CELL CARCINOMA

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/371,572 filed 05-

AUG-2016. The entirety of this application is hereby incorporated by reference for all purposes.

BACKGROUND

Renal cell carcinoma (RCC) accounts for the vast majority of neoplasms of the kidney. See Chow et al., Nat. Rev. Urol. 2010, 7(5): 245 - 257. Diagnosing renal cancer is hindered due to lack of easily observable tumors. Blood in urine is a sign of early kidney cancer. However, kidney stones and urinary tract infections also cause blood to appear in the urine. In addition, some people do not see blood in their urine until the tumors are large or have spread to other parts of the body. Substantial existing intratumoral heterogeneity has profound implications for directed therapeutics and how patients effectively respond to therapy. See Ricketts et al, Nat. Genet. 2014; 46(3): 214 - 215. Therefore, there is a need for reliable methods to detect early stage tumors and the effectiveness of chemotherapy treatments.

The Cancer Genome Atlas (TCGA) Research Network report molecular characterizations of clear cell renal cell carcinoma. Nature 499, 43-49 (2013). Martino et al. report cell-free DNA represents a serum-based diagnostic and prognostic biomarker for RCC. Cancer, 2012, 118:82-90. See also Di Meo et al. Mol Cancer, 2017, 16: 80 and Zill et al. Journal of Clinical Oncology 34(18_suppl):LBA11501-LBA11501 (2016). Nel et al. PLoS One. 2016, l l(4):e0153018.

Several patent applications report biomarkers to assess the severity of cancer. See e.g., US20100222230, US20120251451, WO2012170711, WO2011127219, WO2012174282, WO2012115885, US20070254295, WO2016061064, and WO2012166899.

References herein are not an admission of prior art.

SUMMARY

The present disclosure relates to methods of detecting renal cell carcinoma-associated DNA from a liquid sample of a subject. Tumor-associated DNA carries specific mutations that may be detected and compared to a cancer genome database to assist in diagnosing renal cell carcinoma and treatment planning.

In certain embodiments, this disclosure relates to methods of detecting DNA mutations, said method comprising obtaining sample from blood or urine of a subject; and detecting whether one or more DNA mutations are present in DNA of the sample, wherein detecting whether DNA mutations are present comprises sequencing DNA encoding one or more of the proteins selected from genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL providing DNA sequences. In certain embodiments, the disclosure contemplates detecting one or more splice variants in the DNA sequences.

In certain embodiments, detecting whether DNA mutations are present further comprises comparing the DNA sequences to reference sequences. In certain embodiments, the reference sequences are wild-type sequences, consensus sequences for a nucleic acid encoding the protein, a sequence obtained from the subject determined by sequencing DNA of a non-cancerous cell of the subject or a sequence containing a known recurring DNA mutation.

In certain embodiments, detecting whether DNA mutations are present comprises sequencing DNA encoding proteins selected from genes VHL, PBRMl, SETD2, BAP1 and two DNA mutations are in at least one of the DNA sequences.

In certain embodiments, detecting whether DNA mutations are present consists of sequencing DNA associated with the genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL and two DNA mutations are in at least one of the DNA sequences encoding a single protein.

In certain embodiments, detecting whether DNA mutations are present consists of sequencing DNA associated with all or the genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl , PIK3CA, SETD2, TP53, and VHL and two DNA mutations are in at least two of the DNA sequences encoding a single protein.

In certain embodiments, the DNA mutation in the DNA sequence encoding a protein results in a premature stop codon, a nonsynonymous amino acid change, inframe deletion, fameshift deletion, or splice variant. In certain embodiments, the DNA mutation is selected from those provided for in the tables of Figure 1A, IB, or 1C.

In certain embodiments, this disclosure relates to methods of diagnosing renal cancer, said method comprising: obtaining a urine or blood based sample from a subject and detecting multiple DNA mutations in the subject, said method comprising obtaining sample from the subject; and detecting whether DNA mutations are present in DNA of the sample, wherein detecting whether DNA mutations are present comprises sequencing DNA associated with one or more of the genes selected from BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL; and diagnosing the patient with, at risk of, or at risk of recurring renal cancer when the presence multiple DNA mutation in the sample is detected. In certain embodiments, this disclosure relates to methods for screening DNA for an alteration of a gene selected from BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL which comprises comparing DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL, obtained from a urine or blood based sample of a subject, with wild-type sequences or a sequence with a known DNA mutation of BAP 1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL, wherein a difference in the sequence of the gene of the subject from wild-type indicates an alteration in the DNA of the blood in said subject, and wherein a similarity in the sequence of the gene of the subject to a known DNA mutation indicates an alteration in the DNA of the blood in said subject.

In certain embodiments, said comparing DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL further comprises: hybridizing a wild-type probe or a probe with a sequence that targets a known DNA mutation to DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL isolated from said sample; and detecting the presence of a hybridization product by measuring a label connected to the probe or conformational changes in the probe that are indicative of hybridization to the BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL gene.

In certain embodiments, said comparing DNA sequences of genes BAP1, BRAF, CDKN2A,

FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL further comprises: amplifying by PCR all or part of a DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL from said sample using a set of primers to produce amplified nucleic acids; and sequencing the amplified nucleic acids.

In one embodiment, this disclosure relates to a method of detecting tumor-associated mutations in DNA isolated from a biological sample of a patient by a microfluidic amplification strategy followed by gene sequencing and comparison to a cancer genome database. In specific embodiments, the tumor-associated mutations may be localized to a gene group comprising at least three genes selected from a group of genes encoding: BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL. The biological sample may be selected from the group comprising serum, blood, plasma, or urine.

In another embodiment, this disclosure relates to a method of determining the presence or severity of renal cell carcinoma in a subject comprising detecting circulating tumor DNA in a biological sample by identifying mutations in a tumor-associated gene group. In specific embodiments, the tumor-associated mutations may be localized to a gene group comprising at least three genes selected from a group of genes encoding: BAP1 , BRAF, CDK 2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1 , PIK3CA, SETD2, TP53, or VHL. In certain embodiments, the mutations detected may comprise inframe deletions, frameshift deletions, frameshift insertions, nonsynonymous substitutions, splice variants, or stopgain substitutions in any of these genes. In another specific embodiment, the mutations are detected through a gene microarray that hybridizes with tumor-associated DNA from these genes.

