NZ584815A

NZ584815A - 3.4 kb mitochondrial dna deletion for use in the detection of cancer

Info

Publication number: NZ584815A
Application number: NZ584815A
Authority: NZ
Inventors: Ryan Parr; Robert Thayer; Gabriel Dakubo; Jennifer Creed; Kerry Robinson; Andrea Maggrah; Brian Reguly
Original assignee: Mitomics Inc
Priority date: 2007-09-26
Filing date: 2007-09-26
Publication date: 2012-07-27
Also published as: CA2700941A1; JP2010539908A; CN101874119B; CN101874119A; EP2634267A1; WO2009039601A1; JP5518715B2; KR20100069670A; AU2007359381A1; HK1145343A1; EP2203570A1; EP2203570A4; EP2203570B1; EP2634267B1; AU2007359381B2; KR101449562B1; ES2484044T3; SG185254A1; ES2539001T3

Abstract

Disclosed is a method of detecting a cancer in an individual comprising; a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic add sequence spanning approximately residues 10744 to 14124 of the mtDNA genome; c) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value, wherein the known reference value is: (i) the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and/or (ii) the amount of the deletion in a reference value of mtDNA from known cancerous tissue or body fluid; - wherein, if the reference value is (i), an elevated amount of the deletion in the biological sample compared to the reference value is indicative of cancer; - and wherein, if the reference value is (ii), a similar level of the deletion in the biological sample compared to the reference value is indicative of cancer; - with the proviso that the cancer is not prostate cancer. Further disclosed is a method of monitoring an individual for the development of a cancer comprising; a) extracting mtDNA from 11 biological sample from the individual: b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 to 14124 of the mtDNA genomic; c) repeating steps a) and b) over a duration of time; d) wherein an increasing, level of the deletion over the duration of time is indicative or cancer; - with the proviso that the cancer is not prostate cancer.

Description

<div class="application article clearfix" id="description"> 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 WO 2009/039601 PCT/CA2007/001711 3.4 kb Mitochondrial DNA Deletion for use in the Detection of Cancer CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of PCT application no. PCT/CA2006/000652 filed on April 18, 2006 which claims priority from U.S. provisional application nos. 60/672,016 filed April 18, 2005, 60/721,522 filed September 29, 2005, and 60/789,872 filed April 7, 2006. The entire disclosures of these applications are incorporated herein by reference. FIELD OF THE INVENTION:

[0002] This invention is related to the field of mitochondrial genomics. In particular it is related to a 3.4 kb deletion in the mitochondrial genome and its utility as an indicator of cancer. DESCRIPTION OF THE PRIOR ART

[0003] Mitochondrial DNA (MtDNA) as a Diagnostic Tool

[0004] MtDNA sequence dynamics are important diagnostic tools. Mutations in mtDNA are often preliminary indicators of developing disease, often associated with nuclear mutations, and act as biomarkers specifically related to: disease, such as but not limited to, tissue damage and cancer from smoking and exposure to second hand tobacco smoke (Lee et al., 1998; Wei, 1998); longevity, based on accumulation of mitochondrial genome mutations beginning around 20 years of age and increasing thereafter (von Wurmb, 1998); metastatic disease caused by mutation or exposure to carcinogens, mutagens, ultraviolet radiation (Birch-Machin, 2000); osteoarthritis; cardiovascular, Alzheimer, Parkinson disease (Shoffiier et al., 1993; Sherratt et al., 1997;Zhang et al, 1998); age associated hearing loss (Seidman et al., 1997); optic nerve degeneration and cardiac dysrhythmia (Brown et al., 1997; Wallace et al., 1988); chronic progressive external exophthalmoplegia (Taniike et al., 1992); atherosclerosis (Bogliolo et al., 1999); papillary thyroid carcinomas and thyroid tumours (Yeh et al., 2000); as well as others (e.g. Naviaux, 1997; Chinnery and Turnbull, 1999).

