US20110172113A1 - Aberrant mitochondrial dna, associated fusion transcripts and hybridization probes therefor - Google Patents

Aberrant mitochondrial dna, associated fusion transcripts and hybridization probes therefor Download PDF

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US20110172113A1
US20110172113A1 US12/935,181 US93518109A US2011172113A1 US 20110172113 A1 US20110172113 A1 US 20110172113A1 US 93518109 A US93518109 A US 93518109A US 2011172113 A1 US2011172113 A1 US 2011172113A1
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cancer
transcript
mitochondrial
mtdna
mitochondrial fusion
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Ryan Parr
Brian Reguly
Gabriel Dakubo
Jennifer Creed
Kerry Robinson
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MDNA LIFE SCIENCES Inc
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Mitomics Inc
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    • 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
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    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of mitochondrial genomics.
  • the invention relates to the identification and use of mitochondrial genome fusion transcripts and probes that hybridize thereto.
  • the mitochondrial genome is a compact yet critical sequence of nucleic acids.
  • Mitochondrial DNA or “mtDNA”, comprises a small genome of 16,569 nucleic acid base pairs (bp) (Anderson et al., 1981; Andrews et al., 1999) in contrast to the immense nuclear genome of 3.3 billion by (haploid). Its genetic complement is substantially smaller than that of its nuclear cell mate (0.0005%). However, individual cells carry anywhere from 10 3 to 10 4 mitochondria depending on specific cellular functions (Singh and Modica-Napolitano 2002). Communication or chemical signalling routinely occurs between the nuclear and mitochondrial genomes (Sherratt et al., 1997).
  • the mitochondrial genome codes for a complement of 24 genes, including 2 rRNAs and 22 tRNAs that ensure correct translation of the remaining 13 genes which are vital to electron transport (see FIG. 1 ).
  • the mitochondrial genome is dependent on seventy nuclear encoded proteins to accomplish the oxidation and reduction reactions necessary for this vital function, in addition to the thirteen polypeptides supplied by the mitochondrial genome. Both nuclear and mitochondrial proteins form complexes spanning the inner mitochondrial membrane and collectively generate 80-90% of the chemical fuel adenosine triphosphate, or ATP, required for cellular metabolism.
  • mitochondria In addition to energy production, mitochondria play a central role in other metabolic pathways as well. A critical function of the mitochondria is mediation of cell death, or apoptosis (see Green and Kroemer, 2005). Essentially, there are signal pathways which permeabilize the outer mitochondrial membrane, or in addition, the inner mitochondrial membrane as well. When particular mitochondrial proteins are released into the cytosol, non-reversible cell death is set in motion. This process highlights the multi-functional role that some mitochondrial proteins have. These multi-tasking proteins suggest that there are other mitochondrial proteins as well which may have alternate functions.
  • the mitochondrial genome is unusual in that it is a circular, intron-less DNA molecule.
  • the genome is interspersed with repeat motifs which flank specific lengths of sequences. Sequences between these repeats are prone to deletion under circumstances which are not well understood. Given the number of repeats in the mitochondrial genome, there are many possible deletions. The best known example is the 4977 “common deletion.” This deletion has been associated with several purported conditions and diseases and is thought to increase in frequency with aging (Dai et al., 2004; Ro et al., 2003; Barron et al., 2001; Lewis et al., 2000; Muller-Hocker, 1998; Porteous et al., 1998) ( FIG. 4 ).
  • mitochondrial deletions are merely deleterious by-products of damage to the mitochondrial genome by such agents as reactive oxygen species and UVR. (Krishnan et al 2008, Nature Genetics). Further, though it is recognized that high levels of mtDNA deletions can have severe consequences on the cell's ability to produce energy in the form of ATP as a result of missing gene sequences necessary for cellular respiration, it is not anticipated that these deleted mitochondrial molecules may be a component of downstream pathways, have an intended functional role, and possibly may be more aptly viewed as alternate natural forms of the recognized genes of the mitochondria as has been anticipated by the Applicant.
  • mtDNA The sequence dynamics of mtDNA are important diagnostic tools. Mutations in mtDNA are often preliminary indicators of developing disease. For example, it has been demonstrated that point mutations in the mitochondrial genome are characteristic of tumour foci in the prostate. This trend also extends to normal appearing tissue both adjacent to and distant from tumour tissue (Parr et al. 2006). This suggests that mitochondrial mutations occur early in the malignant transformation pathway.
  • the frequency of a 3.4 kb mitochondrial deletion has excellent utility in discriminating between benign and malignant prostate tissues (Maki et al. 2008).
  • Mitochondrial fusion transcripts have been reported previously in the literature, first in soybeans (Morgens et al. 1984) and then later in two patients with Kearns-Sayre Syndrome, a rare neuromuscular disorder (Nakase et al 1990). Importantly, these transcripts were not found to have (or investigated regarding) association with any human cancers.
  • An object of the present invention to provide aberrant mitochondrial DNA, associated fusion transcripts and hybridization probes therefor.
  • an isolated mitochondrial fusion transcript associated with cancer in accordance with an aspect of the invention, there is provided an isolated mitochondrial fusion transcript associated with cancer.
