CA2719718A1 - Aberrant mitochondrial dna, associated fusion transcripts and hybridization probes therefor - Google Patents

Abstract

The present invention provides novel mitochondrial fusion transcripts and the parent mutated mtDNA molecules that are useful for predicting, diagnosing and/or monitoring cancer.
Hybridization probes complementary thereto for use in the methods of the invention are also provided.

Description

Ref: 102222/00053 1 ABERRANT MITOCHONDRIAL DNA, ASSOCIATED FUSION TRANSCRIPTS AND

2 HYBRIDIZATION PROBES THEREFOR

3 FIELD OF THE INVENTION

4 [0001] The present invention relates to the field of mitochondrial genomics.
In one aspect, the invention relates to the identification and use of mitochondrial genome fusion 6 transcripts and probes that hybridize thereto.

8 [0002] Mitochondrial Genome 9 [0003] 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 11 pairs (bp) (Anderson et al., 1981; Andrews et al., 1999) in contrast to the immense nuclear 12 genome of 3.3 billion bp (haploid). Its genetic complement is substantially smaller than that 13 of its nuclear cell mate (0.0005%). However, individual cells carry anywhere from 103 to 104 14 mitochondria depending on specific cellular functions (Singh and Modica-Napolitano 2002).
Communication or chemical signalling routinely occurs between the nuclear and 16 mitochondrial genomes (Sherratt et al., 1997). Moreover, specific nuclear components are 17 responsible for the maintenance and integrity of mitochondrial sequences (Croteau et al., 18 1999). All mtDNA genomes in a given individual are identical due to the clonal expansion of 19 mitochondria within the ovum, once fertilization has occurred. However mutagenic events can induce sequence diversity reflected as somatic mutations. These mutations may 21 accumulate in different tissues throughout the body in a condition known as heteroplasmy.
22 [0004] Mitochondrial Proteome 23 [0005] About 3,000 nuclear genes are required to construct, operate and maintain 24 mitochondria, with only thirty-seven of these coded by the mitochondrial genome, indicating heavy mitochondrial dependence on nuclear loci. The mitochondrial genome codes for a 26 complement of 24 genes, including 2 rRNAs and 22 tRNAs that ensure correct translation of 27 the remaining 13 genes which are vital to electron transport (see Figure 1). The 28 mitochondrial genome is dependent on seventy nuclear encoded proteins to accomplish the 29 oxidation and reduction reactions necessary for this vital function, in addition to the thirteen polypeptides supplied by the mitochondrial genome. Both nuclear and mitochondrial 31 proteins form complexes spanning the inner mitochondrial membrane and collectively 32 generate 80-90% of the chemical fuel adenosine triphosphate, or ATP, required for cellular 33 metabolism. In addition to energy production, mitochondria play a central role in other 34 metabolic pathways as well. A critical function of the mitochondria is mediation of cell death, Ref: 102222/00053 1 or apoptosis (see Green and Kroemer, 2005). Essentially, there are signal pathways which 2 permeabilize the outer mitochondrial membrane, or in addition, the inner mitochondrial 3 membrane as well. When particular mitochondrial proteins are released into the cytosol, 4 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 6 other mitochondrial proteins as well which may have alternate functions.

7 [0006] Mitochondrial Fusion Transcriptome 8 [0007] The mitochondrial genome is unusual in that it is a circular, intron-less DNA
9 molecule. The genome is interspersed with repeat motifs which flank specific lengths of sequences. Sequences between these repeats are prone to deletion under circumstances 11 which are not well understood. Given the number of repeats in the mitochondrial genome, 12 there are many possible deletions. The best known example is the 4977 "common deletion."
13 This deletion has been associated with several purported conditions and diseases and is 14 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) (Figure 4). The current 16 thinking in the field of mitochondrial genomics is that mitochondrial deletions are merely 17 deleterious by-products of damage to the mitochondrial genome by such agents as reactive 18 oxygen species and UVR. (Krishnan et al 2008, Nature Genetics). Further, though it is 19 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 21 necessary for cellular respiration, it is not anticipated that these deleted mitochondrial 22 molecules may be a component of downstream pathways, have an intended functional role, 23 and possibly may be more aptly viewed as alternate natural forms of the recognized genes 24 of the mitochondria as has been anticipated by the Applicant.

[0008] The sequence dynamics of mtDNA are important diagnostic tools.
Mutations in 26 mtDNA are often preliminary indicators of developing disease. For example, it has been 27 demonstrated that point mutations in the mitochondrial genome are characteristic of tumour 28 foci in the prostate. This trend also extends to normal appearing tissue both adjacent to and 29 distant from tumour tissue (Parr et al. 2006). This suggests that mitochondrial mutations occur early in the malignant transformation pathway.

31 [0009] For example, the frequency of a 3.4kb mitochondrial deletion has excellent utility 32 in discriminating between benign and malignant prostate tissues (Maki et al. 2008).

33 [0010] Mitochondrial fusion transcripts have been reported previously in the literature, 34 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 Ref: 102222/00053 1 transcripts were not found to have (or investigated regarding) association with any human 2 cancers.

4 [0011] An object of the present invention to provide aberrant mitochondrial DNA, associated fusion transcripts and hybridization probes therefor.

6 [0012] In accordance with an aspect of the invention, there is provided an isolated 7 mitochondrial fusion transcript associated with cancer.

8 [0013] In accordance with an aspect of the invention, there is provided a mitochondrial 9 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.

11 [0014] In accordance with another aspect of the invention, there is provided an isolated 12 mtDNA encoding a fusion transcript of the invention.

13 [0015] In accordance with another aspect of the invention, there is provided a 14 hybridization probe having a nucleic acid sequence complementary to at least a portion of a mitochondrial fusion transcript or an mtDNA of the invention.

16 [0016] In accordance with another aspect of the invention, there is provided a method of 17 detecting a cancer in a mammal, the method comprising assaying a tissue sample from the 18 mammal for the presence of at least one mitochondrial fusion transcript associated with 19 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 21 to the invention.

22 [0017] In accordance with another aspect of the invention, there is provided a method of 23 detecting a cancer in a mammal, the method comprising assaying a tissue sample from the 24 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 26 complementary to at least a portion of an mtDNA according to the invention.

27 [0018] In accordance with another aspect of the invention, there is provided a kit for 28 conducting an assay for detecting the presence of a cancer in a mammal, said kit comprising 29 at least one hybridization probe complementary to at least a portion of a fusion transcript or an mtDNA of the invention.

31 [0019] In accordance with another aspect of the invention, there is provided a screening 32 tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial fusion 33 transcripts for identification of those associated with cancer.

Ref: 102222/00053 1 [0020] In accordance with another aspect of the invention, there is provided a screening 2 tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial DNAs 3 corresponding to mitochondrial fusion transcripts for identification of those associated with 4 cancer.

[0021] In accordance with another aspect of the invention, there is provided a screening 6 tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of 7 mitochondrial fusion transcripts for identification of those associated with cancer.

8 [0022] In accordance with another aspect of the invention, there is provided a screening 9 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 11 those associated with cancer.

13 [0023] The embodiments of the invention will now be described by way of example only 14 with reference to the appended drawings wherein:

[0024] Figure 1 is an illustration showing mitochondrial coding genes.

16 [0025] Figure 2 shows polyadenalated fusion transcripts in prostate samples invoked by 17 the loss of the 3.4kb deletion.

18 [0026] Figure 3 shows polyadenalated fusion transcripts in prostate samples invoked by 19 the loss of the 4977kb common deletion.

[0027] Figure 4 shows polyadenalated fusion transcripts in breast samples invoked by 21 the loss of the 3.4 kb segment from the mtgenome.

22 [0028] Figures 5a and 5b show an example of a mitochondrial DNA region before and 23 after splicing of genes.

24 [0029] Figures 6a to 6g illustrate the results for transcripts 2, 3, 8, 9, 10, 11 and 12 of the invention in the identification of colorectal cancer tumours.

26 [0030] Figures 7a to 7d illustrate the results for transcripts 6, 8, 10 and 20 of the 27 invention in the identification of lung cancer tumours.

28 [0031] Figures 8a to 8g illustrate the results for transcripts 6, 10, 11, 14, 15, 16 and 20 of 29 the invention in the identification of melanomas.

[0032] Figures 9a to 9h illustrate the results for transcripts 1, 2, 3, 6, 11, 12, 15 and 20 of 31 the invention in the identification of ovarian cancer.

Ref: 102222/00053 1 [0033] Figures 10 to 18 illustrate the results for transcripts 2, 3, 4, 11, 12, 13, 15, 16 and 2 20 of the invention in the identification of testicular cancer.

[0034] The present invention provides novel mitochondrial fusion transcripts and the 6 parent mutated mtDNA molecules that are useful for predicting, diagnosing and/or 7 monitoring cancer. The invention further provides hybridization probes for the detection of 8 fusion transcripts and associated mtDNA molecules and the use of such probes.

9 [0035] Definitions [0036] Unless defined otherwise, all technical and scientific terms used herein have the 11 same meaning as commonly understood by one of ordinary skill in the art to which this 12 invention belongs.

13 [0037] As used herein, "aberration" or "mutation" encompasses any modification in the 14 wild type mitochondrial DNA sequence that results in a fusion transcript and includes, without limitation, insertions, translocations, deletions, duplications, recombinations, 16 rearrangements or combinations thereof.

17 [0038] As defined herein, "biological sample" refers to a tissue or bodily fluid containing 18 cells from which a molecule of interest can be obtained. For example, the biological sample 19 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 21 fluid and the like. The biological sample may be a surgical specimen or a biopsy specimen.
22 The biological sample can be used either directly as obtained from the source or following a 23 pre-treatment to modify the character of the sample. Thus, the biological sample can be pre-24 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, 26 concentrating liquids, inactivating interfering components, adding reagents, and the like.

27 [0039] A "continuous" transcript is a fusion transcript that keeps the reading frame from 28 the beginning to the end of both spliced genes. An "end" transcript is a fusion transcript that 29 results in a premature termination codon before the original termination codon of a second spliced gene.

31 [0040] As used herein, "mitochondrial DNA" or "mtDNA" is DNA present in mitochondria.
32 [0041] As used herein, the expression "mitochondrial fusion transcript" or "fusion 33 transcript" refers to an RNA transcription product produced as a result of the transcription of

5 Ref: 102222/00053 1 a mutated mitochondrial DNA sequence wherein such mutations may comprise 2 mitochondrial deletions and other large-scale mitochondrial DNA
rearrangements.
3 [0042] Computer Analysis and Sequence Targetting 4 [0043] As discussed above, mitochondrial fusion transcripts have been reported in soybeans (Morgens et al. 1984) and in humans suffering from a rare neuromuscular disorder

6 (Nakase et al 1990). Fusion transcripts associated with human cancer have not, however,

7 been described.