In yet another embodiment, this disclosure relates to a method of predicting the responsiveness of a renal cell carcinoma to a drug based on sequencing circulating tumor DNA in a biological sample to identify mutations in a tumor-associated gene group. In specific embodiments, the drug is a tyrosine kinase inhibitor including, but not limited to, sunitinib and pazopanib, or targeting the Von Hippel-Lindau/Hypoxia-inducible factor alpha (VHL-HIFla) angiogenesis pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows a table of data indicating FFPE and serum samples have mutations in common.

Figure IB shows a table of data indicating FFPE and frozen tumor samples share mutations. Figure 1C shows a table of somatic mutations detected in tumor samples of the 22 patients with ccRCC. Patients are sorted by stage. Mutation rate expressed as percent of mutations relative to whole blood. *Patients with metastatic disease and/or death in follow-up period. - = no mutation present. SNV = single nucleotide variant. AA = amino acid.

Figure 2A shows recurrence-free survival curves of TCGA ccRCC data for patients with BAP1 mutations with (n=28) or without (n=14) concurrent mutations in VHL, BAP1 and/or SETD2. The difference is not significant for recurrence-free survival.

Figure 2B shows overall survival curves of TCGA ccRCC data for patients with BAP1 mutations with (n=28) or without (n=14) concurrent mutations in VHL, BAP1 and/or SETD2. The difference is significant in overall (p=0.045) but not recurrence-free survival (p=0.15).

Figure 3 A shows a table of data on somatic tumor-derived mutations detected in pre- nephrectomy liquid biopsy in patients with ccRCC (with or without other histologic subtypes). Mutations were identified based on the criteria that the mutation was not present in the buffy coat DNA, the mutation was present in at least 10% of the sequence, and if overlapping sequence was available, the mutation was present in the overlapping sequences.

Figure 3B shows a table of data on somatic tumor-derived mutations detected in pre- nephrectomy liquid biopsy in patients with ccRCC (with or without other histologic subtypes). Mutations were identified based on the criteria that the mutation was not present in the buffy coat DNA, the mutation was present in at least 10% of the sequence, and if overlapping sequence was available, the mutation was present in the overlapping sequences.

Figure 4A show data generated from prototype genomic liquid biopsy analysis pipeline.

Histogram showing range of gene mutational frequencies in a cohort vs. TCGA RCC cohort mutational frequencies. Mutational frequencies range is created using different thresholds for mutation detection for allele frequency (5 or 10% of reads show a variant) and read depth (20 or 50 reads covering a mutated base).

Figure 4B show a heatmap showing how a liquid biopsy can be used to characterize tumor heterogeneity. The heatmap shows allele frequencies (darker = higher allele frequencies) of 9 somatic variants (X axis) across 14 sequencing datasets for a single patient, including buffy coat/normal (N), serum (S), fresh frozen tumor (T), and ten distinct samples from across FFPE block of tumor (F). Arrows highlight where variants found in serum are unique to a particular FFPE sample, showing that serum can capture mutations across a tumor that fresh frozen tumor cannot.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Unless defined otherwise, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The transitional term "comprising", which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or steps, e.g., does not exclude the presence of terminal nucleotides. The transitional phrase "consisting of excludes any additional nucleotides, elements, steps, or ingredients not specified in the claim.

The term "renal cell carcinoma", as used herein, refers to a kidney cancer that originates in the lining of the proximal convoluted tubule of the kidney. The term encompass both clear cell renal carcinoma and papillary renal carcinoma.

The term "gene microarray", as used herein, refers to a collection of nucleotide sequences attached to a solid surface, used to measure the expression levels of large numbers of genes or to genotype multiple regions of a genome.

The term "hybridizing", as used herein, refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed "substantially complementary"). In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence. Appropriate conditions enabling specific hybridization of single stranded nucleic acid molecules of varying complementarity are well known in the art.

"Amplification" is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo- ) specificity. Template specificity is frequently described in terms of "target" specificity. Target sequences are "targets" in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by the choice of enzyme.

Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Q_replicase, MDV-1 RNA is the specific template for the replicase (Kacian et al, Proc. Natl. Acad. Sci. USA, 69:3038 (1972)). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al, Nature, 228:227 (1970)). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (Wu and Wallace, Genomics, 4:560 (1989)). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press (1989)).

The term "amplifiable nucleic acid" refers to nucleic acids that may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template. "

The term "sample template" refers to nucleic acid originating from a sample that is analyzed for the presence of "target" (defined below). In contrast, "background template" is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

The term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

The term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, that is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "label" or "reporter molecule," so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems.

In certain embodiments, it is not intended that the present disclosure be limited to any particular detection system or label. In certain embodiments, the disclosure contemplates the use of nucleotide probes to detect the presence of a particular DNA sequence or mutation in a sample or to identify an otherwise unknown DNA sequence. Hybridization can be used to detect the presence of a nucleic acid. Because hybridization occurs in a predictable manner between complementary strands, it is possible to detect the presence of a nucleic acid of interest in a sample. A label can be attached to or incorporated into a nucleic acid strand of a known sequence, i.e., probe, which will fully or partially hybridize with a complementary sequence of interest, i.e., target. Once the probe is hybridized with the target, a detectable signal is generated either from the label itself (referred to as "direct detection") or from a secondary chemical agent that is bound to the label (referred to as "indirect detection"). If a signal is detected from the sample after all unhybridized probes have been removed, detection of the signal implies the presence of a target in that sample. Contemplated labels include, but are not limited to, fluorescent labels, electron-dense reagents, nucleotide derivatives that contain biotin, iminobiotin, lipoic acid or radioactive labels, isotopes of hydrogen, phosphorus, carbon, fluorine or iodine. Labels are typically attached covalently to probe, e.g., through a pyrimidine or purine ring. However, there may also be a secondary probe that binds the detection probe. Labels include molecules that specifically interact uniquely with proteins such as avidin, antibodies, or enzymes capable of depositing reaction products which are capable of indicating the presence, location, or quantity of a probe.

The term "target," when used in reference to the polymerase chain reaction, refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the "target" is sought to be sorted out from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within the target sequence.

The term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683, 195, 4,683,202, and 4,965,188, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ² P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

The terms "PCR product," "PCR fragment," and "amplification product" refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

The term "amplification reagents" refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification except for primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

The term "reverse-transcriptase" or "RT-PCR" refers to a type of PCR where the starting material is mRNA. The starting mRNA is enzymatically converted to complementary DNA or

"cDNA" using a reverse transcriptase enzyme. The cDNA is then used as a "template" for a "PCR" reaction The term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein, through "translation" of mRNA.