  • a mitochondrial fusion protein corresponding to the above fusion transcript, having a sequence as set forth in any one of SEQ ID NOs: 34 to 49 and 52.
  • an isolated mtDNA encoding a fusion transcript of the invention is provided.
  • a hybridization probe having a nucleic acid sequence complementary to at least a portion of a mitochondrial fusion transcript or an mtDNA of the invention.
  • a method of detecting a cancer in a mammal comprising assaying a tissue sample from the mammal for the presence of at least one mitochondrial fusion transcript associated with cancer by hybridizing the sample with at least one hybridization probe having a nucleic acid sequence complementary to at least a portion of a mitochondrial fusion transcript according to the invention.
  • a method of detecting a cancer in a mammal comprising assaying a tissue sample from the mammal for the presence of at least one aberrant mtDNA associated with cancer by hybridizing the sample with at least one hybridization probe having a nucleic acid sequence complementary to at least a portion of an mtDNA according to the invention.
  • kits for conducting an assay for detecting the presence of a cancer in a mammal comprising at least one hybridization probe complementary to at least a portion of a fusion transcript or an mtDNA of the invention.
  • a screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial fusion transcripts for identification of those associated with cancer.
  • a screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial DNAs corresponding to mitochondrial fusion transcripts for identification of those associated with cancer.
  • a screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial fusion transcripts for identification of those associated with cancer.
  • a screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial DNAs corresponding to mitochondrial fusion transcripts for identification of those associated with cancer.
  • FIG. 1 is an illustration showing mitochondrial coding genes.
  • FIG. 2 shows polyadenalated fusion transcripts in prostate samples invoked by the loss of the 3.4 kb deletion.
  • FIG. 3 shows polyadenalated fusion transcripts in prostate samples invoked by the loss of the 4977 kb common deletion.
  • FIG. 4 shows polyadenalated fusion transcripts in breast samples invoked by the loss of the 3.4 kb segment from the mtgenome.
  • FIGS. 5 a and 5 b show an example of a mitochondrial DNA region before and after splicing of genes.
  • FIGS. 6 a to 6 g illustrate the results for transcripts 2, 3, 8, 9, 10, 11 and 12 of the invention in the identification of colorectal cancer tumours.
  • FIGS. 7 a to 7 d illustrate the results for transcripts 6, 8, 10 and 20 of the invention in the identification of lung cancer tumours.
  • FIGS. 8 a to 8 g illustrate the results for transcripts 6, 10, 11, 14, 15, 16 and 20 of the invention in the identification of melanomas.
  • FIGS. 9 a to 9 h illustrate the results for transcripts 1, 2, 3, 6, 11, 12, 15 and 20 of the invention in the identification of ovarian cancer.
  • FIGS. 10 to 18 illustrate the results for transcripts 2, 3, 4, 11, 12, 13, 15, 16 and 20 of the invention in the identification of testicular cancer.
  • the present invention provides novel mitochondrial fusion transcripts and the parent mutated mtDNA molecules that are useful for predicting, diagnosing and/or monitoring cancer.
  • the invention further provides hybridization probes for the detection of fusion transcripts and associated mtDNA molecules and the use of such probes.
  • “aberration” or “mutation” encompasses any modification in the wild type mitochondrial DNA sequence that results in a fusion transcript and includes, without limitation, insertions, translocations, deletions, duplications, recombinations, rearrangements or combinations thereof.
  • biological sample refers to a tissue or bodily fluid containing cells from which a molecule of interest can be obtained.
  • the biological sample can be derived from tissue such as prostate, breast, colorectal, lung and skin, or from blood, saliva, cerebral spinal fluid, sputa, urine, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like.
  • the biological sample may be a surgical specimen or a biopsy specimen.
  • the biological sample can be used either directly as obtained from the source or following a pre-treatment to modify the character of the sample.
  • the biological sample can be pre-treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, and the like.
  • a “continuous” transcript is a fusion transcript that keeps the reading frame from the beginning to the end of both spliced genes.
  • An “end” transcript is a fusion transcript that results in a premature termination codon before the original termination codon of a second spliced gene.
  • mitochondria DNA As used herein, “mitochondrial DNA” or “mtDNA” is DNA present in mitochondria.
  • mitochondrial fusion transcript refers to an RNA transcription product produced as a result of the transcription of a mutated mitochondrial DNA sequence wherein such mutations may comprise mitochondrial deletions and other large-scale mitochondrial DNA rearrangements.
  • mitochondrial fusion transcripts have been reported in soybeans (Morgens et al. 1984) and in humans suffering from a rare neuromuscular disorder (Nakase et al 1990). Fusion transcripts associated with human cancer have not, however, been described.
  • Mitochondrial DNA (mtDNA) dynamics are an important diagnostic tool. Mutations in mtDNA are often preliminary indicators of developing disease and behave as biomarkers indicative of risk factors associated with disease onset. According to the present invention, large-scale rearrangement mutations in the mitochondrial genome result in the generation of fusion transcripts associated with cancer. Thus, the use of mtDNA encoding such transcripts and probes directed thereto for the detection, diagnosis and monitoring of cancer is provided.
