the reagent. Depending on the nature of the materials comprising the lateral flow membrane, covalent binding may be possible, for example by the use of glutaraldehyde or carbodiimides. In immunoassays, the most common binding pair is the antigen-antibody pair; however, a variety of other binding pairs can be implemented, for example enzymes. Such as ligand-substrate and receptor-ligand.

[0135] In some embodiments, the indicator zone comprises a test line and a control line. The test line may further comprise an immobilized binding reagent. The use of antibodies to develop a test line in an LFD employing a sandwich-type assay may also be used. When applied, they are applied at a ratio of about 1-3 μg / cm across the width of a 1 mm wide strip; thus, the antibody concentration is about 10-30 μg / cm 2 which is about 25-100 times higher than that used in ELISA. Brown, M. C., Antibodies: key to a robust lateral flow immunoassay, in Lateral Flow Immunoassay, HYTRC Wong, Editor. 2009, Humana Press: New York, New York. p. 59-74.

[0136] Furthermore, in some embodiments, the lateral flow assay of the present disclosure can be used to detect multiple analytes in a sample. For example, in a lateral flow assay, the reagent zone can contain multiple labeled reagents, each capable of binding to a different analyte in a liquid sample, or a single labeled reagent capable of binding to an analyte. When multiple labeled reagents are used in a lateral flow assay, the reagents can be differentially labeled to distinguish different types of analytes in a liquid sample. It is also possible to place multiple lines of capture antibodies on the membrane to detect different analytes. A combination of antibodies that detect different epitopes of an analyte can optimize specificity.

[0137] For quality control, lateral flow membranes can typically include a control zone, which includes a control line. The term "control zone" refers to a portion of a test device that includes a binding molecule configured to capture a labeled reagent. In a lateral flow assay, the control zone can be a liquid stream that contacts the detection zone of the carrier such that the labeled reagent is captured on the control line when the liquid sample is transported from the detection zone by capillary action. Detection of the labeled reagent on the control line confirms that the assay is functioning for its intended purpose. Placement of the control line is accomplished using a microprocessor-controlled TLC spotter. A dispenser pump can then expel a volume of reagent across the membrane.

[0138] A typical lateral flow device may also include an absorbent pad. An absorbent pad, as used herein, includes "absorbent material" and refers to a porous material having sufficient absorbent capacity to absorb substantially all of the liquid of the assay reagent and any wash solution, and optionally to initiate capillary action and draw the assay liquid through the test device. Suitable absorbent materials include, for example, nitrocellulose, nitrocellulose blends with polyester or cellulose, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymers, plastic, glass, nylon.

[0139] In some embodiments, the lateral flow membrane comprises one or more substantially fluid-impermeable sheets. One on either side, for example the application zone and the indicator zone a bottom sheet having one or more windows defining a An exemplary lateral flow device may also include a housing. The term "housing" refers to any suitable enclosure for the presently disclosed lateral flow device. Exemplary housings will be known to those skilled in the art. The housing may have, for example, a base portion and a lid portion. The lid portion may include a top wall and a substantially vertical side wall. A rim may project upwardly from the top wall and may further define a recess adapted to collect a sample from a subject. Suitable housings include those provided in U.S. Patent No. 7,052,831 to Fletcher et al. and BD Directigen TM (BD Directigen) EZ RSV Lateral Flow Assay Device This includes those used in

[0140] In some embodiments, target analytes such as EPCAM, H4C5 and / or TTC3 can be measured in whole, unconcentrated, or otherwise untreated biological samples using the presently disclosed methods and devices. In other embodiments, the biological sample may be, for example, concentrated, diluted, filtered, and the like, prior to performing the test. Pretreatment of the urine sample may include diluting the urine sample in an aqueous solution, concentrating the urine sample, filtering the urine sample, or a combination thereof.

[0141] Upon reviewing the subject matter of the present disclosure, one of ordinary skill in the art will recognize that the pretreatment steps can be performed in any particular order, for example in some embodiments the sample can be diluted or concentrated and then filtered, while in other embodiments the sample can be filtered and then diluted or concentrated. In certain embodiments, the presently disclosed method includes filtering the urine sample, for example through a desalting column, to remove molecules that may interfere with the detection of antigens in the urine sample. This step can be performed with or without any further dilution or concentration of the sample.

[0142] Thus, in some embodiments, the lateral flow device is configured to pretreat the biological sample prior to contacting the biological sample with at least one antibody specific for EPCAM, at least one antibody specific for H4C5, and / or at least one antibody specific for TTC3. In certain embodiments, the device is adapted to filter, dilute, or concentrate the biological sample, or a combination thereof. In alternative embodiments, the device can be adapted to remove inhibitors that interfere with the detection of EPCAM and / or H4C5 in biological samples, particularly urine samples.

[0143] In other embodiments, to eliminate the need to process biological samples, different parameters of the test, e.g., incubation time, may be varied to improve the sensitivity and / or specificity of the test. Can be manipulated to increase

[0144] VI. Treatment method

[0145] In another aspect, the present invention relates to a prostate cancer cell line that is actually applied after the measurement / detection of a biomarker. Cancer therapies or therapeutic interventions are provided. In certain embodiments, the therapeutic interventions include prostatectomy, radiation therapy, cryotherapy (also called cryosurgery or cryoablation), hormone therapy, chemotherapy, immunotherapy, and combinations thereof.