Gene expression can be regulated at many stages in the process. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called

"activators" and "repressors," respectively.

The term "anti-cancer agent" or "anti-cancer drug" is any agent, compound or entity that would be capably of negatively affecting the cancer in the subject, for example killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the number of metastatic cells, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of the subject with cancer. Anti-cancer therapy includes biological agents (biotherapy), chemotherapy agents, and radiotherapy agents.

Methods of Detection

The present disclosure relates to methods of detecting renal cell carcinoma-associated DNA from a urine or blood based sample of a subject. Tumor-associated DNA commonly carries specific mutations that can be detected and compared to a cancer genome database to assist in diagnosing renal cell carcinoma and treatment planning.

In certain embodiments, the present disclosure relates to the detection of tumor DNA present in a sample, e.g. plasma, by identifying somatic tumor mutations by sequencing ("liquid biopsy") DNA in the sample. This includes a microfluidic amplification strategy (Fluidigm®) followed by Illumina deep sequencing wherein -50 million bases are sequenced per sample with an average depth of coverage of 600x-2000x designed to interrogate the Cancer Genome Atlas (TCGA) identified genes mutated in RCC.

The homo sapiens BRCA1 associated protein 1 (BAP1), RefSeqGene (LRG 529) on chromosome 3 is reported as NCBI Reference Sequence: NG_031859.1. The ubiquitin carboxyl- terminal hydrolase BAP1 [Homo sapiens] is reported as NCBI Reference Sequence: NP_004647.1.

The homo sapiens B-Raf proto-oncogene, serine/threonine kinase (BRAF), RefSeqGene (LRG_299) on chromosome 7 is reported as NCBI Reference Sequence: NG_007873.3. The serine/threonine-protein kinase B-raf [Homo sapiens] is reported as NCBI Reference Sequence: NP_004324.2.

The homo sapiens cyclin dependent kinase inhibitor 2A (CDKN2A), RefSeqGene (LRG l 1) on chromosome 9 is reported as NCBI Reference Sequence: NG_007485.1. Cyclin-dependent kinase inhibitor 2A isoform pl4ARF [Homo sapiens] is reported as NCBI Reference Sequence: NP_478102.2.

The homo sapiens fibroblast growth factor receptor 3 (FGFR3), RefSeqGene (LRG 1021) on chromosome 4 is reported as NCBI Reference Sequence: NG_012632.1. The fibroblast growth factor receptor 3 isoform 3 precursor [Homo sapiens] is reported as NCBI Reference Sequence: NP 001156685.1. The homo sapiens lysine demethylase 5C (KDM5C), RefSeqGene on chromosome X is reported as NCBI Reference Sequence: NG_008085.1. The lysine-specific demethylase 5C isoform 1 [Homo sapiens] is reported as NCBI Reference Sequence: NP_004178.2.

The homo sapiens KIT proto-oncogene receptor tyrosine kinase (KIT), RefSeqGene (LRG_307) on chromosome 4 is reported as NCBI Reference Sequence: NG_007456.1. The mast stem cell growth factor receptor Kit isoform 1 precursor [Homo sapiens] is reported as NCBI Reference Sequence: NP_000213.1

The homo sapiens MET proto-oncogene, receptor tyrosine kinase (MET), RefSeqGene (LRG_662) on chromosome 7 is reported as NCBI Reference Sequence: NG_008996.1. The hepatocyte growth factor receptor isoform a preproprotein [Homo sapiens] is reported as NCBI Reference Sequence: NP_001120972.1.

The homo sapiens mucin 4, cell surface associated (MUC4), RefSeqGene on chromosome 3 is reported as NCBI Reference Sequence: NG_053117.1. The mucin-4 isoform a precursor [Homo sapiens] is reported as NCBI Reference Sequence: NP_060876.5.

Homo sapiens nuclear factor, erythroid 2 like 2 (NFE2L2), transcript variant 2, mRNA is reported as NCBI Reference Sequence: NM_001145412.3. The nuclear factor erythroid 2-related factor 2 isoform 2 [Homo sapiens] is reported as NCBI Reference Sequence: NP_001138884.1.

The homo sapiens polybromo 1 (PBRM1), RefSeqGene on chromosome 3 is reported as NCBI Reference Sequence: NG_032108.1. The protein polybromo-1 isoform 2 [Homo sapiens] is reported as NCBI Reference Sequence: NP_060783.3.

The homo sapiens phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), RefSeqGene (LRG 310) on chromosome 3 is reported as NCBI Reference Sequence: NG_012113.2. The phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform [Homo sapiens] is reported as NCBI Reference Sequence: NP_006209.2.

The homo sapiens SET domain containing 2 (SETD2), RefSeqGene (LRG 775) on chromosome 3 is reported as NCBI Reference Sequence: NG_032091.1. The histone-lysine N- methyltransferase SETD2 isoform 1 [Homo sapiens] is reported as NCBI Reference Sequence: NP_054878.5.

The homo sapiens tumor protein p53 (TP53), RefSeqGene (LRG 321) on chromosome 17 is reported as NCBI Reference Sequence: NG_017013.2. The cellular tumor antigen p53 isoform a [Homo sapiens] is reported as NCBI Reference Sequence: NP_000537.3.

The homo sapiens von Hippel-Lindau tumor suppressor (VHL), RefSeqGene (LRG 322) on chromosome 3 is reported as NCBI Reference Sequence: NG_008212.3. The von Hippel-Lindau disease tumor suppressor isoform 1 [Homo sapiens] is reported as NCBI Reference Sequence: NP_000542.1.

In one embodiment, this disclosure relates to detecting mutations in DNA encoding proteins associated with genes selected from the group comprising or consisting of BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL from a urine or blood base sample.

In one embodiment, this disclosure relates to detecting at least one mutation in at least one gene selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting at least one mutation in at least two genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least three genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least four genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least five genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least six genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least seven genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least eight genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least nine genes selected from the group comprising BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL. In another embodiment, this disclosure relates to detecting mutations in at least ten genes selected from the group comprising BAP1, BRAF, CDK 2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least eleven genes selected from the group comprising BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least twelve genes selected from the group comprising BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, or VHL.

In another embodiment, this disclosure relates to detecting mutations in at least thirteen genes selected from the group comprising BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, or VHL.

In certain embodiments, this disclosure relates to methods for identifying a subject having increased likelihood of developing or having renal cell carcinoma (RCC) detecting DNA mutations in a urine or blood based sample of a subj ect using methods disclosed herein. In certain embodiments, this disclosure relates to methods for monitoring the progression of renal cell carcinoma (RCC) in a subject by detecting DNA mutations in a urine or blood based sample of a subject using methods disclosed herein.

