WO2012149522A1 - Recurrent chimeric rnas enriched in human prostate cancer as biomarkers - Google Patents

Recurrent chimeric rnas enriched in human prostate cancer as biomarkers Download PDF

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WO2012149522A1
WO2012149522A1 PCT/US2012/035769 US2012035769W WO2012149522A1 WO 2012149522 A1 WO2012149522 A1 WO 2012149522A1 US 2012035769 W US2012035769 W US 2012035769W WO 2012149522 A1 WO2012149522 A1 WO 2012149522A1
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chimeric
cancer
rna
rnas
chimeric rnas
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Laising Yen
Kalpana KANNAN
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Baylor College of Medicine
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Baylor College of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention concerns at least the fields of molecular biology, cell biology, and medicine, including cancer medicine.
  • the present invention concerns diagnostics of cancer.
  • Chimeric RNAs unrelated to chromosomal rearrangements are primarily generated by two transcription-induced mechanisms (1, 2).
  • the first one is "read- through/splicing," where a chimeric RNA begins at the upstream gene and ends at a termination point of the adjacent downstream gene, with the region in between removed by splicing.
  • the second one is “transsplicing.”
  • two separately generated RNAs are spliced together to give rise to a single RNA. Both mechanisms result in fused transcripts that possess sequences from both genes.
  • chimeric RNAs like gene fusions that result from chromosomal rearrangements, are expected to increase the proteomic diversity in cells through chimeric proteins or altered regulation of participating mRNAs.
  • chimeric RNAs Despite their importance, few studies have reported on chimeric RNAs, especially regarding their presence and roles in human cancers (3, 4). The lack of reports of chimeric RNAs may stem from the fact that current models of fusion transcripts primarily emphasize chromosomal rearrangements. Alternatively, chimeric RNAs may not have been observed previously because of methods of analyses such as microarrays that lack the necessary resolution for chimeric RNA discovery.
  • SLC45A3-ELK4 may represent a unique class of transcriptional events with important implications in cancer that may have been previously overlooked. This result raises the possibility that chimeric RNAs in cancers are underinvestigated and mostly unknown.
  • the present invention is directed to methods and compositions related to cancer, including diagnostics and/or treatment of cancer, for example.
  • methods and compositions related to chimeric RNAs associated with the presence of cancer or, in alternative embodiments, lack thereof.
  • the invention concerns identification of the presence of one or more particular chimeric RNAs and its association with cancer or increased risk of cancer in a mammal, including a human, dog, cat, or horse, for example.
  • detection of one or more particular RNAs allows for the detection of a stage of a certain cancer.
  • chimeric RNAs that whose absence is indicative of one or more types of cancer or risk thereof.
  • the chimeric RNAs referred to herein are utilized to determine the stage of a particular cancer, to monitor treatment efficiency, and/or to determine if a cancer has returned.
  • the cancer that is associated with one or more chimeric RNAs may be of any type of cancer, including primary or metastatic.
  • Exemplary types of cancer include prostate, esophageal, lung, breast, brain, melanoma, colon, pancreatic, stomach, kidney, liver, bladder, ovarian, cervical, testicular, rectal, spleen, gall bladder, thyroid, and so forth.
  • the inventors took advantage of the analytical power of paired-end high-throughput sequencing (11, 12) to characterize chimeric RNAs enriched in human prostate cancer as an example of a cancer type.
  • the transcribed mRNA (transcriptome) was sequenced from a cohort of patients with human prostate cancer, yielding 1.3 billion raw sequence reads.
  • This sequencing coverage enabled a "deep" survey of chimeric RNAs expressed from the complex human genome, leading to the validation of 32 recurrent but exemplary chimeric RNAs. Among them, 27 chimeric RNAs have not been described before.
  • At least one of these chimeras is highly cancer enriched, as it is expressed at significantly higher levels in human prostate cancers but present at very low levels in noncancer prostates. This indicates that recurrent chimeric RNAs are more common than previously thought. The fact that there are more chimeric RNAs in cancer than in matched benign samples indicates that in at least certain aspects increased chimeric RNA events represent one of the molecular consequences of cancer.
  • composition of matter comprising one or more isolated chimeric RNAs provided herein, and in specific aspects the chimeric RNA is not TMPRSS2-ERG, SLC45A3-ELK4, ANKRD39-ANKRD23, HARS2- ZMAT2, or SMG5-PAQR6.
  • composition of matter comprising an isolated chimeric TMEM79-SMG5 RNA.
  • TMEM79-SMG5 and SMG5-PAQR6 are two separate chimeric RNAs, although in some embodiments they are a single chimeric RNA that contains three genes: TMEM79-SMG5- PAQR6.
  • composition of matter comprising one or more isolated chimeric RNAs amplifiable by PCR primers provided herein, and in specific aspects the chimeric RNA is not TMPRSS2-ERG, SLC45A3-ELK4, ANKRD39- ANKRD23, HARS2-ZMAT2, or SMG5-PAQR6.
  • a substrate comprising polynucleotides attached thereto, said polynucleotides defined as one or more isolated chimeric RNAs of the invention.
  • said polynucleotides defined as one or more isolated chimeric RNAs of the invention.
  • all or greater than 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polynucleotides attached to the substrate are one or more isolated chimeric RNAs of the invention.
  • a method of detecting the presence of cancer, an increased risk for cancer, or response to cancer therapy in an individual comprising the step of detecting one or more chimeric RNAs of the invention in a sample from the individual.
  • at least one of the chimeric RNAs is TMEM79-SMG5 RNA or GOLMJ-MAKJO RNA.
  • the individual is provided a therapy upon detection of the one or more chimeric RNAs.
  • the chimeric RNA is detected in nucleic acid form, such as by polymerase chain reaction, including wherein the polymerase chain reaction amplifies all or part of the chimeric RNA.
  • the polymerase chain reaction amplifies the junction of the chimeric RNA.
  • the chimeric RNA may be detected by its expressed fusion protein, and in specific cases the fusion protein is detected by antibody.
  • the antibody recognizes the junction of the fusion protein.
  • Chimeric RNA can also be detected by RNA hybridization techniques such as northern blot or in situ hybridization.
  • the sample may be obtained in any suitable manner.
  • the sample comprises serum, urine, prostatic fluid, circulating cancer cells, or biopsy, for example.
  • methods of the invention comprise obtaining a sample from the individual.
  • the analysis of a sample from an individual is performed by the individual or entity that obtained the sample.
  • the analysis of a sample from an individual is performed by an individual or entity other than the individual or entity who obtained the sample.
  • methods of the invention comprise the step of obtaining the sample from an individual.
  • the sample is stored under suitable conditions and the sample is later analyzed by the party that obtained the sample or later analyzed by another party.
  • the individual that has a sample subjected to methods of the invention is also subjected to additional methods of cancer diagnosis.
  • Examples of other methods for prostate cancer include at least analysis of prostate specific antigen, magnetic resonance imaging, analysis of PCA3, and/or analysis of TMPRSS2-ERG.
  • the methods of the invention comprising the step of performing an additional cancer detection step on a sample from the individual, and in some cases it is the same sample used for detection of chimeric RNA or it may be a different sample from the same individual.
  • one or more particular chimeric RNAs are indicative of the presence or recurrence of cancer, or response to a cancer therapy, or monitoring the stage of a cancer regardless of the type of sample that is assessed for the presence of the one or more particular chimeric RNAs. In some embodiments, however, one or more particular chimeric RNAs are indicative of the presence or recurrence of cancer, or response to a cancer therapy, or monitoring the stage of a cancer when assaying a particular type of sample from an individual.
  • one or more chimeric RNAs may be diagnostic if it is present in biopsy only, if it is present in urine sample only, present in serum only, or if it is present in a combination of serum, biopsy and urine sample.
  • composition of matter comprising an isolated chimeric RNA selected from the group consisting of TMEM79-SMG5 RNA, ASTN2- PAPAA RNA, GOLMJ-MAKJ0 RNA and one or more chimeric RNAs in Table 1, 2, 3, 4, or 5.
  • a substrate comprising polynucleotides attached thereto, said polynucleotides defined as one or more of the isolated chimeric RNAs.
  • all or greater than 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polynucleotides attached to the substrate are one or more isolated chimeric RNAs of TMEM79-SMG5 RNA, ASTN2-PAPAA RNA, or GOLMJ-MAKJ0 RNA.
  • a method of detecting the presence of cancer, an increased risk for cancer, or response to cancer therapy in an individual comprising the step of detecting one or more chimeric RNAs in a sample from the individual.
  • the cancer is androgen- sensitive cancer.
  • the cancer is prostate cancer or esophageal cancer.
  • the individual is provided a therapy upon detection of the one or more chimeric RNAs.
  • the chimeric RNA is detected in nucleic acid form, such as by polymerase chain reaction (PCR), including PCR that amplifies all or part of the chimeric RNA or amplifies the junction of the chimeric RNA.
  • PCR polymerase chain reaction
  • a sample comprises serum, urine, prostatic fluid, or biopsy.
  • the chimeric RNA is detected by its expressed fusion protein, for example with detection of the fusion protein by antibody.
  • the antibody recognizes the junction of the fusion protein.
  • Some methods of the invention encompass the step of performing an additional cancer detection step on a sample from the individual.
  • the method further comprises the step of obtaining a sample from the individual.
  • FIG. 1 Strategy for identification and validation of chimeric RNAs. Paired reads are mapped to both the genome and the transcriptome, and if each of the paired reads maps to different genes, then it is considered to be a paired "chimeric" read representative of a putative chimeric event. On the basis of the alignment and location of paired chimeric reads, primers (arrows) are designed to amplify the putative RNA junction from patients' RNAs.
  • RNA junction of the chimera Using the RNA junction as template, previously unmappable reads are aligned to the template and thus "junction" reads are identified. These junction reads have one read aligning to the RNA junction and the other read to one of the genes.
  • FIG. 2 Global analysis of chimeric RNAs.
  • A A total of 2,369 putative chimeric events were identified on the basis of stringent bioinformatic criteria described in Example 9. The frequency of distribution based on reads from all samples for all 2,369 distinct chimeric events is shown. Events chosen for experimental validation are shown in purple.
  • B Comparison of the average number of paired chimeric reads showed that chimeric reads are significantly more abundant in cancer samples than in matched benign samples.
  • FIG. 3 Chimeric RNAs are highly recurrent in prostate cancer. The relative frequency of recurrence of chimeric RNAs in each cancer and matched benign sample is shown. Each vertical column represents data from one patient sample and each horizontal row represents relative abundance for a particular chimeric event in each patient sample. Yellow indicates high occurrence and black low occurrence. C, cancer sample; N, matched benign samples. The white line separates cancer from matched benign samples. The P value obtained by the Kolmogorov- Smirnov test for each chimeric RNA is shown on the right. All data are compared at the same scale except TMPRSS2-ERG, which is normalized to a different scale so that its relative expression level can be compared between samples without color saturation. [0029] FIG. 4.
  • TMPRSS2-ERG is highly expressed in VCaP, whereas the control actin is expressed in all cell lines.
  • C Expression of parental genes in PrEC for those chimeric RNAs shown in B. Although this subset of chimeric RNAs is not expressed in PrEC, the expression of many parental genes is clearly detected.
  • FIG. 5 Schematic of TMEM79-SMG5 chimeric RNA in prostate cancer. Chimeric RNAs can result in a new 5 '-UTR and truncated ORF as in the case of TMEM79-SMG5. In the pictogram, coding exons are represented by blocks connected by horizontal lines
  • FIG. 6 The TMEM79-SMG5 chimera as a potential diagnostic marker.
  • A The relative expression level of the TMEM79-SMG5 chimera (normalized to GAPDH) in PrEC, 18 donors without cancer, and 54 patients with prostate cancer, determined by quantitative RT- PCR. These additional samples were not used for high-throughput sequencing.
  • FIG. 7 Identification of junction by Sanger sequencing and bioinformatics.
  • A RT-PCR products were gel purified and subjected to Sanger sequencing. Shown are examples of traces from Sanger sequencing for three chimeric transcripts: FAM18B2-CDRT4, TSPANl-POMGNTl, and ZNF606-C19orfl8. The identified RNA junction is separated by the thick black line. Traces show that RT-PCR products as a population were homogenous, indicating that there as no variation in RNA junctions in the region defined by the primer pairs.
  • B RNA junction of TSPANl-POMGNTl was first determined by RT-PCR and Sanger sequencing.
  • Previously unmappable reads were then aligned to the determined junction of TSPANl-POMGNTl.
  • the number of reads now mapping to this junction is represented by the blue line.
  • the vertical dashed red line points to the junction site. This mapping supports the RNA junction identified by Sanger sequencing. When the order of genes is switched to generate an artificial RNA junction, reads fail to map to this junction (gray line).
  • FIG. 8 Gene expression levels determined by high-throughput sequencing.
  • RPKM refers to reads per kilobase of exon model per million mapped reads.
  • the x axis represents the expression level of genes in cancer samples and the y axis represents the expression level in matched benign samples. Genes aligned with the diagonal line are expressed at equal levels in cancer and matched benign samples. Red circles indicate parental genes involved in validated chimeric RNAs from this study. Most of the parental genes involved in validated chimeric RNAs are not biased toward highly expressed genes and are not
  • Light blue circles refer to TMEM79 and SMG5 genes.
  • a few examples of long-range PCR assay are shown to determine whether chimeric RNAs are a result of DNA rearrangements.
  • C refers to the DNA from the cancer sample in which the chimeric RNA was expressed and H refers to control human genomic DNA. Absence of size differences between C and H reactions indicates that there were no gross DNA rearrangements.
  • As a positive control for long-range PCR reactions DNA samples and primers were used that were provided by the LA PCR kit from Takara. There was successful amplification of DNA products with a size of 17.5 kb. To ensure that the PCR primers designed for each chimera can amplify the intended DNA fragment, human genomic DNA was used as a positive control.
  • FIG. 10 demonstrates an assay for GOLM1-NAA35 in urine of patient samples that had digital rectal exam. The patient' s urine had an undetectable level of
  • FIG. 11 demonstrates an assay for 24 exemplary chimeric RNAs in prostate cancer patients vs. controls in samples from the exosome of urine from patients that did not have digital rectal exam.
  • a method or device that "comprises,” “has,” “includes” or “contains” one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more elements.
  • a step of a method or an element of a device that "comprises,” “has,” “includes” or “contains” one or more features possesses those one or more features, but is not limited to possessing only those one or more features.
  • another may mean at least a second or more.
  • aspects of the invention may "consist essentially of or “consist of one or more sequences of the invention, for example.
  • Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
  • chimeric RNA refers to transcribed RNAs that contain sequences from two distinct genes.
  • chimeric RNAs are utilized in methods of diagnosis of one or more types of cancer.
  • reagents that target the chimeric RNAs are utilized in the treatment of cancer that has cancer cells having one or more particular chimeric RNAs.
  • chimeric RNAs possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, the inventors obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were unique and highly recurrent in cancer samples.
  • chimeras were present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, indicating their associations with cancer.
  • These chimeras contain discernable 5' and 3' splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, indicating that other factors are involved in their production and regulation.
  • One exemplary chimera
  • TMEM79-SMG5 is highly differentially expressed in human cancer samples and is a useful biomarker, in embodiments of the invention.
  • the prevalence of chimeric RNAs allows the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity.
  • chimeric RNAs are widespread, and increased chimeric RNA events represent a unique class of molecular alteration in cancer.
  • one or more compositions that target one or more chimeric RNAs are employed for the treatment of cancer.
  • the cancer has been determined to have the chimeric RNA, whereas in other aspects the cancer has not been determined to have the chimeric RNA.
  • the composition may be of any kind, so long as it is able to directly or indirectly target the chimeric RNA.
  • the composition that targets the chimeric RNA is a polypeptide or small molecule, for example.
  • the composition that targets the chimeric RNA targets the junction site between the two or more respective components of the chimeric RNA.
  • the composition that targets the chimeric RNA does not target the junction site and targets one or the other of the chimeric components.
  • the composition that targets the chimeric RNA is an antibody that recognizes the chimeric RNA gene product or, in alternative cases, the chimeric RNA itself.
  • the antibody immunologically recognizes the junction site of the gene product.
  • one or more chimeric RNAs are detected in a cancer sample from an individual.
  • the chimeric RNA(s) may be detected by any suitable means.
  • a polynucleotide between 13 and 100 nucleotides preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective.
  • Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained.
  • Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
  • nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples.
  • relatively high stringency conditions For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids.
  • relatively low salt and/or high temperature conditions such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C.
  • Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
  • hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions.
  • Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature.
  • a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C.
  • Hybridization conditions can be readily manipulated depending on the desired results.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl 2 , 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C.
  • nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization.
  • appropriate indicator means include fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected.
  • enzyme tags colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.
  • the probes or primers described herein will be useful as reagents in solution hybridization, as in PCRTM, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase.
  • the test DNA or RNA
  • the test DNA is adsorbed or otherwise affixed to a selected matrix or surface.
  • This fixed, single- stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions.
  • the conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art.
  • hybridization is detected, and/or quantified, by determining the amount of bound label.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et ah, 1989). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • the term "primer,” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double- stranded and/or single- stranded form, although the single- stranded form is preferred.
  • Pairs of primers designed to selectively hybridize to nucleic acids corresponding to one or more chimeric RNAs are contacted with the template nucleic acid under conditions that permit selective hybridization.
  • high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers.
  • hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences.
  • the template- primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
  • PCRTM polymerase chain reaction
  • a reverse transcriptase PCRTM amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et ah, 1989).
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art.
  • Representative methods of RT-PCR are described in U.S. Patent No. 5,882,864.
  • Representative primers for PCR analysis of chimeric RNAs are provided in Table 1.
  • GGAGTAGGCGCGAGCTAAG SEQ ID GTCCATAGTCGCTGGAGGAG (SEQ ID NO:
  • HARS2-ZMAT2 ID NO:50 ID NO: 101
  • KRTCAP3-IFT172 (SEQ ID NO:51) ID NO: 102)
  • NCAPD3-JAM3 (SEQ ID NO:57) ID NO: 108)
  • GPT-PPP1R16A (SEQ ID NO:59) NO: 110)
  • HSP90B1-C12orf73 (SEQ ID NO:63) ID NO: 114)
  • BC035340-MCF2L (SEQ ID NO:67) (SEQ ID NO: 118)
  • BC160930-MC1R (SEQ ID NO:68) ID NO: 119)
  • NCBP1 SEQ ID NO:71
  • SEQ ID NO: 122 SEQ ID NO: 122
  • ELF3 (SEQ ID NO:75) (SEQ ID NO: 126)
  • RNPEP ID NO:76 (SEQ ID NO: 127)
  • GAAGCAAAAGTCCTCCACACT SEQ ID GGCTTCCACATTATAGTACTTGCT
  • MAK10 (SEQ ID NO: 82) (SEQ ID NO: 133)
  • NDUFC1 ID NO:83 (SEQ ID NO: 134)
  • NARG1 ID NO: 84 (SEQ ID NO: 135)
  • NCAPD3 (SEQ ID NO: 85) (SEQ ID NO: 136)
  • JAM3 (SEQ ID NO: 86) (SEQ ID NO: 137)
  • ACTIN NO:87 (SEQ ID NO: 138)
  • TMEM79-SMG5 ID NO:88 (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139) TGCACCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ ID NO: 139)
  • GAPDH primers are from Maher et al, 2009a.
  • LCR ligase chain reaction
  • European Application No. 320 308 incorporated herein by reference in its entirety.
  • U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
  • a method based on PCRTM and oligonucleotide ligase assy (OLA), disclosed in U.S. Patent 5,912,148, may also be used.
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [alpha-thio] -triphosphates in one strand of a restriction site may also be useful in the
  • Displacement Amplification is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety.
  • European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA”), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • ssRNA single- stranded RNA
  • dsDNA double- stranded DNA
  • PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single- stranded DNA ("ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "race” and "one-sided PCR” (Frohman, 1990; Ohara et al, 1989).
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et ah, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
  • Separation of nucleic acids may also be effected by chromatographic techniques known in art.
  • chromatographic techniques There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
  • the amplification products are visualized.
  • a typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
  • detection is by Southern blotting and hybridization with a labeled probe.
  • the techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et ah, 1989).
  • U.S. Patent No. 5,279,721 incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids.
  • the apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
  • DGGE denaturing gradient gel electrophoresis
  • RFLP restriction fragment length polymorphism analysis
  • SSCP single- strand conformation polymorphism analysis
  • mismatch is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single or multiple base point mutations.
  • U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single- stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
  • RNase I in mismatch assays.
  • the use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is reported to cleave three out of four known mismatches. Others have described using the MutS protein or other DNA-repair enzymes for detection of single-base mismatches.