[0146] Prostate removal includes radical prostatectomy, either open (retropubic or perineal) or lateral (laparoscopic, including robotic-assisted), and transurethral resection of the prostate (TURP).

[0147] Radiation therapy can be performed using external beam radiation (three-dimensional conformal radiation therapy (3D-CRT), intensity-modulated radiation therapy (IMRT), stereotactic body radiation therapy (SBRT), proton beam radiation therapy) and brachytherapy (internal radiation) (permanent (low-dose-rate or LDR) brachytherapy or temporary (high-dose-rate or HDR) brachytherapy).

[0148] Hormone therapy (androgen suppression therapy) may include orchiectomy (castration surgery), luteinizing hormone releasing hormone (LHRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin), LHRH antagonists (e.g., degarelix), androgen levels from the adrenal glands (e.g., abiraterone, ketoconazole), and antiandrogens (e.g., flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide), and treatments to lower estrogen.

[0149] Chemotherapy includes treatment with compounds including, but not limited to, docetaxel, cabazitaxel, mitoxantrone, and estramustine.

[0150] Immunotherapies include, but are not limited to, cancer vaccines (e.g., sipuleucel-T), as well as immune checkpoint inhibitors (e.g., PD-1 inhibitors, including pembrolizumab). Exemplary immune checkpoint inhibitors include tremelimumab (a CTLA-4 blocking antibody), anti-OX40, PD-L1 monoclonal antibodies (anti-B7-H1; MEDI4736), MK-3475 (a PD-1 blocking antibody), and PD-L1 inhibitors (anti-B7-H1; MEDI4736). These include nivolumab (anti-PDl antibody), CT-011 (anti-PDl antibody), BY55 monoclonal antibody, AMP224 (anti-PDLl antibody), BMS-936559 (anti-PDLl antibody), MPLDL3280A (anti-PDLl antibody), MSB0010718C (anti-PDLl antibody) and Yervoy / ipilimumab (anti-CTLA-4 checkpoint inhibitor).

[0151] Prostate therapeutic interventions include poly(ADP)-ribose polymerase (PARP) inhibitors (e.g., niraparib (Zedula), olaparib (Lynparza), and rucaparib (Rubraca Includes Rubraca).

[0152] Other therapeutic interventions for prostate cancer include androgen receptor (AR) targeted therapies (e.g., enzalutamide, ARN-509, ODM-201, EPI-001, hydrazinobenzoylcurcumin (HBC), aberaterone, and geleterone). ), and seviteronel), antimicrotubule agents, alkylating agents and anthracenediones.

[0153] In certain embodiments, the therapeutic intervention for prostate cancer includes, but is not limited to, abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, Casodex (bicalutamide), darolutamide, degarelix, docetaxel, Eligard (leuprolide acetate), enzalutamide, Erleada (apalutamide), Pharmagon (degarelix), flutamide, goserelin acetate, Jevtana (cabazitaxel), leuprolide acetate, Lupron (leuprolide acetate), Lupron Depot (Lupron Depot) (leuprolide acetate), Lynparza (olaparib), mitoxantrone hydrochloride, Nilandrone (nilutamide), nilutamide, Nubequo (darolutamide), olaparib, Provenzi (sipuleucel-T), radium-223 chloride, Rubraca (rucaparib cansylate), rucaparib cansylate, sipuleucel-T, Taxotere (docetaxel), Xofigo (radium-223 chloride), Xtandi (enzalutamide), Zola This may include administration of medications including Dex (goserelin acetate) and Zytiga (abiraterone acetate).

[0154] VII. Kit

[0155] In another aspect, the invention provides kits for detecting one or more biomarkers. The exact nature of the components configured in a kit of the invention will depend on its intended purpose. In one embodiment, the kit is configured specifically for human subjects.

[0156] The materials or components to be assembled in the kit can be stored and provided to the practitioner in any convenient and suitable manner that preserves their operability and usefulness. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated, or frozen temperatures. The components are typically contained in suitable packaging material(s). As used herein, the phrase "packaging material" refers to Refers to one or more physical structures used to contain the contents of the kit, such as the compositions of the invention and the like. Packaging materials are sterile, free of contaminants, and The kit is constructed in a conventional manner to provide a free-flowing environment for the kit. As used herein, the term "package" refers to a suitable solid matrix or material, such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. The packaging material generally contains the contents of the kit and / or its components. and / or have external signage indicating purpose.