In certain embodiments, a sample obtained from a subject at a second time point; and comparing the presence of DNA mutation in the same genes as measured in the sample from the first time point with the number of DNA mutations in the biological sample from the second time point; wherein a change in the number of DNA mutations of at least three genes in the selected gene group in the biological sample from the subject at the first time point as compared to the number of DNA mutation of at least three of the same genes in the biological sample from the subject at the second time point indicates an alteration in the rate of progression of RCC in the subject. In such embodiments, if a decrease in the number of DNA mutation from the first time point as compared to the second time point, it indicates in improved prognosis of RCC progression at the second time point as compared to the first time point. Alternatively, if an increase in the level of the number of DNA mutations from the first time point as compared to the second time point indicates in decreased prognosis of RCC progression at the second time point as compared to the first time point.

In some embodiments, a biological sample useful for measuring the level of RCC biomarker is serum, blood, plasma, urine, and/or tissue sample. In altemative embodiments, a tissue sample is a biopsy tissue sample. In further embodiments, a biological sample is selected from a group of blood, serum, plasma, urine, stool, spinal fluid, sputum, nipple aspirates, lymph fluid, extemal secretions of the skin, respiratory tract, intestinal and genitourinary tracts, bile, saliva, milk, tumors, organs and also samples of in vitro cell culture constituents.

In certain embodiments, this disclosure relates to methods of detecting DNA mutations, said method comprising obtaining sample from blood or urine of a subject; and detecting whether one or more DNA mutations are present in DNA of the sample, wherein detecting whether DNA mutations are present comprises sequencing DNA encoding one or more of the proteins selected from BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL providing DNA sequences.

In certain embodiments, detecting whether DNA mutations are present further comprises comparing the DNA sequences to reference sequences. In certain embodiments, the reference sequences are wild-type sequences, consensus sequences for a nucleic acid encoding the protein, a sequence obtained from the subject determined by sequencing DNA of a non-cancerous cell of the subject or a sequence containing a known recurring DNA mutation.

In certain embodiments, detecting whether DNA mutations are present comprises sequencing DNA encoding proteins VHL, PBRMl, SETD2, BAPl and two DNA mutations are in at least one of the DNA sequences encoding a single protein.

In certain embodiments, detecting whether DNA mutations are present consists of sequencing DNA encoding all of the proteins selected from BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL and two DNA mutations are in at least one of the DNA sequences encoding a single protein.

In certain embodiments, detecting whether DNA mutations are present consists of sequencing DNA encoding all of the proteins selected from BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL and two DNA mutations are in at least two of the DNA sequences encoding a single protein.

In certain embodiments, the DNA mutation in the DNA sequence encoding a protein results in a premature stop codon, a nonsynonymous amino acid change, inframe deletion, fameshift deletion, or splice variant. In certain embodiments, the DNA mutation is selected from those provided for in the tables of Figure 1A, IB, or 1C.

In certain embodiments, this disclosure relates to methods of diagnosing renal cancer, said method comprising: obtaining a urine or blood based sample from a subject and detecting multiple DNA mutations in the subject, said method comprising obtaining sample from the subject; and detecting whether DNA mutations are present in DNA of the sample, wherein detecting whether DNA mutations are present comprises sequencing DNA encoding one or more of the proteins selected from BAPl, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL; and diagnosing the patient with, at risk of, or at risk of recurring renal cancer when the presence multiple DNA mutation in the sample is detected.

In certain embodiments, this disclosure relates to methods for screening DNA for an alteration of a gene selected from BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL which comprises comparing DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL, obtained from a urine or blood based sample of a subject, with wild-type sequences or a sequence with a known DNA mutation of BAP 1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL, wherein a difference in the sequence of the gene of the subject from wild-type indicates an alteration in the DNA of the blood in said subject, and wherein a similarity in the sequence of the gene of the subject to a known DNA mutation indicates an alteration in the DNA of the blood in said subject.

In certain embodiments, said comparing DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL further comprises: hybridizing a wild-type probe or a probe with a sequence that targets a known DNA mutation to DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL isolated from said sample; and detecting the presence of a hybridization product by measuring conformational changes in the probe that are indicative of hybridization to the BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL gene.

In certain embodiments, said comparing DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL further comprises: amplifying by PCR all or part of a DNA sequences of genes BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, and VHL from said sample using a set of primers to produce amplified nucleic acids; and sequencing the amplified nucleic acids.

In one embodiment, this disclosure relates to a method of detecting tumor-associated mutations in DNA isolated from a biological sample of a patient by a microfluidic amplification strategy followed by gene sequencing and comparison to a cancer genome database. In specific embodiments, the tumor-associated mutations may be localized to a gene group comprising at least three genes selected from a group of genes encoding: BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRMl, PIK3CA, SETD2, TP53, or VHL. The biological sample may be selected from the group comprising serum, blood, plasma, or urine. In another embodiment, this disclosure relates to a method of determining the presence or severity of renal cell carcinoma in a subject comprising detecting circulating tumor DNA in a biological sample by identifying mutations in a tumor-associated gene group. In specific embodiments, the tumor-associated mutations may be localized to a gene group comprising at least three genes selected from a group of genes encoding: BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, or VHL. In certain embodiments, the mutations detected may comprise inframe deletions, frameshift deletions, frameshift insertions, nonsynonymous substitutions, splice variants, or stopgain substitutions in any of these genes. In another specific embodiment, the mutations are detected through a gene microarray that hybridizes with tumor-associated DNA from these genes.

In certain embodiments, this disclosure relates to methods for identifying a subject having increased likelihood of developing or having renal cell carcinoma (RCC) detecting DNA mutations in a urine or blood based sample of a subject using methods disclosed herein. In certain embodiments, this disclosure relates to methods for monitoring the progression of renal cell carcinoma (RCC) in a subject by detecting DNA mutations in a urine or blood based sample of a subject using methods disclosed herein.

In certain embodiments, this disclosure relates to methods for preventing the progression of renal cell carcinoma (RCC), the method comprising measuring the level of DNA mutations of genes in a sample wherein the gene group comprises of at least three genes selected from a group of genes comprising; BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, and VHL 1 in a sample and assessing the risk of a subject developing or having RCC, wherein a clinician directs the subject to be treated with an appropriate therapy if the subject has, or is at risk of developing RCC.