[0005] Mutations at specific sites of the mitochondrial genome can be associated with certain diseases. For example, mutations at positions 4216, 4217 and 4917 are associated with Leber's Hereditary Optic Neuropathy (LHON) (Mitochondrial Research Society; 21682168.2 1 2 WO 2009/039601 PCT/CA2007/001711 Huoponen (2001); MitoMap). A mutation at 15452 was found in 5/5 patients to be associated with ubiquinol cytochrome c reductase (complex III) deficiency (Valnot et al.1999). 3 [0006] Specifically, these mutations or alterations include point mutations (transitions, 4 transversions), deletions (one base to thousands of bases), inversions, duplications, (one base 5 to thousands of bases), recombinations and insertions (one base to thousands of bases). In 6 addition, specific base pair alterations, deletions, or combinations thereof have been found to 7 be associated with early onset of prostate, skin, and lung cancer, as well as aging (e.g. Polyak 8 et al., 1998), premature aging, exposure to carcinogens (Lee et al., 1998), etc. 9 [0007] Prostate Cancer 10 [0008] Prostate cancer is a frequently diagnosed solid tumour that most likely originates 11 in the prostate epithelium (Huang et al. 1999). In 1997, nearly 10 million American men 12 were screened for prostate specific antigen (PSA), the presence of which suggests prostate 13 cancer (Woodwell, 1999). Indeed, this indicates an even higher number of men screened by 14 an initial digital rectal exam (DRE). In the same year, 31 million men had a DRE 15 (Woodwell, 1999). Moreover, the annual number of newly diagnosed cases of prostate 16 cancer in the United States is estimated at 179,000 (Landis et al., 1999). It is the second 17 most commonly diagnosed cancer and second leading cause of cancer mortality in Canadian 18 men. In 1997 prostate cancer accounted for 19,800 of newly diagnosed cancers in Canadian 19 men (28%) (National Cancer Institute of Canada). It is estimated that 30% to 40% of all men 20 over the age of forty-nine (49) have some cancerous prostate cells, yet only 20% to 25% of 21 these men have a clinically significant form of prostate cancer (SpringNet - CE Connection, 22 internet, www.springnet.com/ce/j803a.htm). Prostate cancer exhibits a wide variety of 23 histological behaviour involving both endogenous and exogenous factors, i.e. socio-economic 24 situations, diet, geography, hormonal imbalance, family history and genetic constitution 25 (Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNA alterations have been 26 previously associated with prostate cancer, the need exists for further markers for the 27 detection of prostate cancer. 28 [0009] 3.4kb mtDNA deletion and the detection of prostate cancer. 29 [0010] In the applicant's pending PCT application bearing publication no. 30 WO/06/111029 (the entire contents of which are incorporated herein by reference) a deletion 31 of a 3379 bp segment of mtDNA was identified through full mitochondrial genome 21682168.2 -2- From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 13 of 35 Received mail@iponz 12 June 2012 amplification of prostate tissue. The 3379 bp deletion (referred lo aw the 3.4 kb deletion) was determined to be located between nucleotides 10744-14124 of the mitochondrial genome. It was determined that the detection of this deletion could be used in Ihe diagnosis of" prostrate cancer when tissue samples arc tested. 10011} T 'he 3.4 kb deletion removes all or part of the following genes from the mtDNA genome: (i) NADU dehydrogenase subunit. 4L, (ii) NADH dehydrogenase subunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNA histidine, (v) tRNA scrine2? and (vi) tRNA lcucine2.

[0012] Breast Cancer

[0013] Breast cancer is a cancer of the glandular breast tissue and is the fifth most common cause of cancer death. In 7005, breast canccr caused 502,000 deaths (7% of cancer deaths; almost 1% of all deaths) worldwide (World Health Organization Cancer Fact Sheet No. 297). Among women worldwide, breast cancer is the most common cancer and the most common cause of cancer death (World Health Organization Canccr Fact Sheet No. 297). Although certain mtDNA alterations have been previously associated with breast cancer, for example in Parrella et al. (Cancer Research: 61, 2001), the need exists for further markers for the detection of breast cancer. SUMMARY Of I l ll;. INVENTION 20 (0014) fn one embodiment, the present invention provides a method of detecting a cancer in an individual comprising; a) obtaining a biological sample from the individual; b) extracting mitochondrial DNA, mtDNA, from the sample; e) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid 25 sequence spanning approximately residues 1 074'I to H1 24 of the mtDNA genome; d) comparing the amount of mtDNA in the .sample having the deletion It.) at least one known reference value, 1001S] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising; 30 a) obtaining a biological sample from the individual; b) extracting mitochondrial DNA, mtDNA, from the sample; c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 to 14124 of the inLDNA genome; - 3 - 10 15 From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 14 of 35 Received mail@iponz 12 June 2012 d) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; wherein an elevated amount of the deletion in the biological sample, compared to the reference sample is indicative of cancer. 5 [0016] In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising; a) obtaining a biological sample from the individual; b) extracting mitochondrial DNA, mtDNA, from the sample; c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid 10 sequence spanning approximately residues 10744 to 14124 of the mtDNA genome; d) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid; wherein a similar level of the deletion in the biological sample compared to the reference sample is indicative of cancer. 15 [0017] Tn one embodiment, the present invention provides a method of monitoring an individual for the development of a cancer comprising; a) obtaining a biological sample; b) extracting mtDNA from the sample; c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid 20 sequence spanning approximately residues 10744 to 14124ofthc mtDNA genome; d) repeating steps a) to e) over a duration of time; e) wherein an increasing level of the deletion over the duration of time is indicative of cancer.