  • mtDNA molecules for use in the methods of the present invention may be derived through the isolation of naturally-occurring mutants or may be based on the complementary sequence of any of the fusion transcripts described herein.
  • Exemplary mtDNA sequences and fusion transcripts are disclosed in Applicant's U.S. priority application No. 61/040,616, herein incorporated in its entirety by reference.
  • Mutant mtDNA sequences according to the present invention may comprise any modification that results in the generation of a fusion transcript.
  • modifications include insertions, translocations, deletions, duplications, recombinations, rearrangements or combinations thereof. While the modification or change can vary greatly in size from only a few bases to several kilobases, preferably the modification results in a substantive deletion or other large-scale genomic aberration.
  • Extraction of DNA to detect the presence of such mutations may take place using art-recognized methods, followed by amplification of all or a region of the mitochondrial genome, and may include sequencing of the mitochondrial genome, as described in Current Protocols in Molecular Biology. Alternatively, crude tissue homogenates may be used as well as techniques not requiring amplification of specific fragments of interest.
  • the step of detecting the mutations can be selected from any technique as is known to those skilled in the art.
  • analyzing mtDNA can comprise selection of targets by branching DNA, sequencing the mtDNA, amplifying mtDNA by PCR, Southern, Northern, Western South-Western blot hybridizations, denaturing HPLC, hybridization to microarrays, biochips or gene chips, molecular marker analysis, biosensors, melting temperature profiling or a combination of any of the above.
  • mtDNA is amplified by PCR prior to sequencing.
  • the method of PCR is well known in the art and may be performed as described in Mullis and Faloona, 1987, Methods Enzymol., 155: 335.
  • PCR products can be sequenced directly or cloned into a vector which is then placed into a bacterial host. Examples of DNA sequencing methods are found in Brumley, R. L. Jr. and Smith, L. M., 1991, Rapid DNA sequencing by horizontal ultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 and Luckey, J.
  • the primer can be prepared using conventional solid-phase synthesis using commercially available equipment, such as that available from Applied Biosystems USA Inc. (Foster City, Calif.), DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.).
  • a junction point of a sequence deletion is first identified. Sequence deletions are primarily identified by direct and indirect repetitive elements which flank the sequence to be deleted at the 5′ and 3′ end. The removal of a section of the nucleotides from the genome followed by the ligation of the genome results in the creation of a novel junction point.
  • the nucleotides of the genes flanking the junction point are determined in order to identify a spliced gene.
  • the spliced gene comprises the initiation codon from the first gene and the termination codon of the second gene, and may be expressed as a continuous transcript, i.e. one that keeps the reading frame from the beginning to the end of both spliced genes. It is also possible that alternate initiation or termination codons contained within the gene sequences may be used as is evidenced by SEQ ID No:2 and SEQ ID No: 17 disclosed herein.
  • Some known mitochondrial deletions discovered to have an open reading frame (ORF) when the rearranged sequences are rejoined at the splice site are provided in Table 1.
  • Exemplary mtDNA molecules for use in the methods of the present invention which have been verified to exist in the lab, are provided below. These mtDNAs are based on modifications of the known mitochondrial genome (SEQ ID NO: 1) and have been assigned a fusion or “FUS” designation, wherein A:B represents the junction point between the last mitochondrial nucleotide of the first spliced gene and the first mitochondrial nucleotide of the second spliced gene.
  • the identification of the spliced genes is provided in parentheses followed by the corresponding sequence identifier.
  • AltMet AltMet
  • (OrigMet) refer to alternate and original translation start sites, respectively.
  • the present invention also provides the use of variants or fragments of these sequences for predicting, diagnosing and/or monitoring cancer.
  • variant refers to a nucleic acid differing from a mtDNA sequence of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to a select mtDNA sequence. Specifically, the variants of the present invention comprise at least one of the nucleotides of the junction point of the spliced genes, and may further comprise one or more nucleotides adjacent thereto. In one embodiment of the invention, the variant sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the mtDNA sequences of the invention, or the complementary strand thereto.
  • fragment refers to a short nucleic acid sequence which is a portion of that contained in the disclosed genomic sequences, or the complementary strand thereto. This portion includes at least one of the nucleotides comprising the junction point of the spliced genes, and may further comprise one or more nucleotides adjacent thereto.
  • the fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
  • a fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases of any one of the mtDNA sequences listed above.
  • “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • These fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are also contemplated.
  • the mtDNA sequences are selected from the group consisting of:
  • probe refers to an oligonucleotide which forms a duplex structure with a sequence in the target nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the target region.
  • the probe may be labeled, according to methods known in the art.
  • hybridization of mtDNA to, for example, an array of oligonucleotides can be used to identify particular mutations, however, any known method of hybridization may be used.
  • probes may be generated directly against exemplary mtDNA fusion molecules of the invention, or to a fragment or variant thereof.
  • sequences set forth in SEQ ID NOs: 2-17 and 51 and those disclosed in Table 1 can be used to design primers or probes that will detect a nucleic acid sequence comprising a fusion sequence of interest.
  • primers or probes which hybridize to these nucleic acid molecules may do so under highly stringent hybridization conditions or lower stringency conditions, such conditions known to those skilled in the art and found, for example, in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6.