  • one or more antibodies may be utilized to detect one or more chimeric RNAs of the invention.
  • the antibody may detect the junction of the different regions of the chimeric RNA or it may detect the region alone.
  • the term "antibody” is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. [0081]
  • the term “antibody” is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single domain antibodies
  • scFv single chain Fv
  • Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al. (1992) Biochem 31: 1579-1584.
  • the oligomerization domain comprises self-associating .alpha.-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
  • the oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126.
  • Antibody-like binding peptidomimetics are also contemplated in the present invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003 Mar;49(2):209-16 describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
  • ABSiPs antibody like binding peptidomimetics
  • MAbs Monoclonal antibodies
  • the invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
  • humanized antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof.
  • the term “humanized” immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin.
  • the non-human immunoglobulin providing the CDR's is called the "donor” and the human immunoglobulin providing the framework is called the "acceptor”.
  • a "humanized antibody” is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin .
  • the present invention further provides antibodies against chimeric RNA fusion proteins, polypeptides and peptides that may be linked to at least one agent to form an antibody conjugate.
  • it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides.
  • a reporter molecule is defined as any moiety which may be detected using an assay.
  • Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art.
  • Sites for binding to biological active molecules in the antibody molecule include sites that reside in the variable domain that can bind pathogens, B- cell superantigens, the T cell co-receptor CD4 and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991).
  • the variable domain is involved in antibody self- binding (Kang et ah, 1988), and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al, 1989).
  • antibody conjugates are those conjugates in which the antibody is linked to a detectable label.
  • Detectable labels are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired.
  • Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, and may be termed "immunotoxins”.
  • Antibody conjugates are generally preferred for use as diagnostic agents.
  • Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging".
  • paramagnetic ions paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.
  • paramagnetic ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper
  • III dysprosium
  • III holmium
  • III holmium
  • III erbium
  • gadolinium is particularly preferred.
  • radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 cobalt, copper 6T , 152 Eu, gallium 6T , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron,
  • Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • Monoclonal antibodies according to the invention may be labeled with technetium 99 " 1 by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column.
  • direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the antibody.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene
  • EDTA diaminetetracetic acid
  • fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY- R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein
  • Isothiocyanate HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET,
  • Another type of antibody conjugates contemplated in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate.
  • suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase.
  • Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
  • Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels.
  • hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction.
  • this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
  • Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983).
  • 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et ah, 1985).
  • the 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et ah, 1989; King et ah, 1989; and Dholakia et ah, 1989) and may be used as antibody binding agents.
  • attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N- chloro-p-toluenesulfonamide
  • tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • a coupling agent such as glutaraldehyde or periodate
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
  • derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated.
  • Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).
  • Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
  • the antibodies are linked to semiconductor nanocrystals such as those described in U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which are incorporated herein in their entireties); as well as PCT Publication No. 99/26299 (published May 27, 1999).
  • exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to those described above, including group II- VI, III- V and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, A1S, A1P, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof.
  • group II- VI, III- V and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, Ca
  • the present invention concerns
  • immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as chimeric RNA gene products or the chimeric RNAs themselves.
  • Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • the immunobinding methods include obtaining a sample suspected of containing chimeric RNA fusion protein, polypeptide and/or peptide, and contacting the sample with an antibody in accordance with the present invention under conditions effective to allow the formation of immunocomplexes.
  • these methods include methods for purifying chimeric RNA fusion proteins, polypeptides and/or peptides from patients' samples.
  • the antibody removes the antigenic chimeric RNA fusion protein from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the chimeric RNA fusion protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which chimeric RNA fusion protein antigen is then collected by removing the chimeric RNA fusion protein or the complex from the column.
  • the immunobinding methods also include methods for detecting and quantifying the amount of a chimeric RNA fusion protein reactive component in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • a sample suspected of containing a chimeric RNA fusion protein and contact the sample with an antibody against the chimeric RNA fusion protein, and then detect and quantify the amount of immune complexes formed under the specific conditions.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection uses two different antibodies.
  • a first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology.
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • the immunodetection methods of the present invention have evident utility in the diagnosis and prognosis of conditions such as various forms of cancer.
  • a biological and/or clinical sample suspected of containing a chimeric RNA gene product fusion protein, polypeptide, peptide mutant is used.
  • immunoassays in their most simple and/or direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.
  • the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the chimeric RNA gene product fusion protein antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non- specifically bound immune complexes, the bound chimeric RNA gene product fusion protein antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the chimeric RNA gene product fusion protein antigen are immobilized onto the well surface and/or then contacted with the antibodies of the invention. After binding and/or washing to remove non-specifically bound immune complexes, the bound antibodies are detected. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the chimeric RNA gene product fusion proteins, polypeptides and/or peptides are immobilized involves the use of antibody competition in the detection.
  • labeled antibodies against chimeric RNA gene product fusion protein are added to the wells, allowed to bind, and/or detected by means of their label.
  • the amount of chimeric RNA gene product fusion protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against chimeric RNA gene product fusion before and/or during incubation with coated wells.
  • chimeric RNA gene product fusion protein acts to reduce the amount of antibody against chimeric RNA gene product fusion protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against chimeric RNA gene product fusion protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are well known in the art.
  • the antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et ah, 1990; Abbondanzo et ah, 1990; Allred et al, 1990).
  • frozen- sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.
  • compositions of the present invention comprise an effective amount of one or more chimeric RNA-targeting agents (such as an antibody or small molecule or siRNA-based drugs) dissolved or dispersed in a pharmaceutically acceptable carrier.
  • chimeric RNA-targeting agents such as an antibody or small molecule or siRNA-based drugs
  • pharmaceutically acceptable carrier such as an antibody or small molecule or siRNA-based drugs
  • pharmaceutically acceptable carrier such as an antibody or small molecule or siRNA-based drugs
  • pharmaceutically acceptable carrier refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of an pharmaceutical composition that contains at least one chimeric RNA-targeting agent or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the chimeric RNA-targeting agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
  • the chimeric RNA-targeting agent may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine,
  • solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semisolid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e. , by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi- solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include chimeric RNA-targeting agent, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic ⁇ i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • the chimeric RNA-targeting agent may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • the actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500
  • microgram/kg/body weight about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the chimeric RNA- targeting agentS are formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a
  • compositions may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • chimeric RNA-targeting agentS may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • aqueous solutions for parenteral administration in an aqueous solution
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound chimeric RNA-targeting agent may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration
  • inhalation intranasal, vaginal, etc.
  • inhalation intranasal, vaginal, etc.
  • compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles.
  • Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the delivery of drugs using intranasal microparticle resins Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts.
  • transmucosal drug delivery in the form of a polytetraf uoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
  • compositions described herein may be comprised in a kit.
  • a chimeric RNA, one or more polynucleotides that hybridize to a chimeric RNA, a substrate having a chimeric RNA or one or more polynucleotides that hybridize to a chimeric RNA, an antibody to a chimeric RNA, and/or a cancer therapeutic and/or a siRNA that targets a chimeric RNA may be comprised in suitable container means in a kit.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
  • RNAs that are expressed in prostate cancer sequenced the transcriptome of 20 cancer samples and 10 matched benign samples from patients with prostate adenocarcinoma who received no preoperative therapy before radical prostatectomy.
  • An Illumina Genome Analyzer II was used for sequencing of these samples to generate output sequences of paired 36-nucleotide reads. In all, 30 lanes of Illumina Genome Analyzer were used to yield ⁇ 1.3 billion raw sequence reads. Using stringent filters (see
  • Example 9 the inventors obtained nearly 500 million reads that were both uniquely mappable to the human genome and met our additional bioinformatic criteria.
  • a strategy for identifying chimeric RNAs was to search for paired "chimeric" reads with each read mapping to a different gene either in the genome or in the transcriptome (FIG. 1). To minimize the cases of false positives, the inventors required each read to map to only one location in the genome even with the tolerance of two mismatches. In addition, an event was considered a chimeric candidate only if it was supported by paired chimeric reads from at least three different patient samples. Cases of overlapping genes and homologous genes with shared sequences were filtered out. This strategy led to the identification of 2,369 putative chimeric events (FIG. 2A).
  • RNAs that are highly recurrent in human cancer samples To identify chimeric RNAs that are highly recurrent in human cancer samples, the inventors required that a candidate chimeric RNA must be present in >50 of cancer samples used for sequencing (i.e., >10 of 20 total human cancer samples). The analyses narrowed down from 2,369 candidates to a set of 32 highly recurrent chimeric RNAs, and these are marked in in dark color in FIG. 2A. Among them, 27 are unique chimeric RNAs that have never been described before (Table 2).
  • TMPRSS2-ERG SLC45A3-ELK
  • ANKRD39-ANKRD23 HARS-ZMAT2
  • SMG5-PAQR6 SMG5-PAQR6
  • RNA are listed in column 7. Eighteen of these chimeric RNAs are likely to have a modified
  • TMEM79-SMG5 the full-length chimeric RNA sequence is provided in the sequence listing.
  • the putative 5'- and 3'-UTRS are shown in lowercase letters and the putative coding region is shown in uppercase letters.
  • the start/stop codons and poly(A) signal are shown in boldface type.
  • the chimeric junction is denoted by an asterisk.
  • the inventors designed specific primer pairs with each primer targeting one parental gene and then used strand- specific RT-PCR to validate the presence of candidate chimeric RNAs in human samples that were used for paired-end sequencing. For all 32 candidates, including the 5 known chimeras that were used as controls, they were able to obtain RT-PCR products, indicating that these chimeric RNAs are indeed expressed at detectable levels.
  • the strand- specific RT-PCR enabled the determination of 5' and 3' chimeric RNA partners. In most cases, the inventors obtained only a single RT-PCR band for each chimera, which was then excised and subjected to traditional Sanger sequencing.
  • 2,369 chimeric RNA candidates includes 1,902 interchromosomal and 467 intrachromosomal chimeric events.
  • these interchromosomal chimeric RNAs are not highly recurrent, and therefore they were not included in the set that was chosen for validation.
  • the unique chimeric RNAs identified in this study averaged 47 paired reads, with SLC16A8-BAIAP2L2 having the highest number, 99. This result can be compared with the 9 paired reads supporting SLC45A3-ELK4, the best- characterized chimeric RNA in prostate cancer.
  • the unique chimeric RNAs identified in this study are not rare events, but recurrent at relatively high levels. However, their relative expression levels are lower than that of TMPRSS2-ERG (481 paired reads) and this can be attributed to TMPRSS2-ERG being the result of a DNA-level rearrangement and the fact that it is driven by a strong androgen-regulated promoter.
  • TMPRSS2-ERG used as the control, which is known to be cancer specific (9).
  • the presence of these chimeric RNAs within the matched benign samples may represent a "field effect" within the histologically normal epithelium such that the benign epithelium may have multifocal premalignant lesions that precede histological changes (17).
  • small foci of cancer may be present in some matched benign samples because the tissue is not evaluated histologically throughout the entire tissue used for RNA extraction.
  • TMEM79-SMG5 The remaining five are unique chimeric RNAs: TMEM79-SMG5, SLC16A8- BAIAP2L2, SLC44A4-EHMT2, BC035340- MCF2L, and TSPAN1-POMGNT1.
  • TMEM79- SMG5 appeared to be most significant, with a P value of 0.004 that compares favorably to that of TMPRSS2-ERG.
  • RT-PCR was used to verify the presence of identified chimeric RNAs in an additional 10 human prostate cancer tissues and 5 normal prostate tissues from organ donors without prostate cancer (these samples were not used for transcriptome sequencing). Because this analysis compares prostate tissues from patients with cancer to those from patients without cancer, tissue impurity or field effects are not a consideration. As seen in Table 3, most of the validated chimeric RNAs were highly recurrent in the additional 10 patients with cancer, confirming the results obtained from the transcriptome sequencing.
  • TMEM79-SMG5 (9 of 10 cancer vs. 0 of 5 noncancer)
  • TMPRSS2-ERG 5 of 10 cancer vs. 0 of 5 noncancer
  • the absence of the TMEM79-SMG5 chimera in donors without cancer raised the question of whether this absence is due to the absence of expression of their participating parental genes. Because chimeric RNAs possess shared sequences with their parental genes, it is difficult to determine whether the reads obtained from transcriptome sequencing were derived from chimeric RNA or parental RNA.
  • chimeric RNAs that were identified are highly recurrent in human prostate cancer samples, the inventors considered that in certain embodiments these chimeric RNAs may also be present in established prostate cell lines. Therefore, the inventors tested the expression of these chimeric RNAs in cancer cell lines of prostate origin, including androgen receptor negative cell lines (PC3 and DU145) and androgen- sensitive cell lines (LNCaP, VCaP, and LAPC4), as well as immortalized prostatic epithelial cells (PNTla), to compare with that of the noncancer primary human prostate epithelial cells (PrEC).
  • the RT- PCR results showed that most of the 32 chimeric RNAs displayed varying expression profile in cancer cell lines (Table 4).
  • RNAs were conspicuously absent in noncancer PrEC (Table 4 and FIG. 4B). These RNAs include TMEM79-SMG5, the same chimera detectable by RT-PCR in patients with cancer but not in donors without cancer.
  • the rest of the group comprises XPA-NCBP1, RASL12-OSTbeta, ELF3-RNPEP, ASTN2-PAPPA, GOLM1- MAK10, BC035340-MCF2L, NCAPD3-JAM3, and the control TMPRSS2-ERG that is known to be highly expressed in VCaP cells (FIG. 4B).
  • Table 4 Expression of chimeric RNAs in prostate cancer cell lines determined by RT-PCR
  • Shadowed indicates presence of the chimera and white indicates absence of expression of the chimera determined by RT-PCR.
  • the standard FISH assay requires a minimum distance between genes in the order of 100-150 kb (8). This assay was possible for TMPRSS2-ERG (distance between genes is 3 Mb) (10). It is, however, not possible to use this assay for the rest of the validated chimeric RNAs on the list, as the distances between the ends of the two genes for these chimeras are ⁇ 30 kb (see column 3 in Table 2). Therefore, to verify whether these unique chimeric RNAs are a result of genomic DNA rearrangement, long-range PCR was performed on genomic DNA from patients who displayed expression of the
  • Embodiments of the invention provide a deep survey of chimeric RNAs expressed in human prostate cancers.
  • the inventors sequenced 20 cancer samples and 10 matched benign samples from patients with prostate adenocarcinoma.
  • the 1.3 billion reads resulting from deep sequencing enabled the identification of 2,369 putative chimeric RNAs using stringent bioinformatic criteria.
  • the inventors chose 46 events for experimental validation and of these, 32 events ( ⁇ 70%) could be verified by RT-PCR and their RNA junctions were determined by Sanger sequencing. Although this 70% validation rate is relatively high, the chimeric RNAs chosen for validation are among the highly recurrent ones. One could determine whether the lower recurrent chimeric RNAs could also be validated at a similar rate.
  • Chimeric RNAs like gene fusions, are expected to increase the proteomic diversity in cells through chimeric proteins or altered regulation of participating mRNAs.
  • the number of chimeric RNAs revealed by deep sequencing appears far greater than that of a few gene fusion events identified in prostate tissue so far (7, 13, 14).
  • all of the validated chimeras in our study are recurrent in at least 50% of the patient samples. This result is in contrast to gene fusion events in prostate cancer that are rare and predominantly nonrecurrent with the exception of TMPRSS2-ERG (14).
  • the prevalence of chimeric RNAs may allow the limited number of human genes to encode a larger number of RNAs and proteins, forming yet an additional layer of cellular complexity.
  • RNA junctions contain a discernable 5' splice site and a 3' splice site at the junction site, indicating that the formation of chimeric RNAs is mediated by splicing. This mediation could result from two known transcription-induced mechanisms: read- through/splicing and trans- splicing. However, on the basis of the RNA sequence information, it is impossible to discern between these two mechanisms. For example, a chimeric RNA joining neighboring genes located on the same strand could theoretically result from a single transcript reading through both genes followed by splicing or by splicing of two separate RNA molecules independently generated from neighboring genes.
  • RNA junction sequence was observed from sequenced RT-PCR bands or from junction reads.
  • Each of the validated recurrent chimeras exhibits consistent and precise RNA junction within the same patient and across all patient samples.
  • random processes should generate more chimeras from highly expressed genes.
  • the identified chimeric RNAs are not biased toward highly expressed genes (FIG. 8).
  • the expression of participating parental genes does not automatically lead to the generation of the corresponding chimeric RNAs.
  • both the parental genes SMG5 and TMEM79 are expressed in cancer samples as well as in samples from donors without cancer, but the presence of their chimeras is largely restricted to cancer samples (FIG. 4A).
  • the absence of parental gene expression does not exclude the generation of the chimeric RNA.
  • RASL12 is not detected in cancer samples but the chimera RASL12-OSTbeta is expressed in cancer samples (FIG. 4A).
  • RNA strand information and junction sequences determined experimentally by RT-PCR enabled the inventors to consider the potential biological
  • Example 10 results in the loss of the first exon of SMG5, with genome sequence from TMEM79 contributing a new 5'-UTR to the truncated SMG5 (FIG. 5 and Table 2). This result may lead to altered function and regulation of SMG5, a protein essential to the nonsense-mediated mRNA decay (NMD) (20).
  • NMD nonsense-mediated mRNA decay
  • chimeric RNAs could function as noncoding RNAs or regulatory RNAs without a protein counterpart.
  • TMEM79-SMG5 is highly recurrent and enriched in cancer samples.
  • RT-PCR analysis showed that the TMEM79-SMG5 chimera is absent in non-cancer PrEC but is expressed only in androgen- sensitive cancer cell lines VCaP and LNCaP, even though all these cell lines are epithelial in origin.
  • quantitative RT-PCR analysis on an extended cohort of patients confirmed that this chimera is highly expressed in patients with cancer, whereas its expression is far lower in donors without cancer and undetectable in PrEC.
  • the most direct utility of the chimera TMEM79-SMG5 is perhaps in its use as diagnostic biomarker. Compared with TMPRSS2-ERG, which can be found in -50% of the patients with cancer, TMEM79-SMG5 is detected by RT-PCR in -90% of the cancer samples tested.
  • TMEM79-SMG5 appears to be largely restricted to cancer. Thus, TMEM79-SMG5 is useful to serve as a biomarker to separate patients with cancer from patients without cancer.
  • RNA junction was first determined by RT-PCR and Sanger sequencing. Junction reads that mapped to the determined junction site were then identified using the criteria outlined herein. As a bioinformatic control, the 5' and 3' sequences flanking the junction sites were switched to create artificial RNA junctions and then searched for possible paired reads that would map to the artificial junctions. As expected, the analysis yielded almost no or a negligible number of reads that map to the artificially created junctions. An example of this analysis is shown in FIG. 7B.
  • RNA samples were obtained from the Baylor Prostate Specialized Programs of Research Excellence (SPORE) Tissue Core. RNA was extracted from samples using the Ribopure kit (Ambion). The human prostate cancer cell lines were maintained as detailed below. Total RNA samples were processed for transcriptome sequencing using the Illumina mRNA-seq protocol. Bioinformatic identification of chimeric RNAs and their corresponding junction reads is detailed below. For validation of chimeric RNAs, RT was performed using superscript II (Invitrogen) and PCR was performed using primers listed herein.
  • the human prostate cancer cell lines, PC3, PNTla, DU145 and LNCaP were maintained in RPMI- 1640 (Cellgro) supplemented with 10% FBS (Invitrogen).
  • LAPC4 was maintained in IMDM medium supplemented with 10 nM R1881 and 10% FBS (Invitrogen).
  • VCaP was maintained in DMEM (Cellgro) supplemented with 10% FBS. All media were also supplemented with antibiotics (penicillin and streptomycin). PrEC was obtained from Lonza and maintained as suggested by the instruction provided. RNA was obtained from these cell lines using TRI reagent (Sigma).
  • RNA Processing for Paired-End Transcriptome Sequencing Total RNA samples with RNA integrity number (RIN) >8 were used for transcriptome sequencing using Illumina mRNA-seq protocol. Briefly, 5 ⁇ g of total RNA was used to isolate mRNA using Sera- mag Magnetic Oligo(dT) beads. mRNA was then fragmented and converted into double- stranded cDNA. Adapters were ligated to the double- stranded cDNA and this library was then size selected to obtain fragments in the range of 200-300 bp. Finally, PCR amplification was performed to obtain the final cDNA library.
  • RIN RNA integrity number
  • a total of 10 nM of the library was then used for 36- nucleotide paired-end sequencing on the Illumina genome analyzer II (GAII) at the Center for Cancer Epigenetics Solexa Sequencing Core located in the University of Texas-M. D. Anderson Cancer Center.