[0018] In one embodiment, the present invention provides a method of defecting a cancer 25 in an individual comprising; a) obtaining a biological sample from the individual; b) extracting mitochondrial DNA, mtDNA, from the sample; c) quantifying the amount of mtDNA in the sample having a sequence corresponding to the sequence identified in SEQ ID NO: 1; 30 d) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 to at least one known rcfcrcnee value, [0018a] T 'hus, in one aspect, the inventions provide a method of detecting a cancer in an individual comprising; - 4, From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 15 of 35 Received mail@iponz 12 June 2012 a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 1074-1 to 14124 of the mtDNA genome; c) comparing the amount of mtDNA in the sample having the deletion to at least one 5 known reference value, wherein the known reference value is: (i) the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and/or (ii) the amount, of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid; - wherein, if the reference value is (i), an elevated amount of the deletion in the biological 10 sample compared to the reference value is indicative of cancer; - find wherein, if the reference value is (ii), a similar level of the deletion in the biological sample compared to the reference value is indicative of cancer; - with the proviso that the cancer is not prostate cancer, 15 [0018b] In another aspect the invention provides a method of detecting a cancer in an individual comprising; a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 to 14124 of the mtDNA genome; 20 c) comparing, the amount of mtDNA in the sample having the deletion to a known reference value; - wherein the step of quantifying is conducted using real-time PCR, the quantifying of the deletion includes first amplifying a target region of mtDNA that, is indicative of the deletion, and quantifying the amount of the amplified target region; 25 - and wherein the known reference value is a cycle threshold; - with the proviso that the cancer is not prostate cancer. 10018c] In another aspect, the invention provides a method of monitoring an individual for the development of a cancer comprising; 30 a.) extracting mtDNA from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence .spanning approximately residues 10744 to 14124 of the nilD'NA genome; c) repeating steps a) and b) over a duration of lime; - 5 - From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 21 of 35 Received mail@iponz 12 June 2012 d) wherein an increasing level of the deletion over the duration of time is indicative of cancer; - with the proviso that the cancer is not. prostate cancer. 5 looisdi In another aspect, the invention provides a method of detecting breast cancer in an individual comprising; a) extracting, mitochondrial DNA, mtDNA, from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid 10 sequence spanning approximately residues 10744 to 14 124 of the mtDNA genome, and wherein the deletion has a nuclcie acid sequence corresponding to the sequence identified in SEQ ID NO: i; c) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value, wherein the known reference value is: (i) the amount of the deletion in a 15 reference sample of mtDNA from known non-cancerous tissue or body fluid; and/or (ii) the amount of the deletion in a reference sample of mtDNA from known canccrous tissue or body fluid; - wherein, if the reference value is (i), an elevated amount of the deletion in the biological sample compared to the reference value is indicative of breast canccr; 20 - and wherein, if the reference value is (ii), a similar level of the deletion in the biological sample compared to the reference value is indicative of breast canccr. BRIEF DESCRIPTION OF THE DRAWINGS 25 [0019] An. embodiment of the invention will now be described by way of example only with reference to the appended drawings wherein: [0020) Figure ! is a schcmatic diagram showing the design and sequence of a primer useful for the detection of the 3.4 kb deletion. 30

[0021] Figure 2 is a graph showing a comparison of cycle threshold between malignant and symptomatic benign participants in the 3.4 kb study. - 5 a - From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 22 of 35 Received mail@iponz 12 June 2012

[0022] Figure 3 is a graph showing cyclc threshold as related to Example 1.

[0023] Figure 4 shows a ROC curve illustrating tine specificity and sensitivity of one embodiment of the present invention. 5

[0024] Figure 5 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.

[0025] Figure 6 shows real-time PGR data relating to 3.4kb mtDNA deletion levels 10 associated with breast cancer.

[0026] Figure 7 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention, 15 [0026a] Figure H is a schematic illustration of the Artificial Neural Network described in Example 3. DETAILED DESCRIPTION OF THE INVENTION 20 [0027] As used herein, "cycle threshold" (CT) is the point al which target amplification using real-time PCR rises above background, as indicated by a .signal such as a fluorescence signal. The CT is inversely related to the quantity of the sequence being investigated. |0028| Ay defined herein, "sensitivity" refers to the fraction of true positives (true 25 positive rate) results obtained using the method of the present invention. - 5b - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 WO 2009/039601 PCT/CA2007/001711

[0029] As defined herein, "specificity" refers to the fraction of false positives (false positive rate) results obtained using the method of the present invention.

[0030] In one embodiment of the present invention, methods are provided for monitoring and diagnosing cancer through the detection and quantification of the aforementioned 3.4 kb mtDNA deletion. For example, the present invention may be used for detecting the presence of pre-neoplasia, neoplasia and progression towards potential malignancy of prostate cancer and breast cancer. In one aspect, the present invention involves the detection and quantification of the 3.4kb mtDNA deletion (SEQ ID NO:l) for the detection, diagnosis, and/or monitoring of cancer. In this method, mtDNA is extracted from a biological sample (for example body tissue, or body fluids such as urine, prostate massage fluid). The extracted mtDNA is then tested in order to determine the levels (ie. quantity) of the 3.4 kb deletion in the sample. In tests conducted by the present inventors, the levels of the deletion were found to be elevated in samples obtained from subjects with cancer when compared to samples obtained from subjects without cancer. Based on the information and data supplied below, the inventors have concluded that elevated levels of the 3.4 kb deletion in the mtDNA is indicative of cancer.

[0031] As disclosed in PCT WO/O6/111029, the 3.4kb deletion spans approximately nucleotides 10744 to 14124 of the mtDNA genome. The mtDNA genome is listed as SEQ ID NO:8 (Genbank accession no. AC 000021). The inventors have determined, as provided by example below, that this deletion is also associated with cancer and in particular prostate and breast cancer. Therefore, such deletion provides an accurate biomarker and, therefore, a valuable tool for the detection, diagnosis, or monitoring of cancer in at least these tissues.