  • the probes of the invention contain a sequence complementary to at least a portion of the aberrant mtDNA comprising the junction point of the spliced genes. This portion includes at least one of the nucleotides involved in the junction point A:B, and may further comprise one or more nucleotides adjacent thereto.
  • the present invention encompasses any suitable targeting mechanism that will select an mtDNA molecule using the nucleotides involved and/or adjacent to the junction point A:B.
  • the probe may be a hybridization probe, the binding of which to a target nucleotide sequence can be detected using a general DNA binding dye such as ethidium bromide, SYBR® Green, SYBR® Gold and the like.
  • the probe can incorporate one or more detectable labels. Detectable labels are molecules or moieties a property or characteristic of which can be detected directly or indirectly and are chosen such that the ability of the probe to hybridize with its target sequence is not affected. Methods of labelling nucleic acid sequences are well-known in the art (see, for example, Ausubel et al., (1997 & updates) Current Protocols in Molecular Biology, Wiley & Sons, New York).
  • Labels suitable for use with the probes of the present invention include those that can be directly detected, such as radioisotopes, fluorophores, chemiluminophores, enzymes, colloidal particles, fluorescent microparticles, and the like.
  • directly detectable labels may require additional components, such as substrates, triggering reagents, light, and the like to enable detection of the label.
  • the present invention also contemplates the use of labels that are detected indirectly.
  • the probes of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
  • a probe of “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases that are complementary to an mtDNA sequence of the invention. Of course, larger probes (e.g., 50, 150, 500, 600, 2000 nucleotides) may be preferable.
  • the probes of the invention will also hybridize to nucleic acid molecules in biological samples, thereby enabling the methods of the invention. Accordingly, in one aspect of the invention, there is provided a hybridization probe for use in the detection of cancer, wherein the probe is complementary to at least a portion of an aberrant mtDNA molecule. In another aspect the present invention provides probes and a use of (or a method of using) such probes for the detection of colorectal cancer, lung cancer, breast cancer, ovarian cancer, testicular, cancer, prostate cancer and/or melanoma skin cancer.
  • Measuring the level of aberrant mtDNA in a biological sample can determine the presence of one or more cancers in a subject.
  • the present invention encompasses methods for predicting, diagnosing or monitoring cancer, comprising obtaining one or more biological samples, extracting mtDNA from the samples, and assaying the samples for aberrant mtDNA by: quantifying the amount of one or more aberrant mtDNA sequences in the sample and comparing the quantity detected with a reference value.
  • the reference value is based on whether the method seeks to predict, diagnose or monitor cancer. Accordingly, the reference value may relate to mtDNA data collected from one or more known non-cancerous biological samples, from one or more known cancerous biological samples, and/or from one or more biological samples taken over time.
  • the invention provides a method of detecting cancer in a mammal, the method comprising assaying a tissue sample from the mammal for the presence of an aberrant mitochondrial DNA described above.
  • the present invention also provides for methods comprising assaying a tissue sample from the mammal by hybridizing the sample with at least one hybridization probe.
  • the probe may be generated against a mutant mitochondrial DNA sequence of the invention as described herein.
  • the invention provides a method as above, wherein the assay comprises:
  • diagnostic imaging assays as described below.
  • the diagnostic assays of the invention can be readily adapted for high-throughput.
  • High-throughput assays provide the advantage of processing many samples simultaneously and significantly decrease the time required to screen a large number of samples.
  • the present invention contemplates the use of the nucleotides of the present invention in high-throughput screening or assays to detect and/or quantitate target nucleotide sequences in a plurality of test samples.
  • the present invention further provides the identification of fusion transcripts and associated hybridization probes useful in methods for predicting, diagnosing and/or monitoring cancer.
  • fusion transcripts and associated hybridization probes useful in methods for predicting, diagnosing and/or monitoring cancer.
  • mtDNAs typically comprise a spliced gene having the initiation codon from the first gene and the termination codon of the second gene.
  • fusion transcripts derived therefrom comprise a junction point associated with the spliced genes.
  • Naturally occurring fusion transcripts can be extracted from a biological sample and identified according to any suitable method known in the art, or may be conducted according to the methods described in the examples.
  • stable polyadenylated fusion transcripts are identified using Oligo(dT) primers that target transcripts with poly-A tails, followed by RT-PCR using primer pairs designed against the target transcript.
  • fusion transcripts were detected using such methods and found useful in predicting, diagnosing and/or monitoring cancer as indicated in the examples.
  • fusion transcripts derived from the ORF sequences identified in Table 1 may be useful in predicting, diagnosing and/or monitoring cancer according to the assays and methods of the present invention.
  • fusion transcripts of like character to those described herein are contemplated for use in the field of clinical oncology.
  • Fusion transcripts can also be produced by recombinant techniques known in the art. Typically this involves transformation (including transfection, transduction, or infection) of a suitable host cell with an expression vector comprising an mtDNA sequence of interest.
  • Variants or fragments of the fusion transcripts identified herein are also provided. Such sequences may adhere to the size limitations and percent identities described above with respect to genomic variants and fragments, or as determined suitable by a skilled technician.