8 [0044] Using the knowledge gained from mapping the large-scale deletions of the

9 human mitochondrial genome associated with cancer, the observation of high frequencies of these deletions, and the evidence in another organism and another disease type of 11 trancriptionally active mutated mtDNA molecules, Applicant hypothesized that such deletions 12 may have importance beyond the DNA molecule and the damage and repair processes as it 13 relates to cancer. To test this hypothesis computer analysis of the mitochondrial genome 14 was conducted, specific for repeat elements, which suggested many potential deletion sites.
Following this initial step identifying unique repeats in the mitochondrial sequence having 16 non-adjacent or non-tandem locations, a filter was then applied to identify those repeats that 17 upon initiating a deletion event in the DNA molecule would then likely reclose or religate to 18 produce a fused DNA sequence having an open reading frame (ORF). A subset of 18 19 molecules were then selected for targetting to investigate whether: 1) they existed in the natural biological state of humans and 2) they had relevance to malignancy.
Results from 21 these investigations are described hereinafter.

22 [0045] Genomic Mutations 23 [0046] Mitochondrial DNA (mtDNA) dynamics are an important diagnostic tool.
Mutations 24 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, 26 large-scale rearrangement mutations in the mitochondrial genome result in the generation of 27 fusion transcripts associated with cancer. Thus, the use of mtDNA encoding such transcripts 28 and probes directed thereto for the detection, diagnosis and monitoring of cancer is 29 provided.

[0047] One of skill in the art will appreciate that the mtDNA molecules for use in the 31 methods of the present invention may be derived through the isolation of naturally-occurring 32 mutants or may be based on the complementary sequence of any of the fusion transcripts 33 described herein. Exemplary mtDNA sequences and fusion transcripts are disclosed in 34 Applicant's U.S. priority application no. 61/040,616, herein incorporated in its entirety by reference.

Ref: 102222/00053 1 [0048] Detection of Mutant Genomic Sequences 2 [0049] Mutant mtDNA sequences according to the present invention may comprise any 3 modification that results in the generation of a fusion transcript. Non-limiting examples of 4 such modifications include insertions, translocations, deletions, duplications, recombinations, rearrangements or combinations thereof. While the modification or change can vary greatly 6 in size from only a few bases to several kilobases, preferably the modification results in a 7 substantive deletion or other large-scale genomic aberration.

8 [0050] Extraction of DNA to detect the presence of such mutations may take place using 9 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 11 Protocols in Molecular Biology. Alternatively, crude tissue homogenates may be used as 12 well as techniques not requiring amplification of specific fragments of interest.

13 [0051] The step of detecting the mutations can be selected from any technique as is 14 known to those skilled in the art. For example, analyzing mtDNA can comprise selection of targets by branching DNA, sequencing the mtDNA, amplifying mtDNA by PCR, Southern, 16 Northern, Western South-Western blot hybridizations, denaturing HPLC, hybridization to 17 microarrays, biochips or gene chips, molecular marker analysis, biosensors, melting 18 temperature profiling or a combination of any of the above.

19 [0052] Any suitable means to sequence mitochondrial DNA may be used.
Preferably, mtDNA is amplified by PCR prior to sequencing. The method of PCR is well known in the art 21 and may be performed as described in Mullis and Faloona, 1987, Methods Enzymol., 155:
22 335. PCR products can be sequenced directly or cloned into a vector which is then placed 23 into a bacterial host. Examples of DNA sequencing methods are found in Brumley, R. L. Jr.
24 and Smith, L.M., 1991, Rapid DNA sequencing by horizontal ultrathin gel electrophoresis, Nucleic Acids Res. 19:4121-4126 and Luckey, J.A., et al, 1993, High speed DNA
26 sequencing by capillary gel electrophoresis, Methods Enzymol. 218: 154-172.
The combined 27 use of PCR and sequencing of mtDNA is described in Hopgood, R., et al, 1992, Strategies 28 for automated sequencing of human mtDNA directly from PCR products, Biotechniques 29 13:82-92 and Tanaka, M. et al, 1996, Automated sequencing of mtDNA, Methods Enzymol.
264:407-421.

31 [0053] Methods of selecting appropriate sequences for preparing various primers are 32 also known in the art. For example, the primer can be prepared using conventional solid-33 phase synthesis using commercially available equipment, such as that available from 34 Applied Biosystems USA Inc. (Foster City, California), DuPont, (Wilmington, Del.), or Milligen (Bedford, Mass.).

Ref: 102222/00053 1 [0054] According to an aspect of the invention, to determine candidate genomic 2 sequences, a junction point of a sequence deletion is first identified.
Sequence deletions are 3 primarily identified by direct and indirect repetitive elements which flank the sequence to be 4 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.

6 [0055] Upon identification of the junction point, the nucleotides of the genes flanking the 7 junction point are determined in order to identify a spliced gene. Typically the spliced gene 8 comprises the initiation codon from the first gene and the termination codon of the second 9 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 11 initiation or termination codons contained within the gene sequences may be used as is 12 evidenced by SEQ ID No:2 and SEQ ID No: 17 disclosed herein. Some known 13 mitochondrial deletions discovered to have an open reading frame (ORF) when the 14 rearranged sequences are rejoined at the splice site are provided in Table 1.

[0056] Exemplary mtDNA molecules for use in the methods of the present invention, 16 which have been verified to exist in the lab, are provided below. These mtDNAs are based 17 on modifications of the known mitochondrial genome (SEQ ID NO: 1) and have been 18 assigned a fusion or "FUS" designation, wherein A:B represents the junction point between 19 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 21 parentheses followed by the corresponding sequence identifier. Where provided below, 22 (AltMet) and (OrigMet) refer to alternate and original translation start sites, respectively.
23 FUS 8469:13447 (AltMet) (ATP synthase FO subunit 8 to NADH dehydrogenase 24 subunit) (SEQ ID No: 2) FUS 10744:14124 (NADH dehydrogenase subunit 4L (ND4L) to NADH
26 dehydrogenase subunit 5 (ND5)) (SEQ ID No: 3) 27 FUS 7974:15496 (Cytochrome c oxidase subunit II (COII) to Cytochrome b (Cytb)) 28 (SEQ ID No: 4) 29 FUS 7992:15730 (Cytochrome c oxidase subunit II (COII) to Cytochrome b (Cytb)) (SEQ ID No: 5) 31 FUS 8210:15339 (Cytochrome c oxidase subunit II (COII) to Cytochrome b (Cytb)) 32 (SEQ ID No: 6) 33 FUS 8828:14896 (ATP synthase FO subunit 6 (ATPase6) to Cytochrome b (Cytb)) 34 (SEQ ID No: 7) FUS 10665:14856 (NADH dehydrogenase subunit 4L (ND4L) to Cytochrome b 36 (Cytb)) (SEQ ID No: 8) Ref: 102222/00053 1 FUS 6075:13799 (Cytochrome c oxidase subunit I (COI) to NADH de hydrogenase 2 subunit 5 (ND5)) (SEQ ID No: 9) 3 FUS 6325:13989 (Cytochrome c oxidase subunit I (COI) to NADH dehydrogenase 4 subunit 5 (ND5)) (SEQ ID No: 10) FUS 7438:13476 (Cytochrome c oxidase subunit I (COI) to NADH dehydrogenase 6 subunit 5 (ND5)) (SEQ ID No: 11) 7 FUS 7775:13532 (Cytochrome c oxidase subunit II (COII) to NADH dehydrogenase 8 subunit 5 (ND5)) (SEQ ID No: 12) 9 FUS 8213:13991 (Cytochrome c oxidase subunit II (COII) to NADH dehydrogenase subunit 5 (ND5)) (SEQ ID No: 13) 11 FUS 9191:12909 (ATP synthase FO subunit 6 (ATPase6) to NADH dehydrogenase 12 subunit 5 (ND5)) (SEQ ID No: 14) 13 FUS 9574:12972 (Cytochrome c oxidase subunit III (COIII) to NADH
dehydrogenase 14 subunit 5 (ND5)) (SEQ ID No: 15) FUS 10367:12829 (NADH dehydrogenase subunit 3 (ND3) to NADH dehydrogenase 16 subunit 5 (ND5)) (SEQ ID No: 16) 17 FUS 8469:13447 (OrigMet) (ATP synthase FO subunit 8 to NADH dehydrogenase 18 subunit) (SEQ ID No: 17) 19 FUS 9144:13816 ((ATP synthase FO subunit 6 (ATPase6) to NADH dehydrogenase subunit 5 (ND5)) (SEQ ID No: 51) 22 [0057] The present invention also provides the use of variants or fragments of these 23 sequences for predicting, diagnosing and/or monitoring cancer.

24 [0058] "Variant", as used herein, refers to a nucleic acid differing from a mtDNA
sequence of the present invention, but retaining essential properties thereof.
Generally, 26 variants are overall closely similar, and, in many regions, identical to a select mtDNA
27 sequence. Specifically, the variants of the present invention comprise at least one of the 28 nucleotides of the junction point of the spliced genes, and may further comprise one or more 29 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
31 sequences of the invention, or the complementary strand thereto.

32 [0059] In the present invention, "fragment" refers to a short nucleic acid sequence which 33 is a portion of that contained in the disclosed genomic sequences, or the complementary 34 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 36 thereto. The fragments of the invention are preferably at least about 15 nt, and more 37 preferably at least about 20 nt, still more preferably at least about 30 nt, and even more 38 preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about Ref: 102222/00053 1 150 nt in length. A fragment "at least 20 nt in length," for example, is intended to include 20 2 or more contiguous bases of any one of the mtDNA sequences listed above. In this context 3 "about" includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, 4 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 6 course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are also contemplated.
7 [0060] Thus, in specific embodiments of the invention, the mtDNA sequences are 8 selected from the group consisting of:

9 SEQ ID NO: 2 (FUS 8469:13447; AltMet) SEQ ID NO: 3 (FUS 10744:14124) 11 SEQ ID NO: 4 (FUS 7974:15496) 12 SEQ ID NO: 5 (FUS 7992:15730) 13 SEQ ID NO: 6 (FUS 8210:15339) 14 SEQ ID NO: 7 (FUS 8828:14896) SEQ ID NO: 8 (FUS 10665:14856) 16 SEQ ID NO: 9 (FUS 6075:13799) 17 SEQ ID NO: 10 (FUS 6325:13989) 18 SEQ ID NO: 11 (FUS 7438:13476) 19 SEQ ID NO: 12 (FUS 7775:13532) SEQ ID NO: 13 (FUS 8213:13991) 21 SEQ ID NO: 14 (FUS 9191:12909) 22 SEQ ID NO: 15 (FUS 9574:12972) 23 SEQ ID NO: 16 (FUS 10367:12829) 24 SEQ ID NO: 17(FUS 8469:13447; OrigMet) SEQ ID NO: 51 (FUS 9144:13816), and 27 fragments or variants thereof.
28 [0061] Probes 29 [0062] Another aspect of the invention is to provide a hybridization probe capable of recognizing an aberrant mtDNA sequence of the invention. As used herein, the term "probe"
31 refers to an oligonucleotide which forms a duplex structure with a sequence in the target 32 nucleic acid, due to complementarity of at least one sequence in the probe with a sequence 33 in the target region. The probe may be labeled, according to methods known in the art.

34 [0063] Once aberrant mtDNA associated with a particular disease is identified, hybridization of mtDNA to, for example, an array of oligonucleotides can be used to identify 36 particular mutations, however, any known method of hybridization may be used.