  • GAII Illumina genome analyzer II
  • Bioinformatic Identification of Chimeric RNAs Our bioinformatics pipeline had several filters to remove false-positive chimeric RNAs.
  • the first step in the pipeline was a quality filter in which only those reads that passed the Illumina/Solexa signal purity filter were used for further analyses.
  • the second step was a uniqueness filter.
  • the inventors required that both ends of paired reads be uniquely mapped to either the genome or the transcriptome; i.e., the inventors required each read to map to only one location in the genome or transcriptome even with the tolerance of two mismatches. This additional stringent step prevented any homolog from being recruited as a chimera.
  • the output of this step was paired reads that we called "unique pairs.”
  • the inventors assigned Entrez gene IDs to each read of the unique pair and looked for each read to be assigned to one unique Entrez ID.
  • Step four of the pipeline involved the comparison of the Entrez gene ID for the unique pairs of reads. Unique pairs where both reads were assigned to the same Entrez gene ID were discarded. Only those unique pairs where each read was assigned a different Entrez gene ID were considered a candidate chimeric event.
  • the UCSC knownGene database March 2006, hgl8 for alignment. Using this bioinformatic pipeline, the inventors obtained ⁇ 6,163 putative chimeric events with at least three supporting paired reads from three different patient samples. Of these, chimeric events that involved either KLK2 or KLK3 were removed as the inventors were unable to validate these events.
  • RNA junctions were accurately defined using RT-PCR and Sanger sequencing and then used as templates to align junction reads. Reads that were earlier unmappable to the genome and the transcriptome were aligned to the PCR amplicon.
  • a paired read was considered a junction read only if it met the following conditions: (i) one read of the paired read mapped to either parental gene of the chimeric RNA; (ii) the junction read should overlap with at least six nucleotides of the sequence on either side of the RNA junction; and (iii) mismatch tolerance was set at two mismatches, but for the six nucleotides flanking the RNA junction, no mismatches were tolerated.
  • FIG. 3 was generated using Mev (v4.5) (Conover, 1971; Saeed et al., 2006). For FIG. 3, supporting paired chimeric reads and junction reads were pooled together and normalized to 10 million mappable reads.
  • strand-specific RT-PCR For strand- specific RT-PCR, the following protocol was performed to avoid endogenous priming. All incubations were performed in a thermal cycler. A total of 500 ng of RNA was incubated with 1 ⁇ L ⁇ of each strand- specific primer (4 ⁇ ) at 70 °C for 5 min and then at 50 °C for 10 min. At the same time, a master mix containing dNTPs, lx superscript buffer, 10 mM DTT, 5 mM magnesium chloride, and RNaseOUT was also incubated at 50 °C for 5 min. The master mix was then quickly added to the RNA-primer mix while trying to keep reactions at 50 °C.
  • the above first- strand synthesis was performed using either the forward primer or the reverse primer (primers listed in the later section). Then, two PCR reactions were performed using the cDNA generated from the first step. The strand was determined on the basis of which reaction yielded a product.
  • Two-step PCR was performed with annealing and extension at 68 °C. Extension times were varied depending on the expected size of products: for 2-3 kb, 3 min; for 4-8 kb, 5 min; and for 10-30 kb, 15 min. Products were run on gels and the sizes of PCR products obtained from cancer samples and control human genomic DNA were compared. A detectable difference in PCR product size would indicate a DNA rearrangement.
  • Quantitative RT-PCR Validation Quantitative RT-PCR was performed using Absolute Blue QPCR SYBR Green low ROX mix (Thermo Scientific) on Applied Biosystems' 7500 real-time PCR system. Oligonucleotide primers used for quantitative RT-PCR are listed in the next section. A final concentration of 100 nM of each primer was used in the PCR. The program used for PCR was as follows: 95 °C for 15 min (1 cycle); and 95 °C for 15 s and 60 °C for 1 min (40 cycles). All assays were performed in triplicate and the results are shown as average fold change relative to GAPDH, which serves as an internal control.
  • Quantitative RT-PCR was done only for the chimera TMEM79-SMG5 in the human samples identified in FIG. 6 and PrEC cells. Quantitative RT-PCR was not performed for all chimeras in cell lines or tissue samples.
  • TMPRSS2-ERG and GAPDH primers are from Maher et al. (2009).
  • the full-length chimeric RNA sequence allows the prediction of fusion protein(s) translated from the chimeric RNA. Antibodies specifically raised to recognize the unique amino acid sequence but not the normal proteins could detect the presence of fusion protein. This would allow specific detection of fusion proteins in patients' blood or urine, for example, for diagnostic or prognostic purposes. As indicated below, the full-length sequence predicts a long and different amino acid sequence for each of the chimeric RNAs.
  • ASTN2-PAPAA and GOLM1-MAK10 were present in human esophageal squamous cell cancer. Importantly, they are highly enriched in cancer vs. normal tissues and thus are useful as biomarkers for identifying esophageal squamous cell cancer patients.
  • ASTN2-PAPAA and GOLM1-MAK10 are important chimeric RNAs, their splicing isoforms were identified in human prostate and esophageal cancer samples. The exact sequences near the chimeric junctions are shown below. These isoforms provide additional options when used to diagnose their presence in patients.
  • isoform 2 (SEQ ID NO:6) [0235] GTGCTAATTTTACAGGGAGAAAC CAGCAGAAACTGAGAGGAGA AGATGACTAC AACATGGATG AAAATGAAGC AGAATCTGAG
  • NC refers to a negative control starting from PicoSI kit without input RNA.
  • GOLM1-MAK10 tested positive in Patient 2.
  • TMPRSS2-ERG tested negative in both patient samples. This indicates that chimeric RNAs are present at a detectable level in urine collected after digital rectal exam.
  • Digital rectal exam requires a visit to the doctor and is an uncomfortable procedure for patients.
  • Urines without digital rectal exam can be collected by a patient himself at home and mailed for analysis.
  • FIG. 11 shows results of the assay for the exemplary chimeric RNAs noted above. Positive controls used previously isolated PCR bands as templates. FIG. 11 shows that the exemplary PCR conditions (30 ng of cDNA as template in a 50 ⁇ PCR reaction) were suitable. Thirteen out of 24 chimeric RNAs were present in the exosomes of urine samples, which indicates that chimeric RNAs are present at a detectable level in urine collected without digital rectal exam.
  • RNAs Two chimeric RNAs (number 4 and number 19) were present in patients, but absent in normal control. Four chimeric RNAs were present in normal control, but absent in cancer patients. One can next evaluate additional chimeric RNA candidates.
  • Table 5 Exemplary chimeric genes associated with prostate cancer Gene

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Abstract

The present invention encompasses embodiments for utilizing chimeric RNAs in association with cancer. In particular embodiments, the presence of cancer or an increased risk for developing cancer is determined upon identification of one or more particular chimeric RNAs. In specific aspects, one or more chimeric RNAs are utilized in diagnosis of prostate cancer. In certain aspects, the chimeric RNA is TMEM79-SMG5 RNA.

Description

RECURRENT CHIMERIC RNAS ENRICHED IN HUMAN PROSTATE CANCER AS
BIOMARKERS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 61/480,005 filed April 28, 2011, which is incorporated by reference herein in its entirety.
[0002] This invention was made with government support under Department of Defense Idea Development Award PC093918 awarded by the Department of Defense. The government has certain rights in the invention.
TECHNICAL FIELD
[0003] The present invention concerns at least the fields of molecular biology, cell biology, and medicine, including cancer medicine. In particular aspects, the present invention concerns diagnostics of cancer.
BACKGROUND OF THE INVENTION
[0004] Chimeric RNAs unrelated to chromosomal rearrangements are primarily generated by two transcription-induced mechanisms (1, 2). The first one is "read- through/splicing," where a chimeric RNA begins at the upstream gene and ends at a termination point of the adjacent downstream gene, with the region in between removed by splicing. The second one is "transsplicing." Here two separately generated RNAs are spliced together to give rise to a single RNA. Both mechanisms result in fused transcripts that possess sequences from both genes. Although the exact cause and regulation of chimeric RNAs are unknown, chimeric RNAs, like gene fusions that result from chromosomal rearrangements, are expected to increase the proteomic diversity in cells through chimeric proteins or altered regulation of participating mRNAs. Despite their importance, few studies have reported on chimeric RNAs, especially regarding their presence and roles in human cancers (3, 4). The lack of reports of chimeric RNAs may stem from the fact that current models of fusion transcripts primarily emphasize chromosomal rearrangements. Alternatively, chimeric RNAs may not have been observed previously because of methods of analyses such as microarrays that lack the necessary resolution for chimeric RNA discovery. Recent advances in highthroughput sequencing technology, which provides single -base resolution of transcribed RNAs, have enabled unique discoveries of chimeric RNAs from chronic myeloid leukemia cell lines and cultured human melanoma (5, 6). In prostate cancer, the same technology led to the identification of SLC45A3-ELK4. This chimeric RNA is significant in several ways. First, it was present at high levels in the urine of patients with prostate cancer (7, 8), pointing to the potential value of chimeric RNAs as noninvasive biomarkers. Second, its expression is androgen regulated and thus a potential target for androgen ablation therapy. Finally and most intriguingly, unlike the fusion gene TMPRSS2- ERG (9, 10) that results from chromosomal rearrangement, the chimera of SLC45A3-ELK4 appears to be generated by RNA processing without DNA-level rearrangement (7, 8). Thus, SLC45A3-ELK4 may represent a unique class of transcriptional events with important implications in cancer that may have been previously overlooked. This result raises the possibility that chimeric RNAs in cancers are underinvestigated and mostly unknown.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention is directed to methods and compositions related to cancer, including diagnostics and/or treatment of cancer, for example. In some embodiments, there are methods and compositions related to chimeric RNAs associated with the presence of cancer or, in alternative embodiments, lack thereof. In certain embodiments, the invention concerns identification of the presence of one or more particular chimeric RNAs and its association with cancer or increased risk of cancer in a mammal, including a human, dog, cat, or horse, for example. In specific embodiments, detection of one or more particular RNAs allows for the detection of a stage of a certain cancer. In some embodiments of the invention, there are chimeric RNAs that are enriched in one or more types of cancer or risk thereof. In particular embodiments, there are chimeric RNAs that whose absence is indicative of one or more types of cancer or risk thereof. In certain embodiments, the chimeric RNAs referred to herein are utilized to determine the stage of a particular cancer, to monitor treatment efficiency, and/or to determine if a cancer has returned.
[0006] The cancer that is associated with one or more chimeric RNAs may be of any type of cancer, including primary or metastatic. Exemplary types of cancer include prostate, esophageal, lung, breast, brain, melanoma, colon, pancreatic, stomach, kidney, liver, bladder, ovarian, cervical, testicular, rectal, spleen, gall bladder, thyroid, and so forth. [0007] In some embodiments of the invention, there are recurrent chimeric RNAs that are enriched in human prostate cancer identified by deep sequencing.
[0008] In embodiments of the invention, the inventors took advantage of the analytical power of paired-end high-throughput sequencing (11, 12) to characterize chimeric RNAs enriched in human prostate cancer as an example of a cancer type. The transcribed mRNA (transcriptome) was sequenced from a cohort of patients with human prostate cancer, yielding 1.3 billion raw sequence reads. This sequencing coverage enabled a "deep" survey of chimeric RNAs expressed from the complex human genome, leading to the validation of 32 recurrent but exemplary chimeric RNAs. Among them, 27 chimeric RNAs have not been described before. Importantly, at least one of these chimeras is highly cancer enriched, as it is expressed at significantly higher levels in human prostate cancers but present at very low levels in noncancer prostates. This indicates that recurrent chimeric RNAs are more common than previously thought. The fact that there are more chimeric RNAs in cancer than in matched benign samples indicates that in at least certain aspects increased chimeric RNA events represent one of the molecular consequences of cancer.
[0009] In some embodiments of the invention, there is a composition of matter comprising one or more isolated chimeric RNAs provided herein, and in specific aspects the chimeric RNA is not TMPRSS2-ERG, SLC45A3-ELK4, ANKRD39-ANKRD23, HARS2- ZMAT2, or SMG5-PAQR6. In particular embodiments, there is a composition of matter comprising an isolated chimeric TMEM79-SMG5 RNA. In some embodiments of the invention, TMEM79-SMG5 and SMG5-PAQR6 are two separate chimeric RNAs, although in some embodiments they are a single chimeric RNA that contains three genes: TMEM79-SMG5- PAQR6.
[0010] In some embodiments of the invention, there is a composition of matter comprising one or more isolated chimeric RNAs amplifiable by PCR primers provided herein, and in specific aspects the chimeric RNA is not TMPRSS2-ERG, SLC45A3-ELK4, ANKRD39- ANKRD23, HARS2-ZMAT2, or SMG5-PAQR6.
[0011] In particular embodiments, there is a substrate comprising polynucleotides attached thereto, said polynucleotides defined as one or more isolated chimeric RNAs of the invention. In at least some cases, all or greater than 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polynucleotides attached to the substrate are one or more isolated chimeric RNAs of the invention.
[0012] In some embodiments of the invention, there is a method of detecting the presence of cancer, an increased risk for cancer, or response to cancer therapy in an individual, comprising the step of detecting one or more chimeric RNAs of the invention in a sample from the individual. In some cases, at least one of the chimeric RNAs is TMEM79-SMG5 RNA or GOLMJ-MAKJO RNA. In particular embodiments of methods of the invention, the individual is provided a therapy upon detection of the one or more chimeric RNAs. In specific embodiments, the chimeric RNA is detected in nucleic acid form, such as by polymerase chain reaction, including wherein the polymerase chain reaction amplifies all or part of the chimeric RNA. In specific embodiments, the polymerase chain reaction amplifies the junction of the chimeric RNA. In some embodiments, the chimeric RNA may be detected by its expressed fusion protein, and in specific cases the fusion protein is detected by antibody. In specific
embodiments, the antibody recognizes the junction of the fusion protein. Chimeric RNA can also be detected by RNA hybridization techniques such as northern blot or in situ hybridization.
[0013] In embodiments of the invention that comprise obtaining a sample from an individual known or suspected to have cancer or have an increased risk of cancer, the sample may be obtained in any suitable manner. In specific embodiments, the sample comprises serum, urine, prostatic fluid, circulating cancer cells, or biopsy, for example.
[0014] In certain embodiments, methods of the invention comprise obtaining a sample from the individual. In specific embodiments of the invention, the analysis of a sample from an individual is performed by the individual or entity that obtained the sample. In certain embodiments, the analysis of a sample from an individual is performed by an individual or entity other than the individual or entity who obtained the sample. In some embodiments, methods of the invention comprise the step of obtaining the sample from an individual. In some cases, the sample is stored under suitable conditions and the sample is later analyzed by the party that obtained the sample or later analyzed by another party. [0015] In certain embodiments, the individual that has a sample subjected to methods of the invention is also subjected to additional methods of cancer diagnosis. Examples of other methods for prostate cancer include at least analysis of prostate specific antigen, magnetic resonance imaging, analysis of PCA3, and/or analysis of TMPRSS2-ERG. In particular embodiments, the methods of the invention comprising the step of performing an additional cancer detection step on a sample from the individual, and in some cases it is the same sample used for detection of chimeric RNA or it may be a different sample from the same individual.
[0016] In particular embodiments of the invention, one or more particular chimeric RNAs are indicative of the presence or recurrence of cancer, or response to a cancer therapy, or monitoring the stage of a cancer regardless of the type of sample that is assessed for the presence of the one or more particular chimeric RNAs. In some embodiments, however, one or more particular chimeric RNAs are indicative of the presence or recurrence of cancer, or response to a cancer therapy, or monitoring the stage of a cancer when assaying a particular type of sample from an individual. In specific embodiments in the exemplary case of prostate cancer, one or more chimeric RNAs may be diagnostic if it is present in biopsy only, if it is present in urine sample only, present in serum only, or if it is present in a combination of serum, biopsy and urine sample.
[0017] In some embodiments, there is a composition of matter comprising an isolated chimeric RNA selected from the group consisting of TMEM79-SMG5 RNA, ASTN2- PAPAA RNA, GOLMJ-MAKJ0 RNA and one or more chimeric RNAs in Table 1, 2, 3, 4, or 5.
[0018] In certain embodiments, there is a substrate comprising polynucleotides attached thereto, said polynucleotides defined as one or more of the isolated chimeric RNAs. In specific embodiments, all or greater than 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polynucleotides attached to the substrate are one or more isolated chimeric RNAs of TMEM79-SMG5 RNA, ASTN2-PAPAA RNA, or GOLMJ-MAKJ0 RNA.
[0019] In some embodiments, there is a method of detecting the presence of cancer, an increased risk for cancer, or response to cancer therapy in an individual, comprising the step of detecting one or more chimeric RNAs in a sample from the individual. In some aspects, the cancer is androgen- sensitive cancer. In certain cases, the cancer is prostate cancer or esophageal cancer. In some embodiments, the individual is provided a therapy upon detection of the one or more chimeric RNAs.
[0020] In particular embodiments, the chimeric RNA is detected in nucleic acid form, such as by polymerase chain reaction (PCR), including PCR that amplifies all or part of the chimeric RNA or amplifies the junction of the chimeric RNA.
[0021] In specific embodiments, a sample comprises serum, urine, prostatic fluid, or biopsy.
[0022] In some cases, the chimeric RNA is detected by its expressed fusion protein, for example with detection of the fusion protein by antibody. In specific embodiments, the antibody recognizes the junction of the fusion protein.
[0023] Some methods of the invention encompass the step of performing an additional cancer detection step on a sample from the individual. In certain aspects, the method further comprises the step of obtaining a sample from the individual.
[0024] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawing, in which:
[0026] FIG. 1. Strategy for identification and validation of chimeric RNAs. Paired reads are mapped to both the genome and the transcriptome, and if each of the paired reads maps to different genes, then it is considered to be a paired "chimeric" read representative of a putative chimeric event. On the basis of the alignment and location of paired chimeric reads, primers (arrows) are designed to amplify the putative RNA junction from patients' RNAs.
RTPCR and Sanger sequencing lead to the identification of the RNA junction of the chimera. Using the RNA junction as template, previously unmappable reads are aligned to the template and thus "junction" reads are identified. These junction reads have one read aligning to the RNA junction and the other read to one of the genes.
[0027] FIG. 2. Global analysis of chimeric RNAs. (A) A total of 2,369 putative chimeric events were identified on the basis of stringent bioinformatic criteria described in Example 9. The frequency of distribution based on reads from all samples for all 2,369 distinct chimeric events is shown. Events chosen for experimental validation are shown in purple. (B) Comparison of the average number of paired chimeric reads showed that chimeric reads are significantly more abundant in cancer samples than in matched benign samples.
[0028] FIG. 3. Chimeric RNAs are highly recurrent in prostate cancer. The relative frequency of recurrence of chimeric RNAs in each cancer and matched benign sample is shown. Each vertical column represents data from one patient sample and each horizontal row represents relative abundance for a particular chimeric event in each patient sample. Yellow indicates high occurrence and black low occurrence. C, cancer sample; N, matched benign samples. The white line separates cancer from matched benign samples. The P value obtained by the Kolmogorov- Smirnov test for each chimeric RNA is shown on the right. All data are compared at the same scale except TMPRSS2-ERG, which is normalized to a different scale so that its relative expression level can be compared between samples without color saturation. [0029] FIG. 4. Examples of chimeric RNA and parental gene expression in additional patient cohorts and prostate cancer cell lines. (A) Results of RT-PCR analysis for the indicated chimeric RNAs and their parental genes in 10 cancer samples (C41-C50) and 5 noncancer donor samples (D1-D5). TMEM79-SMG5 is undetectable in the donors without cancer whereas the parental genes SMG5 and TMEM79 are expressed. (B) Results of RT-PCR analysis for the indicated chimeric RNAs in prostate cell lines PrEC, PNTla, LNCaP, LAPC4, VCaP, DU145, and PC3. This subset of chimeric RNAs is undetectable in PrEC. As expected, TMPRSS2-ERG is highly expressed in VCaP, whereas the control actin is expressed in all cell lines. (C) Expression of parental genes in PrEC for those chimeric RNAs shown in B. Although this subset of chimeric RNAs is not expressed in PrEC, the expression of many parental genes is clearly detected.
[0030] FIG. 5. Schematic of TMEM79-SMG5 chimeric RNA in prostate cancer. Chimeric RNAs can result in a new 5 '-UTR and truncated ORF as in the case of TMEM79-SMG5. In the pictogram, coding exons are represented by blocks connected by horizontal lines
representing introns. The 5'- and 3'-UTRS are represented by shorter blocks. Arrows indicate direction of transcription of parental genes. Dashed lines indicate the boundaries of the sequence contributing to chimeric RNAs. Splice junctions are shown as uppercase letters. The sequences in black are part of the introns removed in the formation of the chimeric RNAs.