  • transcripts 1-16 and 20 are listed below. These sequences, which encode hypothetical fusion proteins, are provided as a further embodiment of the present invention.
  • primers or probes can be developed to target the transcript in a biological sample.
  • primers and probes may be prepared using any known method (as described above) or as set out in the examples provided below.
  • a probe may, for example, be generated for the fusion transcript, and detection technologies, such as QuantiGene 2.0TM by PanomicsTM, used to detect the presence of the transcript in a sample.
  • Primers and probes may be generated directly against exemplary fusion transcripts of the invention, or to a fragment or variant thereof. For instance, the sequences set forth in SEQ ID NOs: 18-33 and 50 as well as those disclosed in Table 1 can be used to design probes that will detect a nucleic acid sequence comprising a fusion sequence of interest.
  • probes designed to hybridize to the fusion transcripts of the invention contain a sequence complementary to at least a portion of the transcript expressing the junction point of the spliced genes. This portion includes at least one of the nucleotides complementary to the expressed junction point, and may further comprise one or more complementary nucleotides adjacent thereto.
  • the present invention encompasses any suitable targeting mechanism that will select a fusion transcript that uses the nucleotides involved and adjacent to the junction point of the spliced genes.
  • transcript probes of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
  • a probe of “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases that are complementary to an mtDNA sequence of the invention. Of course, larger probes (e.g., 50, 150, 500, 600, 2000 nucleotides) may be preferable.
  • the invention provides a hybridization probe for use in the detection of cancer, wherein the probe is complementary to at least a portion of a mitochondrial fusion transcript provided above.
  • the present invention provides probes and a use of (or a method of using) such probes for the detection of colorectal cancer, lung cancer, breast cancer, ovarian cancer, testicular cancer, prostate cancer or melanoma skin cancer.
  • Measuring the level of mitochondrial fusion transcripts in a biological sample can determine the presence of one or more cancers in a subject.
  • the present invention provides methods for predicting, diagnosing or monitoring cancer, comprising obtaining one or more biological samples, extracting mitochondrial RNA from the samples, and assaying the samples for fusion transcripts by: quantifying the amount of one or more fusion transcripts in the sample and comparing the quantity detected with a reference value.
  • the reference value is based on whether the method seeks to predict, diagnose or monitor cancer. Accordingly, the reference value may relate to transcript data collected from one or more known non-cancerous biological samples, from one or more known cancerous biological samples, and/or from one or more biological samples taken over time.
  • the invention provides a method of detecting a cancer in a mammal, the method comprising assaying a tissue sample from said mammal for the presence of at least one fusion transcript of the invention by hybridizing said sample with at least one hybridization probe having a nucleic acid sequence complementary to at least a portion of the mitochondrial fusion transcript.
  • the invention provides a method as above, wherein the assay comprises:
  • the diagnostic assays of the invention may also comprise diagnostic methods and screening tools as described herein and can be readily adapted for high-throughput.
  • the present invention contemplates the use of the fusion transcripts and associated probes of the present invention in high-throughput screening or assays to detect and/or quantitate target nucleotide sequences in a plurality of test samples.
  • Methods and screening tools for diagnosing specific diseases or identifying specific mitochondrial mutations are also herein contemplated. Any known method of hybridization may be used to carry out such methods including, without limitation, probe/primer based technologies such as branched DNA and qPCR, both single-plex and multi-plex.
  • Array technology which has oligonucleotide probes matching the wild type or mutated region, and a control probe, may also be used.
  • Commercially available arrays such as microarrays or gene chips are suitable. These arrays contain thousands of matched and control pairs of probes on a slide or microchip, and are capable of sequencing the entire genome very quickly. Review articles describing the use of microarrays in genome and DNA sequence analysis are available on-line.
  • Screening tools designed to identify targets which are relevant to a given biological condition may include specific arrangements of nucleic acids associated with a particular disease or disorder.
  • a screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial fusion transcripts for identification of those associated with one or more cancers.
  • a screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial DNAs corresponding to mitochondrial fusion transcripts for identification of those associated with one or more cancers.
  • a screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial fusion transcripts for identification of those associated with one or more cancers.
  • a screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial DNAs corresponding to mitochondrial fusion transcripts for identification of those associated with one or more cancers.
  • Imaging techniques as Positron Emission Tomography (PET), contrast Magnetic Resonance Imaging (MRI) or the like. These diagnostic methods are well known to those of skill in the art and are useful in the diagnosis and prognosis of cancer.
  • PET Positron Emission Tomography
  • MRI contrast Magnetic Resonance Imaging
  • the methods of the present invention may further comprise the step of recommending a monitoring regime or course of therapy based on the outcome of one or more assays.
  • This allows clinicians to practice personalized medicine; e.g. cancer therapy, by monitoring the progression of the patient's cancer (such as by recognizing when an initial or subsequent mutation occurs) or treatment (such as by recognizing when a mutation is stabilized).
  • the information can be used to diagnose a pre-cancerous condition or existing cancer condition. Further, by quantitating the amount of aberrant mtDNA in successive samples over time, the progression of a cancer condition can be monitored. For example, data provided by assaying the patient's tissues at one point in time to detect a first set of mutations from wild-type could be compared against data provided from a subsequent assay, to determine if changes in the aberration have occurred.