Ref: 102222/00053 1 [0064] As with the primers of the present invention, probes may be generated directly 2 against exemplary mtDNA fusion molecules of the invention, or to a fragment or variant 3 thereof. For instance, the sequences set forth in SEQ ID NOs: 2-17 and 51 and those 4 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. As would be understood by those of skill 6 in the art, primers or probes which hybridize to these nucleic acid molecules may do so 7 under highly stringent hybridization conditions or lower stringency conditions, such 8 conditions known to those skilled in the art and found, for example, in Current Protocols in 9 Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6.

[0065] In specific embodiments of the invention, the probes of the invention contain a 11 sequence complementary to at least a portion of the aberrant mtDNA
comprising the junction 12 point of the spliced genes. This portion includes at least one of the nucleotides involved in 13 the junction point A:B, and may further comprise one or more nucleotides adjacent thereto.
14 In this regard, the present invention encompasses any suitable targeting mechanism that will select an mtDNA molecule using the nucleotides involved and/or adjacent to the junction 16 point A:B.

17 [0066] Various types of probes known in the art are contemplated by the present 18 invention. For example, the probe may be a hybridization probe, the binding of which to a 19 target nucleotide sequence can be detected using a general DNA binding dye such as ethidium bromide, SYBR Green, SYBR Gold and the like. Alternatively, the probe can 21 incorporate one or more detectable labels. Detectable labels are molecules or moieties a 22 property or characteristic of which can be detected directly or indirectly and are chosen such 23 that the ability of the probe to hybridize with its target sequence is not affected. Methods of 24 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).

26 [0067] Labels suitable for use with the probes of the present invention include those that 27 can be directly detected, such as radioisotopes, fluorophores, chemiluminophores, enzymes, 28 colloidal particles, fluorescent microparticles, and the like. One skilled in the art will 29 understand that directly detectable labels may require additional components, such as substrates, triggering reagents, light, and the like to enable detection of the label. The 31 present invention also contemplates the use of labels that are detected indirectly.

32 [0068] The probes of the invention are preferably at least about 15 nt, and more 33 preferably at least about 20 nt, still more preferably at least about 30 nt, and even more 34 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 Ref: 102222/00053 1 or more contiguous bases that are complementary to an mtDNA sequence of the invention.
2 Of course, larger probes (e.g., 50, 150, 500, 600, 2000 nucleotides) may be preferable.

3 [0069] The probes of the invention will also hybridize to nucleic acid molecules in 4 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 6 cancer, wherein the probe is complementary to at least a portion of an aberrant mtDNA
7 molecule. In another aspect the present invention provides probes and a use of (or a method 8 of using) such probes for the detection of colorectal cancer, lung cancer, breast cancer, 9 ovarian cancer, testicular, cancer, prostate cancer and/or melanoma skin cancer.
[0070] Assays 11 [0071] Measuring the level of aberrant mtDNA in a biological sample can determine the 12 presence of one or more cancers in a subject. The present invention, therefore, 13 encompasses methods for predicting, diagnosing or monitoring cancer, comprising obtaining 14 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
16 sequences in the sample and comparing the quantity detected with a reference value. As 17 would be understood by those of skill in the art, the reference value is based on whether the 18 method seeks to predict, diagnose or monitor cancer. Accordingly, the reference value may 19 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 21 samples taken overtime.

22 [0072] In one aspect, the invention provides a method of detecting cancer in a mammal, 23 the method comprising assaying a tissue sample from the mammal for the presence of an 24 aberrant mitochondrial DNA described above. The present invention also provides for methods comprising assaying a tissue sample from the mammal by hybridizing the sample 26 with at least one hybridization probe. The probe may be generated against a mutant 27 mitochondrial DNA sequence of the invention as described herein.

28 [0073] In another aspect, the invention provides a method as above, wherein the assay 29 comprises:

a) conducting a hybridization reaction using at least one of the probes to allow the at 31 least one probe to hybridize to a complementary aberrant mitochondrial DNA
sequence;

32 b) quantifying the amount of the at least one aberrant mitochondrial DNA
sequence 33 in the sample by quantifying the amount of the mitochondrial DNA hybridized to the at least 34 one probe; and, Ref: 102222/00053 1 c) comparing the amount of the mitochondrial DNA in the sample to at least one 2 known reference value.

3 [0074] Also included in the present invention are methods for predicting, diagnosing or 4 monitoring cancer comprising diagnostic imaging assays as described below.
The diagnostic assays of the invention can be readily adapted for high-throughput. High-throughput assays 6 provide the advantage of processing many samples simultaneously and significantly 7 decrease the time required to screen a large number of samples. The present invention, 8 therefore, contemplates the use of the nucleotides of the present invention in high-9 throughput screening or assays to detect and/or quantitate target nucleotide sequences in a plurality of test samples.

11 [0075] Fusion Transcripts 12 [0076] The present invention further provides the identification of fusion transcripts and 13 associated hybridization probes useful in methods for predicting, diagnosing and/or 14 monitoring cancer. One of skill in the art will appreciate that such molecules may be derived through the isolation of naturally-occurring transcripts or, alternatively, by the recombinant 16 expression of mtDNAs isolated according to the methods of the invention. As discussed, 17 such mtDNAs typically comprise a spliced gene having the initiation codon from the first 18 gene and the termination codon of the second gene. Accordingly, fusion transcripts derived 19 therefrom comprise a junction point associated with the spliced genes.

[0077] Detection of Fusion Transcripts 21 [0078] Naturally occurring fusion transcripts can be extracted from a biological sample 22 and identified according to any suitable method known in the art, or may be conducted 23 according to the methods described in the examples. In one embodiment of the invention, 24 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 26 target transcript.

27 [0079] The following exemplary fusion transcripts were detected using such methods 28 and found useful in predicting, diagnosing and/or monitoring cancer as indicated in the 29 examples. Likewise, 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 31 and methods of the present invention.

32 SEQ ID NO: 18 (Transcripts 1;8469:13447; AltMet) 33 SEQ ID NO: 19 (Transcript 2;10744:14124) 34 SEQ ID NO: 20 (Transcript 3;7974:15496) Ref: 102222/00053 1 SEQ ID NO: 21 (Transcript 4;7992:15730) 2 SEQ ID NO: 22 (Transcript 5;8210:15339) 3 SEQ ID NO: 23 (Transcript 6;8828:14896) 4 SEQ ID NO: 24 (Transcript 7;10665:14856) SEQ ID NO: 25 (Transcript 8;6075:13799) 6 SEQ ID NO: 26 (Transcript 9;6325:13989) 7 SEQ ID NO: 27 (Transcript 10;7438:13476) 8 SEQ ID NO: 28 (Transcript 11;7775:13532) 9 SEQ ID NO: 29 (Transcript 12;8213:13991) SEQ ID NO: 30 (Transcript 14;9191:12909) 11 SEQ ID NO: 31 (Transcript 15;9574:12972) 12 SEQ ID NO: 32 (Transcript 16;10367:12829) 13 SEQ ID NO: 33 (Transcript 20;8469:13447; OrigMet) 14 SEQ ID NO: 50 (Transcript 13; 9144:13816) 16 [0080] Further, fusion transcripts of like character to those described herein are 17 contemplated for use in the field of clinical oncology.

18 [0081] Fusion transcripts can also be produced by recombinant techniques known in the 19 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.

21 [0082] Variants or fragments of the fusion transcripts identified herein are also provided.
22 Such sequences may adhere to the size limitations and percent identities described above 23 with respect to genomic variants and fragments, or as determined suitable by a skilled 24 technician.

[0083] In addition, putative protein sequences corresponding to transcripts 1-16 and 20 26 are listed below. These sequences, which encode hypothetical fusion proteins, are provided 27 as a further embodiment of the present invention.

28 SEQ ID NO: 34 (Transcripts 1) 29 SEQ ID NO: 35 (Transcript 2) SEQ ID NO: 36 (Transcript 3) 31 SEQ ID NO: 37 (Transcript 4) 32 SEQ ID NO: 38 (Transcript 5) 33 SEQ ID NO: 39 (Transcript 6) 34 SEQ ID NO: 40 (Transcript 7) SEQ ID NO: 41 (Transcript 8) 36 SEQ ID NO: 42 (Transcript 9) Ref: 102222/00053 1 SEQ ID NO: 43 (Transcript 10) 2 SEQ ID NO: 44 (Transcript 11) 3 SEQ ID NO: 45 (Transcript 12) 4 SEQ ID NO: 46 (Transcript 14) SEQ ID NO: 47 (Transcript 15) 6 SEQ ID NO: 48 (Transcript 16) 7 SEQ ID NO: 49 (Transcripts 20) 8 SEQ ID NO: 52 (Transcript 13) 9 [0084] Probes [0085] Once a fusion transcript has been characterized, primers or probes can be 11 developed to target the transcript in a biological sample. Such primers and probes may be 12 prepared using any known method (as described above) or as set out in the examples 13 provided below. A probe may, for example, be generated for the fusion transcript, and 14 detection technologies, such as QuantiGene 20TM by Panomics TM, used to detect the presence of the transcript in a sample. Primers and probes may be generated directly 16 against exemplary fusion transcripts of the invention, or to a fragment or variant thereof. For 17 instance, the sequences set forth in SEQ ID NOs: 18-33 and 50 as well as those disclosed in 18 Table 1 can be used to design probes that will detect a nucleic acid sequence comprising a 19 fusion sequence of interest.

[0086] As would be understood by those skilled in the art, probes designed to hybridize 21 to the fusion transcripts of the invention contain a sequence complementary to at least a 22 portion of the transcript expressing the junction point of the spliced genes. This portion 23 includes at least one of the nucleotides complementary to the expressed junction point, and 24 may further comprise one or more complementary nucleotides adjacent thereto. In this regard, the present invention encompasses any suitable targeting mechanism that will select 26 a fusion transcript that uses the nucleotides involved and adjacent to the junction point of the 27 spliced genes.

28 [0087] Various types of probes and methods of labelling known in the art are 29 contemplated for the preparation of transcript probes. Such types and methods have been described above with respect to the detection of genomic sequences. The transcript probes 31 of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, 32 still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at 33 least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A probe of "at least 34 20 nt in length," for example, is intended to include 20 or more contiguous bases that are Ref: 102222/00053 1 complementary to an mtDNA sequence of the invention. Of course, larger probes (e.g., 50, 2 150, 500, 600, 2000 nucleotides) may be preferable.

3 [0088] In one aspect, the invention provides a hybridization probe for use in the 4 detection of cancer, wherein the probe is complementary to at least a portion of a mitochondrial fusion transcript provided above.

6 [0089] In another aspect, the present invention provides probes and a use of (or a 7 method of using) such probes for the detection of colorectal cancer, lung cancer, breast 8 cancer, ovarian cancer, testicular cancer, prostate cancer or melanoma skin cancer.

9 [0090] Assays [0091] Measuring the level of mitochondrial fusion transcripts in a biological sample can 11 determine the presence of one or more cancers in a subject. The present invention, 12 therefore, provides methods for predicting, diagnosing or monitoring cancer, comprising 13 obtaining one or more biological samples, extracting mitochondrial RNA from the samples, 14 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.
16 As would be understood by those of skill in the art, the reference value is based on whether 17 the method seeks to predict, diagnose or monitor cancer. Accordingly, the reference value 18 may relate to transcript data collected from one or more known non-cancerous biological 19 samples, from one or more known cancerous biological samples, and/or from one or more biological samples taken over time.