[0031] FIG. 6. The TMEM79-SMG5 chimera as a potential diagnostic marker. (A) The relative expression level of the TMEM79-SMG5 chimera (normalized to GAPDH) in PrEC, 18 donors without cancer, and 54 patients with prostate cancer, determined by quantitative RT- PCR. These additional samples were not used for high-throughput sequencing. (B) Box plot showing the range of relative expression levels in patients with prostate cancer vs. donors without cancer. TMEM79-SMG5 was expressed at a significantly high level in patients with prostate cancer (P value = 1.048 e-05). Sample minimum and maximum are represented by horizontal lines. The box represents the lower and upper quartile separated by a thick line, which is the median. The circles represent values that are considered to be outliers.
[0032] FIG. 7. Identification of junction by Sanger sequencing and bioinformatics. (A) RT-PCR products were gel purified and subjected to Sanger sequencing. Shown are examples of traces from Sanger sequencing for three chimeric transcripts: FAM18B2-CDRT4, TSPANl-POMGNTl, and ZNF606-C19orfl8. The identified RNA junction is separated by the thick black line. Traces show that RT-PCR products as a population were homogenous, indicating that there as no variation in RNA junctions in the region defined by the primer pairs. (B) RNA junction of TSPANl-POMGNTl was first determined by RT-PCR and Sanger sequencing. Previously unmappable reads were then aligned to the determined junction of TSPANl-POMGNTl. The number of reads now mapping to this junction is represented by the blue line. The vertical dashed red line points to the junction site. This mapping supports the RNA junction identified by Sanger sequencing. When the order of genes is switched to generate an artificial RNA junction, reads fail to map to this junction (gray line).
[0033] FIG. 8. Gene expression levels determined by high-throughput sequencing. RPKM refers to reads per kilobase of exon model per million mapped reads. The x axis represents the expression level of genes in cancer samples and the y axis represents the expression level in matched benign samples. Genes aligned with the diagonal line are expressed at equal levels in cancer and matched benign samples. Red circles indicate parental genes involved in validated chimeric RNAs from this study. Most of the parental genes involved in validated chimeric RNAs are not biased toward highly expressed genes and are not
overexpressed in cancer. Light blue circles refer to TMEM79 and SMG5 genes.
[0034] FIG. 9. Chimeric RNAs are not supported by DNA level rearrangements. A few examples of long-range PCR assay are shown to determine whether chimeric RNAs are a result of DNA rearrangements. C refers to the DNA from the cancer sample in which the chimeric RNA was expressed and H refers to control human genomic DNA. Absence of size differences between C and H reactions indicates that there were no gross DNA rearrangements. As a positive control for long-range PCR reactions, DNA samples and primers were used that were provided by the LA PCR kit from Takara. There was successful amplification of DNA products with a size of 17.5 kb. To ensure that the PCR primers designed for each chimera can amplify the intended DNA fragment, human genomic DNA was used as a positive control.
[0035] FIG. 10 demonstrates an assay for GOLM1-NAA35 in urine of patient samples that had digital rectal exam. The patient' s urine had an undetectable level of
TMPRSS2-ERG. [0036] FIG. 11 demonstrates an assay for 24 exemplary chimeric RNAs in prostate cancer patients vs. controls in samples from the exosome of urine from patients that did not have digital rectal exam.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. The terms "comprise" (and any form of comprise, such as "comprises" and "comprising"), "have" (and any form of have, such as "has" and "having"), "include" (and any form of include, such as "includes" and "including") and "contain" (and any form of contain, such as "contains" and "containing") are open-ended linking verbs. As a result, a method or device that "comprises," "has," "includes" or "contains" one or more steps or elements possesses those one or more steps or elements, but is not limited to possessing only those one or more elements. Likewise, a step of a method or an element of a device that "comprises," "has," "includes" or "contains" one or more features possesses those one or more features, but is not limited to possessing only those one or more features. As used herein "another" may mean at least a second or more. In specific embodiments, aspects of the invention may "consist essentially of or "consist of one or more sequences of the invention, for example. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
[0037] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. [0038] The term "chimeric RNA" as used herein refers to transcribed RNAs that contain sequences from two distinct genes.
II. General Embodiments of the Invention
[0039] In general embodiments of the invention, there are provided methods and compositions concerning chimeric RNAs or associated compositions thereof. In specific embodiments, chimeric RNAs are utilized in methods of diagnosis of one or more types of cancer. In some embodiments, reagents that target the chimeric RNAs are utilized in the treatment of cancer that has cancer cells having one or more particular chimeric RNAs.
[0040] Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, the inventors obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were unique and highly recurrent in cancer samples. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, indicating their associations with cancer. These chimeras contain discernable 5' and 3' splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, indicating that other factors are involved in their production and regulation. One exemplary chimera,
TMEM79-SMG5, is highly differentially expressed in human cancer samples and is a useful biomarker, in embodiments of the invention. The prevalence of chimeric RNAs allows the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. In embodiments of the invention, chimeric RNAs are widespread, and increased chimeric RNA events represent a unique class of molecular alteration in cancer.
[0041] In some aspects of the invention, one or more compositions that target one or more chimeric RNAs are employed for the treatment of cancer. In particular aspects, the cancer has been determined to have the chimeric RNA, whereas in other aspects the cancer has not been determined to have the chimeric RNA. The composition may be of any kind, so long as it is able to directly or indirectly target the chimeric RNA. In specific embodiments, the composition that targets the chimeric RNA is a polypeptide or small molecule, for example. In some embodiments, the composition that targets the chimeric RNA targets the junction site between the two or more respective components of the chimeric RNA. However, in some embodiments the composition that targets the chimeric RNA does not target the junction site and targets one or the other of the chimeric components. In certain embodiments, the composition that targets the chimeric RNA is an antibody that recognizes the chimeric RNA gene product or, in alternative cases, the chimeric RNA itself. In particular aspects of the invention, the antibody immunologically recognizes the junction site of the gene product.
III. Nucleic Acid Detection
[0042] In certain embodiments of the invention, one or more chimeric RNAs are detected in a cancer sample from an individual. The chimeric RNA(s) may be detected by any suitable means.
[0043] 1. Hybridization
[0044] The use of a polynucleotide between 13 and 100 nucleotides, preferably between 17 and 100 nucleotides in length, or in some aspects of the invention up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length are generally preferred, to increase stability and/or selectivity of the hybrid molecules obtained. One will generally prefer to design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.
[0045] Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of DNAs and/or RNAs or to provide primers for amplification of DNA or RNA from samples. Depending on the application envisioned, one would desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of the probe or primers for the target sequence.
[0046] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.
[0047] For certain applications, for example, site-directed mutagenesis, it is appreciated that lower stringency conditions are preferred. Under these conditions,
hybridization may occur even though the sequences of the hybridizing strands are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20°C to about 55°C. Hybridization conditions can be readily manipulated depending on the desired results.
[0048] In other embodiments, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1.0 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, at temperatures ranging from approximately 40°C to about 72°C.
[0049] In certain embodiments, it will be advantageous to employ nucleic acids of defined sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of being detected. In preferred embodiments, one may desire to employ a fluorescent label or an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a detection means that is visibly or spectrophotometrically detectable, to identify specific hybridization with complementary nucleic acid containing samples.
[0050] In general, it is envisioned that the probes or primers described herein will be useful as reagents in solution hybridization, as in PCR™, for detection of expression of corresponding genes, as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single- stranded nucleic acid is then subjected to hybridization with selected probes under desired conditions. The conditions selected will depend on the particular circumstances (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Optimization of hybridization conditions for the particular application of interest is well known to those of skill in the art. After washing of the hybridized molecules to remove non- specifically bound probe molecules, hybridization is detected, and/or quantified, by determining the amount of bound label.
Representative solid phase hybridization methods are disclosed in U.S. Patent Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of hybridization that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,849,481, 5,849,486 and 5,851,772. The relevant portions of these and other references identified in this section of the Specification are incorporated herein by reference.
[0051] 2. Amplification of Nucleic Acids
[0052] Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et ah, 1989). In certain embodiments, analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid. The nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA. [0053] The term "primer," as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process. Typically, primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double- stranded and/or single- stranded form, although the single- stranded form is preferred.
[0054] Pairs of primers designed to selectively hybridize to nucleic acids corresponding to one or more chimeric RNAs are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences. Once hybridized, the template- primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
[0055] The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
[0056] A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al, 1988, each of which is incorporated herein by reference in their entirety.
[0057] A reverse transcriptase PCR™ amplification procedure may be performed to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et ah, 1989). Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent No. 5,882,864. Representative primers for PCR analysis of chimeric RNAs are provided in Table 1.
Forward primer (5'-3') Reverse primer (5'-3')
Chimeric RNA
GGAGTAGGCGCGAGCTAAG (SEQ ID GTCCATAGTCGCTGGAGGAG (SEQ ID
TMPRSS2-ERG NO:39) NO:90)
ACCAGGGCTGCAGGCCGCTACCA (SEQ CTTCCCGTCCTCCGTCTTGTGCTC (SEQ
VPS26B-NCAPD3 ID NO:40) ID NO:91)
GGAGGTGATGAAAATATTATC (SEQ ID GGAAAAGGGAGACGATTGTTC (SEQ
XPA-NCBP1 NO:41) ID NO:92)
GGACTCTGTGTCTCAGTCGCT (SEQ ID TGCTCCCTCCTCCTGCAACACA (SEQ
ANKRD39-ANKRD23 NO:42) ID NO:93)
CAGAGCTTTGATAGCAGCAGCA (SEQ GGTTAAGCCCTGTTTAAAATGCA (SEQ
RASL12-OSTbeta ID NO:43) ID NO:94)
TTCCTCACCGCTGACATGGAG (SEQ ID GAATACTTTATTTGGAGGGAG (SEQ ID
ELF3-RNPEP NO:44) NO:95)
CTGTGGTTTAGAGTCTCTAGCTTAC GTGTTCAACCAGAGACCAAAAC (SEQ
TMEM79-SMG5 (SEQ ID NO:45) ID NO:96)
CAGTCTCTCCGAGTTGGCAACA (SEQ AATGCCTCATCCCTACTCCAGA (SEQ
NARG1-NDUFC1 ID NO:46) ID NO:97)
TTTCCCCCATTCTGTTGCAATG (SEQ ID AGTCATGGAGGGGAGGGTTGTAGA
TSPAN1-POMGNT1 NO:47) (SEQ ID NO:98)
TCTTGGTGGCTGGGTCCAGGTC (SEQ CCTCACTGCCTGCTCCCCTATG (SEQ
SLC44A4-EHMT2 ID NO:48) ID NO:99)
GCCATCCCTCGAGGAAGTTTC (SEQ ID GCATGCATTCCAGTTCTCTAG (SEQ ID
FAM18B2-CDRT4 NO:49) NO: 100)
AGCAGCTATTCTGCATGGGTGCA (SEQ TTTCTCTCTCTTCCGTGAGCCTC (SEQ
HARS2-ZMAT2 ID NO:50) ID NO: 101)
AGGATCATTGGTCTGGTAGCTCAGT AGCCTTGGCTCTCTGGATCCCT (SEQ
KRTCAP3-IFT172 (SEQ ID NO:51) ID NO: 102)
ACCTGGTCGGGGAGGAGACCA (SEQ GCTGCCTGCACACGACAGAGGCCT
MTG1-LOC619207 ID NO:52) (SEQ ID NO: 103)
TCTGTGACCCCACGACCCCTAAGCCA TTCTTGAGGTGCTGGCTGCTGCCCA
NOS1AP-Clor 226 (SEQ ID NO:53) (SEQ ID NO: 104)
CAGAACACGCTCCGAGGATTC (SEQ ID GGACAAGGGTGGCTCCCACCCA (SEQ
C9orfl63-SEC16A NO:54) ID NO: 105)
TTGCCAGGACGCCTTCCCCGA (SEQ ID GGATATGGAAGAACGAAGCCAGGT
DHCR24-Clorfl77 NO:55) (SEQ ID NO: 106)
GCAGATCCTGCCCTACACAC (SEQ ID AGCTGAAGAAGGAACTGCCA (SEQ ID
SLC45A3-ELK4 NO:56) NO: 107)
AGAGCATCAGTGATGTCACGTTTG TGAGCTTTACTCACGTGGCCCT (SEQ
NCAPD3-JAM3 (SEQ ID NO:57) ID NO: 108)
AGGAGAGGCTCCTTGAAGCTGCT (SEQ GACCTGGTGGACTTGAAGAAGT (SEQ
SMG5-PAQR6 ID NO:58) ID NO: 109)
TCACGCCTCTCAATGTACCGCGCCA TGGGCAGTATCTGTGGCTCTC (SEQ ID
GPT-PPP1R16A (SEQ ID NO:59) NO: 110)
GCAAGGCCTGTGTGGTTGGAGAG (SEQ AGTGCCCTATCTTTTCTGCCGCA (SEQ
ASTN2-PAPPA ID NO:60) ID NO: 111)
GTGCTAATTTTACAGGGAGAAAC (SEQ TCAAACCGTGGTGCTGAGGATTC (SEQ
GOLM1-MAK10 ID NO:61) ID NO: 112)
CCATTGCAGAAGTGAGGTCATCCT ATGTTACGAGAGCGGGCTGCGT (SEQ
ACTR8-IL17RB (SEQ ID NO:62) ID NO: 113) TAGGTGGAAGAAGAGCCCGAAGAAGA ACAAGTCTTTCGGTCGGAGAACT (SEQ
HSP90B1-C12orf73 (SEQ ID NO:63) ID NO: 114)
GTGGAAAATCCTGTAGCCAGATGGCT ATCTCGAGGCAGCCCCTTGAATC (SEQ
ZNF606-C19orfl8 (SEQ ID NO:64) ID NO: 115)
ACAGGCCTTCACTTGTGAACATC (SEQ TGGGGAAGATGCTGCGTCCA (SEQ ID
C12orf76-ANKRD13A ID NO:65) NO: 116)
ACCCCAAGTCTCCTTCTCCTGCA (SEQ CAGCAGCAACAGCAGCAGATC (SEQ
SLC16A8-BAIAP2L2 ID NO:66) ID NO: 117)
CCCAGCTTCTGGAACTCACCGGACA CCTGGAAGCAACAGTTATTCTTCCCCA
BC035340-MCF2L (SEQ ID NO:67) (SEQ ID NO: 118)
GCTCCGCAGGCCGTTCACAGCAGTC GGATGCCAGACACTGAATAAAC (SEQ
BC160930-MC1R (SEQ ID NO:68) ID NO: 119)
CCTCACTTAGTGGTTGCTGGCACA ATGGTGTAGCCGTGGGTGGAACTGT
TMEM165-CLOCK (SEQ ID NO:69) (SEQ ID NO: 120)
AAGCTTACTAAAATGCAGGCT (SEQ ID ACAGATCTGTAGCAGGAGGGCA (SEQ
RAPGEF3-SLC48A1 NO:70) ID NO: 121)
Parental genes
TCCTGTCATGCCAGGGAGTCATTCA TACGCTGATGGTAAGACAACCT
NCBP1 (SEQ ID NO:71) (SEQ ID NO: 122)
GTTGTTCATCAACCAGGACCTGT (SEQ GTAAGAAGGCAATCACAGACATGA
XPA ID NO:72) (SEQ ID NO: 123)
TGGACCACAACAGCGAGTCCA (SEQ ID AGGTCTTTCCGGCTGTGTTCT (SEQ
PAPPA NO:73) ID NO: 124)
GTCCAGCACCACTACAACTCTCA (SEQ CAGGGTCCCACAAACTCAATAATGT
ASTN2 ID NO:74) (SEQ ID NO: 125)
CGCAGGTTCTGGACTGGATCAGCT TCAGCTTCTCGTAGGTCATGTTGCT
ELF3 (SEQ ID NO:75) (SEQ ID NO: 126)
GTACACCTGCTTGGAGGCTGCA (SEQ ACAGTTGGGCTAGCTCTTCAGCA
RNPEP ID NO:76) (SEQ ID NO: 127)
GAAGCAAAAGTCCTCCACACT (SEQ ID GGCTTCCACATTATAGTACTTGCT
SMG5 NO:77) (SEQ ID NO: 128)
GCCTGATCTACACACTGCGCT (SEQ ID CAGCATCGACAGCAGTGGCAGA
TMEM79 NO:78) (SEQ ID NO: 129)
TTTGATAGCAGCAGCAGCTACCT (SEQ CCCTTCAGGAGAGTCAGGGTAG
RASL12 ID NO:79) (SEQ ID NO: 130)
GTCTTCACGGCTTCTCTGCCCA (SEQ ID CTCATCCAAATGCAGGACTTCT (SEQ
OSTBETA NO:80) ID NO: 131)
TCCAGGTGGTGAATGAGGAGCCTCA TGTGACACCTTATTAGACACTTCCA
GOLM1 (SEQ ID NO:81) (SEQ ID NO: 132)
GCAGAGAAGGTTGATGCAGCGCT CCAGTCTCTCTCAAACAAGTTTCACA
MAK10 (SEQ ID NO: 82) (SEQ ID NO: 133)
CCTTGCCGTGCTGGAATTCACGT (SEQ AACTCAACAGATGGTGAATCCAGA
NDUFC1 ID NO:83) (SEQ ID NO: 134)
TCGAGATATTCAAGGCTGAAGCA (SEQ ATGCTCTCTGCGCAGGTCGAAGCT
NARG1 ID NO: 84) (SEQ ID NO: 135)
AGAGCACTTCACAGATGAACAGCGA AAGTGAAAGGCAGCACTCCTAAGCT
NCAPD3 (SEQ ID NO: 85) (SEQ ID NO: 136)
TCCAGCAATCGAACCCCAGTGGTACA GGATGTAGTTAACTCCATCTGGT
JAM3 (SEQ ID NO: 86) (SEQ ID NO: 137)
GCTCGTCGTCGACAACGGCTC (SEQ ID CAAACATGATCTGGGTCATCTTCTC
ACTIN NO:87) (SEQ ID NO: 138)
Quantitative RT-PCR
GTTGCAAAGGATGAGGTCAAGT (SEQ CAGCTACCTGACCAGATTGCCACA
TMEM79-SMG5 ID NO:88) (SEQ ID NO: 139) TGCACCACCAACTGCTTAGC (SEQ ID GGCATGGACTGTGGTCATGAG (SEQ
GAPDH NO:89) ID NO: 140)
[0058]
Primers used for validation of chimeric transcripts. GAPDH primers are from Maher et al, 2009a.
[0059] Another method for amplification is ligase chain reaction ("LCR"), disclosed in European Application No. 320 308, incorporated herein by reference in its entirety. U.S. Patent 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence. A method based on PCR™ and oligonucleotide ligase assy (OLA), disclosed in U.S. Patent 5,912,148, may also be used.
[0060] Alternative methods for amplification of target nucleic acid sequences that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB
Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety.
[0061] Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as an amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence which may then be detected.
[0062] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'- [alpha-thio] -triphosphates in one strand of a restriction site may also be useful in the
amplification of nucleic acids in the present invention (Walker et al., 1992). Strand
Displacement Amplification (SDA), disclosed in U.S. Patent No. 5,916,779, is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
[0063] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Application WO 88/10315, incorporated herein by reference in their entirety). European Application No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention.
[0064] PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter region/primer sequence to a target single- stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "race" and "one-sided PCR" (Frohman, 1990; Ohara et al, 1989).
[0065] 3. Detection of Nucleic Acids
[0066] Following any amplification, it may be desirable to separate the amplification product from the template and/or the excess primer. In one embodiment, amplification products, or any polynucleotides, are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods (Sambrook et ah, 1989). Separated amplification products may be cut out and eluted from the gel for further manipulation. Using low melting point agarose gels, the separated band may be removed by heating the gel, followed by extraction of the nucleic acid.
[0067] Separation of nucleic acids may also be effected by chromatographic techniques known in art. There are many kinds of chromatography which may be used in the practice of the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, molecular sieve, reverse-phase, column, paper, thin-layer, and gas chromatography as well as HPLC.
[0068] In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under the appropriate excitatory spectra. [0069] In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled. In another embodiment, the probe is conjugated to a binding partner, such as an antibody or biotin, or another binding partner carrying a detectable moiety.
[0070] In particular embodiments, detection is by Southern blotting and hybridization with a labeled probe. The techniques involved in Southern blotting are well known to those of skill in the art (see Sambrook et ah, 1989). One example of the foregoing is described in U.S. Patent No. 5,279,721, incorporated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids. The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention.
[0071] Other methods of nucleic acid detection that may be used in the practice of the instant invention are disclosed in U.S. Patent Nos. 5,840,873, 5,843,640, 5,843,651,
5,846,708, 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is incorporated herein by reference.