[0032] The deletion results in the creation of two deletion monomers, one of 3.4kb in size (small sublimon) and one of approximately 12.6kb in size (large sublimon). The occurrence of the deletion may be detected by either identifying the presence of the small sublimon, or by determining that the 3.4 kb sequence has been deleted from the large sublimon.

[0033] As discussed above, the deletion is approximately 3379 bp, and comprises genes encoding NADH dehydrogenase subunit 4L, NADH dehydrogenase subunit 4, NADH dehydrogenase subunit 5, tRNA histidine, tRNAserine2, and tRNA leucine2.

[0034] In one embodiment, samples of, for example prostate tissue, prostate massage fluid, urine or breast tissue, are obtained from an individual and tested over a period of time 21682168.2 -6- WO 2009/039601 PCT/CA2007/001711 1 (eg. years) in order to monitor the genesis or progression of cancer. Increasing levels of the 2 3.4 kb deletion over time could be indicative of the beginning or progression of cancer. 3 [0035] Age related accumulation of the 3.4 kb mtDNA deletion may predispose an 4 individual to, for example, prostate cancer or breast cancer, which is prevalent in middle aged 5 and older men, and middle aged and older women, respectively. According to one aspect of 6 the invention, a method is provided wherein regular cancer screening may take place by 7 monitoring over time the amount of the 3.4 kb deletion in body tissues such as breast tissue or 8 body fluids such as prostate massage fluid, or urine. 9 [0036] The system and method of the present invention may be used to detect cancer at 10 an early stage, and before any histological abnormalities. For example, the system and 11 method of the present invention may be used to detect pre-neoplasia in breast tissue. 12 [0037] The following primer sequences are preferred for the detection of the 3.4 kb 13 deletion: 14 3.4 forward (binds to bases 10729-10743/14125-14139 of the mtDNA genome) 5'- 15 TAGACT ACGTAC AT ACT AACCCTACTCCT A-3' (SEQ ID NO: 2); 16 3.4 reverse (binds to bases 14361-14379 of the mtDNA genome) 5'- 17 GAGGTAGGATTGGTGCTGT-3' (SEQ ID NO: 3). 18 [0038] In one embodiment of the present invention, a pair of amplification primers 19 are used to amplify a target region indicative of the presence of the 3.4 kb deletion. In this 20 embodiment, one of the pair of amplification primers overlaps a spliced region of mtDNA 21 after deletion of the 3.4 kb sequence has occurred (ie. a splice at a position between 10743 22 and 14125 of the mtDNA genome). Therefore, extension of the overlapping primer can only 23 occur if the 3.4 kb section is deleted. 24 [0039] In another embodiment of the present invention, a pair of amplification primers 25 are used to amplify a target region associated with the deleted 3.4 kb sequence. The deleted 26 3.4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. In this 27 embodiment, one of the pair of amplification primers overlaps the rejoining site of the ends of 28 the 3.4 kb sequence. Thus, an increase in the amount of the 3.4 kb molecule detected in a 21682168.2 -7- WO 2009/039601 PCT/CA2007/001711 1 sample is indicative of cancer. The below primer pair is preferred for the detection of the 2 deleted 3.4 kb nucleic acid. 3 Forward 14115/10755 5'-CCCACTCATCACCTAAACCTAC-3' (SEQ ID NO: 9) 4 Reverse 10980R 5'-GGTAGGAGTCAGGTAGTTAG-3' (SEQ ID NO: 10). 5 [0040] In one aspect of the invention, a kit for diagnosing cancer, for example prostate or 6 breast cancer, comprising means for extraction of mtDNA, primers having the nucleic acid 7 sequences recited in SEQ ID NOS: 2 and 3, or SEQ ID NOS: 9 and 10, reagents and 8 instructions, is provided. 9 [0041] Another aspect of the invention provides methods for confirming or refuting the 10 presence of a cancer biopsy test from a biopsy sample (eg. prostate or breast cancer), 11 comprising: obtaining non-cancerous tissue from a biopsy sample; and detecting and 12 quantifying the amount of the 3.4 kb mtDNA deletion in the non-diseased tissue. 13 [0042] In one embodiment the present invention provides a method for screening 14 individuals for prostate or breast cancer from a body fluid sample comprising; obtaining a 15 body fluid sample, and detecting and quantifying the level of the 3.4 kb mtDNA deletion in 16 the body fluid. 17 [0043] Although real-time quantitative PCR methods, as described in the examples 18 below, represent the preferred means for detecting and quantifying the presence or absence of 19 the 3.4kb deletion, other methods that would be well known to an individual of skill in the art 20 could also be utilized. For example quantification of the deletion could be made using Bio- 21 Rad's Bioplex™ System and Suspension Array technology. Generally, the method requires 22 amplification and quantification of sequences using any known methods. 23 [0044] The examples provided below illustrate that not only can this deletion be used for 24 the detection of prostate cancer in prostate tissue, but can also be used to detect the presence 25 of cancer in other biological samples, for example prostate massage fluid, urine, and breast 26 tissue. Based on the findings in these examples, the 3.4 kb mtDNA deletion may be used as a 27 biomarker for cancer. 28 [0045] The various examples provided illustrate a difference in the amount of mtDNA 29 having the 3.4 kb deletion between samples obtained from subjects having cancer, and 21682168.2 -8- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 WO 2009/039601 PCT/CA2007/001711 subjects without cancer. The amount of the 3.4 kb deletion was found to be higher in the samples obtained from subjects having cancer. This determination was made by comparing the amount of the 3.4 kb deletion in the test samples with amounts from known cancer cells and/or known non-cancer cells.