21 [0092] In one aspect, the invention provides a method of detecting a cancer in a 22 mammal, the method comprising assaying a tissue sample from said mammal for the 23 presence of at least one fusion transcript of the invention by hybridizing said sample with at 24 least one hybridization probe having a nucleic acid sequence complementary to at least a portion of the mitochondrial fusion transcript.

26 [0093] In another aspect, the invention provides a method as above, wherein the assay 27 comprises:

28 a) conducting a hybridization reaction using at least one of the above-noted probes to 29 allow the at least one probe to hybridize to a complementary mitochondrial fusion transcript;
b) quantifying the amount of the at least one mitochondrial fusion transcript in the 31 sample by quantifying the amount of the transcript hybridized to the at least one probe; and, 32 c) comparing the amount of the mitochondrial fusion transcript in the sample to at 33 least one known reference value.

Ref: 102222/00053 1 [0094] As discussed above, the diagnostic assays of the invention may also comprise 2 diagnostic methods and screening tools as described herein and can be readily adapted for 3 high-throughput. The present invention, therefore, contemplates the use of the fusion 4 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.
6 [0095] Diagnostic Methods and Screening Tools 7 [0096] Methods and screening tools for diagnosing specific diseases or identifying 8 specific mitochondrial mutations are also herein contemplated. Any known method of 9 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 11 multi-plex. Array technology, which has oligonucleotide probes matching the wild type or 12 mutated region, and a control probe, may also be used. Commercially available arrays such 13 as microarrays or gene chips are suitable. These arrays contain thousands of matched and 14 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
16 sequence analysis are available on-line.

17 [0097] Screening tools designed to identify targets which are relevant to a given 18 biological condition may include specific arrangements of nucleic acids associated with a 19 particular disease or disorder. Thus, in accordance with one embodiment of the invention, there is provided a screening tool comprised of a microarray having 10's, 100's, or 1000's of 21 mitochondrial fusion transcripts for identification of those associated with one or more 22 cancers. In accordance with another embodiment, there is provided a screening tool 23 comprised of a microarray having 10's, 100's, or 1000's of mitochondrial DNAs 24 corresponding to mitochondrial fusion transcripts for identification of those associated with one or more cancers. In a further embodiment, there is provided a screening tool comprised 26 of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial fusion 27 transcripts for identification of those associated with one or more cancers. In yet another 28 embodiment of the invention, there is provided a screening tool comprised of a multiplexed 29 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 31 cancers.

32 [0098] Approaches useful in the field of clinical oncology are also herein contemplated 33 and may include such diagnostic imaging techniques as Positron Emission Tomography 34 (PET), contrast Magnetic Resonance Imaging (MRI) or the like. These diagnostic methods Ref: 102222/00053 1 are well known to those of skill in the art and are useful in the diagnosis and prognosis of 2 cancer.

3 [0099] Diagnostic Monitoring 4 [00100] 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 6 more assays. This allows clinicians to practice personalized medicine; e.g.
cancer therapy, 7 by monitoring the progression of the patient's cancer (such as by recognizing when an initial 8 or subsequent mutation occurs) or treatment (such as by recognizing when a mutation is 9 stabilized).

[00101] With knowledge of the boundaries of the sequence variation in hand, the 11 information can be used to diagnose a pre-cancerous condition or existing cancer condition.
12 Further, by quantitating the amount of aberrant mtDNA in successive samples over time, the 13 progression of a cancer condition can be monitored. For example, data provided by assaying 14 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 16 aberration have occurred.

17 [00102] Where a mutation is found in an individual who has not yet developed symptoms 18 of cancer, the mutation may be indicative of a genetic susceptibility to develop a cancer 19 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, 21 of the cancer condition in the patient's family history and the presence of other risk factors, 22 such as exposure to environmental factors and whether the patient's cells also carry a 23 mutation of another sort.

24 [00103] Biological Sample [00104] The present invention provides for diagnostic tests which involve obtaining or 26 collecting one or more biological samples. In the context of the present invention, "biological 27 sample" refers to a tissue or bodily fluid containing cells from which mtDNA and mtRNA can 28 be obtained. For example, the biological sample can be derived from tissue including, but 29 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 31 fluid, peritoneal fluid, amniotic fluid and the like. The biological sample may be obtained from 32 a cancerous or non-cancerous tissue and may be, but is not limited to, a surgical specimen 33 or a biopsy specimen.

Ref: 102222/00053 1 [00105] The biological sample can be used either directly as obtained from the source or 2 following a pre-treatment to modify the character of the sample. Thus, the biological sample 3 can be pre-treated prior to use by, for example, preparing plasma or serum from blood, 4 disrupting cells, preparing liquids from solid materials, diluting viscous fluids, filtering liquids, distilling liquids, concentrating liquids, inactivating interfering components, adding reagents, 6 and the like.

7 [00106] One skilled in the art will understand that more than one sample type may be 8 assayed at a single time (i.e. for the detection of more than one cancer).
Furthermore, where 9 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 11 test period. In this regard, biological samples may be taken once only, or at regular intervals 12 such as biweekly, monthly, semi-annually or annually.

13 [00107] Kits 14 [00108] The present invention provides diagnostic/screening kits for detecting cancer in a clinical environment. Such kits may include one or more sampling means, in combination 16 with one or more probes according to the present invention.

17 [00109] The kits can optionally include reagents required to conduct a diagnostic assay, 18 such as buffers, salts, detection reagents, and the like. Other components, such as buffers 19 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 21 further comprise reagents suitable for the reconstitution of the lyophilised components.

22 [00110] Where appropriate, the kit may also contain reaction vessels, mixing vessels and 23 other components that facilitate the preparation of the test sample. The kit may also 24 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.

26 [00111] In one embodiment of the invention there is provided a kit for diagnosing cancer 27 comprising sampling means and a hybridization probe of the invention.

28 [00112] Various aspects of the invention will be described by illustration using the 29 following examples. The examples provided herein serve only to illustrate certain specific embodiments of the invention and are not intended to limit the scope of the invention in any 31 way.

Ref: 102222/00053 2 [00113] Example 1: Detection of Mitochondrial Fusion Transcripts 3 [00114] The mitochondrial 4977 "common deletion" and a 3.4kb deletion previously 4 identified by the present Applicant in PCT application no. PCT/CA2007/001711 (the entire contents of which are incorporated by reference) result in unique open reading frames 6 having active transcripts as identified by oligo-dT selection in prostate tissue (Figures 2 and 7 3). Examination of breast tissue samples also reveals the presence of a stable 8 polyadenylated fusion transcript resulting from the 3.4kb deletion (Figure 4).

9 [00115] Reverse transcriptase-PCR protocol for deletion transcript detection [00116] RNA isolation cDNA synthesis 11 [00117] Total RNA was isolated from snap frozen prostate and breast tissue samples 12 (both malignant and normal samples adjacent to tumours) using the AurumTM
Total RNA
13 Fatty and Fibrous Tissue kit (Bio-Rad, Hercules, CA) following the manufacturer's 14 instructions. Since in this experiment, genomic DNA contamination was to be avoided, a DNase I treatment step was included, using methods as commonly known in the art. RNA
16 quantity and quality were determined with an ND-1000 spectrophotometer (NanoDrop 17 technologies). From a starting material of about 100g, total RNA
concentrations varied from 18 100 - 1000ng/ul with a 260/280 ratio between 1.89 - 2.10. RNA
concentrations were 19 adjusted to 100ng/ul and 2u1 of each template were used for first strand DNA synthesis with SuperScriptTM First-Strand Synthesis System for RT-PCR (Invitrogen) following the 21 manufacturer's instructions. In order to identify stable polyadenylated fusion transcripts, 22 Oligo(dT) primers that target transcripts with poly-A tails were used.

23 [00118] PCR

24 [00119] Real time PCR was performed using 5u1 of each cDNA template with the iQT""
SYBR Green Supermix (Bio-Rad, Hercules, CA) on DNA Engine Opticon 2 Continuous 26 Fluorescence Detection System (Bio-Rad, Hercules, CA). The primer pairs targeting the 27 4977bp deletion are; 8416F 5'- CCTTACACTATTCCTCATCAC- 3', 13637R 5'-28 TGACCTGTTAGGGTGAGAAG - 3', and those for the 3.4 kb deletion are; ND4LF 5'-29 TCGCTCACACCTCATATCCTC -3', ND5R 5'- TGTGATTAGGAGTAGGGTTAGG -3'. The reaction cocktail included: 2X SYBR Green Supermix (100mM KCL, 40mM Tris-HCI, pH
31 8.4, 0.4mM of each dNTP [dATP, dCTP, dGTP, and dTTP], iTagTM DNA
polymerase, 50 32 units/m1, 6mM MgC12, SYBR Green 1, 20nM flourescein, and stabilizers), 250nM each of 33 primers, and ddH2O. PCR cycling parameters were as follows; (1) 95 C for 2 min, (2) 95 C
34 for 30 sec, (3) 55 C (for the 4977bp deletion) and 63 C (for the 3.4 kb deletion) for 30 sec , Ref: 102222/00053 1 (4) 72 C for 45 sec, (5) plate read, followed by 39 cycles of steps 3 to 5, and final incubation 2 at 4 C. Apart from cycling threshold and melting curve analysis, samples were run on 3 agarose gels for specific visualization of amplification products (see Figures 2 to 4).

4 [00120] Figure 2 is an agarose gel showing polyadenalated fusion transcripts in prostate samples invoked by the loss of 3.4kb from the mitochondrial genome. Legend for Figure 2:
6 B-blank, Lanes 1-6 transcripts detected in cDNA; lanes 7-12 no reverse transcriptase (RT) 7 controls for samples in lanes 1-6.

8 [00121] Figure 3 shows polyadenalated fusion transcripts in prostate samples invoked by 9 the loss of the 4977kb common deletion. Legend for Figure 3: B-blank, Lanes transcripts detected in cDNA; lanes 7-12 no RT controls for samples in lanes 1-6.

11 [00122] Figure 4 shows polyadenalated fusion transcripts in breast samples invoked by 12 the loss of 3.4kb from the mtgenome. Legend for Figure 4: Lanes 2-8 transcripts from breast 13 cDNAs; lane 9 negative (water) control; lanes 10 and 11, negative, no RT, controls for 14 samples in lanes 2 and 3.

[00123] These results demonstrate the existence of stable mitochondrial fusion 16 transcripts.

17 [00124] Example 2: Identification and Targetting of Fusion Products 18 Various hybridization probes were designed to detect, and further demonstrate the presence 19 of novel transcripts resulting from mutated mitochondrial genomes, such as the 3.4kb deletion. For this purpose, a single-plex branched DNA platform for quantitative gene 21 expression analysis (QuantiGene 2.0TM, PanomicsTM) was utilized. The specific deletions 22 and sequences listed in this example are based on their relative positions with the entire 23 mtDNA genome, which is recited in SEQ ID NO: 1. The nucleic acid sequences of the four 24 transcript to which the probes were designed in this example are identified herein as follows:
Transcript 1 (SEQ ID NO: 18), Transcript 2 (SEQ ID NO: 19), Transcript 3 (SEQ
ID NO: 20) 26 and Transcript 4 (SEQ ID NO: 21).