  • the mutation may be indicative of a genetic susceptibility to develop a cancer condition.
  • a determination of susceptibility to disease or diagnosis of its presence can further be evaluated on a qualitative basis based on information concerning the prevalence, if any, of the cancer condition in the patient's family history and the presence of other risk factors, such as exposure to environmental factors and whether the patient's cells also carry a mutation of another sort.
  • biological sample refers to a tissue or bodily fluid containing cells from which mtDNA and mtRNA can be obtained.
  • the biological sample can be derived from tissue including, but not limited to, skin, lung, breast, prostate, nervous, muscle, heart, stomach, colon, rectal tissue and the like; or from blood, saliva, cerebral spinal fluid, sputa, urine, mucous, synovial fluid, peritoneal fluid, amniotic fluid and the like.
  • the biological sample may be obtained from a cancerous or non-cancerous tissue and may be, but is not limited to, a surgical specimen or a biopsy specimen.
  • the biological sample can be used either directly as obtained from the source or following a pre-treatment to modify the character of the sample.
  • the biological sample can be pre-treated prior to use by, for example, preparing plasma or serum from blood, disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, and the like.
  • sample type may be assayed at a single time (i.e. for the detection of more than one cancer).
  • a course of collections are required, for example, for the monitoring of cancer over time, a given sample may be diagnosed alone or together with other samples taken throughout a test period.
  • biological samples may be taken once only, or at regular intervals such as biweekly, monthly, semi-annually or annually.
  • kits for detecting cancer in a clinical environment.
  • kits may include one or more sampling means, in combination with one or more probes according to the present invention.
  • kits can optionally include reagents required to conduct a diagnostic assay, such as buffers, salts, detection reagents, and the like.
  • Other components such as buffers and solutions for the isolation and/or treatment of a biological sample, may also be included in the kit.
  • One or more of the components of the kit may be lyophilised and the kit may further comprise reagents suitable for the reconstitution of the lyophilised components.
  • the kit may also contain reaction vessels, mixing vessels and other components that facilitate the preparation of the test sample.
  • the kit may also optionally include instructions for use, which may be provided in paper form or in computer-readable form, such as a disc, CD, DVD or the like.
  • kits for diagnosing cancer comprising sampling means and a hybridization probe of the invention.
  • the mitochondrial 4977 “common deletion” and a 3.4 kb deletion previously identified by the present Applicant in PCT application no. PCT/CA2007/001711 result in unique open reading frames having active transcripts as identified by oligo-dT selection in prostate tissue ( FIGS. 2 and 3 ). Examination of breast tissue samples also reveals the presence of a stable polyadenylated fusion transcript resulting from the 3.4 kb deletion ( FIG. 4 ).
  • RNA concentrations were adjusted to 100 ng/ul and 2 ul of each template were used for first strand DNA synthesis with SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen) following the manufacturer's instructions.
  • Oligo(dT) primers that target transcripts with poly-A tails were used.
  • the reaction cocktail included: 2 ⁇ SYBR® Green Supermix (100 mM KCL, 40 mM Tris-HCl, pH 8.4, 0.4 mM of each dNTP [dATP, dCTP, dGTP, and dTTP], iTaqTM DNA polymerase, 50 units/ml, 6 mM MgCl 2 , SYBR® Green 1, 20 nM flourescein, and stabilizers), 250 nM each of primers, and ddH 2 O.
  • PCR cycling parameters were as follows; (1) 95° C. for 2 min, (2) 95° C. for 30 sec, (3) 55° C. (for the 4977 bp deletion) and 63° C.
  • FIG. 2 is an agarose gel showing polyadenalated fusion transcripts in prostate samples invoked by the loss of 3.4 kb from the mitochondrial genome.
  • B-blank Lanes 1-6 transcripts detected in cDNA; lanes 7-12 no reverse transcriptase (RT) controls for samples in lanes 1-6.
  • RT reverse transcriptase
  • FIG. 3 shows polyadenalated fusion transcripts in prostate samples invoked by the loss of the 4977 kb common deletion.
  • B-blank Lanes 1-6 transcripts detected in cDNA; lanes 7-12 no RT controls for samples in lanes 1-6.
  • FIG. 4 shows polyadenalated fusion transcripts in breast samples invoked by the loss of 3.4 kb from the mtgenome.
  • Lanes 2-8 transcripts from breast cDNAs Lanes 2-8 transcripts from breast cDNAs; lane 9 negative (water) control; lanes 10 and 11, negative, no RT, controls for samples in lanes 2 and 3.
  • ND4L NADH dehydrogenase subunit 4L
  • ND5 NADH dehydrogenase subunit 5
  • the repetitive elements occur at positions 10745-10754 in ND4L and 14124-14133 in ND5.
  • SEQ ID NO: 3 is the complementary DNA sequence to the RNA transcript (SEQ ID NO: 19) detected in the manner described above.
  • transcript 1 is a fusion transcript between ATPase 8 and ND5 associated with positions 8469:13447 (SEQ ID NO: 18).
  • Transcripts 3 and 4 are fusion transcripts between COII and Cytb associated with nucleotide positions 7974:15496 and 7992:15730 respectively.