[0157] In various embodiments, the present invention relates to a method for detecting a cellular component comprising the steps of: (a) mass spectrometry, antibody techniques, antibodies, lectins, , nucleic acid aptamer method, nucleic acid aptamer, immunoassay, ELISA, immunoprecipitation, SISCAPA, Western blot, PCR (qPCR, digital PCR, etc.) (b) one or more internal standards suitable for measuring one or more of the biomarkers included; and Kits containing reagents and instructions for pull processing, preparation and biomarker measurement / detection (c) measuring a biomarker in a sample obtained from the subject. The kit may further include instructions for using the kit to

[0158] In certain embodiments, the kit contains reagents necessary for sample processing and performance of the assay, hi specific embodiments, the assay is an immunoassay, such as an ELISA. Thus, in certain embodiments, the kit includes a substrate (e.g., a 96-well polystyrene plate) for performing the assay. The substrate can be coated with an antibody specific for the biomarker protein. In further embodiments, the kit can include a detection antibody, including, for example, a polyclonal antibody specific for the biomarker protein conjugated to a detectable moiety or label (e.g., horseradish peroxidase). The kit can also include a standard (e.g., a human protein standard). The kit can also include one or more of a buffer diluent, a calibrator diluent, a wash buffer concentrate, a color reagent, a stop solution, and a plate sealer (e.g., an adhesive strip). can.

[0159] In certain embodiments, the kit may include a solid support such as a chip, a microtiter plate (e.g., a 96-well plate), beads, or a resin with a protein biomarker capture reagent. The kit may include a protein biomarker, such as an antibody, and a secondary antibody-signal complex, such as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody and tetramethylbenzidine (TMB) as a substrate for HRP, to detect the protein biomarker. In other embodiments, the kit may include magnetic beads conjugated to the antibody (or a separate container thereof for later conjugation). The kit may further include a detection antibody, e.g., a biotinylated antibody or a lectin, that can be detected using, for example, streptavidin labeled with a labeled fluorescent marker, such as phycoerythrin. The kit may be configured to perform the assay in a singleplex or multiplex format.

[0160] The kit may be provided as an immunochromatographic strip containing a membrane with immobilized antibody and a means for detection, e.g., gold particle-conjugated antibody, where the membrane includes NC membrane and PVDF membrane. The kit may include a sample application pad, a gold particle-conjugated antibody temporarily immobilized on a glass fiber filter, a nitrocellulose membrane with immobilized antibody bands and secondary antibody bands, and an absorbent pad are placed in series to maintain continuous capillary flow of the sample.

[0161] In a specific embodiment, the kit comprises: (a) a nucleic acid sequence specific for a biomarker protein of interest; (b) magnetic beads for conjugation to antibodies that specifically bind to the biotinylated peptide of interest; (c) a monoclonal antibody that specifically binds to a marker protein; (d) a biotinylated lectin that specifically binds to the biomarker protein of interest; and (e) a streptavidin-labeled fluorescent marker.

[0162] In certain embodiments, the subject is provided with a biological sample (e.g., urine) from the patient, and a kit for detecting the relevant protein biomarkers bound to the antibodies and / or lectins. Specifically, the method comprises the steps of: (i) collecting serum from a patient; (ii) adding urine from the patient to a diagnostic kit; and (iii) detecting the protein biomarker conjugated to the antibody / lectin. If the biomarker is present in the sample, the antibody / lectin , binds to the sample or a portion thereof. In other kit and diagnostic embodiments, urine is not collected from the patient (i.e., has already been collected). In other embodiments, the sample may include urine, blood, plasma sweat, tissue, blood, or a clinical sample.

[0163] The kit can also include a wash solution or instructions for making a wash solution, where the combination of capture reagent and wash solution is used for subsequent detection, e.g., antibodies / lectins or proteins. Quantitative analysis allows for capture of the protein biomarkers on a solid support. In further embodiments, the kit can include instructions for appropriate operating parameters in the form of a label or separate insert. For example, the instructions can inform the consumer on how to collect the sample, etc. In yet another embodiment, the kit can include one or more containers with protein biomarker samples, and can include calibration or standardization. Detection of the markers described herein can be accomplished using lateral flow assays.

[0164] In certain embodiments, the target proteins of the present invention can be captured and concentrated using nanoparticles. (R) Proteins can be captured and concentrated using Nanotrap technology (Ceres Nanosciences, Inc., Manassas, VA). In brief, the NanoTrap platform offers a novel approach to protein enrichment by enabling the enrichment of target proteins, the removal of high abundance analytes, and the implementation of an innovative, one-step collection workflow. Reduce pre-analytical variability by preventing degradation of highly labile analytes in the elution stream. Multiple analytes isolated from a single sample are concentrated and eluted in a small volume, increasing the efficiency by up to 100-fold or more, depending on the starting sample volume. can be effectively amplified (Shafagati, 2014; Shafagati, 2013; Longo et al., 2009), A significant improvement in downstream analytical sensitivity is achieved.

[0165] In certain embodiments, the kit comprises the reagents and components necessary to perform an electrochemiluminescence ELISA. Contains ingredients.

[0166] In certain embodiments, the kits include lateral flow devices, dipsticks, assay sticks with immunochromatographic detection displays, and the use of any such devices known to those skilled in the art. In certain embodiments, the reagents and / or detection components may be immobilized on the device itself (i.e., the dipstick).

[0167] In some embodiments, the kit comprises one or more nucleic acid markers described herein (eccDNA In some embodiments, the present invention includes reagents that allow for quantification of RNA, such as mRNA, circRNA, IncRNA, etc. In one embodiment, the kit comprises: (i) at least one reagent that allows for quantification (e.g., determination of abundance, concentration or level) of an expression product of one or more nucleic acid markers in a biological sample; and optionally (ii) instructions for using at least one reagent. The composition may further comprise reagents for detection / measurement of biomarkers.