[0046] Example 1: 3.4 kb Deletion in the mtDNA of Prostate Tissue

[0047] A deletion of approximately 3.4 kilobases (kb) was identified through full mitochondrial genome amplification of fresh frozen prostate tissue. Using linear regression, the size of the deletion was estimated to be between 3000 base pairs (bp) and 3500 bp. Two possible candidate deletions were identified using Mitomap™ (Brandon, M. C., Lott, M. T., Nguyen, K. C., Spolim, S., Navathe, S. B., Baldi, P. & Wallace, D. C., MITOMAP: a human mitochondrial genome database—2004 update. Nucleic Acids Research 33 (Database Issue):D611-613, 2005; www.mitomap.org), the 3397 bp deletion at 9574-12972, and the 3379 bp deletion at 10744-14124. In order to determine which of the two deletions was associated with prostate cancer, if either, a forward primer which bridged the deletion junction was developed for each of the two candidates, ensuring that the primer extended further than the repeat regions that flank the deletions. Figure 1 is a schematic diagram showing the design and sequence of the primer (ie. SEQ ED NO: 2). Positive amplification results for the amplicon corresponding to the 3379 bp deletion (referred to as the 3.4 kb deletion) at 10744-14124 were obtained.

[0048] As indicated above, the 3.4 kb deletion removes all or part of the following genes: (i) NADH dehydrogenase subunit 4L, (ii) NADH dehydrogenase subunit 4, (iii) NADH dehydrogenase subunit 5, (iv) tRNA histidine, (v) tRNA serine2, and (vi) tRNA leucine2.

[0049] The 3.4kb deletion was determined to be present in 91% of 33 fresh frozen prostate samples. With the specific deletion primers, formalin fixed tissues were tested in order increase the n value.

[0050] The present investigators sequenced entire mitochondrial genomes from 32 tissue samples microdissected by laser capture microdisection and 12 needle biopsies from histologically normal prostates. Archived tissue sections from each of these samples were used for the following study. 1-2 serial sections were removed from each sample. DNA was extracted from each sample in its entirety rather than as a microdissection. Thus, each sample consisted of a mixture of glandular prostate tissue as well as stromal prostate tissue. 21682168.2 -9- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 WO 2009/039601 PCT/CA2007/001711 This extraction was performed using Qiagen's QIAamp™ DNA Mini Kit (Cat # 51304). Following extraction the samples were quantified using a Nano-Drop™ spectrophotometer and the concentrations were subsequently normalized to 2ng/ul. Each sample was amplified using 20ng input DNA and an iQ™ SYBR Green Supermix™ kit (Bio-Rad Laboratories Inc.) Reactions were run on an Opticon® 2 two colour real-time PCR system (MJ Research).

[0051] As shown in Figure 2, a distinct difference was observed in cycle threshold and, by extension, quantity of the deletion between the malignant prostate samples and the symptomatic benign prostate samples. Malignant samples exhibited a consistently earlier cycle threshold than the benign samples.

[0052] Example 2: 3.4kb Deletion Blinded Study - Comparison of Cycle Threshold

[0053] An additional 21 prostate tissue samples were selected, 10 of which were benign and 11 of which were malignant. The pathological status was determined by needle biopsies conducted by a qualified pathologist. The samples were blinded such that the present investigators were unaware of their pathological status when they conducted this test. The present investigators were able to predict pathological status correctly in 81% of the cases by examining the cycle threshold. Of the 4 incorrect calls, two were malignant samples that were determined to be benign and 2 were benign samples that were determined to be malignant. Follow-up clinical information for the 2 individuals in the latter scenario was requested from the physician to determine if they had been diagnosed with prostate cancer subsequent to the needle biopsy results used for this study. One of the individuals who originally produced a benign sample but was predicted by this study to have a malignancy subsequently produced a malignant sample. As a result, one of the false positives became a true positive. Therefore, pathological status was predicted correctly in 86% of the cases examined in this study. The ultimate positive predictive value (PPV, where PPV=true positives/(true positives+false positives)) for this study was 91% and the negative predictive value (NPV, where NPV=true negatives/(true negatives+false negatives)) was 80%.

[0054] Example 3: 3.4kb Deletion Study - Methods (n=76)

[0055] Seventy-six prostate tissue samples were examined for the 3.4 kb deletion in this study. All tissue samples were formalin-fixed, 25 being malignant, 12 being normal, and 39 having benign prostatic disease as shown histologically. Of the latter group more then half 21682168.2 - 10- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 WO 2009/039601 PCT/CA2007/001711 had hyperplasia. All specimens were needle biopsies taken from the investigators' tissue archives.