  • Table 3 provides a summary of the relationships between the various sequences used in this example. Table 3 includes the detected fusion transcript and the DNA sequence complementary to the fusion transcript detected.
  • fusion transcripts 1 to 4 Two prostate tissue samples from one patient were analyzed to assess the quantitative difference of the novel predicted fusion transcripts.
  • Table 2 The results of the experiment are provided in Table 2 below, wherein “Homog 1” refers to the homogenate of frozen prostate tumour tissue from a patient and “Homog 2” refers to the homogenate of frozen normal prostate tissue adjacent to the tumour of the patient. These samples were processed according to the manufacturer's protocol ( QuantiGene® Sample Processing Kit for Fresh or Frozen Animal Tissues; and QuantiGene® 2.0 Reagent System User Manual ) starting with 25.8 mg of Homog 1 and 28.9 mg of Homog 2 (the assay setup is shown in Tables 5a and 5b).
  • Example 4 Using the same protocol from Example 3 but focusing only on Transcript 2, the novel fusion transcript associated with the 3.4 kb mtgenome deletion, analyses were conducted on two samples of breast tumour tissue and two samples of tumour-free tissues adjacent to those tumours, as well as three samples of prostate tumour tissue, one sample comprising adjacent tumour-free tissue. Results for this example are provided in Table 4.
  • the prostate tumour tissue sample having a corresponding normal tissue section demonstrated a similar pattern to the prostate sample analyzed in Example 3 in that the tumour tissue had approximately 2 times the amount of the fusion transcript than did the normal adjacent tissue.
  • the breast tumour samples demonstrated a marked increase in the fusion transcript levels when compared to the adjacent non-tumour tissues. A 1:100 dilution of the homogenate was used for this analysis as it performed most reproducibly in the experiment cited in Example 3.
  • transcripts of the invention were prepared comprising nine control (benign) tissue samples (samples 1 to 9) and ten tumour (malignant) tissue samples (samples 10 to 19). The samples were homogenized according to the manufacturer's recommendations (Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual). Seven target transcripts and one housekeeper transcript were prepared in the manner as outlined above in previous examples. The characteristics of the transcripts are summarized as follows:
  • transcripts 2 and 3 are the same as those discussed above with respect to Examples 3 and 4.
  • the analysis of the data comprised the following steps:
  • FIGS. 6 a to 6 g comprise plots of the log 2 a RLU ⁇ log 2 h RLU against sample number. Also illustrated are the respective ROC (Receiver Operating Characteristic) curves determined from the results for each transcript.
  • ROC Receiveiver Operating Characteristic
  • Transcript 2 There exists a statistically significant difference between the means (p ⁇ 0.10) of the normal and malignant groups (p>0.09), using a cutoff value of 3.6129 as demonstrated by the ROC curve results in a sensitivity of 60% and specificity of 89% and the area under the curve is 0.73 indicating fair test accuracy.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • transcripts 2, 3, 8, 9, 10, 11, and 12 were also found to have utility in the detection of colorectal cancer and in distinguishing malignant from normal colorectal tissue.
  • transcripts 2 and 3 were also found to have utility in the detection of prostate cancer.
  • Transcript 2 was also found to have utility in the detection of breast cancer.
  • Transcript 11 was also found to have utility in the detection of melanoma skin cancer.
  • Transcript 10 was also found to have utility in the detection of lung cancer and melanoma.
  • Transcript 8 was also found to have utility in the detection of lung cancer. Any of the 7 transcripts listed may be used individually or in combination as a tool for the detection of characterization of colorectal cancer in a clinical setting.
  • Example 5 This study sought to determine the effectiveness of several transcripts of the invention in the detection of lung cancer.
  • nine control (benign) tissue samples (samples 1 to 9) and ten tumour (malignant) tissue samples (samples 10 to 19) were homogenized according to the manufacturer's recommendations (Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual).
  • Homogenates were diluted 1:8 and the quantity of 4 target transcripts and 1 housekeeper transcript was measured in Relative Luminenscence Units RLU on a GlomaxTM Multi Detection System (Promega). All samples were assayed in triplicate for each transcript. Background measurements (no template) were done in triplicate as well.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • transcripts 6, 8, 10, and 20 of the invention are useful in the detection of lung cancer tumours and the distinction between malignant and normal lung tissues. Any of these three transcripts may be used for the detection or characterization of lung cancer in a clinical setting.
  • the 14 tissue samples used in this example had the following characteristics:
  • transcripts 10 and 11 were also used in Example 5.
  • the analysis of data was performed according to the method described in Example 5. The results are illustrated in FIGS. 8 a - 8 g.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • transcripts 6, 10, 11, 14, 15, 16 and 20 of the invention illustrate the utility of transcripts 6, 10, 11, 14, 15, 16 and 20 of the invention in the detection of malignant melanomas.
  • transcripts 10 and 11 were also found have utility in detecting colorectal cancer while transcript 6 has utility in the detection of lung cancer.
  • a transcript summary by disease is provided at Table 6.
  • samples 1 to 10 The samples were homogenized according to the manufacturer's recommendations (Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual). Eight target transcripts and one housekeeper transcript were prepared in the manner as outlined above in previous examples.