[0168] Nucleic acid-based detection kits may include primers or probes that specifically hybridize to the target polynucleotide. The kit may further include a target biomarker polynucleotide to be used as a positive control. Also, depending on the nucleic acid amplification technique used, various polymerases (reverse transcriptase, Taq, Sequenase) may be used to provide the necessary reaction mixture for amplification. TM (Sequenase), DNA ligase, etc.), deoxyribonuclease Enzymes suitable for amplifying nucleic acids may also be included, including nucleotides and buffers. Such kits will also generally contain, in appropriate means, separate containers for each individual reagent and enzyme, and for each primer or probe.

[0169] In even more specific embodiments, the kit comprises one or more of the nucleic acid biomarkers described herein. The kit may further include substrates and other reagents necessary to perform PCR (e.g., quantitative real-time PCR, digital PCR). The kit may be configured to perform singleplex or multiplex PCR. The kit may further include instructions for carrying out PCR. In embodiments, a biological sample obtained from a subject may be manipulated to extract nucleic acids. In further embodiments, the nucleic acids are contacted with primers that specifically bind to target biomarkers to form primer:biomarker complexes. One or more The complex can be amplified and detected / quantified / measured to determine the level of the above biomarkers. The subject can then be identified as having myocardial damage based on a comparison of the measured levels of one or more biomarkers to one or more reference controls.

[0170] The reagents described herein may optionally be associated with a detectable label and may be present in a microfluidic card, chip or chamber, examples or assays described below, examples of which may be present in a microarray or kit format adapted for use with the RT-PCR, Q PCR, digital PCR techniques described herein.

[0171] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are illustrative only, and are not limiting of the remainder of the disclosure in any way whatsoever. EXAMPLES

[0172] The following examples are illustrative of the compounds, compositions, articles, devices, and / or methods described and claimed herein. The present invention is presented to provide one of ordinary skill in the art with a complete disclosure and description of how the methods are made and evaluated, and they are intended to be purely illustrative and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for herein. Unless otherwise specified, parts are parts by weight, temperatures are degrees Celsius or ambient, and pressures are at or near atmospheric pressure. There are numerous variations and combinations of reaction conditions, examples being component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the purity and yield of products obtained from the described processes. Only reasonable and routine experimentation is required to optimize such process conditions.

[0173] The objective of the present invention is to detect invasive cancer from its indolent forms in PCa patients. The objective of the present invention is to develop a sensitive, specific, and cost-effective non-invasive molecular diagnostic test for screening for PCa. coding and non-coding RNAs 1-2 , disease-specific SNPs 3 , metabolites and lipids 2,4 Identify markers The key clinical and laboratory discoveries to date have focused on the treatment of prostate cancer. Identification of a panel of urinary enriched RNAs and metabolites in patients. In a statistically significant patient population, urinary specific RNAs (coding and non-coding), extracellular DNA (eccDNA) and metabolites were identified. 4 We will develop a PCa detection assay using several biotechnologies. Although many companies have developed FDA-approved molecular markers for PCa (PSA, PCA3, etc.), failed to produce the expected results. One of the major limitations of existing PCa markers is Dependence on a single biomarker (PSA or PCA3). Cancer is heterogeneous in many phases. Cancer is recognized as a multi-step process involving extensive genomic and epigenomic changes. The complexity of cancer demands multivariate assays for accurate diagnosis, prognosis, and treatment monitoring. Multivariate gene expression assays have recently been proven feasible (Oncotype DX and MammaPrint for determining whether chemotherapy is required in breast cancer). However, these tests are expensive and many require extensive biopsies and specialized specimen collection processes. The goal of this invention is to develop an accurate and reproducible multivariate assay for detecting invasive forms of PCa from readily available tissue and urine samples. The major benefit of a multiplex assay for clinical use is that it has the "power" to be highly accurate. The establishment of a molecular assay that matches the accuracy of invasive procedures will shift the paradigm of clinical practice. The inventors demonstrate that a urine-based combinatorial multi-analyte (RNAs and eccDNA) assay can differentiate invasive PCa from its non-invasive forms. We strongly believe that this is a powerful approach to detection.

[0174] Example 1 E3 ubiquitin-protein ligase, tetratricopeptide repeat domain 3 (TTC3 ), H4 clustered histone 5 (H4C5), and epithelial cell adhesion molecule (EpCAM) are novel urine enriched liquid biopsy biomarkers for detecting prostate cancer in men.

[0175] Prostate cancer (PCa) is the most common cancer in men, with 250,000 cases of prostate cancer diagnosed annually in the United States (1). Although a largely indolent disease (low-grade, low-stage disease), PCa remains the second leading cause of cancer deaths in men. In recent years, a variety of treatment options (surgery, The combination of chemotherapy, radiation, and androgen deprivation therapy (TRT) has improved survival in patients with PCa. The median age at presentation improved (2). Although the incidence rate has decreased overall, the incidence of advanced stage PCa increased from 4% to 6% from 2014 to 2018. Furthermore, the incidence of PCa and The overall decrease in mortality was due to extensive scrutiny of patient management (3). Biomarker testing can better characterize tumor phenotypes. Prostate-specific antigen (PSA) is the most clinically accepted serum biomarker used for PCa, but men with benign prostatic hyperplasia or prostatitis tend to have high levels of PSA. The specificity is limited by the presence of two expected screens in the United States and Europe. Screening trials have shown consistent benefit in overall patient survival from PSA screening. failed to demonstrate a concordant benefit (4, 5). The combined application of imaging and biomarkers found in serum, urine, and tissues has become increasingly mature in emerging clinical diagnostics (6). Therefore, the identification of significant and reliable biomarkers related to the detection and monitoring disease progression of PCa will be important in guiding clinical decision-making.