[0056] Prostate Specimens

[0057] A tapelift was performed on each slide using Prep-Strips (Catalogue Number LCM0207) from Arcturus Bioscience Inc. This allowed the removal of any particulate matter or non-adhering tissue from the slide prior to DNA extraction. With the tissue still on the slides, the slides were rinsed with PBS (Phosphate Buffered Saline Solution) to remove as much fixative as possible. The 1-2 needle biopsy sections on the slides were scraped into sterile microcentrifuge tubes using individually wrapped, sterilized surgical razor blades. DNA was then isolated and purified using a QIAamp® DNA Mini Kit (Qiagen, Cat. # 51304) according to manufacturer's specifications. A negative extract control was processed in parallel with the slide extractions as a quality control checkpoint. The total concentration of DNA and purity ratio for each sample was determined by spectrophotometry (Nano-Drop™ ND-1000) and dilutions of 2ng/|il were prepared for the purpose of Quantitative Polymerase Chain Reaction (qPCR).

[0058] Primers (Oligonucleotides)

[0059] Purified oligonucleotide primers were chemically synthesized by Invitrogen (California, USA). The sequences of the primers and the expected sizes of the PCR products amplified are listed in Table 1. In addition, PCR analysis for mtDNA deletions included positive controls (DNA from a source known to carry the mutant mtDNA). Each primer set with the exception of TNF (tumor necrosis factor) were checked against a mitochondria-free rho 0 cell line to confirm the absence of pseudogene coamplification.

[0060] Table 1 Amplification Primers. Primer Pair Position Amplified 5'- 3' Length of amplified product (base pairs) 3.4 Deletion Real- 10729-14379 (less 3379bp at 273 Time 10744-14124) 12s mtDNA 708-945 238 TNF 3756-3886 131 21682168.2 - 11 - WO 2009/039601 PCT/CA2007/001711 3.4 forward (10729-10743 - 14125-14139) 5 'T AGACT ACGT AC AT ACT AACCCT ACTCCT A-3' SEQ ID NO: 2 3.4 reverse (14361-14379) 5'-GAGGTAGGATTGGTGCTGT-3' SEQ ID NO: 3 12s forward (708-728 ) 5'-CGTTCCAGTGAGTTCACCCTC-3" SEQ ID NO: 4 12s reverse (923-945) 5'-CACTCTTTACGCCGGCTTCTATT-3' SEQ ID NO: 5 TNF forward (3756-3775) 5' -CCTGCCCCAATCCCTTTATT-3' SEQ ID NO: 6 TNF reverse (3866-3886) 5'-GGTTTCGAAGTGGTGGTCTTG-3'SEQ ID NO: 7 1 2 [0061] Real-Time Polymerase Chain Reaction 3 [0062] Three separate PCRs were performed on each sample. Each reaction was 25ja.l 4 total volume and included template DNA, one pair of primers (12s or 3.4 Deletion or TNF ), 5 an iQ™ SYBR Green Supermix™ kit (Catalogue Number 170-8882, Bio-Rad Laboratories 6 Inc.) and distilled deionized water (ddH20). The TNF (tumor necrosis factor) comprised 7 single copy nuclear gene primers, and 12s comprised total mitochondrial genome primers. 8 The volume and concentrations for template DNA, primers, and reaction buffer are listed 9 below. 10 [0063] Table 2 qPCR Components. Reagent Concentration per Reaction Volume per Reaction Reaction Buffer IX 12.5(0-1 Primer (forward 250nM 0.0625(il of each and reverse) 100 umole stock ddH20 N/A 2.375.JJ.1 Template DNA 20ng 10.0p.l Total 25(0.1 11 12 [0064] The cycling parameters for each amplicon are listed in Table 3. 13 [0065] Table 3 Cycling Parameters. Step Temperature (°C) Duration 1 95 3 min 2 95 30 sec 3 66 (3.4 deletion primers) or 61.5 (12s primers) or 30 sec 21682168.2 -12- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 WO 2009/039601 PCT/CA2007/001711 61.5 (TNF primers) 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve 50°C - 110°C reading every 1°C 3 sec Repeat steps 2-5, 44 times for a total of 45 cycles.

[0066] Thermal cycling, real-time detection and analysis of the reactions was carried out using a DNA Engine Opticon® 2 Continuous Fluorescence Detection System equipped with Intuitive Opticon Monitor™ software (MJ Research Inc.). The standard curve method was utilized for DNA quantification. A set of serial dilutions (106, 105, 104, 103, 102, 101) of three purified PCR generated templates, one product for the 3.4 deletion, one for the 12s primers, and one for TNF. From this, three different standard curves were generated showing the number of copies of total mtDNA (12s amplicon-total mitochondrial genome primers), the amount of mtDNA having the 3.4 kb deletion, or total nuclear DNA (TNF-single copy nuclear gene primers). The Cr values of the samples were then converted to the number of DNA copies by comparing the sample Ct to that of the standards. The 3.4 deletion was considered to be absent or at low levels if the deletion was not detected within 37 cycles.