[0072] 4. Other Assays
[0073] Other methods for genetic screening may be used within the scope of the present invention, for example, to detect mutations in genomic DNA, cDNA and/or RNA samples. Methods used to detect point mutations include denaturing gradient gel electrophoresis ("DGGE"), restriction fragment length polymorphism analysis ("RFLP"), chemical or enzymatic cleavage methods, direct sequencing of target regions amplified by PCR™ (see above), single- strand conformation polymorphism analysis ("SSCP") and other methods well known in the art.
[0074] One method of screening for point mutations is based on RNase cleavage of base pair mismatches in RNA/DNA or RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definition thus includes mismatches due to insertion/deletion mutations, as well as single or multiple base point mutations.
[0075] U.S. Patent No. 4,946,773 describes an RNase A mismatch cleavage assay that involves annealing single- stranded DNA or RNA test samples to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNase A. For the detection of mismatches, the single-stranded products of the RNase A treatment, electrophoretically separated according to size, are compared to similarly treated control duplexes. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive.
[0076] Other investigators have described the use of RNase I in mismatch assays. The use of RNase I for mismatch detection is described in literature from Promega Biotech. Promega markets a kit containing RNase I that is reported to cleave three out of four known mismatches. Others have described using the MutS protein or other DNA-repair enzymes for detection of single-base mismatches.
[0077] Alternative methods for detection of deletion, insertion or substitution mutations that may be used in the practice of the present invention are disclosed in U.S. Patent Nos. 5,849,483, 5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which is incorporated herein by reference in its entirety.
IV. Immunological Reagents and Methods
[0078] 1. Antibodies
[0079] In certain aspects of the invention, one or more antibodies may be utilized to detect one or more chimeric RNAs of the invention. The antibody may detect the junction of the different regions of the chimeric RNA or it may detect the region alone.
[0080] As used herein, the term "antibody" is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. [0081] The term "antibody" is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab')2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art
(See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference).
[0082] "Mini-antibodies" or "minibodies" are also contemplated for use with the present invention. Minibodies are sFv polypeptide chains which include oligomerization domains at their C-termini, separated from the sFv by a hinge region. Pack et al. (1992) Biochem 31: 1579-1584. The oligomerization domain comprises self-associating .alpha.-helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds. The oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
Generally, minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126.
[0083] Antibody-like binding peptidomimetics are also contemplated in the present invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003 Mar;49(2):209-16 describe "antibody like binding peptidomimetics" (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
[0084] Monoclonal antibodies (MAbs) are recognized to have certain
advantages, e.g., reproducibility and large-scale production, and their use is generally preferred. The invention thus provides monoclonal antibodies of the human, murine, monkey, rat, hamster, rabbit and even chicken origin. Due to the ease of preparation and ready availability of reagents, murine monoclonal antibodies will often be preferred.
[0085] However, "humanized" antibodies are also contemplated, as are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. As used herein, the term "humanized" immunoglobulin refers to an immunoglobulin comprising a human framework region and one or more CDR's from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin .
[0086] 2. Antibody Conjugates
[0087] The present invention further provides antibodies against chimeric RNA fusion proteins, polypeptides and peptides that may be linked to at least one agent to form an antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostic or therapeutic agents, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule. Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules which have been attached to antibodies include toxins, anti-tumor agents, therapeutic enzymes, radio-labeled nucleotides, antiviral agents, chelating agents, cytokines, growth factors, and oligo- or poly-nucleotides. By contrast, a reporter molecule is defined as any moiety which may be detected using an assay. Non-limiting examples of reporter molecules which have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
[0088] Any antibody of sufficient selectivity, specificity or affinity may be employed as the basis for an antibody conjugate. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind pathogens, B- cell superantigens, the T cell co-receptor CD4 and the HIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann et al., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al., 1993; Kreier et al., 1991). In addition, the variable domain is involved in antibody self- binding (Kang et ah, 1988), and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al, 1989).
[0089] Certain examples of antibody conjugates are those conjugates in which the antibody is linked to a detectable label. "Detectable labels" are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the antibody to which they are attached to be detected, and/or further quantified if desired. Another such example is the formation of a conjugate comprising an antibody linked to a cytotoxic or anti-cellular agent, and may be termed "immunotoxins".
[0090] Antibody conjugates are generally preferred for use as diagnostic agents. Antibody diagnostics generally fall within two classes, those for use in in vitro diagnostics, such as in a variety of immunoassays, and/or those for use in vivo diagnostic protocols, generally known as "antibody-directed imaging".
[0091] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, for e.g., U.S. Patent Nos. 5,021,236; 4,938,948; and
4,472,509, each incorporated herein by reference). The imaging moieties used can be
paramagnetic ions; radioactive isotopes; fluorochromes; NMR-detectable substances; X-ray imaging.
[0092] In the case of paramagnetic ions, one might mention by way of example ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper
(II) , neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium
(III) , dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred. Ions useful in other contexts, such as X-ray imaging, include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
[0093] In the case of radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine211, 14carbon, 51chromium, 36chlorine, 57cobalt, 58cobalt, copper 6T , 152 Eu, gallium 6T , 3 hydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron,
32phosphorus, rhenium186, rhenium188, 75selenium, 35sulphur, technicium99"1 and/or yttrium90. 125I is often being preferred for use in certain embodiments, and technicium99"1 and/or indium111 are also often preferred due to their low energy and suitability for long range detection. Radioactively labeled monoclonal antibodies of the present invention may be produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium and/or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium99"1 by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column. Alternatively, direct labeling techniques may be used, e.g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody.
Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylene
diaminetetracetic acid (EDTA).
[0094] Among the fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY- R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein
Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET,
Tetramethylrhodamine, and/or Texas Red.
[0095] Another type of antibody conjugates contemplated in the present invention are those intended primarily for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. Preferred secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each incorporated herein by reference.
[0096] Yet another known method of site-specific attachment of molecules to antibodies comprises the reaction of antibodies with hapten-based affinity labels. Essentially, hapten-based affinity labels react with amino acids in the antigen binding site, thereby destroying this site and blocking specific antigen reaction. However, this may not be advantageous since it results in loss of antigen binding by the antibody conjugate.
[0097] Molecules containing azido groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et ah, 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins (Khatoon et ah, 1989; King et ah, 1989; and Dholakia et ah, 1989) and may be used as antibody binding agents.
[0098] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a
diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N- chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the antibody (U.S. Patent Nos. 4,472,509 and 4,938,948, each incorporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Patent No. 4,938,948, imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate.
[0099] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin, using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region have also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostically and therapeutically promising antibodies which are currently in clinical evaluation.
[0100] In another embodiment of the invention, the antibodies are linked to semiconductor nanocrystals such as those described in U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which are incorporated herein in their entireties); as well as PCT Publication No. 99/26299 (published May 27, 1999). In particular, exemplary materials for use as semiconductor nanocrystals in the biological and chemical assays of the present invention include, but are not limited to those described above, including group II- VI, III- V and group IV semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, A1S, A1P, AlSb, PbS, PbSe, Ge and Si and ternary and quaternary mixtures thereof. Methods for linking semiconductor nanocrystals to antibodies are described in U.S. Patent Nos. 6,630,307 and 6,274,323.
[0101] 3. Immunodetection Methods
[0102] In still further embodiments, the present invention concerns
immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as chimeric RNA gene products or the chimeric RNAs themselves. Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle MH and Ben-Zeev O, 1999; Gulbis B and Galand P, 1993; De Jager R et al., 1993; and Nakamura et al., 1987, each incorporated herein by reference.
[0103] In general, the immunobinding methods include obtaining a sample suspected of containing chimeric RNA fusion protein, polypeptide and/or peptide, and contacting the sample with an antibody in accordance with the present invention under conditions effective to allow the formation of immunocomplexes.
[0104] These methods include methods for purifying chimeric RNA fusion proteins, polypeptides and/or peptides from patients' samples. In these instances, the antibody removes the antigenic chimeric RNA fusion protein from a sample. The antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the chimeric RNA fusion protein antigenic component will be applied to the immobilized antibody. The unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody, which chimeric RNA fusion protein antigen is then collected by removing the chimeric RNA fusion protein or the complex from the column.
[0105] The immunobinding methods also include methods for detecting and quantifying the amount of a chimeric RNA fusion protein reactive component in a sample and the detection and quantification of any immune complexes formed during the binding process. Here, one would obtain a sample suspected of containing a chimeric RNA fusion protein and contact the sample with an antibody against the chimeric RNA fusion protein, and then detect and quantify the amount of immune complexes formed under the specific conditions.
[0106] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any chimeric RNA gene product fusion protein antigens present. After this time, the sample-antibody composition, such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non- specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
[0107] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. U.S. Patents concerning the use of such labels include 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art. [0108] The antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody. In these cases, the second binding ligand may be linked to a detectable label. The second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non- specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
[0109] Further methods include the detection of primary immune complexes by a two step approach. A second binding ligand, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
[0110] One method of immunodetection uses two different antibodies. A first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution containing the first step antibody.
If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced which is macroscopically visible.
[0111] Another known method of immunodetection takes advantage of the immuno-PCR (Polymerase Chain Reaction) methodology. The PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls. At least in theory, the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
[0112] The immunodetection methods of the present invention have evident utility in the diagnosis and prognosis of conditions such as various forms of cancer. Here, a biological and/or clinical sample suspected of containing a chimeric RNA gene product fusion protein, polypeptide, peptide mutant is used.
[0113] In the clinical diagnosis and/or monitoring of patients with various forms of disease, such as cancer, the detection of chimeric RNA gene products and/or an alteration in the levels of chimeric RNA gene products in comparison to the levels in a corresponding biological sample from a normal subject is indicative of a patient with cancer. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive.
[0114] 1. ELISAs
[0115] As detailed above, immunoassays, in their most simple and/or direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art.
Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and/or western blotting, dot blotting, FACS analyses, and/or the like may also be used.
[0116] In one exemplary ELISA, the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the chimeric RNA gene product fusion protein antigen, such as a clinical sample, is added to the wells. After binding and/or washing to remove non- specifically bound immune complexes, the bound chimeric RNA gene product fusion protein antigen may be detected. Detection is generally achieved by the addition of another antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
[0117] In another exemplary ELISA, the samples suspected of containing the chimeric RNA gene product fusion protein antigen are immobilized onto the well surface and/or then contacted with the antibodies of the invention. After binding and/or washing to remove non-specifically bound immune complexes, the bound antibodies are detected. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
[0118] Another ELISA in which the chimeric RNA gene product fusion proteins, polypeptides and/or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against chimeric RNA gene product fusion protein are added to the wells, allowed to bind, and/or detected by means of their label. The amount of chimeric RNA gene product fusion protein antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against chimeric RNA gene product fusion before and/or during incubation with coated wells. The presence of chimeric RNA gene product fusion protein in the sample acts to reduce the amount of antibody against chimeric RNA gene product fusion protein available for binding to the well and thus reduces the ultimate signal. This is also appropriate for detecting antibodies against chimeric RNA gene product fusion protein in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
[0119] Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non- specifically bound species, and detecting the bound immune complexes. These are well known in the art.
[0120] 2. Immunohistochemistry
[0121] The antibodies of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC). The method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et ah, 1990; Abbondanzo et ah, 1990; Allred et al, 1990).
[0122] Briefly, frozen- sections may be prepared by rehydrating 50 ng of frozen "pulverized" tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
[0123] Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.
V. Pharmaceutical Preparations
[0124] In some embodiments of the invention, a cancer is treated with an agent that targets a particular chimeric RNA, and in at least some cases the cancer is identified as having one or more particular chimeric RNAs. Pharmaceutical compositions of the present invention comprise an effective amount of one or more chimeric RNA-targeting agents (such as an antibody or small molecule or siRNA-based drugs) dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one chimeric RNA-targeting agent or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical
Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of
Biological Standards.
[0125] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
[0126] The chimeric RNA-targeting agent may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). [0127] The chimeric RNA-targeting agent may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine,
trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
[0128] Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semisolid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
[0129] In accordance with the present invention, the composition is combined with the carrier in any convenient and practical manner, i.e. , by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art. [0130] In a specific embodiment of the present invention, the composition is combined or mixed thoroughly with a semi- solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
[0131] In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include chimeric RNA-targeting agent, one or more lipids, and an aqueous solvent. As used herein, the term "lipid" will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term "lipid" is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic {i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods of the present invention.
[0132] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the chimeric RNA-targeting agent may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.
[0133] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
[0134] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
[0135] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.
[0136] A. Alimentary Compositions and Formulations [0137] In preferred embodiments of the present invention, the chimeric RNA- targeting agentS are formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
[0138] In certain embodiments, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety). The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.
[0139] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a
therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
[0140] Additional formulations which are suitable for other modes of alimentary administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
[0141] B. Parenteral Compositions and Formulations
[0142] In further embodiments, chimeric RNA-targeting agentS may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).. [0143] Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety). In all cases the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
[0144] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
[0145] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. A powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
[0146] C. Miscellaneous Pharmaceutical Compositions and Formulations
[0147] In other preferred embodiments of the invention, the active compound chimeric RNA-targeting agent may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration
(intranasal, vaginal, etc.) and/or inhalation.
[0148] Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture. Transdermal administration of the present invention may also comprise the use of a "patch". For example, the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
[0149] In certain embodiments, the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871, specifically incorporated herein by reference in its entirety) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetraf uoroetheylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety).
[0150] The term aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.
VI. Kits of the Invention
[0151] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, a chimeric RNA, one or more polynucleotides that hybridize to a chimeric RNA, a substrate having a chimeric RNA or one or more polynucleotides that hybridize to a chimeric RNA, an antibody to a chimeric RNA, and/or a cancer therapeutic and/or a siRNA that targets a chimeric RNA may be comprised in suitable container means in a kit.
[0152] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing components in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
[0153] However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be
reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
[0154] The kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.
EXAMPLES
[0155] The following examples are included to demonstrate some embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute some modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1
IDENTIFICATION OF NOVEL RECURRENT CHIMERIC RNAS IN PROSTATE CANCER
[0156] To identify chimeric RNAs that are expressed in prostate cancer, the inventors sequenced the transcriptome of 20 cancer samples and 10 matched benign samples from patients with prostate adenocarcinoma who received no preoperative therapy before radical prostatectomy. An Illumina Genome Analyzer II was used for sequencing of these samples to generate output sequences of paired 36-nucleotide reads. In all, 30 lanes of Illumina Genome Analyzer were used to yield ~1.3 billion raw sequence reads. Using stringent filters (see
Example 9), the inventors obtained nearly 500 million reads that were both uniquely mappable to the human genome and met our additional bioinformatic criteria.
[0157] A strategy for identifying chimeric RNAs was to search for paired "chimeric" reads with each read mapping to a different gene either in the genome or in the transcriptome (FIG. 1). To minimize the cases of false positives, the inventors required each read to map to only one location in the genome even with the tolerance of two mismatches. In addition, an event was considered a chimeric candidate only if it was supported by paired chimeric reads from at least three different patient samples. Cases of overlapping genes and homologous genes with shared sequences were filtered out. This strategy led to the identification of 2,369 putative chimeric events (FIG. 2A). Analysis of paired chimeric reads against a patient's age indicates that there is no apparent correlation between age and the number of chimeric reads either in cancer or in matched benign samples. However, paired chimeric reads appear to be more abundant in cancer than in matched benign samples (P value = 0.00014) (FIG. 2B). Together, the results indicate that chimeric RNAs are widespread and occur more frequently in cancer.
[0158] To identify chimeric RNAs that are highly recurrent in human cancer samples, the inventors required that a candidate chimeric RNA must be present in >50 of cancer samples used for sequencing (i.e., >10 of 20 total human cancer samples). The analyses narrowed down from 2,369 candidates to a set of 32 highly recurrent chimeric RNAs, and these are marked in in dark color in FIG. 2A. Among them, 27 are unique chimeric RNAs that have never been described before (Table 2). The remaining 5, TMPRSS2-ERG, SLC45A3-ELK, ANKRD39-ANKRD23, HARS-ZMAT2, and SMG5-PAQR6, are previously known chimeric RNAs in prostate cancer (7, 8, 13, 14), indicating that the procedure is able to "rediscover" previously verified chimeric RNAs.