27 [00125] An example of a continuous transcript from the 3.4kb mitochondrial genome 28 deletion occurs with the genes ND4L (NADH dehydrogenase subunit 4L) and ND5 (NADH
29 dehydrogenase subunit 5). A probe having a complementary sequence to SEQ ID
NO: 19, was used to detect transcript 2. The repetitive elements occur at positions 10745-10754 in 31 ND4L and 14124-14133 in ND5.

32 [00126] The 3.4kb deletion results in the removal of the 3' end of ND4L, the full ND4 33 gene, tRNA histidine, tRNA serine2, tRNA leucine2, and the majority of the 5' end of ND5 34 (see Figure 5a), resulting in a gene splice of ND4L and ND5 with a junction point of Ref: 102222/00053 1 10744(ND4L):14124(ND5) (Figure 5b). SEQ ID NO: 3 is the complementary DNA
sequence 2 to the RNA transcript (SEQ ID NO: 19) detected in the manner described above.

3 [00127] Similarly, transcript 1 is a fusion transcript between ATPase 8 and 4 associated with positions 8469:13447 (SEQ ID NO: 18). Transcripts 3 and 4 (SEQ ID NO: 20 and SEQ ID NO: 21, respectively) are fusion transcripts between COII and Cytb associated 6 with nucleotide positions 7974:15496 and 7992:15730 respectively. Table 3 provides a 7 summary of the relationships between the various sequences used in this example. Table 3 8 includes the detected fusion transcript and the DNA sequence complementary to the fusion 9 transcript detected.

[00128] Example 3: Application to Prostate Cancer 11 [00129] Using the four fusion transcripts, i.e. transcripts 1 to 4, discussed above, two 12 prostate tissue samples from one patient were analyzed to assess the quantitative difference 13 of the novel predicted fusion transcripts. The results of the experiment are provided in Table 14 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 16 to the tumour of the patient. These samples were processed according to the 17 manufacturer's protocol (QuantiGene Sample Processing Kit for Fresh or Frozen Animal 18 Tissues; and QuantiGene 2.0 Reagent System User Manual) starting with 25.8 mg of 19 Homog 1 and 28.9 mg of Homog 2 (the assay setup is shown in Tables 5a and 5b).

[00130] Clearly demonstrated is an increased presence of mitochondrial fusion transcripts 21 in prostate cancer tissue compared to normal adjacent prostate tissue. The fusion transcript 22 is present in the normal tissue, although at much lower levels. The relative luminescence 23 units (RLU) generated by hybridization of a probe to a target transcript are directly 24 proportional to the abundance of each transcript. Table 2 also indicates the coefficients of variation, CV, expressed as a percentage, of the readings taken for the samples. The CV
26 comprises the Standard deviation divided by the average of the values. The significance of 27 such stably transcribed mitochondrial gene products in cancer tissue has implications in 28 disease evolution and progression.

29 [00131] Example 4: Application to Breast Cancer [00132] Using the same protocol from Example 3 but focusing only on Transcript 2, the 31 novel fusion transcript associated with the 3.4kb mtgenome deletion, analyses were 32 conducted on two samples of breast tumour tissue and two samples of tumour-free tissues 33 adjacent to those tumours, as well as three samples of prostate tumour tissue, one sample 34 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 Ref: 102222/00053 1 demonstrated a similar pattern to the prostate sample analyzed in Example 3 in that the 2 tumour tissue had approximately 2 times the amount of the fusion transcript than did the 3 normal adjacent tissue. The breast tumour samples demonstrated a marked increase in the 4 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 6 experiment cited in Example 3.

7 [00133] Thus, the above discussed results illustrate the application of the transcripts of 8 the invention in the detection of tumours of both prostate and breast tissue.

9 [00134] Example 5: Application to Colorectal Cancer [00135] This study sought to determine the effectiveness of several transcripts of the 11 invention in detecting colorectal cancer. A total of 19 samples were prepared comprising 12 nine control (benign) tissue samples (samples 1 to 9) and ten tumour (malignant) tissue 13 samples (samples 10 to 19). The samples were homogenized according to the 14 manufacturer's recommendations (Quantigene Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual). Seven target 16 transcripts and one housekeeper transcript were prepared in the manner as outlined above 17 in previous examples. The characteristics of the transcripts are summarized as follows:

18 [00136] Table 7: Characteristics of Breast Cancer Transcripts Transcript ID Junction Site Gene Junction 2 10744:14124 ND4L:ND5 3 7974:15496 COII:Cytb

10 7438:13476 COI:ND5

11 7775:13532 COII:ND5

12 8213:13991 COII:ND5 Peptidylpropyl isomerase B (PPIB) N/A N/A
("housekeeper") [00137] It is noted that transcripts 2 and 3 are the same as those discussed above with 21 respect to Examples 3 and 4.

22 [00138] Homogenates were prepared using approximately 25mg of tissue from OCT
23 blocks and diluted 1:1 for transcripts 2 and 4, and 1:8 for transcripts 10 and 11. The quantity 24 of the transcripts was measured in Relative Luminenscence Units RLU on a GlomaxTM Multi Detection System (Promega). All samples were assayed in triplicate for each transcript.
26 Background measurements (no template) were done in triplicate as well. The analysis 27 accounted for background by subtracting the lower limit from the RLU values for the Ref: 102222/00053 1 samples. Input RNA was accounted for by using the formula 1092 a RLU - 1092 h RLU where 2 a is the target fusion transcript and h is the housekeeper transcript.

3 [00139] The analysis of the data comprised the following steps:
4 a) Establish CV's (coefficients of variation) for triplicate assays;
acceptable if <_ 15%.
b) Establish average RLU value for triplicate assays of target fusion transcript(a) and 6 housekeeper transcript (h).
7 c) Establish lower limit from triplicate value of background RLU (I).
8 d) Subtract lower limit (I) from (a).
9 e) Calculate 1092 a RLU - 1092 h RLU.
[00140] Summary of Results:

11 [00141] The results of the above analysis are illustrated in Figures 6a to 6g, which 12 comprise plots of the 1092 a RLU - 1092 h RLU against sample number. Also illustrated are

13 the respective ROC (Receiver Operating Characteristic) curves determined from the results

14 for each transcript.

[00142] Transcript 2: There exists a statistically significant difference between the 16 means (p<0.10) of the normal and malignant groups (p>0.09), using a cutoff value of 3.6129 17 as demonstrated by the ROC curve results in a sensitivity of 60% and specificity of 89% and 18 the area under the curve is 0.73 indicating fair test accuracy. The threshold value chosen 19 may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

21 [00143] Transcript 3: There exists a statistically significant difference between the 22 means (p<0.05) of the normal and malignant groups (p=0.03), using a cutoff value of 4.0813 23 as demonstrated by the ROC curve results in a sensitivity of 60% and specificity of 78% and 24 the area under the curve is 0.79 indicating fair to good test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 26 particular application.

27 [00144] Transcript 8: There exists a statistically significant difference between the 28 means (p<0.1) of the normal and malignant groups (p=0.06). Using a cutoff value of -6.0975 29 as demonstrated by the ROC curve results in a sensitivity of 60% and specificity of 89% and the area under the curve is 0.76 indicating fair test accuracy. The threshold value chosen 31 may be adjusted to increase either the specificity or sensitivity of the test for a particular 32 application.

33 [00145] Transcript 9: There exists a statistically significant difference between the 34 means (p<0.1) of the normal and malignant groups (p=0.06). Using a cutoff value of -Ref: 102222/00053 1 7.5555 as demonstrated by the ROC curve results in a sensitivity of 60% and specificity of 2 89% and the area under the curve is 0.76 indicating fair to good test accuracy. The 3 threshold value chosen may be adjusted to increase either the specificity or sensitivity of the 4 test for a particular application.

[00146] Transcript 10: There is a statistically significant difference between the 6 means (p:50.01) of the normal and malignant groups (p=0.01). Using a cutoff value of -7 3.8272as demonstrated by the ROC curve results in a sensitivity of 90% and specificity of 8 67% and the area under the curve is 0.84, indicating good test accuracy. The threshold 9 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

11 [00147] Transcript 11: There exists a statistically significant difference between the 12 means (p<0.1) of the normal and malignant groups (p=0.06), using a cutoff value of 3.1753 13 as demonstrated by the ROC curve results in a sensitivity of 70% and specificity of 78% and 14 the area under the curve is 0.76 indicating fair to good test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 16 particular application.

17 [00148] Transcript 12: There exists a statistically significant difference between the 18 means (p<0.1) of the normal and malignant groups (p=0.06), using a cut-off value of 3.2626 19 as demonstrated by the ROC curve results in a sensitivity of 70% and specificity of 78% and the area under the curve is 0.76 indicating fair to good test accuracy. The threshold value 21 chosen may be adjusted to increase either the specificity or sensitivity of the test for a 22 particular application.
23 [00149] Conclusions:

24 [00150] The above results illustrate the utility of transcripts 2, 3, 8, 9, 10, 11, and 12 in the detection of colorectal cancer and in distinguishing malignant from normal colorectal tissue.
26 As indicated above, transcripts 2 and 3 were also found to have utility in the detection of 27 prostate cancer. Transcript 2 was also found to have utility in the detection of breast cancer.
28 Transcript 11 was also found to have utility in the detection of melanoma skin cancer.
29 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 31 transcripts listed may be used individually or in combination as a tool for the detection of 32 characterization of colorectal cancer in a clinical setting.

33 [00151] Example 6: Application to Lung Cancer Ref: 102222/00053 1 [00152] This study sought to determine the effectiveness of several transcripts of the 2 invention in the detection of lung cancer. As in Example 5, nine control (benign) tissue 3 samples (samples 1 to 9) and ten tumour (malignant) tissue samples (samples 10 to 19) 4 were homogenized according to the manufacturer's recommendations (Quantigene Sample Processing Kit for Fresh or Frozen Animal Tissues; and Quantigene 2.0 Reagent 6 System User Manual). Homogenates were diluted 1:8 and the quantity of 4 target 7 transcripts and 1 housekeeper transcript was measured in Relative Luminenscence Units 8 RLU on a GlomaxTM Multi Detection System (Promega). All samples were assayed in 9 triplicate for each transcript. Background measurements (no template) were done in triplicate as well.