In certain embodiments, this disclosure relates to arrays comprising solid platforms, including nanochips and beads comprising in known positions on the array antisense nucleic acid sequences to fragments of at most 14 different genes, wherein at least three of the 14 genes are selected from BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, and VHL.

An assay for detecting BRAF V600E mutation (T→A at gene position 1799) in human DNA is reported in Miotke et al. (2015) Enzyme-Free Detection of Mutations in Cancer DNA Using Synthetic Oligonucleotide Probes and Fluorescence Microscopy. PLoS ONE 10(8): eOl 36720. Target DNA is enriched using a gene specific 120mer enrichment probe, followed by fluorescent detection with EvaGreen dye and a second mutation specific, shorter LNA/DNA capture probe. By using serial dilutions of mutant/wild-type cell line DNA mixtures, the assay is capable for using fluorescent sensing of a clinically relevant SNP or other mutation at low concentrations and high sample complexity. One can use fluorescence microscopy specifically detect low concentrations of DNA containing the target mutation.

In certain embodiments, this disclosure contemplates a probe configured to hybridize wild- type sequences and DNA mutations disclosed herein having LNA (locked nucleic acids), i.e., the ribose ring is connected by a methylene bridge (orange) between the 2'-0 and 4'-C atoms.

Methods of Treatment

In certain embodiments, this disclosure provides for methods of treating subjects identified, using the methods disclosed herein, to be at risk of developing or afflicted with RCC, wherein the biological sample obtained from the subject has DNA mutations as disclosed herein as compared to the same genes analyzed in a reference sample.

In certain embodiments, this disclosure provides for methods for selecting a therapeutic regimen or determining if a certain therapeutic regimen is more appropriate for a subject identified as having RCC or at increased risk of developing RCC as identified by the methods as disclosed herein. For example, an aggressive anti-cancer therapeutic regime can be perused in which a subject identified with RCC, where the subject is administered a therapeutically effective amount of an anticancer agent to treat the RCC. In alternative embodiments, a prophylactic anti-cancer therapeutic regimen can be pursued in a subject identified to have increased likelihood of developing RCC, where the subject is administered a prophylactic dose or maintenance dose of an anti-cancer agent to prevent the development of RCC. In alternative embodiments, a subject can be monitored for RCC using the methods as disclosed herein, and if on a first (i.e. initial) testing the subject is identified as having RCC, the subject can be administered an anti-cancer therapy, and on a second (i.e. follow-up testing), the subject is identified as not having RCC or having decreased DNA mutations as analyzed in the first testing, the subject can be administered an anti-cancer therapy at a maintenance dose.

In yet another embodiment, this disclosure relates to a method of predicting the responsiveness of a renal cell carcinoma to a drug based on sequencing circulating tumor DNA in a biological sample to identify mutations in a tumor-associated gene group. In specific embodiments, the drug is a tyrosine kinase inhibitor including, but not limited to, sunitinib and pazopanib, or targeting the Von Hippel-Lindau/Hypoxia-inducible factor alpha (VHL-HIFla) angiogenesis pathway.

In general, a therapy is considered to "treat" RCC if it provides one or more of the following treatment outcomes: reduce or delay recurrence of the RCC after the initial therapy; increase median survival time or decrease metastases. The method is particularly suited to determining which subjects will be responsive or experience a positive treatment outcome to a chemotherapeutic regimen. In some embodiments, an anti-cancer therapy is, for example but not limited to administration of a chemotherapeutic agents such as fluoropyrimidine drug such as 5-FU or a platinum drug such as oxaliplatin or cisplatin. Alternatively, the chemotherapy includes administration of a topoisomerase inhibitor such as irinotecan. In a yet further embodiment, the therapy comprises co-administration of an antibody (as broadly defined herein), ligand or small molecule that binds the Epidermal Growth Factor Receptor (EGFR) or Programmed cell death protein 1 or ligand (PD-1/PD-1L).

Treatment can include prophylaxis, including agents which slow or reduce the risk of RCC in a subject. In other embodiments, the treatments are any means to prevent the proliferation of RCC cancerous cells. In some embodiments, the treatment is an agent which suppresses the EGF-EGFR pathway, for example but not limited to inhibitors and agents of EGFR. Inhibitors of EGFR include, but are not limited to, tyrosine kinase inhibitors such as quinazolines, such as PID 153035, 4-(3- chloroanilino) quinazoline, or CP-358,774, pyridopyrimi dines, pyrimidopyrimi dines, pyrrolopyrimi dines, such as CGP 59326, CGP 60261 and CGP 62706, and pyrazolopyrimi dines, 4- (phenylamino)-7H-pyrrolo[2,3-d]pyrimi dines (Traxler et al., (1996) J. Med Chem 39:2285-2292), curcumin (diferuloyl methane) (Laxmin arayana, et al, (1995), Carcinogen 16: 1741-1745), 4,5-bis(4- fluoroanilino)phthalimide (Buchdunger et al. (1995) Clin. Cancer Res. 1 :813-821; Dinney et al. (1997) Clin. Cancer Res. 3: 161-168); tyrphostins containing nitrothiophene moieties (Brunton et al. (1996) Anti Cancer Drug Design 11 :265-295); the protein kinase inhibitor ZD-1 839 (AstraZeneca); CP-358774 (Pfizer, Inc.); PD-01 83805 (Warner-Lambert), EKB-569 (Torrance et al, Nature Medicine, Vol. 6, No. 9, September 2000, p. 1024), HKI-272 and HKI-357 (Wyeth); or as described in International patent application WO05/018677 (Wyeth); WO99/09016 (American Cyanamid); WO98/43960 (American Cyanamid); WO 98/14451 ; WO 98/02434; W097/38983 (Warener Labert); WO99/06378 (Warner Lambert); WO99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc.); W096/33978 (Zeneca); W096/33977 (Zeneca); and WO96/33980 (Zeneca), WO 95/19970; U.S. Pat. App. Nos. 2005/0101618 assigned to Pfizer, 2005/0101617, 20050090500 assigned to OSI Pharmaceuticals, Inc.; all herein incorporated by reference.