[0159] Table 2. Validated chimeric RNAs in human prostate cancer
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Full length chimeric RNA sequence for TMEM79-SMG5:
5'
gagagtaagaaggctgcgaggagagctcacacatccttggtttgtatgaagaaaaggacagtaccctctaccagggccatcccctc attg acaaacagg aag ag aagg acagtaccctcaaccaggccccccaaccttg ag acctcatctctatatttttttaattttaattatttat ttatttgtttgagacggagtctcactctgtcacccaggctggagtgcaatggcgcgatcttggctcactgcaacctccgcctcccaggttc aagcgattctcctgcctccgcctcctgaatagctgggattacagatgcccaccaccacacccggctaattttttgtatttttagtagacctg gggtttcaccttgttggccaagctggtctcgagctcctgacattgtgatccacccacctcagcctcccaaagtgctgggattacaggcgt gagccactacacccggctatttatttttttgagatgaagtctagctctgtcgtccaggctagagtacagtgacacgatcttgtgtcactgca atctccacctcctggactcaagcgattctcctgcctcagcctcctcagtagctgggactacaggtgtgcaccaccatgcccagctaatttt tgtatttttagtagagacagggtttcaccatattggccaggctggtctcgaactcctgacctcaagtgatctgccacctcggcctctcaaa gtgctgagattacaggcatgagccactgcacccagccttgtctctattttgaaaaaatatatctatttttaagttgtacatttggttacaggg ggtgttcaaccagagaccaaaacagctacctgaccagattgccacaggagtggctgcaatgggtgcattgatctagatgacacctg aaagccttcttgttccatatggttatatatgtctctgaaatcggcttccatctgctgaccagctgag*ggctgtggtggaggctgtgcatcga cttgacctcatcctttgcaacaaaactgcttatcaagaagtattcaaaccagaaaacattagcctgaggaacaagctgcgtgagctct gcgtcaagcttATGTTCCTGCACCCAGTGGACTATGGGAGAAAGGCTGAGGAGCTGCTGTGGA GAAAGGTATACTATGAAGTTATCCAGCTTATCAAGACTAACAAAAAGCACATCCACAGCCG GAGCACTTTGGAATGTGCCTACAGGACGCACCTGGTTGCTGGTATTGGCTTCTACCAGCA TCTCCTTCTCTATATCCAGTCCCACTACCAGCTGGAACTGCAGTGCTGCATCGACTGGACC CATGTCACTGACCCCCTCATAGGATGCAAGAAGCCAGTGTCTGCCTCAGGGAAGGAGATG GATTGGGCACAGATGGCATGTCACCGATGTCTGGTGTATCTGGGGGATTTGTCCCGATAT CAGAATGAATTAGCTGGCGTAGATACCGAGCTGCTAGCCGAGAGATTTTACTACCAAGCC CTGTCAGTAGCTCCTCAGATTGGAATGCCCTTCAATCAGCTGGGCACCCTGGCAGGCAGC AAGTACTATAATGTGGAAGCCATGTATTGCTACCTGCGCTGCATCCAGTCAGAAGTGTCCT TTGAGGGAGCCTATGGGAACCTCAAGCGGCTGTATGACAAGGCAGCCAAAATGTACCACC AACTGAAGAAGTGTGAGACTCGGAAACTGTCTCCTGGCAAAAAGCGATGTAAAGACATTAA AAGGTTGCTAGTGAACTTTATGTATCTGCAAAGCCTCCTACAGCCCAAAAGCAGCTCCGTG GACTCAGAGCTGACCTCACTTTGCCAGTCAGTCCTGGAGGACTTCAACCTCTGCCTCTTCT ACCTGCCCTCCTCACCCAACCTCAGCCTGGCCAGTGAGGATGAGGAGGAGTATGAGAGT GGATATGCTTTCCTCCCGGACCTTCTCATCTTTCAAATGGTCATCATCTGCCTTATGTGTGT GCACAGCTTGGAGAGAGCAGGATCCAAGCAGTACAGTGCAGCCATTGCCTTCACCCTGGC CCTCTTTTCCCACCTCGTCAATCATGTCAACATACGGCTGCAGGCTGAGCTGGAAGAGGG CGAGAATCCCGTCCCGGCATTCCAGAGTGATGGCACAGATGAACCAGAGTCCAAGGAACC TGTGGAGAAAGAGGAGGAGCCAGATCCTGAGCCTCCTCCTGTAACACCCCAAGTGGGTGA GGGCAGAAAGAGCCGTAAGTTCTCTCGCCTCTCCTGTCTCCGCCGTCGCCGCCACCCACC CAAAGTTGGTGATGACAGTGACCTGAGTGAAGGCTTTGAATCGGACTCAAGCCATGACTC AGCCCGGGCCAGTGAGGGCTCAGACAGTGGCTCTGACAAGAGTCTTGAAGGTGGGGGAA CGGCCTTTGATGCTGAAACAGACTCGGAAATGAATAGCCAGGAGTCCCGATCAGACTTGG AAGATATGGAGGAAGAGGAGGGGACACGGTCACCAACCCTGGAGCCCCCTCGGGGCAGA TCAGAGGCTCCCGATTCCCTCAATGGCCCACTGGGCCCCAGTGAGGCTAGCATTGCCAGC AATCTACAAGCCATGTCCACCCAGATGTTCCAGACTAAGCGCTGCTTCCGACTGGCCCCC ACCTTTAGCAACCTGCTCCTCCAGCCCACCACCAACCCTCATACCTCGGCCAGCCACAGG CCTTGCGTCAATGGGGATGTAGACAAGCCTTCAGAGCCAGCCTCTGAGGAGGGCTCTGAG TCGGAGGGGAGTGAGTCCAGTGGACGCTCCTGTCGGAATGAGCGCAGCATCCAGGAGAA GCTTCAGGTCCTGATGGCCGAAGGTCTGCTTCCTGCTGTGAAAGTCTTCCTGGACTGGCT
TCGGACCAACCCCGACCTCATCATCGTGTGTGCGCAGAGCTCTCAAAGTCTGTGGAACCG
CCTGTCTGTGTTGCTGAATCTGTTGCCTGCTGCTGGTGAACTCCAGGAGTCTGGCCTGGC
CTTGTGTCCTGAGGTCCAAGATCTTCTTGAAGGTTGTGAACTGCCTGACCTCCCCTCTAGC
CTTCTGCTCCCAGAGGACATGGCTCTTCGTAACCTGCCCCCGCTCCGAGCTGCCCACAGA
CGCTTTAACTTTGACACGGATCGGCCCCTGCTCAGCACCTTAGAGGAGTCAGTGGTGCGC
ATCTGCTGCATCCGCAGCTTTGGTCATTTCATCGCCCGCCTGCAAGGCAGCATCCTGCAG
TTCAACCCAGAGGTTGGCATCTTCGTCAGCATTGCCCAGTCTGAGCAGGAGAGCCTGCTG
CAGCAGGCCCAGGCACAGTTCCGAATGGCACAGGAGGAAGCTCGTCGGAACAGGCTCAT
GAGAGACATGGCTCAGCTACGACTTCAGCTCGAAGTGTCTCAGCTGGAGGGCAGCCTGCA
GCAGCCCAAGGCCCAGTCAGCCATGTCTCCCTACCTCGTCCCTGACACCCAGGCCCTCTG
CCACCATCTCCCTGTCATCCGCCAACTGGCCACCAGTGGCCGCTTCATTGTCATCATCCCA
AGGACAGTGATCGATGGCCTGGATTTGCTGAAGAAGGAACACCCAGGGGCCCGGGATGG
GATTCGGTACCTGGAGGCAGAGTTTAAAAAAGGAAACAGGTACATTCGCTGCCAGAAAGA
GGTGGGAAAGAGCTTTGAGCGGCATAAGCTGAAGAGGCAGGATGCAGATGCCTGGACTC
TCTATAAGATCCTAGACAGCTGCAAACAGCTGACTCTGGCCCAGGGGGCAGGTGAGGAG
GATCCGAGTGGCATGGTGACCATCATCACAGGCCTTCCACTGGACAACCCCAGCGTGCTT
TCAGGCCCCATGCAGGCAGCCCTGCAGGCCGCTGCCCACGCCAGTGTGGACATCAAGAA
TGTTCTGGACTTCTACAAGCAGTGGAAGGAAATTGGTTGAtactgacccccaggccctgcagtggggctg actccagatctctcctgccctccctggcagccaggaccagcacctgtagtcaccccaccacacgcagactcatgcacgcacacagg agggaggcctagctgctcagaggctgcagggagggcccaggagccggctgggagggtggggtccctttgttgccaagacgttagg aaagcgaggaaagtgcttggattaggagagtcttgtgggcccctggccagccttcctgcctcagctcccctgctgtctccaggggcag gtggtaggcatgggtacctgcatttcactggaatgggttcttggatctctgaggggaaggaacagcaaaagaggcccttcttcctcacc caagatgcagggtggttggggccaggagtttggaccctctaggtcttgggggaagagctgggtaatacctggtgtctgagtgattctct gcagacccttcccctcctcaaggatcacccatcctcctttcagccccctttatggggaccaggcagctctggagccagccacagggg ctgttagagaagcaaggcctggagtggcctgcaccgagtagcagggtcagggttcgtgtgctcctcctcctgctgcaggggctgcac atcccattgccccacttctgctttgtgtctccctctgtctagcttccagggcagggagcaggccccacctagggctgcaggcagtctggc ctgtgccagcacggtctcctgtgcccaccagccccacaggtgctgtgctttgtgctcttggctgctgtgctgggacagaatgggatgcca ggaagagaagaaagggggtgcagtctgaggccaccaccccccttcctatctaagggagggctgaagacaaggggccggcattc agtgggcagcagaaaggagaggctccttgaagctgctcagtcagaggcccccgtccctccttttgccttccgcaggactgaagacct gaaggggctggcttttggagtgttgaggtgaatatctgggagcagagatcatgaatagctcagggcagtgaatggcgcaccaagag cagggctgtgtgtgggaggctgcagccaggattgcctcagctcctccccctcaggctgggaggatagcacaggctaggggctcggg gtggagggtctcagctctgctgcccccaccccagtactagcctagcttcccaagctgtggcttagaggatagttggcttcctgcctctctc ctctaaaatagcaagtctgggaaatcctggggtgagtggagtcaccccactcccagttgctggcagagactgagactaaagcatca cttaataaaccccccaagccca 3'
[0160] The 32 validated chimeric RNAs and the number of paired chimeric and junction reads supporting the corresponding chimeric events are shown. Total reads are the sum of paired chimeric reads and junction reads. Potential consequences as the results of chimeric
RNA are listed in column 7. Eighteen of these chimeric RNAs are likely to have a modified
ORF that can lead to unique protein sequences. The last column shows the experimentally determined RT-PCR sequence including the RNA junction. The junction is denoted by an asterisk. For TMPRSS2-ERG and SLC45A3-ELK4, the most frequently observed chimeric RNA sequence is shown and the other splice variants are not shown. The distance (Dis) between ends of parental genes is also shown for each of the 32 indicated chimeric RNAs. Chr:
chromosome. For TMEM79-SMG5, the full-length chimeric RNA sequence is provided in the sequence listing. The putative 5'- and 3'-UTRS are shown in lowercase letters and the putative coding region is shown in uppercase letters. The start/stop codons and poly(A) signal are shown in boldface type. The chimeric junction is denoted by an asterisk.
[0161] To confirm that these identified chimeric RNAs are expressed, the inventors designed specific primer pairs with each primer targeting one parental gene and then used strand- specific RT-PCR to validate the presence of candidate chimeric RNAs in human samples that were used for paired-end sequencing. For all 32 candidates, including the 5 known chimeras that were used as controls, they were able to obtain RT-PCR products, indicating that these chimeric RNAs are indeed expressed at detectable levels. In addition, the strand- specific RT-PCR enabled the determination of 5' and 3' chimeric RNA partners. In most cases, the inventors obtained only a single RT-PCR band for each chimera, which was then excised and subjected to traditional Sanger sequencing. This sequencing led to the identification of the exact junction of the chimeric RNAs (column 8 in Table 2). The results from Sanger sequencing also showed no apparent variation in the RNA junction for each excised band (FIG. 7A), indicating that chimeric RNAs were generated by a precise mechanism. The newly obtained junction sequences enabled the inventors to search among previously unmappable reads for paired "junction" reads that could now be specifically mapped to a junction site with one read and to a parental gene with another read (see strategy in FIG. 1). For each of the 32 chimeric RNAs, they were able to identify the corresponding junction reads. Together, the results identified and confirmed the junction sequences and strongly support the presence of the corresponding chimeric RNAs in patients' tissues.
[0162] In addition to those putative chimeras chosen to validate, there were 6 additional chimeras that could not be validated by RT-PCR. There were also 8 additional chimeras with their RNA sequences extending continuously from one gene to the next without a break or new junctions. These chimeras are considered as simple read-through transcripts, not as chimeric RNAs. None of these chimeras are included in the 32 set described earlier. In all, of a total of 46 putative chimeric events chosen for validation, 32 were experimentally validated as chimeric RNAs with confirmed junctions. Interestingly, all of these 32 chimeric RNAs are intrachromosomal events, with 4 separated by one or more genes, whereas the remaining 28 involve neighboring genes. It is important to note that the list of 2,369 chimeric RNA candidates includes 1,902 interchromosomal and 467 intrachromosomal chimeric events. However, these interchromosomal chimeric RNAs are not highly recurrent, and therefore they were not included in the set that was chosen for validation.
[0163] It is known that high-throughput sequencing of the transcriptome can accurately detect gene expression levels over a wide dynamic range (15, 16). The analysis of gene expression levels using sequence data showed that the parental genes involved in 32 chimeric RNAs are not biased toward highly expressed genes (FIG. 8). Therefore, the chimeric RNAs identified are not simply the result of random "transcriptional leakage" or artifacts of cDNA library construction, as both would generate more events in highly expressed genes. Furthermore, most of the parental genes involved in chimeric RNAs appear to be not overexpressed in cancer vs. matched benign samples. Thus, the higher recurrence of chimeric RNAs in cancer samples cannot simply be attributed to higher expression levels of the parental genes in cancer samples. To estimate the relative expression levels of these chimeric RNAs, the inventors combined all paired chimeric reads and junction reads associated with each chimera and compared them with those of previously known chimeric RNAs in prostate cancer.
[0164] As shown in column 6 of Table 2, the unique chimeric RNAs identified in this study averaged 47 paired reads, with SLC16A8-BAIAP2L2 having the highest number, 99. This result can be compared with the 9 paired reads supporting SLC45A3-ELK4, the best- characterized chimeric RNA in prostate cancer. Thus, the unique chimeric RNAs identified in this study are not rare events, but recurrent at relatively high levels. However, their relative expression levels are lower than that of TMPRSS2-ERG (481 paired reads) and this can be attributed to TMPRSS2-ERG being the result of a DNA-level rearrangement and the fact that it is driven by a strong androgen-regulated promoter.
EXAMPLE 2
RECURRENCE OF CHIMERIC RNAS IN CANCER VS. MATCHED BENIGN TISSUE
[0165] To evaluate their differential expression in cancer vs. matched benign tissues, the inventors plotted the number of supporting chimeric reads and junction reads for each chimeric RNA and its occurrence for each patient. As shown in FIG. 3, most of the verified chimeric RNAs appeared to be highly recurrent in cancer samples, although many of them also appeared in matched benign tissues from the radical prostatectomy specimens, albeit with lower frequency. This includes TMPRSS2-ERG used as the control, which is known to be cancer specific (9). The presence of these chimeric RNAs within the matched benign samples may represent a "field effect" within the histologically normal epithelium such that the benign epithelium may have multifocal premalignant lesions that precede histological changes (17). Alternatively, small foci of cancer may be present in some matched benign samples because the tissue is not evaluated histologically throughout the entire tissue used for RNA extraction.
Nevertheless, the sequencing data of matched benign tissues enabled the valuable comparison of chimeric RNAs to that of cancer. To determine which chimeric RNAs are differentially expressed in cancer tissue, the inventors used the nonparametric Kolmogorov-Smirnov test and identified seven chimeric RNAs with a P value more significant than that of TMPRSS2-ERG (P =0.046) (FIG. 3). These include the chimera previously known to be elevated in prostate cancer tissue (SLC45A3-ELK4, P=0.027) (7,8) or found in cancer cell lines (ANKRD39-ANKRD23, P=0.046) (13). The remaining five are unique chimeric RNAs: TMEM79-SMG5, SLC16A8- BAIAP2L2, SLC44A4-EHMT2, BC035340- MCF2L, and TSPAN1-POMGNT1. Among them, TMEM79- SMG5 appeared to be most significant, with a P value of 0.004 that compares favorably to that of TMPRSS2-ERG.
[0166] To further evaluate the differential expression of unique chimeric RNAs without the confounding issue of tissue impurity, RT-PCR was used to verify the presence of identified chimeric RNAs in an additional 10 human prostate cancer tissues and 5 normal prostate tissues from organ donors without prostate cancer (these samples were not used for transcriptome sequencing). Because this analysis compares prostate tissues from patients with cancer to those from patients without cancer, tissue impurity or field effects are not a consideration. As seen in Table 3, most of the validated chimeric RNAs were highly recurrent in the additional 10 patients with cancer, confirming the results obtained from the transcriptome sequencing.
[0167] Table 3. Recurrence frequency of chimeric RNAs in additional patient cohorts
Figure imgf000056_0001
BC160930-MC1R 9 3
C12orf76-ANKRD13A 5 5
XPA-NCBP1 10 3
[0168] Importantly, two of the chimeric RNAs were found only in patients with cancer, but were undetectable in the donors without cancer, suggesting that their expression is enriched in cancer cells. These include TMEM79-SMG5 (9 of 10 cancer vs. 0 of 5 noncancer) and the known cancer-specific TMPRSS2-ERG (5 of 10 cancer vs. 0 of 5 noncancer). The absence of the TMEM79-SMG5 chimera in donors without cancer raised the question of whether this absence is due to the absence of expression of their participating parental genes. Because chimeric RNAs possess shared sequences with their parental genes, it is difficult to determine whether the reads obtained from transcriptome sequencing were derived from chimeric RNA or parental RNA. Therefore, to answer this question, we designed PCR primers targeting specific segments of participating parental mRNA that are absent in chimeric RNAs. The results of two chimeras (TMEM79-SMG5 and RASL12-OSTbeta), shown in FIG.4A, indicate that the parental genes were clearly expressed in donors without cancer. The expression of parental TMEM79 and SMG5, however, did not lead to a detectable level of chimera expression in donors without cancer. This result is in contrast to that seen in the cancer tissues where the parental mRNAs and chimera TMEM79-SMG5 were both clearly expressed (FIG. 4A).
[0169] Furthermore, the absence of expression of the 5 '-parental gene does not exclude the generation of the chimeric RNA. This result is seen in RASL12-OSTbeta, where the 5'-parental gene RASL12 is not detected in cancer samples by RT-PCR; however, the chimera is expressed in cancer samples (FIG. 4A).
EXAMPLE 3
EXPRESSION OF CHIMERIC RNAS IN PROSTATE CANCER CELL LINES
[0170] Because the chimeric RNAs that were identified are highly recurrent in human prostate cancer samples, the inventors considered that in certain embodiments these chimeric RNAs may also be present in established prostate cell lines. Therefore, the inventors tested the expression of these chimeric RNAs in cancer cell lines of prostate origin, including androgen receptor negative cell lines (PC3 and DU145) and androgen- sensitive cell lines (LNCaP, VCaP, and LAPC4), as well as immortalized prostatic epithelial cells (PNTla), to compare with that of the noncancer primary human prostate epithelial cells (PrEC). The RT- PCR results showed that most of the 32 chimeric RNAs displayed varying expression profile in cancer cell lines (Table 4). However, 9 chimeric RNAs were conspicuously absent in noncancer PrEC (Table 4 and FIG. 4B). These RNAs include TMEM79-SMG5, the same chimera detectable by RT-PCR in patients with cancer but not in donors without cancer. The rest of the group comprises XPA-NCBP1, RASL12-OSTbeta, ELF3-RNPEP, ASTN2-PAPPA, GOLM1- MAK10, BC035340-MCF2L, NCAPD3-JAM3, and the control TMPRSS2-ERG that is known to be highly expressed in VCaP cells (FIG. 4B).
[0171] Table 4. Expression of chimeric RNAs in prostate cancer cell lines determined by RT-PCR
Figure imgf000058_0001
[0172] Shadowed indicates presence of the chimera and white indicates absence of expression of the chimera determined by RT-PCR.
[0173] The absence of these chimeric RNAs in PrEC also raised the question of whether this absence is because their parental genes are not expressed. The RT-PCR results, however, showed that many of the parental genes were expressed in PrEC (FIG. 4C). For example, TMEM79 and SMG5 are both expressed; however, the corresponding chimeric RNA is undetectable. Similarly, both ELF3 and RNPEP are expressed in PrEC but not the chimeric RNA. Hence, the absence of these chimeric RNAs in PrEC cannot be attributed simply to the absence of expression of participating parental genes.
EXAMPLE 4
MECHANISMS RESPONSIBLE FOR GENERATION OF CHIMERIC RNAS
[0174] To validate DNA rearrangements, the standard FISH assay requires a minimum distance between genes in the order of 100-150 kb (8). This assay was possible for TMPRSS2-ERG (distance between genes is 3 Mb) (10). It is, however, not possible to use this assay for the rest of the validated chimeric RNAs on the list, as the distances between the ends of the two genes for these chimeras are <30 kb (see column 3 in Table 2). Therefore, to verify whether these unique chimeric RNAs are a result of genomic DNA rearrangement, long-range PCR was performed on genomic DNA from patients who displayed expression of the
corresponding chimeric RNAs. The expected PCR product size from genomic DNA for the majority of the chimeras is <30 kb, and this size is within the detection range of long-range PCR. The results, however, showed that there is no evidence for gross genomic rearrangement for the 27 unique chimeric RNAs (examples shown in FIG. 9), indicating that these chimeras are likely the result of RNA-level events similar to SLC45A3-ELK4 (7, 8). Intriguingly, further sequence analyses indicated that almost all of the validated chimeras contain a discernable 5' splice site and a 3' splice site at the RNA junction (see example in FIG. 5), indicating that the formation of these chimeras is mediated by splicing. To test whether the parental genes involved in chimeras prefer a certain range of intergenic distance, the inventors calculated the distances between the ends of the two participating parental genes on the genome.
[0175] As shown in column 3 of Table 2, parental genes of the chimeric RNAs tend to reside closer on the genome with a median distance of 2 kb compared with the known median of 48 kb for adjacent genes in the entire genome (18). The observation of splice sites at RNA junctions, together with the fact that the chimera formation prefers neighboring genes with short distances, and the lack of evidence of DNA-level rearrangements indicates that they may be generated by either read-through/splicing or transsplicing.
EXAMPLE 5
TMEM79-SMG5 AS AN EXEMPLARY DIAGNOSTIC TOOL
[0176] The results from three different analyses, (i) transcriptome sequencing of human cancer and matched benign samples, (ii) RT-PCR of human cancer and non-cancer donor samples, and (iii) RT-PCR of prostate cancer cell lines and primary prostate cells, all independently indicate that the chimera TMEM79-SMG5 is highly recurrent and is enriched in cancer. To further evaluate this chimeric RNA as a diagnostic tool, the inventors performed quantitative RT-PCR analysis in an extended cohort of human prostate samples from 54 patients with prostate cancer and 18 donors without cancer. In parallel, the inventors also performed quantitative RT-PCR in PrEC, which represents primary prostate epithelium cells pooled from donors without cancer. As shown in FIG. 6, the chimera is expressed at a distinctly higher level in most samples from patients with cancer than in donors without cancer (P value= 1.048 e-05), and it is undetectable in PrEC. The pronounced difference between expression levels of this chimera in patients with cancer and donors without cancer and its complete absence in PrEC indicate that TMEM79-SMG5 is a valuable indicator to differentiate between patients with cancer and patients without cancer. EXAMPLE 6
SIGNIFICANCE OF CERTAIN EMBODIMENTS OF THE INVENTION
[0177] Embodiments of the invention provide a deep survey of chimeric RNAs expressed in human prostate cancers. The inventors sequenced 20 cancer samples and 10 matched benign samples from patients with prostate adenocarcinoma. The 1.3 billion reads resulting from deep sequencing enabled the identification of 2,369 putative chimeric RNAs using stringent bioinformatic criteria. Altogether, the inventors chose 46 events for experimental validation and of these, 32 events (~70%) could be verified by RT-PCR and their RNA junctions were determined by Sanger sequencing. Although this 70% validation rate is relatively high, the chimeric RNAs chosen for validation are among the highly recurrent ones. One could determine whether the lower recurrent chimeric RNAs could also be validated at a similar rate. Because the results were derived from 1.3 billion reads with each chimeric candidate supported by at least 3 different patient samples, increasing the sequencing depth or sample numbers should further increase the number of recurrent chimeric RNAs identified while reducing the incidence of false positives. If a significant portion of these 2,369 chimeric events are true, they would involve up to 3,144 parental genes that correspond to ~14% of the total human protein- coding genes.