11 [00153] The following transcripts were prepared for this example:
12 [00154] Table 8: Characteristics of Lung Cancer Transcripts Transcript ID Junction Site Gene Junction 6 8828:14896 ATPase6:Cytb 8 6075:13799 COI:ND5 10 7438:13476 COI:ND5 8469:13447 ATPase8:ND5 Peptidylpropyl isomerase B (PPIB) N/A N/A
("housekeeper") 14 [00155] The tissue samples used in this example had the following characteristics:

15 [00156] Table 9: Characteristics of Lung Cancer Samples Sample Malignant Comments (source of tissue) 1 NO interstitial lung disease 2 NO emphysema 3 NO aneurysm 4 NO bronchopneumonia, COPD
malignant neoplasm in liver, origin unknown, calcified granulomas in 5 NO lung 6 NO 12 hours post mortem, mild emphysema 12 hours post mortem, large B cell lymphoma, pulmonary edema, 7 NO pneumonia 8 NO pneumonia, edema, alveolar damage 9 NO congestion and edema 10 YES adenocarcinoma, non-small cell 11 YES small cell 12 YES squamous cell carcinoma, NSC, emphysema 13 YES adenocarcinoma, lung cancer, nsc, metastatic 14 YES squamous cell carcinoma, non-small cell 15 YES mixed squamous and adenocarcinoma

16 YES non-small cell carcinoma, squamous Ref: 102222/00053

17 YES small cell carcinoma

18 YES adenocarcinoma, lung cancer, nsc

19 YES adenocarcinoma, lung cancer, nsc, metastatic 2 [00157] The analysis of data was performed according to the method described in 3 Example 5. The results are illustrated in figures 7a, 7b, 7c and 7d.

4 [00158] Summary of Results:

[00159] Transcript 6: There exists a statistically significant difference between the 6 means (p<0.1) of the normal (benign) and malignant groups (p=0.06), using a cutoff value of 7 -6.5691 as demonstrated by the ROC curve results in a sensitivity of 80% and specificity of 8 71 % and the area under the curve is 0.77, indicating fair test accuracy.
The threshold value 9 chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

11 [00160] Transcript 8: The difference between the means of the normal and 12 malignant groups is statistically significant, p<0.05 (p=0.02). Using a cutoff value of -9.6166 13 as demonstrated by the ROC curve results in a sensitivity of 90% and specificity of 86% and 14 the area under the curve is 0.86 indicating good test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular 16 application.

17 [00161] Transcript 10: The difference between the means of the normal and 18 malignant groups is statistically significant, p:50.01 (p=0.01). Using a cutoff value of -19 10.6717 as demonstrated by the ROC curve results in a sensitivity of 90%
and specificity of 86% and the area under the curve is 0.89 indicating good test accuracy. The threshold 21 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 22 particular application.

23 [00162] Transcript 20: The difference between the means of the normal and 24 malignant groups is statistically significant, p:50.1 (p=0.1). Using a cutoff value of 2.5071 as demonstrated by the ROC curve results in a sensitivity of 70% and specificity of 71 % and the 26 area under the curve is 0.74 indicating fair test accuracy. The threshold value chosen may 27 be adjusted to increase either the specificity or sensitivity of the test for a particular 28 application.

29 [00163] Conclusions:

[00164] The results from example 6 illustrate the utility of transcripts 6, 8, 10, and 20 of 31 the invention in the detection of lung cancer tumours and the distinction between malignant Ref: 102222/00053 1 and normal lung tissues. Any of these three transcripts may be used for the detection or 2 characterization of lung cancer in a clinical setting.

3 [00165] Example 7: Application to Melanoma 4 [00166] This study sought to determine the effectiveness of several transcripts of the invention in the detection of melanomas. In this study a total of 14 samples were used, 6 comprising five control (benign) tissue samples and nine malignant tissue samples. All 7 samples were formalin fixed, paraffin embedded (FFPE). The FFPE tissue samples were 8 sectioned into tubes and homogenized according to the manufacturer's recommendations 9 (Quantigene 2.0 Sample Processing Kit for FFPE Samples; and Quantigene 2.0 Reagent System User Manual) such that each sample approximated 20 microns prior to 11 homogenization. Homogenates were diluted 1:4 and the quantity of 7 target transcripts and 12 1 housekeeper transcript was measured in Relative Luminenscence Units RLU
on a 13 GlomaxTM Multi Detection System (Promega). All samples were assayed in triplicate for 14 each transcript. Background measurements (no template) were done in triplicate as well.
[00167] The 14 tissue samples used in this example had the following characteristics:
16 [00168] Table 10: Characteristics of Melanoma Cancer Samples Sample Malignant Comments (source of tissue) 1 NO breast reduction tissue (skin) 2 NO breast reduction tissue (skin) 3 NO breast reduction tissue (skin) 4 NO breast reduction tissue (skin) 5 NO breast reduction tissue (skin) 6 YES lentigo maligna, (melanoma in situ) invasive melanoma not present 7 YES invasive malignant melanoma 8 YES nodular melanoma, pT3b, associated features of lentigo maligna residual superficial spreading invasive malignant melanoma, Clark's 9 YES level II
10 YES superficial spreading malignant melanoma, Clark's Level II
11 YES nodular malignant melanoma, Clark's level IV
superficial spreading malignant melanoma in situ, no evidence of 12 YES invasion superficial spreading malignant melanoma, Clark's level II, focally 13 YES present vertical phase 14 YES superficial spreading malignant melanoma in situ, Clark's level I

18 [00169] The following transcripts were prepared for this example:
19 [00170] Table 11: Characteristics of Melanoma Cancer Transcripts Ref: 102222/00053 Transcript ID Junction Site GeneJunction 6 8828:4896 ATPase6:Cytb 7438:13476 COI:ND5 11 7775:13532 COll:ND5 14 9191:12909 ATPase6:ND5 9574:12972 COlll:ND5 16 10367:12829 ND3:ND5 8469:13447 ATPase8:ND5 Peptidylpropyl isomerase B (PPIB) N/A N/A
("housekeeper") 3 [00171] As indicated, transcripts 10 and 11 were also used in Example 5. The analysis of 4 data was performed according to the method described in Example 5. The results are 5 illustrated in figures 8a -8g.

6 [00172] Summary of Results:

7 [00173] Transcript 6: There exists a statistically significant difference between the 8 means (p:50.01) of the normal and malignant groups (p=0.01). Further, using a cutoff value 9 of -5.9531 as demonstrated by the ROC curve results in a sensitivity of 89%
and specificity 10 of 80% and the area under the curve is 0.96, indicating very good test accuracy. The 11 threshold value chosen may be adjusted to increase either the specificity or sensitivity of the 12 test for a particular application.

13 [00174] Transcript 10: There exists a statistically significant difference between the 14 means (p:50.05) of the normal and malignant groups (p=0.05), using a cutoff value of -15 4.7572as demonstrated by the ROC curve results in a sensitivity of 89% and specificity of 16 40% and the area under the curve is 0.82, indicating good test accuracy.
The threshold 17 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 18 particular application.

19 [00175] Transcript 11: There exists a statistically significant difference between the

20 means (p<0.05) of the normal and malignant groups (p=0.02). Further, using a cutoff value

21 of 1.6762 as demonstrated by the ROC curve results in a sensitivity of 78%
and specificity of

22 100% and the area under the curve is 0.89, indicating good test accuracy.
The threshold

23 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a

24 particular application.

[00176] Transcript 14: There exists a statistically significant difference between the 26 means (p:50.05) of the normal and malignant groups (p=0.05). Further, using a cutoff value Ref: 102222/00053 1 of -4.9118 as demonstrated by the ROC curve results in a sensitivity of 89%
and specificity 2 of 60% and the area under the curve is 0.82, indicating good test accuracy.
The threshold 3 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 4 particular application.

[00177] Transcript 15: There exists a statistically significant difference between the 6 means (p<0.1) of the normal and malignant groups (p=0.07), using a cutoff value of -7 7.3107as demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 8 67% and the area under the curve is 0.80, indicating good test accuracy. The threshold 9 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

11 [00178] Transcript 16: There exists a statistically significant difference between the 12 means (p<0.05) of the normal and malignant groups (p=0.03). Further, using a cutoff value 13 of -1 0.5963as demonstrated by the ROC curve results in a sensitivity of 89% and specificity 14 of 80% and the area under the curve is 0.878, indicating good test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 16 particular application.

17 [00179] Transcript 20: There exists a statistically significant difference between the 18 means (p<0.05) of the normal and malignant groups (p=0.04). Further, using a cutoff value 19 of -8.3543as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 80% and the area under the curve is 0.89, indicating good test accuracy.
The threshold 21 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 22 particular application.

23 [00180] Conclusions:

24 [00181] The results from example 7 illustrate the utility of transcripts 6, 10, 11, 14, 15, 16 and 20 of the invention in the detection of malignant melanomas. As indicated above, 26 transcripts 10 andl1were also found have utility in detecting colorectal cancer while 27 transcript 6 has utility in the detection of lung cancer. A transcript summary by disease is 28 provided at Table 6.

29 [00182] Example 8: Application to Ovarian Cancer [00183] This study sought to determine the effectiveness of several transcripts of the 31 invention in detecting ovarian cancer. A total of 20 samples were prepared comprising ten 32 control (benign) tissue samples (samples 1 to 10) and ten tumour (malignant) tissue samples 33 (samples 11 to 20). The samples were homogenized according to the manufacturer's 34 recommendations (Quantigene Sample Processing Kit for Fresh or Frozen Animal Tissues;

Ref: 102222/00053 1 and Quantigene 2.0 Reagent System User Manual). Eight target transcripts and one 2 housekeeper transcript were prepared in the manner as outlined above in previous 3 examples.

4 [00184] The 20 tissue samples used in this example had the following characteristics:
[00185] Table 12: Characteristics of Ovarian Cancer Samples Sample Diagnosis Comments 1 Normal follicular cyst 2 Normal fibroma 3 Normal No pathological change in ovaries 4 Normal follicular cysts 5 Normal cellular fibroma 6 Normal benign follicular and simple cysts 7 Normal leiomyomata, corpora albicantia 8 Normal copora albicantia and an epithelial inclusions cysts 9 Normal corpora albicantia Normal corpora albicantia, surface inclusion cysts, follicullar cysts 11 Malignant high grade poorly differentiated papillary serous carcinoma involving omentum 12 Malignant endometrioid adenocarcinoma, well to moderately differentiated with focal serous differentiation 13 Malignant papillary serous carcinoma 14 Malignant mixed epithelial carcinoma predominantly papillary serous carcinoma Malignant High grade: serous carcinoma, papillary and solid growth patterns 16 Malignant High Grade (3/3) Papillary serous carcinoma 17 Malignant papillary serous carcinoma, high nuclear grade 18 Malignant Papillary serous cystadenocarcinomas Grade:lll 19 Malignant poorly differentiated papillary serous carcinoma Malignant Well-differentiated adnocarcinoma, Endometrioid type, Grade 1 7 [00186] The characteristics of the transcripts are summarized as follows:
8 [00187] Table 13: Characteristics of Ovarian Cancer Transcripts Transcript ID Junction Site Gene Junction 1 8469:13447 ATPase8:ND5 2 10744:14124 ND4L:ND5 3 7974:15496 COll:Cytb 6 8828:14896 ATPase6:Cytb 11 7775:13532 COll:ND5 12 8213:13991 COll:ND5 15 9574:12972 COlll:ND5 20 8469:13447 ATPase8:ND5 Ref: 102222/00053 Ribosomal Protein Large PO (LRP) N/A N/A
Housekeeper 1 [00188] It is noted that transcripts 1, 2, 3, 6, 11, 12, 15 and 20 are the same as those 2 discussed above with respect to Examples 3-7.