[0176] An ideal biomarker for clinical use should have three main characteristics: 1) a safe and easy means of measurement, preferably non-invasive; 2) high sensitivity, specificity, and positive and negative predictive value for its intended outcome; and 3) clinical utility. The aim of the present invention is to improve decision-making capabilities in relation to physiological parameters. Urine, as a non-invasive and easily accessible biological fluid, is emerging as an essential source of biomarkers, especially in the early diagnosis and post-treatment monitoring of tumors (7). The researchers used genomic DNA sequences obtained from urine samples of PCa patients and healthy controls that displayed abnormal gene signatures. Integrated analysis of RNAs and metabolites in PCa can help to differentiate between PCa and normal individuals (8). Recently, several studies have revolved around urinary biomarker tests that help provide even more specificity, including SelectMDx (DLX1, HOXC6) (9), ExoDx Prostate IntelScore (EPI) (SPDEF, ERG, PCA3) (10), and Michigan Prostate Score (PCA3, PSA, TMPSS2:ERG) (11). These tests are supported by the National Although it is endorsed by the National Comprehensive Cancer Network Guidelines, results have been mixed ( 12 ).

[0177] Thus, this example evaluates the diagnostic accuracy of urinary multivariate biomarkers for PCa as part of a National Cancer Institute's Early Detection Research Network (EDRN)-defined Phase II biomarker study. (13) The assay is unique in that, in some embodiments, it does not require a prior prostate exam and urine can be easily collected as part of the basic clinical workflow. Urine dipsticks are widely used as screening and diagnostic tools for disease as a simple, inexpensive and rapid test.

[0178] material and method

[0179] Study population .the Johns Hopkins University School of Medicine The Human Research Ethics Committee and IRB protocol of the University of California, San Luis, School of Medicine approved the research for this study (reference number 237998). PCa patients were enrolled at the AdventHealth Global Robotics Institute between September 2021 and July 2022. Global Robotics Institute, Celebration, FL Patients were recruited through the University of Florida, Gainesville, FL, USA. Initial diagnosis of PCa was based on transrectal ultrasound-guided prostate biopsy and confirmed by histological and immunohistochemical examination of resected tissue. Urine was collected before and after resection by robotic-assisted radical prostatectomy. Patients with BPH were recruited through the University of Florida, Gainesville, FL, USA. Normal controls were recruited through the Johns Hopkins University School of Medicine. The normal controls were volunteers recruited from the University of Tokyo. They had no personal or family history of prostate cancer and did not have any significant lower urinary tract symptoms. Clinicopathological data were collected from the histopathology reports of prostate biopsies. Parameters collected included preoperative PSA, Patient demographics included the number of cores detected, the number of cores positive for cancer, and the overall ISUP Grade Group. Written informed consent was obtained in all cases. Patient demographics are displayed in Table 2.

[0180] Urine sample collectionFirst and mid-morning urine was collected after waking up. Urine samples were immediately processed by adding urine storage solution (Norgen Bioteck) and kept at room temperature until centrifugation to separate exfoliated cells in the urine samples. For urine dipstick assay, three drops of fresh urine were used before further processing of the urine samples. Exfoliated cells from urine samples were used for total RNA purification using miRNeasy mini kit (Qiagen). Total RNA was subjected to quantitative real-time PCR to identify gene expression. Cell-free The urine was applied to the ELISA assay.

[0181] ELISA Soluble EpCAM levels were measured in urine samples using the Human EpCAM DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions. Urine samples were vortexed at room temperature and centrifuged at 1000 g for 10 min. To each well of the assay, 100 μl of urine supernatant was added, as well as two-fold dilutions of the 1 ng / ml standard. A seven-point calibration curve was constructed using an EnVision 2105 Microscope. A plate reader (PerkinElmer) was used to measure the wavelength at 450 nm (570 nm). EpCAM concentrations (pg / ml) were determined using a two-parameter linear curve. The standard deviations were fitted and multiplied by the dilution factor. All measurements were performed in duplicate.