[0178] Because only a subset of the human genome is expressed in any given differentiated tissue such as prostate, the percentage of "transcribed" genome that is involved in chimeric RNAs could be significantly higher. The deep- sequencing results therefore indicate that chimeric RNAs may be more prevalent in human cells than previously thought. The results thus significantly extend the observation made by a recent in silico analysis of ESTs and cDNAs in the National Center for Biotechnology Information databases that independently estimated that as much as 5% of the human genome could transcribe chimeric sequences (19). The biological consequences of chimeric RNAs would be similar to that of gene fusions resulting from DNA rearrangement, as both mechanisms generate chimeric transcripts that possess sequences from two genes. Chimeric RNAs, like gene fusions, are expected to increase the proteomic diversity in cells through chimeric proteins or altered regulation of participating mRNAs. However, the number of chimeric RNAs revealed by deep sequencing appears far greater than that of a few gene fusion events identified in prostate tissue so far (7, 13, 14). Furthermore, all of the validated chimeras in our study are recurrent in at least 50% of the patient samples. This result is in contrast to gene fusion events in prostate cancer that are rare and predominantly nonrecurrent with the exception of TMPRSS2-ERG (14). The prevalence of chimeric RNAs may allow the limited number of human genes to encode a larger number of RNAs and proteins, forming yet an additional layer of cellular complexity. Importantly, the exemplary results indicate that the number of total chimeric RNAs appeared significantly higher in cancer than in matched benign tissues (Fig. 2B; P value = 0.00014), raising the possibility that increased chimeric RNA events could represent one of the molecular consequences of cancer.
[0179] PCR analysis of patients' genomic DNA indicates that all these validated chimeric RNAs are likely generated in the absence of DNA-level rearrangements. Furthermore, sequence analyses indicated that almost all of the RNA junctions contain a discernable 5' splice site and a 3' splice site at the junction site, indicating that the formation of chimeric RNAs is mediated by splicing. This mediation could result from two known transcription-induced mechanisms: read- through/splicing and trans- splicing. However, on the basis of the RNA sequence information, it is impossible to discern between these two mechanisms. For example, a chimeric RNA joining neighboring genes located on the same strand could theoretically result from a single transcript reading through both genes followed by splicing or by splicing of two separate RNA molecules independently generated from neighboring genes.
[0180] Intriguingly, with the exception of TMPRSS2-ERG, SLC45A3- ELK4, and ANKRD39-ANKRD23, the set of recurrent chimeric RNAs that were identified in human prostate cancer tissues has virtually no overlap with the set of chimeric RNAs identified in prostate cancer cell lines using similar sequencing methods (7, 13). This result is not because the recurrent chimeric RNAs found in tissues are absent in cancer cell lines. In fact, the inventors experimentally demonstrated that all of the 27 unique recurrent chimeras identified in the study displayed expression in at least one or more prostate cancer cell lines. However, the reverse is not true, as the chimeric RNAs reported from cell line studies are largely absent from the extended list of 2,369 putative chimeric events. This result is unlikely due to the depth of sequencing as highly recurrent chimeric RNAs will appear frequently in every study.
[0181] More likely, the difference between these studies reflects the differences between cancer cell lines and tissue samples, and the chimeric RNA species prevailing in human cancer issues appeared to be dramatically different from those present in cancer cell lines.
Because the composition of chimeric RNA species and their recurrence in this study are derived from human cancer tissues directly, the identified chimeras could be more relevant to tumor biology and may have more valuable clinical utilities. Several observations indicate that the generation of recurrent chimeric RNAs enriched in cancer is not the result of stochastic processes. First, no variation in RNA junction sequence was observed from sequenced RT-PCR bands or from junction reads.
[0182] Each of the validated recurrent chimeras exhibits consistent and precise RNA junction within the same patient and across all patient samples. Second, random processes should generate more chimeras from highly expressed genes. However, the identified chimeric RNAs are not biased toward highly expressed genes (FIG. 8). Third, the expression of participating parental genes does not automatically lead to the generation of the corresponding chimeric RNAs. For example, both the parental genes SMG5 and TMEM79 are expressed in cancer samples as well as in samples from donors without cancer, but the presence of their chimeras is largely restricted to cancer samples (FIG. 4A). Furthermore, the absence of parental gene expression does not exclude the generation of the chimeric RNA. For example, RASL12 is not detected in cancer samples but the chimera RASL12-OSTbeta is expressed in cancer samples (FIG. 4A). Together, this indicates that the generation of chimeric RNAs, whether through trans- splicing or read-through/splicing, is largely independent of the expression of parental genes and is tightly regulated. This result indicates that other factors are involved in the production and regulation of chimeric RNAs, in certain aspects.
[0183] The RNA strand information and junction sequences determined experimentally by RT-PCR enabled the inventors to consider the potential biological
consequences of these chimeric RNAs. The analysis indicated that 18 of the 32 identified chimeric RNAs could lead to unique protein sequences due to fused or truncated protein-coding regions, and almost all would have either new 5'- or 3'-UTRs that could lead to altered gene regulation (column 7 in Table 2). For example, the recurrent chimera TMEM79-SMG5
(Example 10; full-length sequence is provided in the sequence listing) results in the loss of the first exon of SMG5, with genome sequence from TMEM79 contributing a new 5'-UTR to the truncated SMG5 (FIG. 5 and Table 2). This result may lead to altered function and regulation of SMG5, a protein essential to the nonsense-mediated mRNA decay (NMD) (20). However, it is also possible that chimeric RNAs could function as noncoding RNAs or regulatory RNAs without a protein counterpart.
[0184] The results indicate that the chimera TMEM79-SMG5 is highly recurrent and enriched in cancer samples. First, RT-PCR analysis showed that the TMEM79-SMG5 chimera is absent in non-cancer PrEC but is expressed only in androgen- sensitive cancer cell lines VCaP and LNCaP, even though all these cell lines are epithelial in origin. Second, quantitative RT-PCR analysis on an extended cohort of patients confirmed that this chimera is highly expressed in patients with cancer, whereas its expression is far lower in donors without cancer and undetectable in PrEC. The most direct utility of the chimera TMEM79-SMG5 is perhaps in its use as diagnostic biomarker. Compared with TMPRSS2-ERG, which can be found in -50% of the patients with cancer, TMEM79-SMG5 is detected by RT-PCR in -90% of the cancer samples tested.
[0185] Compared with SLC45A3-ELK4, which is expressed at a relatively high level in patients without cancer (FIG. 4A), TMEM79-SMG5 appears to be largely restricted to cancer. Thus, TMEM79-SMG5 is useful to serve as a biomarker to separate patients with cancer from patients without cancer.
EXAMPLE 7
QUALITY CONTROL FOR READS OBTAINED FROM SEQUENCING
[0186] Reads obtained from sequencing were analyzed and determined to be of good quality on the basis of three factors. First, reads were equally spread throughout the entire length of mRNA transcripts except for the extreme 5' and 3' ends similar to that of the standard output of paired-end sequencing, suggesting that there is no positional bias within the transcripts (Mortazavi et al., 2008). Second, the use of the four nucleotides in all positions of sequencing reads showed a similar distribution to those from previous studies (Wang et al., 2008), suggesting that there were no aberrations in the sequencing methods. Finally,—71% of the reads mapped to exons, 19% of the reads mapped to introns, and 10% of the reads mapped to the intergenic region. These results are comparable to those of published studies using similar sequencing protocols (Wang et al., 2008). The protocol was designed to enrich for fragments in the 200- to 300-bp range. Analysis of the cDNA fragment size of the paired reads confirmed that the highest density of reads was in the 250- bp size as expected. Together, the results indicate that the RNA processing and paired-end sequencing have successfully generated high-quality reads from patient samples, with close to 500 million reads that passed the additional criteria and uniquely mapped to the human genome.
EXAMPLE 8
BIOINFORMATIC CONTROL FOR IDENTIFICATION OF RNA JUNCTION READS
[0187] RNA junction was first determined by RT-PCR and Sanger sequencing. Junction reads that mapped to the determined junction site were then identified using the criteria outlined herein. As a bioinformatic control, the 5' and 3' sequences flanking the junction sites were switched to create artificial RNA junctions and then searched for possible paired reads that would map to the artificial junctions. As expected, the analysis yielded almost no or a negligible number of reads that map to the artificially created junctions. An example of this analysis is shown in FIG. 7B.
EXAMPLE 9
MATERIALS AND METHODS
[0188] All radical prostatectomy tissue samples were obtained from the Baylor Prostate Specialized Programs of Research Excellence (SPORE) Tissue Core. RNA was extracted from samples using the Ribopure kit (Ambion). The human prostate cancer cell lines were maintained as detailed below. Total RNA samples were processed for transcriptome sequencing using the Illumina mRNA-seq protocol. Bioinformatic identification of chimeric RNAs and their corresponding junction reads is detailed below. For validation of chimeric RNAs, RT was performed using superscript II (Invitrogen) and PCR was performed using primers listed herein.
[0189] Human Prostate Cancer Samples and Cell Lines. All radical prostatectomy tissue samples were obtained from the Baylor Prostate Specialized Programs of Research
Excellence (SPORE) Tissue Core and collected from fresh radical prostatectomy specimens after obtaining informed consent under an institutional review board-approved protocol. The pathological status was confirmed before processing, and the tumor samples had a tumor cell percentage of >80% with Gleason scores of 6-9. RNA was extracted from cancer samples, matched benign samples, and noncancer donor samples using the Ribopure kit (Ambion). The human prostate cancer cell lines, PC3, PNTla, DU145 and LNCaP were maintained in RPMI- 1640 (Cellgro) supplemented with 10% FBS (Invitrogen). LAPC4 was maintained in IMDM medium supplemented with 10 nM R1881 and 10% FBS (Invitrogen). VCaP was maintained in DMEM (Cellgro) supplemented with 10% FBS. All media were also supplemented with antibiotics (penicillin and streptomycin). PrEC was obtained from Lonza and maintained as suggested by the instruction provided. RNA was obtained from these cell lines using TRI reagent (Sigma).
[0190] RNA Processing for Paired-End Transcriptome Sequencing. Total RNA samples with RNA integrity number (RIN) >8 were used for transcriptome sequencing using Illumina mRNA-seq protocol. Briefly, 5 μg of total RNA was used to isolate mRNA using Sera- mag Magnetic Oligo(dT) beads. mRNA was then fragmented and converted into double- stranded cDNA. Adapters were ligated to the double- stranded cDNA and this library was then size selected to obtain fragments in the range of 200-300 bp. Finally, PCR amplification was performed to obtain the final cDNA library. A total of 10 nM of the library was then used for 36- nucleotide paired-end sequencing on the Illumina genome analyzer II (GAII) at the Center for Cancer Epigenetics Solexa Sequencing Core located in the University of Texas-M. D. Anderson Cancer Center.
[0191] Bioinformatic Identification of Chimeric RNAs. Our bioinformatics pipeline had several filters to remove false-positive chimeric RNAs. The first step in the pipeline was a quality filter in which only those reads that passed the Illumina/Solexa signal purity filter were used for further analyses. The second step was a uniqueness filter. In this step, the inventors required that both ends of paired reads be uniquely mapped to either the genome or the transcriptome; i.e., the inventors required each read to map to only one location in the genome or transcriptome even with the tolerance of two mismatches. This additional stringent step prevented any homolog from being recruited as a chimera. The output of this step was paired reads that we called "unique pairs." In the third step, the inventors assigned Entrez gene IDs to each read of the unique pair and looked for each read to be assigned to one unique Entrez ID.
Those reads that were assigned to multiple Entrez IDs were discarded. Step four of the pipeline involved the comparison of the Entrez gene ID for the unique pairs of reads. Unique pairs where both reads were assigned to the same Entrez gene ID were discarded. Only those unique pairs where each read was assigned a different Entrez gene ID were considered a candidate chimeric event. To achieve a comprehensive annotation of the human genome, we chose the UCSC knownGene database (March 2006, hgl8) for alignment. Using this bioinformatic pipeline, the inventors obtained ~6,163 putative chimeric events with at least three supporting paired reads from three different patient samples. Of these, chimeric events that involved either KLK2 or KLK3 were removed as the inventors were unable to validate these events. This removal resulted in a final list of 2,369 putative chimeric events. For the list of validated chimeric RNAs (FIG. 3), the inventors also checked them using hgl9 gene annotation and found none of them to be transcriptional isoforms in hgl9.
[0192] To pick out the most recurrent chimeric RNAs, the inventors used a cutoff requiring events to be present in at least 10 of 20 cancer samples. This cutoff resulted in 46 chimeric events that were used for further analysis. These chimeric events were ranked by a one- tailed Kolmogorov-Smirnov test (Conover, 1971), a statistical test that takes into account the difference in accumulative expression levels between cancer and matched benign samples.
[0193] Identification of Junction Reads. RNA junctions were accurately defined using RT-PCR and Sanger sequencing and then used as templates to align junction reads. Reads that were earlier unmappable to the genome and the transcriptome were aligned to the PCR amplicon. A paired read was considered a junction read only if it met the following conditions: (i) one read of the paired read mapped to either parental gene of the chimeric RNA; (ii) the junction read should overlap with at least six nucleotides of the sequence on either side of the RNA junction; and (iii) mismatch tolerance was set at two mismatches, but for the six nucleotides flanking the RNA junction, no mismatches were tolerated. FIG. 3 was generated using Mev (v4.5) (Conover, 1971; Saeed et al., 2006). For FIG. 3, supporting paired chimeric reads and junction reads were pooled together and normalized to 10 million mappable reads.
[0194] Determination of Reads per Kilobase of Exon Model per Million Mapped Reads (RPKM). Reads uniquely aligned to the genome were collected and mapped to the transcriptome. RPKM was calculated as described in Mortazavi et al. (2008). RT-PCR. A total of 1 μg of RNA was used for each reverse transcription reaction. RNA was incubated with Oligo(dT) and dNTPs and denatured at 70 °C. This process was followed by the addition of a master mix containing lx superscript buffer, 10 mM DTT, 5 mM magnesium chloride,
RNaseOUT, and Superscript III reverse transcriptase. Reactions were then incubated at 50 °C for 50 min. Reactions were terminated by incubation at 85 °C for 5 min. cDNA was then treated with RNase- H for 20 min at 37 °C. A total of 1 of cDNA was used as template for PCR using the primers listed in the later section. PCR master mixes included 3% DMSO and PCR was done using a standard three-step protocol with annealing temperature of 56 °C. The products of RT-PCR were gel purified and sequenced by Sanger sequencing to identify the exact fusion junctions of the candidate events. A minus-RT reaction was also conducted and used as a negative control.
[0195] For strand- specific RT-PCR, the following protocol was performed to avoid endogenous priming. All incubations were performed in a thermal cycler. A total of 500 ng of RNA was incubated with 1 μL· of each strand- specific primer (4 μΜ) at 70 °C for 5 min and then at 50 °C for 10 min. At the same time, a master mix containing dNTPs, lx superscript buffer, 10 mM DTT, 5 mM magnesium chloride, and RNaseOUT was also incubated at 50 °C for 5 min. The master mix was then quickly added to the RNA-primer mix while trying to keep reactions at 50 °C. A total of 1 μϊ^ of superscript III reverse transcriptase was also added and the reactions were incubated at 50 °C for 50 min. Reactions were terminated by heating the contents to 85 °C for 10 min. As before, RNA was removed from cDNA by incubation with RNase-H. A total of 1 μL· of the cDNA was then used in the subsequent PCR. To identify the RNA strand, the above first- strand synthesis was performed using either the forward primer or the reverse primer (primers listed in the later section). Then, two PCR reactions were performed using the cDNA generated from the first step. The strand was determined on the basis of which reaction yielded a product.
[0196] Long-Range PCR. Using the primers listed in the later section, we performed long-range PCR on 200 ng of genomic DNA. The LA PCR kit (Takara) was used for these reactions and reactions were performed according to the manufacturer's protocols. As a positive control for long-range PCR reactions, DNA samples and primers were used that were provided by the LA PCR kit. To ensure that the PCR primers designed for each chimera can amplify the intended DNA fragment, we used human genomic DNA (Promega) as a control.
Two-step PCR was performed with annealing and extension at 68 °C. Extension times were varied depending on the expected size of products: for 2-3 kb, 3 min; for 4-8 kb, 5 min; and for 10-30 kb, 15 min. Products were run on gels and the sizes of PCR products obtained from cancer samples and control human genomic DNA were compared. A detectable difference in PCR product size would indicate a DNA rearrangement.
[0197] Quantitative RT-PCR Validation. Quantitative RT-PCR was performed using Absolute Blue QPCR SYBR Green low ROX mix (Thermo Scientific) on Applied Biosystems' 7500 real-time PCR system. Oligonucleotide primers used for quantitative RT-PCR are listed in the next section. A final concentration of 100 nM of each primer was used in the PCR. The program used for PCR was as follows: 95 °C for 15 min (1 cycle); and 95 °C for 15 s and 60 °C for 1 min (40 cycles). All assays were performed in triplicate and the results are shown as average fold change relative to GAPDH, which serves as an internal control.
Quantitative RT-PCR was done only for the chimera TMEM79-SMG5 in the human samples identified in FIG. 6 and PrEC cells. Quantitative RT-PCR was not performed for all chimeras in cell lines or tissue samples.
[0198] Primers used for validation of chimeric transcripts. TMPRSS2-ERG and GAPDH primers are from Maher et al. (2009).
EXAMPLE 10
SPECIFIC EXEMPLARY CHIMERIC RNAs
[0199] Full-length chimeric RNA sequences for GOLM 1 -MAR 10 and TMEM79- SMG5 are provided in SEQ ID NO: 1 and SEQ ID NO:2
[0200] The full-length chimeric RNA sequence allows the prediction of fusion protein(s) translated from the chimeric RNA. Antibodies specifically raised to recognize the unique amino acid sequence but not the normal proteins could detect the presence of fusion protein. This would allow specific detection of fusion proteins in patients' blood or urine, for example, for diagnostic or prognostic purposes. As indicated below, the full-length sequence predicts a long and different amino acid sequence for each of the chimeric RNAs.
[0201] Full length chimeric RNA sequence for GOLM 1 -MAR 10 (SEP ID NO: l): [0202]
ATGATGGGCTTGGGAAACGGGCGTCGCAGCATGAAGTCGCCGCCCCTCGTGCTGGC
CGCCCTGGTGGCCTGCATCATCGTCTTGGGCTTCAACTACTGGATTGCGAGCTCCCG
GAGCGTGGACCTCCAGACACGGATCATGGAGCTGGAAGGCAGGGTCCGCAGGGCG
GCTGCAGAGAGAGGCGCCGTGGAGCTGAAGAAGAACGAGTTCCAGGGAGAGCTGG
AGAAGCAGCGGGAGCAGCTTGACAAAATCCAGTCCAGCCACAACTTCCAGCTGGAG
AGCGTCAACAAGCTGTACCAGGACGAAAAGGCGGTTTTGGTGAATAACATCACCAC
AGGTGAGAGGCTCATCCGAGTGCTGCAAGACCAGTTAAAGACCCTGCAGAGGAATT
ACGGCAGGCTGCAGCAGGATGTCCTCCAGTTTCAGAAGAACCAGACCAACCTGGAG
AGGAAGTTCTCCTACGACCTGAGCCAGTGCATCAATCAGATGAAGGAGGTGAAGGA
ACAGTGTGAGGAGCGAATAGAAGAGGTCACCAAAAAGGGGAATGAAGCTGTAGCT
TCCAGAGACCTGAGTGAAAACAACGACCAGAGACAGCAGCTCCAAGCCCTCAGTGA
GCCTCAGCCCAGGCTGCAGGCAGCAGGCCTGCCACACACAGAGGTGCCACAAGGG
AAGGGAAACGTGCTTGGTAACAGCAAGTCCCAGACACCAGCCCCCAGTTCCGAAGT
GGTTTTGGATTCAAAGAGACAAGTTGAGAAAGAGGAAACCAATGAGATCCAGGTG
GTGAATGAGGAGCCTCAGAGGGACAGGCTGCCGCAGGAGCCAGGCCGGGAGCAGG
TGGTGGAAGACAGACCTGTAGGTGGAAGAGGCTTCGGGGGAGCCGGAGAACTGGG
CCAGACCCCACAGGTGCAGGCTGCCCTGTCAGTGAGCCAGGAAAATCCAGAGATGG
AGGGCCCTGAGCGAGACCAGCTTGTCATCCCCGACGGACAGGAGGAGGAGCAGGA
AGCTGCCGGGGAAGGGAGAAACCAGCAGAAACTGAGAGAGAGTGACTACAACATG
GATGAAAATGAAGCAGAATCTGAGACAGACAAGCAAGCAGCCCTGGCAGGGAATG
ACAGAAACATAGATG*GACACATGAGTGAAGGGGACAACTGGAATGCATCCTTTTC
TTCTCATATATCTTGTTTATTTTGTCGCTCCTGAaagggaacaactactctgccgaagaacatcttttatcaa ctccaaagctcctgaagtgaatcctcagcaccacggtttga
[0203] Bold: Start and stop codon
[0204] Underlined: GOLM1 sequence
[0205] Not underlined: MAK10 sequence
[0206] Upper case: coding sequence
[0207] Lower case: UTR sequence [0208] The chimeric junction is denoted by an asterisk.