3 [00189] Homogenates were prepared using approximately 25mg of frozen tissue and 4 diluted 1:4. The quantity of the transcripts was measured in Relative Luminenscence Units RLU on a GlomaxTM Multi Detection System (Promega). All samples were assayed in 6 triplicate for each transcript. Background measurements (no template) were done in 7 triplicate as well. The analysis accounted for background by subtracting the lower limit from 8 the RLU values for the samples. Input RNA was accounted for by using the formula 1092 a 9 RLU - 1092 h RLU where a is the target fusion transcript and h is the housekeeper transcript.
[00190] The analysis of the data comprised the following steps:
11 a) Establish CV's (coefficients of variation) for triplicate assays;
acceptable if <_ 15%.
12 b) Establish average RLU value for triplicate assays of target fusion transcript(a) and 13 housekeeper transcript (h).
14 c) Establish lower limit from triplicate value of background RLU (I).
d) Subtract lower limit (I) from (a).
16 e) Calculate 1092 a RLU - 1092 h RLU.
17 [00191] Summary of Results:

18 [00192] The results of the above analysis are illustrated in Figures 9a to 9h, which 19 comprise plots of the 1092 a RLU - 1092 h RLU against sample number. Also illustrated are the respective ROC (Receiver Operating Characteristic) curves determined from the results 21 for each transcript.

22 [00193] Transcript 1: There exists a statistically significant difference between the 23 means (p<0.05) of the normal and malignant groups (p=0.002). Using a cutoff value of -24 11.1503 as demonstrated by the ROC curve results in a sensitivity of 90%
and specificity of 80% and the area under the curve is 0.91 indicating very good test accuracy.
The threshold 26 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 27 particular application.

28 [00194] Transcript 2: There exists a statistically significant difference between the 29 means (p<0.01) of the normal and malignant groups (p=0.001). Using a cutoff value of 0.6962 as demonstrated by the ROC curve results in a sensitivity of 90% and specificity of 31 100% and the area under the curve is 0.96 indicating very good test accuracy. The 32 threshold value chosen may be adjusted to increase either the specificity or sensitivity of the 33 test for a particular application.

Ref: 102222/00053 1 [00195] Transcript 3: There exists a statistically significant difference between the 2 means (p<0.01) of the normal and malignant groups (p=0.000). Using a cutoff value of 3 0.6754 as demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 4 100% and the area under the curve is 1.00 indicating excellent test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 6 particular application.

7 [00196] Transcript 6: There exists a statistically significant difference between the 8 means (p<0.01) of the normal and malignant groups (p=0.007). Using a cutoff value of -9 9.6479 as demonstrated by the ROC curve results in a sensitivity of 90% and specificity of 70% and the area under the curve is 0.86 indicating good test accuracy. The threshold 11 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 12 particular application.

13 [00197] Transcript 11: There is a statistically significant difference between the 14 means (p<0.01) of the normal and malignant groups (p=0.000). Using a cutoff value of -1.3794 demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 16 90% and the area under the curve is 0.99, indicating excellent test accuracy. The threshold 17 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 18 particular application.

19 [00198] Transcript 12: There exists a statistically significant difference between the means (p<0.01) of the normal and malignant groups (p=0.001). Using a cutoff value of -21 1.2379 as demonstrated by the ROC curve results in a sensitivity of 90% and specificity of 22 100% and the area under the curve is 0.96 indicating excellent test accuracy. The threshold 23 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 24 particular application.

[00199] Transcript 15: There exists a statistically significant difference between the 26 means (p<0.05) of the normal and malignant groups (p=0.023). Using a cut-off value of -27 8.6926 as demonstrated by the ROC curve results in a sensitivity of 70% and specificity of 28 80% and the area under the curve is 0.80 indicating good test accuracy. The threshold 29 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

31 [00200] Transcript 20: There exists a statistically significant difference between the 32 means (p<0.01) of the normal and malignant groups (p=0.000). Using a cut-off value of 33 0.6521 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 34 100% and the area under the curve is 0.76 indicating fair to good test accuracy. The Ref: 102222/00053 1 threshold value chosen may be adjusted to increase either the specificity or sensitivity of the 2 test for a particular application.

3 [00201] Conclusions:

4 [00202] The above results illustrate the utility of transcripts 1, 2, 3, 6, 11, 12, 15, and 20 in the detection of ovarian cancer and in distinguishing malignant from normal ovarian tissue.
6 Transcripts 1, 2 and 3 were also found to have utility in the detection of prostate cancer.
7 Transcript 6 was also found to have utility in the detection of melanoma and lung cancer.
8 Transcript 11 was also found to have utility in the detection of melanoma skin cancer, 9 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 11 utility in the detection of melanoma and testicular cancer. Transcript 20 was also found to 12 have utility in the detection of colorectal cancer, melanoma, and testicular cancer. Any of 13 the 8 transcripts listed may be used individually or in combination as a tool for the detection 14 or characterization of ovarian cancer in a clinical setting.

[00203] Example 9: Application to Testicular Cancer 16 [00204] This study sought to determine the effectiveness of several transcripts of the 17 invention in detecting testicular cancer. A total of 17 samples were prepared comprising 18 eight control (benign) tissue samples (samples 1 to 8) and 9 tumour (malignant) tissue 19 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 21 the manufacturer's recommendations (Quantigene Sample Processing Kit for Fresh or 22 Frozen Animal Tissues; and Quantigene 2.0 Reagent System User Manual). 10 target 23 transcripts and one housekeeper transcript were prepared in the manner as outlined above 24 in previous examples.

[00205] The 17 tissue samples used in this example had the following characteristics:
26 [00206] Table 14: Characteristics of Testicular Cancer Samples Sample General Stratified Diagnosis Malignant Diagnosis 1 Benign Benign 2 Benign Benign 3 Benign Benign 4 Benign Benign 5 Benign Benign 6 Benign Benign 7 Benign Benign 8 Benign Benign 9 Malignant Non-Ref: 102222/00053 Seminoma Malignant Non-Seminoma 11 Malignant Non-Seminoma 12 Malignant Non-Seminoma 13 Malignant Non-Seminoma 14 Malignant Seminoma Malignant Seminoma 16 Malignant Seminoma 17 Malignant Seminoma 1 [00207] The characteristics of the transcripts are summarized as follows:
2 [00208] Table 15: Characteristics of Testicular Cancer Transcripts Transcript ID Junction Site Gene Junction 2 10744:14124 ND4L:ND5 3 7974:15496 COII:Cytb 4 7992:15730 COII:Cytb 11 7775:13532 COII:ND5 12 8213:13991 COII:ND5 13 9144:13816 ATPase6:ND5 15 9574:12972 COIII:ND5 16 10367:12829 ND3:ND5 8469:13447 ATPase8:ND5 Peptidylpropyl isomerase B (PPIB) N/A N/A

4 [00209] It is noted that transcripts 2, 3, 4, 7, 11, 12, 15, 16 and 20 are the same as those 5 discussed above with respect to Examples 3-8.

6 [00210] Homogenates were prepared using approximately 25mg of frozen tissue and 7 diluted 1:4. The quantity of the transcripts was measured in Relative Luminenscence Units 8 RLU on a GlomaxTM Multi Detection System (Promega). All samples were assayed in 9 triplicate for each transcript. Background measurements (no template) were done in 10 triplicate as well. The analysis accounted for background by subtracting the lower limit from 11 the RLU values for the samples. Input RNA was accounted for by using the formula 1092 a 12 RLU - 1092 h RLU where a is the target fusion transcript and h is the housekeeper transcript.
13 [00211] The analysis of the data comprised the following steps:
14 a) Establish CV's (coefficients of variation) for triplicate assays;
acceptable if <_ 15%.

Ref: 102222/00053 1 b) Establish average RLU value for triplicate assays of target fusion transcript(a) and 2 housekeeper transcript (h).
3 c) Establish lower limit from triplicate value of background RLU (I).
4 d) Subtract lower limit (I) from (a).
e) Calculate 1092 a RLU - 1092 h RLU.
6 [00212] Summary of Results:

7 [00213] The results of the above analysis are illustrated in Figures 10 to 18, which 8 comprise plots of the 1092 a RLU - 1092 h RLU against sample number. Also illustrated are 9 the respective ROC (Receiver Operating Characteristic) curves determined from the results for each transcript.

11 [00214] While some transcripts distinguish between benign and malignant testicular 12 tissue, others demonstrate distinction between the tumour subtypes of seminoma and non-13 seminoma and/or benign testicular tissue. It is therefore anticipated that combining 14 transcripts from each class will facilitate not only detection of testicular cancer but also classification into subtype of seminoma or non-seminomas.

16 [00215] Transcript 2: There exists a statistically significant difference between the 17 means (p<0.05) of the normal group and the malignant seminomas (p=0.02).
Using a cutoff 18 value of 1.5621 as demonstrated by the ROC curve results in a sensitivity of 100% and 19 specificity of 100% and the area under the curve is 1.00 indicating excellent test accuracy.
There also exists a statistically significant difference between the means (p<0.05) of the 21 malignant seminomas and the malignant non-seminomas (p=0.024). Using a cutoff value of 22 2.1006 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 23 80% and the area under the curve is 0.90 indicating excellent test accuracy. The threshold 24 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

26 [00216] Transcript 3: There exists a statistically significant difference between the 27 means (p<0.05) of the normal group and the malignant seminomas (p=0.018).
Using a cutoff 28 value of 0.969 as demonstrated by the ROC curve results in a sensitivity of 100% and 29 specificity of 87.5% and the area under the curve is 0.969 indicating excellent accuracy.
There also exists a statistically significant difference between the means (p<0.05) of the 31 malignant seminomas and the malignant non-seminomas (p=0.017). Using a cutoff value of 32 1.8181 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 33 80% and the area under the curve is 0.9 indicating excellent test accuracy.
The threshold 34 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

Ref: 102222/00053 1 [00217] Transcript 4: There exists a statistically significant difference between the 2 means (p<0.05) of the normal and malignant groups (p=0.034). Using a cutoff value of -3 9.7628 as demonstrated by the ROC curve results in a sensitivity of 67% and specificity of 4 100% and the area under the curve is 0.833 indicating good test accuracy.
The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 6 particular application.

7 [00218] Transcript 11: There exists a statistically significant difference between the 8 means (p<0.05) of the normal group and the malignant seminomas (p=0.016).
Using a cutoff 9 value of 0.732 as demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 100% and the area under the curve is 1.00 indicating excellent test accuracy.
11 There also exists a statistically significant difference between the means (p<0.05) of the 12 malignant seminomas and the malignant non-seminomas (p=0.016). Using a cutoff value of 13 0.9884 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 14 80% and the area under the curve is 0.90 indicating excellent test accuracy. The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 16 particular application.

17 [00219] Transcript 12: There exists a statistically significant difference between the 18 means (p<0.1) of the normal group and the malignant seminomas (p=0.056).
Using a cutoff 19 value of 1.5361 as demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 87.5% and the area under the curve is 0.969 indicating excellent test accuracy.
21 There also exists a statistically significant difference between the means (p<0.05) of the 22 malignant seminomas and the malignant non-seminomas (p=0.044). Using a cutoff value of 23 1.6039 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 24 80% and the area under the curve is 0.9 indicating excellent test accuracy.
The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a 26 particular application.