In some embodiments, the anti-cancer treatment comprises the administration of a chemotherapeutic drug selected from the group consisting of fluoropyrimidine (e.g., 5-FU), oxaliplatin, CPT-11, (e.g., irinotecan) a platinum drug or an anti EGFR antibody, such as the cetuximab antibody or a combination of such therapies, alone or in combination with surgical resection of the tumor. In yet a further aspect, the treatment compresses radiation therapy and/or surgical resection of the tumor masses. In one embodiment, the present invention encompasses administering to a subject identified as having, or increased risk of developing RCC an anti-cancer combination therapy where combinations of anti-cancer agents are used, such as for example Taxol, cyclophosphamide, cisplatin, gancyclovir and the like. Anti-cancer therapies are well known in the art and are encompassed for use in the methods of the present invention. Chemotherapy includes, but is not limited to an alkylating agent, mitotic inhibitor, antibiotic, or antimetabolite, anti-angliogenic agents etc. The chemotherapy can comprise administration of CPT-11, temozolomide, or a platinum compound. Radiotherapy can include, for example, x-ray irradiation, γ-irradiation, or microwaves.

The compounds used in connection with the treatment methods of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual subject, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The pharmaceutically "effective amount" for purposes herein is thus determined by such considerations as are known in the art. The amount must be effective to achieve improvement including, but not limited to, improved survival rate or more rapid recovery, or improvement or elimination of symptoms and other indicators as are selected as appropriate measures by those skilled in the art.

The methods of the present invention are useful for the early detection of subjects susceptible to developing RCC. Thus, treatment may be initiated early, e.g. before or at the beginning of the onset of symptoms, for example before the onset of RCC. In alternative embodiments, the treatment may be administered to a subject that has, or is at risk of developing RCC. In alternative embodiments, the treatment may be administered prior to, during, concurrent or post the development of RCC. The effective amount or dosage required at these early stages will typically be lower than those needed at later stages of disease where the symptoms of RCC are severe. Such dosages are known to those of skill in the art and can be determined by a physician.

In some embodiments, where a subject is identified as having increased risk of having or developing RCC using DNA mutation and methods as disclosed herein, a clinician can recommended a treatment regimen to reduce or lower the DNA mutations in the subject.

In another embodiment, a subject with identified as having or at risk of developing RCC using the methods as disclosed herein can be monitored for DNA mutations in a biological sample before, during and after an anti-cancer therapy or treatment regimen, and where a subject is identified to not have lowered number of DNA mutations after a period of time of being administered such a treatment regimen, then the treatment regimen could be modified, for example the subject could be administered (i) a different anti-cancer therapy or anti-cancer drug (ii) a different amount such as in increased amount or dose of an anti-cancer therapy or anti-cancer drug or (iii) a combination of anti-cancer therapies etc. Kits

In some embodiments, the present disclosure provides for diagnostic methods for determining the likelihood of a subject having or developing RCC by detecting one or more DNA mutations as listed in Table 1A, B, and C. In some embodiments, the methods use probes or primers comprising nucleotide sequences which are complementary to the DNA mutations, or subgroup thereof in any combination. Accordingly, the disclosure provides kits for performing these methods.

The kit can comprise at least two probes or two primer-pairs which are capable of specifically hybridizing to at least two DNA mutations and instructions for use. Preferred kits amplify a portion of at least 2 mutations, or at least 3-5, or about 5-7, or about 7-9 or about 9-11 mutations as disclosed in Table 1A, B, and C. Such kits are suitable for detection of mutations by, for example, fluorescence detection, by electrochemical detection, or by other detection.

Oligonucleotides, whether used as probes or primers, contained in a kit can be detectably labeled. Labels can be detected either directly, for example for fluorescent labels, or indirectly. Indirect detection can include any detection method known to one of skill in the art, including biotin- avidin interactions, antibody binding and the like. Fluorescently labeled oligonucleotides also can contain a quenching molecule such as in the case of molecule beacons. Oligonucleotides can be bound to a surface. In one embodiment, the preferred surface is silica or glass. In another embodiment, the surface is a metal electrode.

An array comprising polynucleotide binding probes to the wild-type genes and/or mutations as disclosed herein are useful in the methods as disclosed herein. An array can be made of any conventional substrate. Moreover, the array can be in any shape that can be read, including rectangular and spheroid. Preferred substrates are any suitable rigid or semi-rigid support including membranes, filter, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the probes are bound. Preferably, the substrates are optically transparent. EXAMPLES

Blood sampling for liquid biopsy of renal cell carcinoma overcomes sampling error associated with biopsy for molecular genotyping

The objective of this study was to determine if blood-based analysis of somatic mutations in circulating free DNA could overcome the problem of sampling error intrinsic to traditional tissue based biopsy in clear cell RCC (ccRCC).

The gene panel was selected by focusing on genes reported in the Catalogue Of Somatic Mutations In Cancer (COSMIC) and The Cancer Genome Atlas (TCGA) to be associated with RCC, along with a few druggable gene targets. The final gene panel included the 14 following genes: BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, VHL. Samples were obtained from patients who underwent nephrectomy for pathologically- confirmed ccRCC. Illumina HiSeq deep sequencing was performed using DNA purified from multiple tumor samples (initially frozen, followed by formalin-fixed paraffin-embedded) and whole blood. Whole blood was separated into serum and buffy coat for leukocyte control DNA. The formalin-fixed paraffin-embedded tissues were cored after confirmation by a board-certified, fellowship-trained genitourinary pathologist to be distinct ccRCC tumor populations. Circulating free DNA was purified from serum or urine using modification of a commercially available Qiagen kit. The three patients presented here were chosen because they had one or multiple somatic mutation(s) not detected in the frozen biopsy but present in serum and/or urine (See figure 1 A and IB). Intratumor genetic heterogeneity limits the ability of a single tumor sample (such as frozen section or biopsy) to identify key genetic tumor markers. Blood and/or urine may serve as a source of tumor-derived DNA that can be detected by focused Illumina sequencing of the genes most frequently mutated in ccRCC,

Prognostic value of simultaneous chromosome 3p mutations in RCC

Concurrent chromosome 3p mutations in primary tumor predicts worse outcome. A high throughput sequencing protocol for ccRCC specimens was developed using Fluidigm Access Array with Illumina MiSeq sequencing. A gene sequencing panel was developed of 14 genes (BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, VHL). For this study mutations on chromosome 3p (VHL, PBRM1, SETD2, BAP1) were evaluated.