[0067] The determination of malignancy is based upon the quantity of the 3.4kb deletion present in the normalized sample as indicated by the location of the cycle threshold. This location may be either absolute, as in greater than 25 cycles but less than 35 cycles, or more likely a ratio between the total mitochondrial DNA present as indicated by the 12s amplicon, , and the 3.4kb deletion. This may be expressed as a per cent of the total mitochondrial DNA. The number of cells, as represented by the TNF amplicon, may be incorporated to refine the distinction between benign and malignant tissues.

[0068] In order to automate the analyses of these samples, bioinformatics tools were employed. The three variables that were considered for these analyses were the cycle threshold Ct of Tumour Necrosis Factor (TNF), total pecies of mitochondria that contain those specific primer sites, and those mitochondria that harbour the deletion of interest.

[0069] Cluster Analysis

[0070] The clustering was not normalized nor were logarithmic functions used due to the similar and small range of data. 21682168.2 - 13 - From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 17 of 35 Received mail@iponz 12 June 2012 10071} Figure 3 shows the actual movement and trends of the data. The x-axis is Che patient number and the y-axis is the cycle threshold obtained from real time PCR.

[0072] It is important to note thai the higher the cycle threshold is, the lower amount of 5 the deletion is present. |0073] The general trend shown in Figure 3 is based upon the differcnccs/ratios between the variables of Deletion, Total, and T.NF. The deletion is low to absent for the benign/normal samples (right side) and increases (toward the left) with abnormal benign and malignant samples. 1 0 The abnormal benign and malignant samples begin to differentiate themselves from each other based on the cycle threshold rat io of Deletion to TNF, [0(>74] Supervised Learning 15 [0075] Supervised learning is based on the system trying to predict outcomes for known samples. Half of the delta was used to train and the other half to test: the algorithm. Supervised learning compares its predictions to the target answer and "learns" from its mistakes. But, if the predicted output is higher or lower than the actual outcome in the data, the error is propagated back through the system and the weights are adjusted accordingly, 20

[0076] Data SET: 5% to 35% - Benign 35% to 65% - Hyperplasia 65% to 95% - Malignant 25 [0077] Artificial Neural Network (ANN) Algorithm (shown schematically in Figure 8), Half of Data set used for Training ANN Other half used to compare the accuracyAccuraey - Compare expected data set with obtained data set -■> 86,6% - 14- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 WO 2009/039601 PCT/CA2007/001711

[0078] Supervised Learning of Deletion Data using Artificial Neural Network (ANN)

[0079] Three Classifications: Benign Hyperplasia Malignant

[0080] Three variables for each classification were used based on Real Time PCR Cycle Threshold Cj: Tumour Necrosis Factor (TNF) - Nuclear copy control. Total Mitochondria - Mitochondria copy control Deletion - Mitochondria in the deleted state.

[0081] Results:

[0082] Half of data set is used to train the ANN, and the remaining half is used to compare the accuracy. Three Classification Accuracy = 86.6% Positive Predictive Value (PPV); Benign to Malignant = 88.2% Negative Predictive Value (NPV) Benign to Malignant = 76.5%

[0083] Example 4: 3.4 kb Deletion in mtDNA Associated with Breast Cancer

[0084] 18 samples were tested from malignant and benign breast tissue, 9 being malignant and 9 being benign, for the presence of the aforementioned 3.4 kb deletion. Samples were classified as either malignant or benign using conventional histopathological analysis.

[0085] DNA was isolated and purified from the samples using a QIAamp® DNA Mini Kit (Qiagen, Cat. # 51304) according to manufacturer's specifications.

[0086] Purified oligonucleotide primers were chemically synthesized by Invitrogen (California, USA). The sequences of the primers and the expected sizes of the PCR products amplified are listed in Table 1 above. 21682168.2 - 15 - WO 2009/039601 PCT/CA2007/001711 1 [0087] Real-Time Polymerase Chain Reaction 2 [0088] Three separate PCRs were performed on each sample. Each reaction was 25|il 3 total volume and included template DNA, one pair of primers (12s or 3.4 Deletion or TNF ), 4 an iQ™ SYBR Green Supermix kit (Catalogue Number 170-8882, Bio-Rad Laboratories 5 Inc.) and distilled deionized water (ddH20). The TNF (tumor necrosis factor) comprised 6 single copy nuclear gene primers, and 12s comprised total mitochondrial genome primers. 7 The volume and concentrations for template DNA, primers, and reaction buffer are listed 8 below: 9 [0089] Table 4 qPCR Components. Reagent Concentration per Reaction Volume per Reaction Reaction Buffer IX 12.5p,l Primer (forward 250nM 0.0625(il of each and reverse) 100 p.mole stock ddH20 N/A 2.375.^1 Template DNA 20ng lO.Ofjl Total 25jal 10 11 [0090] The cycling parameters for each amplicon are listed in Table 5. 12 [0091] Table 5 Cycling Parameters. Step Temperature (°C) Duration 1 95 3 min 2 95 30 sec 3 66 (3.4 deletion primers) or 61.5 (12s primers) or 61.5 (TNF primers) 30 sec 4 72 30 sec 5 Plate Read 6 72 10 min 7 Melting Curve 50°C -110°C reading every 1°C 3 sec Repeat steps 2-5, 44 times for a total of 45 cycles. 21682168.2 - 16- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 WO 2009/039601 PCT/CA2007/001711