[0182] RNA extraction of exfoliated cells, reverse transcription, preliminary amplification and quantitative real-time PCRTotal RNA from exfoliated cells from urine samples was extracted using QIAzol lysis reagent and the miRNeasy mini kit (Qiagen). Samples were treated with DNase I (Qiagen) and RNA concentrations were measured using a NanoDrop 8000 (Thermo Scientific). cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. The total volume of preamplification was 50 μl for each sample. Reactions contained 25 μl preamplification mastermix, 24 μl cDNA, 1 μl pooled primers, with a final concentration of 10 nM for each primer. Then, 14 cycles of cDNA preamplification were performed according to the following schedule: 95 °C for 10 min, 95 °C for 15 s, and 58 °C for 4 min. Gene expression was quantified using SYBR Green Master Mix (Applied Biosystems). β-actin was detected by PCR using a SYBR Green Master Mix (Applied Biosystems). Quantitative real-time PCR was performed using QuantStudio 5 (Applied Biosystems). Relative changes in gene expression were calculated using the 2 -ΔCT law The analysis was carried out using

[0183] cell culture PCa cell lines PC3 and LNCaP were cultured at the American Type Culture Collection (American Mycoplasma-associated proteins were obtained from the American Type Culture Collection (ATCC) and cells were cultured in Ham's F-12K and RPMI 1640 medium (Gibco) with 10% FBS and 1% penicillin / streptomycin, respectively. Cell lines were authenticated by STR profiling and routinely tested for mycoplasma contamination throughout the study.

[0184] Cell transfection Cells were transiently transfected with silencer-selected siRNAs for EpCAM (s8370, s8371, s8372) or TTC3 (s14475, s14476, s14477) or silencer-selected negative control #1 siRNA (Invitrogen) at 20 nM. Infections were performed using Lipofectamine RNAiMAX Reagent (Invitrogen). Assays were performed 48 h (for qPCR) or 72 h (for Western blot) after transfection unless stated.

[0185] Cell proliferation assay Cells were seeded in 96-well plates at 3000 cells per well. EpCAM and TTC3 siRNAs were added the following day. After 24, 48, and 72 h of incubation, cell proliferation was measured by cellTiter 96 AQueous non-radioactive cell proliferation assay (Promega). Absorbance values ​​were measured at 490 nm using an EnVision 2105 microplate reader (PerkinElmer).

[0186] This study examined the excretion rate of 100% in pre- and post-prostatectomy men with PCa. Urine (50 ml) from exfoliated cells and debris shed in urine was used as a control. RNA from exfoliated cells and debris shed in urine was isolated and RNA sequencing was performed using the Illumina Next-seq 550 platform. We employed advanced computational and machine learning approaches to identify candidate biomarkers for men with PCa. The TCGA database was interrogated to validate the PCa-specific expression of the identified RNAs in tumor tissues. Two RNA markers were further tested by qPCR, and one was Soluble protein markers in urine were measured by immunoassay.

[0187] The study included 106 men with PCa and 88 control men. The presence of ≥1 RNA markers (TTC3, H4C5) and protein marker (EpCAM) were identified and validated in urine as potential candidate biomarkers for PCa detection. These markers were tested and Using qPCR for TTC3, H4C5, and ELISA assays for EpCAM with even higher specificity and sensitivity were developed (Table 1). Results outperformed known urinary tract markers, PCA3 and SPDEF (Figure 31). TTC3, H4C5, and EpCAM markers were reduced to low or undetectable levels in post-prostatectomy compared to pre-prostatectomy men with PCa. shRNA knockdown of TTC3 and EpCAM in androgen-sensitive and -insensitive cells induces biological changes, suggesting their relevance to prostate cancer.

[0188] Thus, in a particular embodiment, the present invention relates to EpCAM (protein), as well as TTC3 and Highly accurate identification of three urine-based biomarkers for PCa detection, including H4C5(RNA) and H4C5(RNA) In certain embodiments, the urine liquid biopsy biomarker assay comprises a highly accurate panel of three urine-based biomarker assays that distinguish between PCa and healthy men. The assay further provides a panel of three urine-based biomarker assays that further differentiate between PCa and healthy men with elevated PSA levels. It has been associated with improved identification of patients with high-grade prostate cancer and could reduce the overall number of unnecessary biopsies.