[0209] The full-length chimeric RNA predicts a fusion protein.
[0210] Full length chimeric RNA sequence for TMEM79-SMG5 (SEP ID NO:2 :
[0211]
5'gagagtaagaaggctgcgaggagagctcacacatccttggtttgtatgaagaaaaggacagtaccctctaccagggccatcccctcatt gacaaacaggaagagaaggacagtaccctcaaccaggccccccaaccttgagacctcatctctatatttttttaattttaattatttatttatttgtt tgagacggagtctcactctgtcacccaggctggagtgcaatggcgcgatcttggctcactgcaacctccgcctcccaggttcaagcgattct cctgcctccgcctcctgaatagctgggattacagatgcccaccaccacacccggctaattttttgtatttttagtagacctggggtttcaccttgt tggccaagctggtctcgagctcctgacattgtgatccacccacctcagcctcccaaagtgctgggattacaggcgtgagccactacacccg gctatttatttttttgagatgaagtctagctctgtcgtccaggctagagtacagtgacacgatcttgtgtcactgcaatctccacctcctggactca agcgattctcctgcctcagcctcctcagtagctgggactacaggtgtgcaccaccatgcccagctaatttttgtatttttagtagagacagggtt tcaccatattggccaggctggtctcgaactcctgacctcaagtgatctgccacctcggcctctcaaagtgctgagattacaggcatgagcca ctgcacccagccttgtctctattttgaaaaaatatatctatttttaagttgtacatttggttacagggggtgttcaaccagagaccaaaacagctac ctgaccagattgccacaggagtggctgcaatgggtgcattgatctagatgacacctgaaagccttcttgttccatatggttatatatgtctctga aatcggcttccatctgctgaccagctgag*ggctgtggtggaggctgtgcatcgacttgacctcatcctttgcaacaaaactgcttatcaaga agtattcaaaccagaaaacattagcctgaggaacaagctgcgtgagctctgcgtcaagcttATGTTCCTGCACCCAGTGG
ACTATGGGAGAAAGGCTGAGGAGCTGCTGTGGAGAAAGGTATACTATGAAGTTATC
CAGCTTATCAAGACTAACAAAAAGCACATCCACAGCCGGAGCACTTTGGAATGTGC
CTACAGGACGCACCTGGTTGCTGGTATTGGCTTCTACCAGCATCTCCTTCTCTATATC
CAGTCCCACTACCAGCTGGAACTGCAGTGCTGCATCGACTGGACCCATGTCACTGAC
CCCCTCATAGGATGCAAGAAGCCAGTGTCTGCCTCAGGGAAGGAGATGGATTGGGC
ACAGATGGCATGTCACCGATGTCTGGTGTATCTGGGGGATTTGTCCCGATATCAGAA
TGAATTAGCTGGCGTAGATACCGAGCTGCTAGCCGAGAGATTTTACTACCAAGCCCT
GTCAGTAGCTCCTCAGATTGGAATGCCCTTCAATCAGCTGGGCACCCTGGCAGGCA
GCAAGTACTATAATGTGGAAGCCATGTATTGCTACCTGCGCTGCATCCAGTCAGAA
GTGTCCTTTGAGGGAGCCTATGGGAACCTCAAGCGGCTGTATGACAAGGCAGCCAA
AATGTACCACCAACTGAAGAAGTGTGAGACTCGGAAACTGTCTCCTGGCAAAAAGC
GATGTAAAGACATTAAAAGGTTGCTAGTGAACTTTATGTATCTGCAAAGCCTCCTAC
AGCCCAAAAGCAGCTCCGTGGACTCAGAGCTGACCTCACTTTGCCAGTCAGTCCTG
GAGGACTTCAACCTCTGCCTCTTCTACCTGCCCTCCTCACCCAACCTCAGCCTGGCC AGTGAGGATGAGGAGGAGTATGAGAGTGGATATGCTTTCCTCCCGGACCTTCTCAT CTTTCAAATGGTCATCATCTGCCTTATGTGTGTGCACAGCTTGGAGAGAGCAGGATC CAAGCAGTACAGTGCAGCCATTGCCTTCACCCTGGCCCTCTTTTCCCACCTCGTCAA TCATGTCAACATACGGCTGCAGGCTGAGCTGGAAGAGGGCGAGAATCCCGTCCCGG CATTCCAGAGTGATGGCACAGATGAACCAGAGTCCAAGGAACCTGTGGAGAAAGA GGAGGAGCCAGATCCTGAGCCTCCTCCTGTAACACCCCAAGTGGGTGAGGGCAGAA AGAGCCGTAAGTTCTCTCGCCTCTCCTGTCTCCGCCGTCGCCGCCACCCACCCAAAG TTGGTGATGACAGTGACCTGAGTGAAGGCTTTGAATCGGACTCAAGCCATGACTCA GCCCGGGCCAGTGAGGGCTCAGACAGTGGCTCTGACAAGAGTCTTGAAGGTGGGGG AACGGCCTTTGATGCTGAAACAGACTCGGAAATGAATAGCCAGGAGTCCCGATCAG ACTTGGAAGATATGGAGGAAGAGGAGGGGACACGGTCACCAACCCTGGAGCCCCC TCGGGGCAGATCAGAGGCTCCCGATTCCCTCAATGGCCCACTGGGCCCCAGTGAGG CTAGCATTGCCAGCAATCTACAAGCCATGTCCACCCAGATGTTCCAGACTAAGCGCT GCTTCCGACTGGCCCCCACCTTTAGCAACCTGCTCCTCCAGCCCACCACCAACCCTC ATACCTCGGCCAGCCACAGGCCTTGCGTCAATGGGGATGTAGACAAGCCTTCAGAG CCAGCCTCTGAGGAGGGCTCTGAGTCGGAGGGGAGTGAGTCCAGTGGACGCTCCTG TCGGAATGAGCGCAGCATCCAGGAGAAGCTTCAGGTCCTGATGGCCGAAGGTCTGC TTCCTGCTGTGAAAGTCTTCCTGGACTGGCTTCGGACCAACCCCGACCTCATCATCG TGTGTGCGCAGAGCTCTCAAAGTCTGTGGAACCGCCTGTCTGTGTTGCTGAATCTGT TGCCTGCTGCTGGTGAACTCCAGGAGTCTGGCCTGGCCTTGTGTCCTGAGGTCCAAG ATCTTCTTGAAGGTTGTGAACTGCCTGACCTCCCCTCTAGCCTTCTGCTCCCAGAGG ACATGGCTCTTCGTAACCTGCCCCCGCTCCGAGCTGCCCACAGACGCTTTAACTTTG ACACGGATCGGCCCCTGCTCAGCACCTTAGAGGAGTCAGTGGTGCGCATCTGCTGC ATCCGCAGCTTTGGTCATTTCATCGCCCGCCTGCAAGGCAGCATCCTGCAGTTCAAC CCAGAGGTTGGCATCTTCGTCAGCATTGCCCAGTCTGAGCAGGAGAGCCTGCTGCA GCAGGCCCAGGCACAGTTCCGAATGGCACAGGAGGAAGCTCGTCGGAACAGGCTC ATGAGAGACATGGCTCAGCTACGACTTCAGCTCGAAGTGTCTCAGCTGGAGGGCAG CCTGCAGCAGCCCAAGGCCCAGTCAGCCATGTCTCCCTACCTCGTCCCTGACACCCA GGCCCTCTGCCACCATCTCCCTGTCATCCGCCAACTGGCCACCAGTGGCCGCTTCAT TGTCATCATCCCAAGGACAGTGATCGATGGCCTGGATTTGCTGAAGAAGGAACACC CAGGGGCCCGGGATGGGATTCGGTACCTGGAGGCAGAGTTTAAAAAAGGAAACAG GTACATTCGCTGCCAGAAAGAGGTGGGAAAGAGCTTTGAGCGGCATAAGCTGAAGA GGCAGGATGCAGATGCCTGGACTCTCTATAAGATCCTAGACAGCTGCAAACAGCTG
ACTCTGGCCCAGGGGGCAGGTGAGGAGGATCCGAGTGGCATGGTGACCATCATCAC
AGGCCTTCCACTGGACAACCCCAGCGTGCTTTCAGGCCCCATGCAGGCAGCCCTGC
AGGCCGCTGCCCACGCCAGTGTGGACATCAAGAATGTTCTGGACTTCTACAAGCAG
TGGAAGGAAATTGGTTGAtactgacccccaggccctgcagtggggctgactccagatctctcctgccctccctggcagc caggaccagcacctgtagtcaccccaccacacgcagactcatgcacgcacacaggagggaggcctagctgctcagaggctgcaggga gggcccaggagccggctgggagggtggggtccctttgttgccaagacgttaggaaagcgaggaaagtgcttggattaggagagtcttgt gggcccctggccagccttcctgcctcagctcccctgctgtctccaggggcaggtggtaggcatgggtacctgcatttcactggaatgggttc ttggatctctgaggggaaggaacagcaaaagaggcccttcttcctcacccaagatgcagggtggttggggccaggagtttggaccctcta ggtcttgggggaagagctgggtaatacctggtgtctgagtgattctctgcagacccttcccctcctcaaggatcacccatcctcctttcagccc cctttatggggaccaggcagctctggagccagccacaggggctgttagagaagcaaggcctggagtggcctgcaccgagtagcagggt cagggttcgtgtgctcctcctcctgctgcaggggctgcacatcccattgccccacttctgctttgtgtctccctctgtctagcttccagggcagg gagcaggccccacctagggctgcaggcagtctggcctgtgccagcacggtctcctgtgcccaccagccccacaggtgctgtgctttgtgc tcttggctgctgtgctgggacagaatgggatgccaggaagagaagaaagggggtgcagtctgaggccaccaccccccttcctatctaagg gagggctgaagacaaggggccggcattcagtgggcagcagaaaggagaggctccttgaagctgctcagtcagaggcccccgtccctcc ttttgccttccgcaggactgaagacctgaaggggctggcttttggagtgttgaggtgaatatctgggagcagagatcatgaatagctcaggg cagtgaatggcgcaccaagagcagggctgtgtgtgggaggctgcagccaggattgcctcagctcctccccctcaggctgggaggatagc acaggctaggggctcggggtggagggtctcagctctgctgcccccaccccagtactagcctagcttcccaagctgtggcttagaggatagt tggcttcctgcctctctcctctaaaatagcaagtctgggaaatcctggggtgagtggagtcaccccactcccagttgctggcagagactgag actaaagcatcacttaataaaccccccaagccca 3 '
[0212] Underlined: TMEM79 sequence
[0213] Not underlined: SMG5 sequence
[0214] The 5' and 3' UTRs are shown in lower cases while the coding region is shown in capital letters. The start/stop codons and poly- A signal are shown in bold. The chimeric junction is denoted by an asterisk. The full-length chimeric RNA predicts a truncated SMG5 protein. EXAMPLE 11
ASTN2-PAPAA AND GOLM1-MAK10 ARE ENRICHED IN ESOPHAGEAL
SQUAMOUS CELL CANCER
[0215] In addition to their presence in human prostate cancer, ASTN2-PAPAA and GOLM1-MAK10 were present in human esophageal squamous cell cancer. Importantly, they are highly enriched in cancer vs. normal tissues and thus are useful as biomarkers for identifying esophageal squamous cell cancer patients.
EXAMPLE 12
NEW ALTERNATIVE SPLICING ISOFORMS IDENTIFIED FOR THE FOLLOWING TWO
CHIMERIC RNAS
[0216] Because ASTN2-PAPAA and GOLM1-MAK10 are important chimeric RNAs, their splicing isoforms were identified in human prostate and esophageal cancer samples. The exact sequences near the chimeric junctions are shown below. These isoforms provide additional options when used to diagnose their presence in patients.
[0217] ASTN2-PAPAA
[0218] isoform 1 (SEQ ID NO:3)
[0219]
AGTGCCCTATCTTTTCTGCCGCAGCGAGGAGGTCCGGCCTGCAGGCATGGTGTGGTA
TAGCATCCTCAAGGACACCAAAATCACGTGTGAGGAGAAGATGGTGTCAATGGCCC
GAAACAC*GAGCCAGTGTCCTGGGCTAAACACAAGAGTGCTGATTCCCACTGTAAG
TTACAGTGAAGAACTTCTGCTATCTGAGGGCATGTGTTTTCATCTTCAAAAAAGGAT
GGACAGTCCCCATGAACCTTCCCTCTCCAACCACACAGGCCTTGC
[0220] Chimeric junction marked by *
[0221] Underlined: ASTN2
[0222] Not Underlined: PAPAA [0223] isoform 2 (SEQ ID NO:4)
[0224]
AGTGCCCTATCTTTTCTGCCGCAGCGAGGAGGTCCGGCCTGCAGGCATGGTGTGGTA TAGCATCCTCAAGGACACCAAAATCACGT*GrAGAGACCrCrGGrCrAGrCrrGCrCCr GAA GTAGA GCTGCTA TGTGA CCTTGA GCAAGCCGTTCA CCCTCTGTGGGCCCTGGGTTA T CCA TGTGCCCCCA CCA CA CTAGGGGA GA TCTA TGTGTCA TGGA TGTGAAA CA TTA TA TCAG A TGA TGA CTCA G*G AGCC AGTGTCCTGGGCTA A AC AC A AG AGTGCTG ATTCCC ACTGT AAGTTACAGTGAAGAACTTCTGCTATCTGAGGGCATGTGTTTTCATCTTCAAAAAAG GATGGACAGTCCCCATGAACCTTCCCTCTCCAACCACACAGGCCTTGC
[0225] Chimeric junctions marked by *
[0226] Underlined: ASTN2
[0227] Not Underlined: PAPAA
[0228] GOLM1-NAA35
[0229] isoform 1 (SEQ ID NO:5)
[0230] GTGCTAATTTTACAGGGAGAAAC CAGCAGAAACTG
AGAGGAGAAG ATGACTACAA CATGGATGAA AATGAAGCAG AATCTGAGAC AGACAAGCAA GCAGCCCTGG CAGGGAATGA CAGAAACATA GATG* GTTTAT TTTGTCCGCA AGTGAAAGGG AAGAACTACT CTGCCGAAGA TCATCTTTTA TCAACTCCAA
AGCTCCTGAAGTGAATCCTCAGCACCACGGTTTGAAACAGTTATGCTCCCAGTGATC ACCTTGGTCATGATTTCTTCCAAGAAGACAGACCTCATTCCAATTTGATGTTTAA
[0231] Chimeric junction marked by *
[0232] Underlined: GOLM1
[0233] Not Underlined: NAA35
[0234] isoform 2 (SEQ ID NO:6) [0235] GTGCTAATTTTACAGGGAGAAAC CAGCAGAAACTGAGAGGAGA AGATGACTAC AACATGGATG AAAATGAAGC AGAATCTGAG
AC AG AC A AGC A AGCAGCCCTGGCAGGG A ATG AC AG A A AC AT AG ATG* GA CA CA TGA GTGAAGGGGACAACTAAGATGCAGC CTTTTCTTCTCAAA TA TCA G GTTTATTTTGT CCGCAAGTGAAAGGGAAGAACTACTCTGCCGAAGATCATCTTTTATCAACTCCAAA GCTCCTGAAGTGAATCCTCAGCACCACGGTTTGAAACAGTTATGCTCCCAGTGATCA CCTTGGTCATGATTTCTTCCAAGAAGACAGACCTCATTCCAATTTGATGTTTAA
[0236] Chimeric junction marked by *
[0237] Underlined: GOLM1
[0238] Not Underlined: NAA35
EXAMPLE 13
CHIMERIC RNAS ARE DETECTABLE IN PROSTATE CANCER PATIENT URINE
[0239] The present example concerns evaluation of two chimeric RNAs in exosomes isolated from patients' urine after digital rectal exam (FIG. 10). NC refers to a negative control starting from PicoSI kit without input RNA. GOLM1-MAK10 tested positive in Patient 2. TMPRSS2-ERG tested negative in both patient samples. This indicates that chimeric RNAs are present at a detectable level in urine collected after digital rectal exam. Digital rectal exam requires a visit to the doctor and is an uncomfortable procedure for patients. Urines without digital rectal exam can be collected by a patient himself at home and mailed for analysis.
[0240] There was evaluation of 24 exemplary chimeric RNAs in exosome isolated from human urine without digital rectal exam.
Figure imgf000076_0001
Figure imgf000077_0001
[0241] FIG. 11 shows results of the assay for the exemplary chimeric RNAs noted above. Positive controls used previously isolated PCR bands as templates. FIG. 11 shows that the exemplary PCR conditions (30 ng of cDNA as template in a 50 μΐ PCR reaction) were suitable. Thirteen out of 24 chimeric RNAs were present in the exosomes of urine samples, which indicates that chimeric RNAs are present at a detectable level in urine collected without digital rectal exam.
[0242] Two chimeric RNAs (number 4 and number 19) were present in patients, but absent in normal control. Four chimeric RNAs were present in normal control, but absent in cancer patients. One can next evaluate additional chimeric RNA candidates.
[0243] In Table 5, the following chimeric genes have a 40% recurrent rate in prostate cancer and in certain embodiments of the invention, one or more of these chimeric genes are analyzed in methods of the invention.
[0244] Table 5: Exemplary chimeric genes associated with prostate cancer Gene
ID Gene name
Gene 1 Gene 2 Gene 1 Gene 2
1153 55009 CIRBP C19orf24
1674 2316 DES FLNA
1938 2316 EEF2 FLNA
2170 51538 FABP3 ZCCHC17
2316 4627 FLNA MYH9
2316 4629 FLNA MYH11
23524 378938 SRRM2 MALAT1
26505 200539 CNNM3 ANKRD23
3329 3336 HSPD1 HSPE1
4261 28955 CIITA DEXI
4629 23524 MYH11 SRRM2
4691 23524 NCL SRRM2
55003 347744 PAK1IP1 C6orf52
55721 149069 IQCC DCDC2B
5696 6890 PSMB8 TAP1
6448 79092 SGSH CARMA2
6876 51092 TAGLN SIDT2
7917 57827 BAT3 C6orf47
811 23524 CALR SRRM2
8675 79716 STX16 NPEPL1
8717 55336 TRADD FBXL8
8878 11282 SQSTM1 MGAT4B
968 9526 CD68 MPDU1
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[0347] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims

CLAIMS What is claimed is:
1. As a composition of matter, an isolated chimeric RNA selected from the group consisting of TMEM79-SMG5 RNA, ASTN2- PAPAA RNA, GOLMJ-MAKJ0 RNA and one or more chimeric RNAs in Table 5.
2. A substrate comprising polynucleotides attached thereto, said
polynucleotides defined as one or more isolated chimeric RNAs from claim 1.
3. The substrate of claim 2, wherein all or greater than 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% of the polynucleotides attached to the substrate are one or more isolated chimeric RNAs of TMEM79-SMG5 RNA, ASTN2-PAPAA RNA, or GOLMJ-MAKJ0 RNA.
4. A method of detecting the presence of cancer, an increased risk for cancer, or response to cancer therapy in an individual, comprising the step of detecting one or more chimeric RNAs of claim 1 in a sample from the individual.
5. The method of claim 4, wherein the cancer is androgen-sensitive cancer.
6. The method of claim 4, wherein the cancer is prostate cancer or esophageal cancer.
7. The method of claim 4, wherein the individual is provided a
therapy upon detection of the one or more chimeric RNAs.
8. The method of claim 4, wherein the chimeric RNA is detected in nucleic acid form.
9. The method of claim 8, wherein the detecting step comprises polymerase chain reaction.
10. The method of claim 9, wherein the polymerase chain reaction amplifies all or part of the chimeric RNA.
11. The method of claim 9, wherein the polymerase chain reaction amplifies the junction of the chimeric RNA.
12. The method of claim 4, wherein the sample comprises serum, urine, prostatic fluid, or biopsy.
13. The method of claim 4, wherein the chimeric RNA is detected by its expressed fusion protein.
14. The method of claim 13, wherein the method comprises detection of the fusion protein by antibody.
15. The method of claim 14, wherein the antibody recognizes the junction of the fusion protein.
16. The method of claim 4, further comprising the step of performing an additional cancer detection step on a sample from the individual.
17. The method of claim 4, further comprising the step of obtaining the sample from the individual.
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