  • the 20 tissue samples used in this example had the following characteristics:
  • transcripts 1, 2, 3, 6, 11, 12, 15 and 20 are the same as those discussed above with respect to Examples 3-7.
  • the analysis of the data comprised the following steps:
  • FIGS. 9 a to 9 h comprise plots of the log 2 a RLU ⁇ log 2 h RLU against sample number. Also illustrated are the respective ROC (Receiver Operating Characteristic) curves determined from the results for each transcript.
  • ROC Receiveiver Operating Characteristic
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • transcripts 1, 2, 3, 6, 11, 12, 15, and 20 were also found to have utility in the detection of prostate cancer.
  • Transcripts 1, 2 and 3 were also found to have utility in the detection of prostate cancer.
  • Transcript 6 was also found to have utility in the detection of melanoma and lung cancer.
  • Transcript 11 was also found to have utility in the detection of melanoma skin cancer, colorectal cancer and testicular cancer.
  • Transcript 12 was also found to have utility in the detection of colorectal cancer and testicular cancer.
  • Transcript 15 was also found to have utility in the detection of melanoma and testicular cancer.
  • Transcript 20 was also found to have utility in the detection of colorectal cancer, melanoma, and testicular cancer. Any of the 8 transcripts listed may be used individually or in combination as a tool for the detection or characterization of ovarian cancer in a clinical setting.
  • samples 1 to 8 were prepared comprising eight control (benign) tissue samples (samples 1 to 8) and 9 tumour (malignant) tissue samples (samples 9 to 17), 5 of the malignant samples were non-seminomas (samples 9-13) and 4 were seminomas (samples 14-17).
  • the samples were homogenized according to the manufacturer's recommendations (Quantigene® Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual). 10 target transcripts and one housekeeper transcript were prepared in the manner as outlined above in previous examples.
  • the 17 tissue samples used in this example had the following characteristics:
  • transcripts 2, 3, 4, 7, 11, 12, 15, 16 and 20 are the same as those discussed above with respect to Examples 3-8.
  • the analysis of the data comprised the following steps:
  • FIGS. 10 to 18 comprise plots of the log 2 a RLU ⁇ log 2 h RLU against sample number. Also illustrated are the respective ROC (Receiver Operating Characteristic) curves determined from the results for each transcript.
  • ROC Receiveiver Operating Characteristic
  • transcripts distinguish between benign and malignant testicular tissue, others demonstrate distinction between the tumour subtypes of seminoma and non-seminoma and/or benign testicular tissue. It is therefore anticipated that combining transcripts from each class will facilitate not only detection of testicular cancer but also classification into subtype of seminoma or non-seminomas.
  • the threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.
  • transcripts 2, 3, 4, 11, 12, 13, 15, 16, and 20 were also found to have utility in the detection of testicular cancer, and testicular cancer subtypes, and in distinguishing malignant from normal testicular tissue.
  • Transcript 2 was also found to have utility in the detection of prostate, breast, colorectal and ovarian cancer.
  • Transcript 3 was also found to have utility in the detection of prostate, breast, melanoma, colorectal, and ovarian cancers.
  • Transcript 4 was also found to have utility in the detection of prostate and colorectal cancers.
  • Transcript 11 was also found to have utility in the detection of colorectal, melanoma, and ovarian cancers.
  • Transcript 12 was also found to have utility in the detection of colorectal and ovarian cancers.
  • Transcript 15 was also found to have utility in the detection of melanoma and ovarian cancers.
  • Transcript 16 was also found to have utility in the detection of melanoma skin cancer.
  • Transcript 20 was also found to have utility in the detection of colorectal cancer, melanoma, and ovarian cancer. Any of the 9 transcripts listed may be used individually or in combination as a tool for the detection or characterization of testicular cancer in a clinical setting.
  • the invention provides a kit for conducting an assay for determining the presence of cancer in a tissue sample.
  • the kit includes the required reagents for conducting the assay as described above.
  • the kit includes one or more containers containing one or more hybridization probes corresponding to transcripts 1 to 17, and 20 described above.
  • the reagents for conducting the assay may include any necessary buffers, salts, detection reagents etc.
  • the kit may include any necessary sample collection devices, containers etc. for obtaining the needed tissue samples, reagents or materials to prepare the tissue samples for example by homogenization or nucleic acid extraction, and for conducting the subject assay or assays.
  • the kit may also include control tissues or samples to establish or validate acceptable values for diseased or non-diseased tissues.
  • Used Qiagen TissueRuptor Used 40 ul homogenate supernatant, 20, 10 and 5 ul for dilution
  • Homogenate1 Tumour tissue from the tumorous Prostate Homogenate2- Used 29 mg of tissue to homogenize in 700 ul H soln with PK.
  • Used Qiagen TissueRuptor Used 40 ul homogenate supernatant, 20, 10 and 5 ul for dilution
  • Homogenate2 Normal tissue from the tumorous Prostate RNA dilution was made as below. RNA was from Prostate Normal from Ambion. Assay was done in duplicates.

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AU2009227935A1 (en) 2009-10-01
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US8715960B2 (en) 2014-05-06
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