[0189] References 1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer statistics, 2022. CA Cancer J Clin. Shands) 2022 Jan;72(l):7-33. 2. Litwin MS, Tan HJ. The Diagnosis and Treatment of Prostate Cancer: A Review. JAMA. 2017 Jun 27;317(24):2532-2542. 3. Gulati R, Tsodikov A, Etzioni R, Hunter-Merrill RA, Gore JL, Mariotto AB, Cooperaberg MR. Expected population impacts of discontinued prostate-specific antigen screening. Hibiki).Cancer.2014 Nov 15;120(22):3519-26. 4. Andriole GL, Crawford ED, Grubb RL 3rd, Buys SS, Chia D, Church TR, Fouad MN, Gelmann EP, Kvale PA, Reding DJ, Weissfeld JL, Yokochi LA, O'Brien B, Clapp JD, Rathmel JM, Riley TL, Hayes RB, Kramer BS, Izmirlian G, Miller AB, Pinsky PF, Prorok PC, Gohagan JK, Berg CD; PLCO Project Team; Project Team).Mortality results from a randomized prostate-cancer screening trial.N Engl J Med.2009 Mar 26;360(l 3):1310-9. 5. Schroder FH, Hugosson J, Roobol MJ, Tammela TL, Ciatto S, Nelen V, Kwiatkowski M, Lujan M, LiljaH, Zappa M, Denis LJ, Recker F, Berenguer A , Maattanen L, Bangma CH, Aus G, Villers A, Rebillard X, van der Kwast T, Blijenberg BG, Moss SM, de Koning HJ, Auvinen a;ERSPC Investigators Screening and prostate-cancer mortality in a randomized European Screening and prostate cancer mortality in a randomized European study. N Engl J Med. 2009 Mar 26;360(13):1320-8. 6. Cucchiara V, Cooperberg MR, Dall'Era M, Lin DW, Montorsi F, Schalken JA, Evans CP, Genomic Markers in Prostate Cancer Decision Making. Genomic markers in cancer decision making. Eur Urol. 2018 Apr;73(4):572-582. 7. Bax C, Lotesoriere BJ, Sironi S, Capelli L.Review and Comparison of Cancer Biomarker Trends in Urine as a Basis for New Diagnostic Pathways. Cancers(Basel). 2019 Aug 25;11(9):1244. 8. Lee B, Mahmud I, Marchica J, Derezinski P, Qi F, Wang F, Joshi P, Valerio F, Rivera I, Patel V, Pavlovich CP, Garrett TJ, Schroth GP, Sun Y, Perera RJ.Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery. Sci Rep. Reports) 2020 Feb 28;10(1):3716. 9. Van Neste L, Hendriks RJ, Dijkstra S, Trooskens G, Cornel EB, Jannink SA, de Jong H, Hessels D, Smit FP, Melchers WJ, Leyten GH, de Reijke TM, Vergunst H, Kil P, Knaischeer BC, Hulsbergen-van de Kaa CA, Mulders PF, van Oort IM, Van Criekinge W, Schalken JA.Detection of High-grade Prostate Cancer Using a Urinary Molecular Biomarker-Based Risk Score. Eur Urol. 2016 Nov;70(5):740-748. 10. McKiernan J, Donovan MJ, O'Neill V, Bentink S, Noerholm M, Belzer S, Skog J, Kattan MW, Partin A, Andriole G, Brown G, Wei JT, Thompson IM Jr, Carroll PA Novel Urine Exosome Gene Expression Assay to Predict High-grade Prostate Cancer at Initial Biopsy. JAMA Oncol. 2016 Jul 1;2(7):882-9. 11. Sanda MG, Feng Z, Howard DH, Tomlins SA, Sokoll LJ, Chan DW, Regan MM, Groskopf J, Chipman J, Patil DH, Salami SS, Scherr DS, Kagan J, Srivastava. S, Thompson IM Jr, Siddiqui J, Fan J, Joon AY, Bantis LE, Rubin MA, Chinnayian AM, Wei JT; and the EDRN-PCA3 Study Group, Bidair M, Kibel A, Lin DW, Lotan Y, Partin A, Taneja S. Association Between Combined TMPRSS2:ERG and PCAJ RNA Urinary Testing and Detection of Aggressive Prostate Cancer. JAMA Oncol.2017 Aug 1;3(8):1085-1093. 12. Carroll PH, Mohler JL. NCCN Guidelines Updates: Prostate Cancer NCCN Guidelines Update: Prostate Cancer and Prostate Cancer Early Detection. J Natl Compr Cane Netw. 2018 May 16(5S):620-623. 13. Pepe MS, Etzioni R, Feng Z, Potter JD, Thompson ML, Thornquist M ,Winget M,Yasui Y.Phases of biomarker development for early detection of cancer.J Natl Cancer Inst.2001 Jul 18;93(14) :1054-61.

[0190] [Table 1]

[0191] [Table 2]

[0192] [Table 3]

[0193] [Table 4]

[0194] [Table 5-1] [Table 5-2]

[0195] Example 2 : Application of a group of urinary enriched RNAs (coding and noncoding) as PCa biomarkers. PCa is the leading cause of cancer death among men in the United States, with over 3.6 million men alive with prostate cancer. However, many new Diagnosed prostate cancer is indolent and clinically insignificant with low metastatic potential. Therefore, the development of non-invasive and accurate markers for early detection to distinguish indolent from aggressive cancers is timely. 1,2,4 and preliminary results Based on this, a panel of urinary enriched RNAs (mRNAs, IncRNAs, and circRNAs) may be useful for PCa detection. PCa-upregulated RNA panel (mRNA, Circular and long non-coding RNAs and eccDNA will be measured by qPCR and digital PCR in samples from a statistically significant number of patients, given the power requirements of the patient cohort: (a) urine samples from high- and low-grade PCa patients; (b) urine samples from non-cancerous but high PSA individuals (i.e., benign prostatic hyperplasia, prostatitis, etc.) and (c) urine samples from control healthy individuals. The panel of RNA signatures is useful for establishing novel PCa non-invasive tests. To develop an ELISA-based method to test these markers in the clinic, did.

[0196] Example 3 : Multivariant biomarker testing by integrating PCa-specific RNA signatures We developed a multivariate logistic regression model to identify PCa-specific RNAs in urine compared to normal healthy individuals. The RNA data was combined with comprehensive gene expression analysis to investigate complex gene networks for better PCa diagnosis. Multivariate logistic regression models have been used to predict PCa. It has been developed as a novel and powerful single molecule test for PCa detection in men. Multianalyte markers (mRNAs, circRNAs, IncRNAs and eccDNAs) will be applied in patient samples.