27 [00220] Transcript 13: There exists a statistically significant difference between the 28 means (p<0.05) of the normal group and the malignant group (p=0.019). Using a cutoff value 29 of -9.8751 as demonstrated by the ROC curve results in a sensitivity of 87.5% and specificity of 78% and the area under the curve is 0.875 indicating very good test accuracy. There also 31 exists a statistically significant difference between the means (p<0.01) of the malignant non-32 seminomas and the benign group (p=0.000). Using a cutoff value of -13.9519 as 33 demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 87.5% and 34 the area under the curve is 0.975 indicating excellent test accuracy. There also exists a statistically significant difference between the means (p<0.01) of the malignant seminomas 36 and the malignant non-seminomas (p=0.001). Using a cutoff value of -15.8501 as Ref: 102222/00053 1 demonstrated by the ROC curve results in a sensitivity of 100% and specificity of 100% and 2 the area under the curve is 1.00 indicating excellent test accuracy. The threshold value 3 chosen may be adjusted to increase either the specificity or sensitivity of the test for a 4 particular application.

[00221] Transcript 15: There exists a statistically significant difference between the 6 means (p<0.1) of the normal and malignant groups (p=0.065). Using a cut-off value of -7 5.4916 as demonstrated by the ROC curve results in a sensitivity of 75% and specificity of 8 89% and the area under the curve is 0.833 indicating good test accuracy. The threshold 9 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

11 [00222] Transcript 16: There exists a statistically significant difference between the 12 means (p<0.05) of the normal and malignant groups including both seminomas and non-13 seminomas(p=0.037). Using a cut-off value of -6.448 as demonstrated by the ROC curve 14 results in a sensitivity of 89% and specificity of 75% and the area under the curve is 0.806 indicating good test accuracy. There also exists a statistically significant difference between 16 the means (p<0.05) of the normal and malignant seminomas (p=0.037). Using a cut-off 17 value of -7.4575 as demonstrated by the ROC curve results in a sensitivity of 100% and 18 specificity of 87.5% and the area under the curve is 0.938 indicating excellent test accuracy.
19 The threshold value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

21 [00223] Transcript 20: There exists a statistically significant difference between the 22 means (p<0.01) of the normal group and the malignant seminomas (p=0.006).
Using a cutoff 23 value of 1.8364 as demonstrated by the ROC curve results in a sensitivity of 100% and 24 specificity of 100% and the area under the curve is 1.00 indicating excellent test accuracy.
There also exists a statistically significant difference between the means (p<0.01) of the 26 malignant seminomas and the malignant non-seminomas (p=0.004). Using a cutoff value of 27 1.6065 as demonstrated by the ROC curve results in a sensitivity of 100%
and specificity of 28 100% and the area under the curve is 1.00 indicating excellent test accuracy. The threshold 29 value chosen may be adjusted to increase either the specificity or sensitivity of the test for a particular application.

31 [00224] Conclusions:

32 [00225] The above results illustrate the utility of transcripts 2, 3, 4, 11, 12, 13, 15, 16, and 33 20 in the detection of testicular cancer, and testicular cancer subtypes, and in distinguishing 34 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 Ref: 102222/00053 1 have utility in the detection of prostate, breast, melanoma, colorectal, and ovarian cancers.
2 Transcript 4 was also found to have utility in the detection of prostate and colorectal cancers.
3 Transcript 11 was also found to have utility in the detection of colorectal, melanoma, and 4 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 6 melanoma and ovarian cancers. Transcript 16 was also found to have utility in the detection 7 of melanoma skin cancer. Transcript 20 was also found to have utility in the detection of 8 colorectal cancer, melanoma, and ovarian cancer. Any of the 9 transcripts listed may be 9 used individually or in combination as a tool for the detection or characterization of testicular cancer in a clinical setting.

11 [00226] In one aspect, the invention provides a kit for conducting an assay for 12 determining the presence of cancer in a tissue sample. The kit includes the required 13 reagents for conducting the assay as described above. In particular, the kit includes one or 14 more containers containing one or more hybridization probes corresponding to transcripts 1 to 17, and 20 described above. As will be understood, the reagents for conducting the assay 16 may include any necessary buffers, salts, detection reagents etc. Further, the kit may 17 include any necessary sample collection devices, containers etc. for obtaining the needed 18 tissue samples, reagents or materials to prepare the tissue samples for example by 19 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 21 values for diseased or non-diseased tissues.

22 [00227] Although the invention has been described with reference to certain specific 23 embodiments, various modifications thereof will be apparent to those skilled in the art 24 without departing from the spirit and scope of the invention as outlined in the claims appended hereto. All documents (articles, manuals, patent applications etc) referred to in 26 the present application are incorporated herein in their entirety by reference.

27 [00228] Bibliography 28 [00229] The following references, amongst others, were cited in the foregoing description.
29 The entire contents of these references are incorporated herein by way of reference thereto.
Author Journal Title Volume Date Anderson et al Nature Sequence and Organization of the Human 290(5806):457-Mitochondrial Genome 65 Andrews et al Nat Genet Reanalysis and revision of the Cambridge 23(2):147 reference sequence for human mitochondrial DNA.
Modica- Expert Rev Mitochondria as targets for detection and 4:1-19 2002 Napolitano et al Mol Med treatment of cancer Ref: 102222/00053 Sherratt et al Clin Sci (Lond) Mitochondrial DNA defects: a widening 92(3):225-clinical spectrum of disorders.
Croteau et al Mutat Res Mitochondrial DNA repair pathways. 434(3):137-48 1999 Green and J Clin Invest Pharmacological manipulation of cell death: 115(10):

Kroemer clinical applications in sight? 2617 Dai et al Acta Correlation of cochlear blood supply with 24(2):130-6 2004 Otolaryngol mitochondrial DNA common deletion in presbyacusis.
Ro et al Muscle Nerve Deleted 4977-bp mitochondrial DNA 28(6):737-43 2003 mutation is associated with sporadic amyotrophic lateral sclerosis: a hospital-based case-control study.
Barron et al Invest Mitochondrial abnormalities in ageing 42(12):3016-22 2001 Ophthalmol macular photoreceptors.
Vis Sci Lewis et al J Pathol Detection of damage to the mitochondrial 191(3):274-81 genome in the oncocytic cells of Warthin's tumour.
Muller-Hocker Mod Pathol The common 4977 base pair deletion of 11(3):295-301.

et al mitochondrial DNA preferentially accumulates in the cardiac conduction system of patients with Kearns-Sayre syndrome.
Porteous et al Eur J Biochem Bioenergetic consequences of accumulating 257(1):192-201 1998 the common 4977-bp mitochondrial DNA
deletion.
Parr et al J Mol Diagn Somatic mitochondrial DNA mutations in 8(3):312-9. 2006 prostate cancer and normal appearing adjacent glands in comparison to age-matched prostate samples without malignant histology.
Maki et al Am J Clin Mitochondrial genome deletion aids in the 129(1):57-66 Pathol identification of false- and true-negative prostate needle core biopsy specimens.
Nakase et al Am J Hum Transcription and translation of deleted 46(3):418-27.

Genet mitochondrial genomes in Kearns-Sayre syndrome: implications for pathogenesis.
Libura et al Blood Therapy-related acute myeloid leukemia- 105(5):2124-31 2005 like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion.
Meyer et al Proc Natl Diagnostic tool for the identification of MLL 102(2):449-Acad Sci U S rearrangements including unknown partner A genes.
Eguchi et al Genes MLL chimeric protein activation renders 45(8):754-60 2006 Chromosomes cells vulnerable to chromosomal damage:
Cancer an explanation for the very short latency of infant leukemia.
Hayashi et al Proc Natl Introduction of disease-related 88: 10614- 1991 Acad Sci U S mitochondrial DNA deletions into HeLa cells 10618 A lacking mitochondrial DNA results in mitochondrial dysfunction M

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Claims (23)

Claims:

1. An isolated mitochondrial fusion transcript associated with cancer.

2. The mitochondrial fusion transcript of claim 1, wherein the transcript comprises an insertion, translocation, deletion, duplication, recombination, rearrangement or combination thereof.

3. The mitochondrial fusion transcript of claim 2, wherein the transcript comprises a deletion.

4. The mitochondrial fusion transcript of claim 3, wherein the transcript comprises a sequence as set forth in any one of SEQ ID NOs:18 to 33 or 50.

5. The mitochondrial fusion transcript of claim 3, wherein the transcript comprises a sequence as set forth in any one of SEQ ID NOs: 18-21, 23, 25-33 or 50.

6. The mitochondrial fusion transcript of claim 3, wherein the transcript comprises the expressed RNA transcript of a deletion sequence set out in Table 1.

7. A mitochondrial fusion protein corresponding to the fusion transcript of claim 4 and having a sequence as set forth in any one of SEQ ID NOs: 34 to 49 and 52.

8. An isolated mitochondrial DNA (mtDNA) encoding the fusion transcript of claim 1.

9. The isolated mtDNA of claim 8 having a sequence as set forth in any one of SEQ ID
NOs: 2-17 or 51.

10. A hybridization probe having a nucleic acid sequence complementary to at least a portion of the mitochondrial fusion transcript according to any one of claims 1 to 6 or the mtDNA of claim 8 or 9.

11. A method of detecting a cancer in a mammal, the method 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 the mitochondrial fusion transcript according to any one of claims 1 to 6.

12. A method of detecting a cancer in a mammal, the method 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 the mtDNA according to claim 7 or 8.

13. The method of claim 11 or 12, wherein the cancer is selected from the group consisting of prostate cancer, testicular cancer, ovarian cancer, breast cancer, colorectal cancer, lung cancer, melanoma skin cancer and combinations thereof.

14. The method of claim 13, wherein the assay comprises:
a) conducting a hybridization reaction using at least one of said probes to allow said at least one probe to hybridize to a complementary mitochondrial fusion transcript or mtDNA;
b) quantifying the amount of the at least one mitochondrial fusion transcript or mtDNA
in said sample by quantifying the amount of said transcript or mtDNA
hybridized to said at least one probe; and, c) comparing the amount of the mitochondrial fusion transcript or mtDNA in the sample to at least one known reference value.

15. The method of claim 14, wherein the assay is carried out using diagnostic imaging technology.

16. The method of claim 15, wherein the diagnostic imaging technology comprises high throughput microarray analysis.

17. The method of claim 14, wherein the assay is carried out using branched DNA
technology.

18. The method of claim 14, wherein the assay is carried out using PCR.

19. A kit for conducting an assay for detecting the presence of a cancer in a mammal, said kit comprising at least one hybridization probe complementary to at least a portion of the fusion transcript of any one of claims 1 to 6 or the mtDNA of claim 8 or 9.

20. A screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial fusion transcripts according to any one of claims 1 to 6 for identification of those associated with cancer.

21. A screening tool comprised of a microarray having 10's, 100's, or 1000's of mitochondrial DNAs according to claim 8 or 9 for identification of those associated with cancer.

22. A screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial fusion transcripts according to any one of claims 1 to 6 for identification of those associated with cancer.

23. A screening tool comprised of a multiplexed branched DNA assay having 10's, 100's, or 1000's of mitochondrial DNAs according to claim 8 or 9 for identification of those associated with cancer.