Frozen tissue and whole blood banked from 21 patients undergoing a nephrectomy for renal mass were used to isolate genomic DNA using Qiagen Gentra Puregene Tissue kit and FlexiGene DNA Kit (whole blood). Tumor samples and pair whole blood DNA of 21 patients with ccRCC underwent deep sequencing. Briefly, 50 ng of DNA per sample was used for amplicon preparation according to the Fluidgm Access Array standard protocol. See Wang et al. Somatic Mutation Screening Using Archival Formalin-Fixed, Paraffin-Embedded Tissues by Fluidigm Multiplex PCR and Illumina Sequencing, J Mol Diagn, 2015, 17(5): 521-532. Amplicon libraries were quantitated using Q-PCR and Agilent Bioanalyzer and run on the Illumina MiSeq instrument using the v2 kit. FASTQ data files for each specimen were analyzed using a combination of the amplicon-aware (AA) tools, the GEMINI tool suite, and a local Galaxy server at George Washington University. Read alignment and variant calling were done using the AA tools, and GEMINI was used for variant annotation and filtering. A Galaxy workflow was created to run the entire analysis pipeline automatically.

The mean post-operative follow-up time for the cohort was 52.5 months. Of the 21 patients,

4 were metastatic at time of surgery and an additional 5 patients later developed metastasis. The cohort was divided into those with mutations in 2 or more of the chr3p genes compared to those with 1 or none. All 4 cases that were metastatic at time of surgery, 4 of the 5 patients subsequently diagnosed with metastatic spread, and all 6 patients who died within the follow-up period had mutations in 2 or more of these chr3p genes (Figure 1C). The difference in overall survival (p=0.027), but not grade (p=0.68), stage (p=0.43), or age (p=0.48), was significantly different between the two groups. These findings suggest that co-existing somatic mutations in VHL, PBRMl, SETD2, and/or BAP1, are predictive of poor prognosis in ccRCC.

Because of the limited size of our cohort, the TCGA dataset was utilized to verify our findings. Data from the TCGA dataset Kidney Renal Clear Cell Carcinoma "sequenced tumors" (TCGA, Nature 2013) was analyzed using cBioportal. The query included VHL, PBRMl, SETD2 and BAP1 to assess mutation exclusivity and co-occurrence. Overall and recurrence-free survival status was obtained based on clinical data downloaded from the TCGA portal.

Overall and recurrence-free survival data was compared using log-rank survival analysis of Kaplan-Meier survival curves in JMP 12 (JMP®, Version 12. SAS Institute Inc., Cary, NC) with median survival analyzed. In the TCGA cohort, after excluding cases with only mutations in VHL and PBRMl (n=72), the patients with concurrent chromosome 3p gene mutations (n=62) had significantly decreased survival overall (3.26 v. 9.74 years; p=0.0001) (Figure 2B) and recurrence- free survival (2.10 v. 6.42 years; p=0.0001) (Figure 2A) relative to the rest of the cohort (n=362). The difference in survival was independent of total mutational frequency and BAP1 mutations. In addition, among those with BAP1 mutations, those with additional chromosome 3p mutations (n=28) had decreased overall survival (p=0.045) compared to others with BAP1 mutations alone

(n=14). In conclusion, the presence of pathologic mutations in 2 or more of these 4 chromosome 3p genes is associated with a worse prognosis in patients with ccRCC. Liquid biopsy using the existing TCGA gene panel prior to surgery for renal mass excision.

Operating procedures for all components of the liquid biopsy were developed based on comparing various procedures with the quality of the end sequence data. During this evolution, the proportion of patients giving high quality, high concentration DNA from plasma has increased and having passed this critical QC analysis the proportion of those DNAs that give high quality sequence has also increased. The current protocol involves drawing 40 cc of blood into cell-free DNA BCT tubes (Streck tubes) designed to stabilize cell free DNA. Blood is mixed by inverting several times then placed on ice for transportation to the lab across the street. Plasma is processed within 2 hours as follows: centrifugation at 1300 g for 20 minutes, the plasma layer is carefully transferred to a separate, clean and sterile 15 mL conical tube and the plasma centrifuged for 10 minutes at 5000 g. The buffy coat layer is removed and stored at -80 °C for future purification of genomic DNA. The plasma is transferred centrifuged a final time at 5000 g for 10 min. These additional steps are to ensure the removal of cells that contain contaminating germline DNA. The final plasma sample is aliquotted and stored at -80 °C for future cfDNA purification.

CfDNA purification is accomplished using QIAamp-Circulating Nucleic Acid Kit (Qiagen, Mansfield, MA) with modifications. Briefly, after thawing, the plasma is spun at 5000 g for 20 min to remove any particulate matter that resulted from the freeze thaw process. The cfDNA from the remaining plasma (10-20 mL) is purified modifying the QIAamp protocol to accommodate the volume of plasma. CfDNA is eluted in 70 ul of molecular grade water and concentrated to a volume of approximately 20 ul. CfDNA concentration is determined using Qubit dsDNA HS Assay Kit (ThermoFisher, Waltham, MA). The cfDNA is stored at -20 °C until batched and sent to the genomics core for sequencing library preparation. Frozen aliquots are thawed on ice and pipetted into 96 well plates at a concentration of at least 4 ng/ul and total DNA of 50-65 ng for efficient loading into the Fluidigm microfluidic amplification chip for preparation of sequencing libraries with subsequent sequencing on the Illumina platform.

Pre-nephrectomy liquid biopsy

Of 22 patients whose plasma DNA and buffy coat DNA gave high quality deep-sequence, 17 (77%) were found to have at least one pathogenic mutation that fulfilled all our criteria for a genuine tumor-derived signal. The 22 successful amplifications included 13 ccRCC, 1 papillary RCC 1 chromophobe RCC, 5ccRCC in combination with a second cell type, 1 unclassified RCC and 1 metastatic melanoma. For ccRCC specifically (alone or in combination), 14/18 (78%) had at least one mutation detected in the plasma. Concentrating on these 14 ccRCCs, the number of mutations ranged between 3 and 17 per patient (Figure 3 A and 3B). If these preliminary data extend to the general population with renal cell carcinoma the inventors would expect 77% of all patients with a lesion detectable by current imaging to have a positive test (detectable pathologic mutation in plasma cfDNA not seen in the inherited genome). This is a substantially greater sensitivity than the current literature reports. In the largest series reported to date, only 5 RCC patients were evaluated and the authors report that ctDNA was detected in 2 of 5 (40%) of advanced cases (C Bettegowda et al, 2014, Sci Transl Med 6, 224ra24). In that same report, they examined 223 patients with localized cancer of all types and found that ctDNA were found in 49 to 78% of all patients with localized cancer. Compared to this series, our detection sensitivity is at the very top of their range.