[0197] The main impact and innovative aspect of the present invention is based on the non-invasive nature of a diverse panel of RNAs to detect aggressive PCa, which cannot be done with current single biomarker tests (PCA3 or PSA). The present invention will be complemented by additional candidates as they become available to further improve the performance. Combinatorial "multi-RNA" based molecular markers - Develop a panel.

[0198] All proposed manufacturers are required to test with free-flow urine. Prostate massage is possible and not necessary (e.g. PCA3 test).

[0199] References 1. Lee B, Mazar J, Aftab MN, Qi F, Shelley J, Li JL, Govindarajan S, Valerio F, Rivera I, Thurn T, Tran TA, Kameh D, Patel V, and Perera RJ. Long noncoding RNAs as putative biomarkers for prostate cancer detection. Long non-coding RNAs as putative biomarkers. J. Mol Diagn. (Journal of Molecular Diagnostics 2014;16:615-26. 2. Mouraviev V, Lee B, Patel V, Albala D, Johansen TE, Partin A, Ross A and Perera RJ. Clinical prospects of long noncoding RNAs as novel biomarkers and therapeutic targets in prostate cancer. Clinical testing of long non-coding RNAs as cancer markers and therapeutic targets). Prostate Cancer Prostatic Dis. 2016;19:14-20. 3. Lee B, Li JL, Marchica J, Mercola M, Patel V and Perera RJ. Mapping genetic variability in mature miRNAs and miRNA binding sites in prostate cancer. Mapping genetic variability at mature miRNAs and miRNA binding sites in adenocarcinoma. J Hum Genet. 2021. 4. Lee B, Mahmud I, Marchica J, Derezinski P, Qi F, Wang F, Joshi P, Valerio F, Rivera I, Patel V, Pavlovich CP, Garrett TJ, Schroth GP, Sun Y and Perera RJ. .Integrated RNA and metabolite profiling of urine liquid biopsies for prostate cancer biomarker discovery.Sci Rep. 2020;10:3716.

Claims

1. A method for identifying a patient as having invasive prostate cancer, comprising detecting overexpression of epithelial cell adhesion molecule (EpCAM), H4 clustered histone 5 (H4C5), and tetratricopeptide repeat domain 3 (TTC3) in a sample obtained from the patient compared to a control.

2. The method of claim 1, wherein the detecting step comprises detecting EpCAM protein levels in a urine sample.

3. The method of claim 1, wherein the detecting step comprises detecting ribonucleic acid (RNA) levels of H4C5 and TTC3.

4. 10. The method of claim 1, wherein the method distinguishes between aggressive prostate cancer, indolent prostate cancer, benign prostatic hyperplasia, and prostatitis.

5. 10. The method of claim 1, further comprising detecting overexpression of one or more messenger ribonucleic acid (mRNA), circular RNA (circRNA), extracellular DNA (eccDNA), and long non-coding RNA (lncRNA) compared to a control.

6. The method of claim 5, wherein the mRNA comprises one or more of RIDA, H1-4, H4C2 and H4C3.

7. 10. The method of claim 1, further comprising detecting one or more metabolites selected from the group consisting of asparagine, aspartate, glycerate, citrate, isocitrate, glutamate, itaconate, malate, meglutol, cisaconitate, isoleucine, leucine, pantothenate, glutamine, nicotinate, threonine, ketoglutarate, alpha-ketoisovaleric acid (KIVA), cysteine, 3P-glycerate, xanthine, and hypoxanthine.

8. A method for identifying a patient as having invasive prostate cancer, comprising detecting overexpression of one or more proteins, mRNAs, circRNAs, eccDNAs, and lncRNAs in a sample obtained from the patient relative to a control.

9. The method of claim 8, wherein the protein comprises EPCAM.

10. The method of claim 8, wherein the mRNA comprises one or more of H4C5, TTC3, RIDA, H1-4, H4C2 and H4C3.

11. 9. The method of claim 5 or 8, wherein the circRNAs include one or more of circ842, circ3266, circ1809, circ1979, circ645, and circ1607.

12. eccDNA contains one or more of the following: chr22: 50276214-50276428; chr20: 2236337-2236458; chr6: 54059859-54063911; chr16: 85975027-85975617; chr3: 5565190-5565271; chr10: 130300872- 130301712; chr11: 58900903-59058535; chr22: 44599233-49967822; chr17: 69961543-69 961943;chr18:9809075-9809266;chr17:80024303-80024653;chrY:10945178-11295108; chr7:65038315-65873352;chr1:21669328-93846973;chr6:168914322-168914396;chr6 :35786783-35799011;chr6:26305559-28597426;chr9:34681483-34681981;chr1:20769 8916-207868701; chr8: 57203766-57210492; chr16: 20504588-20504731; chr3: 67362279 -136250669;chr1:248404181-248404245;chr12:1574344-1574491;chr7:66728585-7396 7789; chr1:55002895-55003057; and chr16:89907819-89908811.

13. 9. The method of claim 5 or 8, wherein the lncRNA comprises lnc-CCDC125-13 and / or ZNF667-AS1.

14. 10. The method of claim 2 or 8, wherein the detecting step comprises a lateral flow assay.

15. 11. The method of any of claims 1-10, wherein the sample comprises free flow urine and / or prostate massage urine.

16. 11. The method of claim 3 or 10, wherein the sample comprises blood or serum.

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