Blood sampling for liquid biopsy overcomes sampling error associated with analysis of a single piece of tumor

Samples were obtained from patients who underwent nephrectomy for ccRCC. Illumina HiSeq deep sequencing was performed using DNA purified from multiple tumor samples (frozen and FFPE), and whole blood. In addition, circulating free DNA was purified from serum or urine. The gene panel was selected by focusing on genes reported in the Catalogue Of Somatic Mutations In Cancer (COSMIC) and The Cancer Genome Atlas (TCGA) to be associated with RCC, along with a few druggable gene targets. The final gene panel included the 14 following genes: BAP1, BRAF, CDKN2A, FGFR3, KDM5C, KIT, MET, MUC4, NFE2L2, PBRM1, PIK3CA, SETD2, TP53, VHL. The three patients were chosen because they had a somatic mutation detected in serum or urine that was not found on sequencing DNA from the frozen section biopsy.

Two of the three patients (19 and 26) had a technically satisfactory sequencing from FFPE, while a third (patient 32) did not. Patient 19 had tumor frozen tissue sequenced as well as lymphocytes, serum and urine and 10 separate tumor foci from fixed tissues. A single FFPE tumor focus (F7) was found to harbor the original mutation detected in serum (KDM5C gene). No other tumor foci had this mutation detected. Patient 19 also had an additional variant in a second gene (SETD2) that appears in serum and one FFPE sample only. Patient 26 had tumor frozen tissue sequenced as well as lymphocytes, serum and urine and 9 separate tumor foci from fixed tissues. A single FFPE tumor focus (F6) was found to harbor the original serum detected mutation (KIT gene). In addition, 3 mutations that were found on frozen section were also detected in various other tumor foci from FFPE tissue. These included a single VHL frame shift mutation that was present in the frozen section and an additional 5 FFPE tumor foci, (Fl, F2, F5, F6, F7). A SETD2 mutation in the frozen section was detected in two FFPE foci (F2, F6), and a PBMR1 mutation was found in the frozen section and two FFPE foci (F5, F6). In order to compare the results of liquid biopsy with the same genetic analysis of primary tumor the TCGA gene panel was ran on blood and tissue from the same patients. One of the first findings was that there were mutations in the blood that were not seen in the single frozen piece of tissue. Because of the well-known heterogeneity that RCC exhibits inventors went back to the FFPE blocks in two of the cases (patients 19 and 26) where there was discordance between serum and tissue. See Figure 4B. Patient 19 had tumor frozen tissue sequenced as well as lymphocytes, serum and urine and 10 separate tumor foci from formalin fixed paraffin embedded (FFPE) tissues. A single FFPE tumor focus (F7) was found to harbor the original mutation detected in serum (KDM5C gene; Lys961Glu). No other tumor foci had this mutation detected. Patient 26 had tumor frozen tissue sequenced as well as lymphocytes, serum and urine and 9 separate tumor foci from FFPE. A single FFPE tumor focus (F6) was found to harbor the original serum detected mutation (KIT gene; Trp246Ter). In addition, 3 mutations that were found on frozen section were also detected in various other tumor foci from FFPE tissue. These included a single VHL frame shift mutation (Tyrl 12ValfsTer45) that was present in the frozen section and an additional 5 FFPE tumor foci, (Fl, F2, F5, F6, F7). A SETD2 splice site mutation in the frozen section was detected in two FFPE foci (F2, F6), and a PBMR1 missense mutation (Vall012Ile) was found in the frozen section and two FFPE foci (F5, F6).

Liquid Biopsy for Renal Cell Carcinoma

Patients with solid renal tumors and healthy controls gave 40 mL blood and plasma cell free

DNA was prepared. A multiplex bar coded polymerase chain reaction amplification using the Fluidigm Access Array was performed to prepare sequencing libraries for the Illumina HiSeq platform. Galaxy workflow was used to identify mutations and results were compared to buffy coat sequencing. The following genes were queried: VHL, PBRM1, SETD2, BAP1, KDM5C, KIT, NFE2L2, MET, TP53, CDKN2A, FGFR3, PIK3CA, BRAF, MUC4. Criteria for calling mutations included adequate frequency by overall count and percentage of reads, identification in all overlapping sequences, and presence of buffy coat for comparison with <0.5% containing the mutation.

Thirty preoperative test patients with RCC and 32 healthy controls were analyzed using the gene panel. Of the 32 patients analyzed in the healthy control cohort, 27 (84%) failed to yield sequence of the genes of interest. Of the preoperative RCC patients, 20/30 (67%) had detectable somatic mutations, resulting in nonsynonymous, frameshift, stopgain, or splice site mutations, compared to 1/32 (3.1%) controls. Mutations were detected in both early and advanced stage disease, including a patient with a 1.1 x 0.7 x 0.5 cm tumor. Mutations were seen in all genes assayed. These data demonstrate feasibility of gene-specific whole exome sequencing of ctDNA for diagnosis of RCC in patients with solid renal tumors. The majority of RCC patients of various stages and histology had ctDNA detected in a single preoperative blood sample. A single control gave a positive test. Non-invasive detection of RCC shows promise for not only initial diagnosis but also disease monitoring and guidance of targeted therapies throughout a wide spectrum of disease severity, including small lesions.

Targeted therapy gene panel for use in liquid biopsy for patients undergoing targeted therapy for advanced or metatstatic RCC

In order to identify additional genes that may predict response to sunitinib and pazopanib, inventors queried the Drug-Gene Interaction database (DGIdb) curated by Washington University. The DGIdb is designed to return the rank ordered list of genes that when altered will alter the response to the drug in question. The results of the TCGA data were added on the number of ccRCC cases that have alterations in each individual gene. Interestingly, the gene that received the top score for sunitinib was the KIT gene. Although the KIT gene mutations are rare in the TCGA ccRCC dataset (The Cancer Genome Atlas Research Network, 2013, Nature 499:43-49), which has 3 mutations out of 424 tumors, KIT mutations were identified in 6 of the 14 ccRCC cases (43%) that had pre- nephrectomy liquid biopsy (see data Table 3A and 3B). Two of the 6 cases with KIT mutations had two KIT mutations, but it has not been determined if these mutations are biallelic. KIT mutations in our liquid biopsy series included missense, splice site and frameshift mutations. These data suggest that the liquid biopsy may be detecting subclonal populations that may not be evident in the TCGA dataset derived solely from frozen tumor sections. As another example of the use of DGIdb, while MTOR only achieves a DGIdb score of 2, it is very frequently mutated in ccRCC, thus MTOR will be on the panel interrogating response to TKIs.