[0092] Thermal cycling, real-time detection and analysis of the reactions was carried out using a DNA Engine Opticon® 2 Continuous Fluorescence Detection System equipped with Intuitive Opticon Monitor™ software (MJ Research Inc.). The standard curve method was utilized for DNA quantification. A set of serial dilutions (106, 105, 104, 103, 102, 101) of three purified PCR generated templates were performed, one product for the 3.4 deletion, one for the 12s primers, and one for TNF. From this, three different standard curves were generated showing the number of copies of total mtDNA (12s amplicon-total mitochondrial genome primers), 3.4 deletion or total nuclear DNA (TNF-single copy nuclear gene primers). The Ct values of the samples were then converted to the number of DNA copies by comparing the sample Cj to that of the standards.

[0093] The determination of malignancy was based upon the quantity of the 3.4kb deletion present in the normalized sample as indicated by the location of the cycle threshold. This location may be either absolute, as in greater than 25 cycles but less than 30 cycles, or more likely a ratio between the total mitochondrial DNA present as indicated by the 12s amplicon, and the 3.4kb deletion. This may be expressed as a percent of the total mitochondrial DNA.

[0094] In order to automate the analyses of these samples, bioinformatics tools were employed. The three variables that were considered for these analyses were the cycle threshold Ct of Tumour Necrosis Factor (TNF), total species of mitochondria that contain those specific primer sites, and those mitochondria that harbour the deletion of interest.

[0095] Table 6 and figure 7 show the difference in the mean Ct scores for samples from malignant tissue and benign tissue. The mean Ct value for normal tissue was 30.5889, while the mean CTfor malignant tissue was 27.8533 thereby illustrating a difference in the quantity of mtDNA having the 3.4 kb deletion in malignant breast tissue compared to normal breast tissue.

[0096] Table 6 Mean values for Ct scores Group Statistics Std. Error GRP N Mean Std. Deviation Mean del3.4 normal 9 30.5889 2.53897 .84632 malignant 9 27.8533 2.52253 .84084 21682168.2 - 17- 1 2 3 4 5 6 7 8 9 10 11 12 13 WO 2009/039601 PCT/CA2007/001711

[0097] Figure 8 is an ROC curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for breast cancer when testing breast tissue. These results were obtained using a cutoff Ct of 29.1900. The sensitivity of the marker at this Ct was 77.8%, while the specificity was 77.8%.

[0098] Table 7 shows the calculation of the area under the curve for the present example. As a measure of the accuracy of the test.

[0099] Table 7 Results Showing Area Under the Curve Area Under the Curve Test Result Variable(s): de!3.4 Area Std. Error9 Asymptotic Sig.b Asymptotic 95% Confidence Interval Lower Bound Upper Bound .790 .112 .038 .570 1.010 a Underthe nonparametric assumption b. Null hypothesis: true area = 0.5

[00100] The determination of the cutoff Ct of 29.1900 is shown in table 8 below. The results listed in table 8 show that a cutoff Ct of 29.1900 provided the highest sensitivity and specificity at 78% and 78% respectively.

[00101] Table 8: Determination of Ct cutoff. Coordinates of the Curve Test Result Variable(s): de!3.4 Positive if Less T h an or Equal T oa Sen s ti vi ty 1 - Specificity 2-4. ©OOO . OOO .OOO 25.6800 .111 .OOO 25.7700 .22 2 .OOO 25.9250 . 33 3 .OOO 26.2050 . -444 .OOO 26 . 84O O .55© .OOO 27 .4800 .55© .111 28. 1 600 .55© .222 28.8800 . ©© 7 .222 29. 1900 . 77 8 .222 29.4600 .77 8 . 33 3 29.8*750 ;.778 ;.444 ;30.5850 ;.77 8 ;.55© ;31 .2200 ;.77 8 ;.©67 ;31 .5000 ;.88 9 ;.©©7 ;31 .7650 ;.889 ;.778 ;32.9900 ;1 .OOO ;.778 ;34. 335O ;1 .OOO ;.889 ;35.6400 ;1 .OOO ;1 .OOO ;a- The smallest cutoff value isthe minimum observed test va I ue mi nus 1 , a nd the la rg est cutoff value isthe maximum observed test value plus 1. All the other cutoff val ues are the averages of two consecutive ordered observed test values. ;21682168.2 ;- 18- ;1 ;2 ;3 ;4 ;5 ;6 ;7 ;8 ;9 ;10 ;11 ;12 ;13 ;14 ;15 ;16 ;17 ;18 ;19 ;20 ;21 ;22 ;23 ;24 ;25 ;26 ;27 ;28 ;29 ;WO 2009/039601 PCT/CA2007/001711 ;[00102] Example 5: The 3.4kb Deletion in the Prostate Massage Fluid of Individuals with Prostate Cancer as Compared to the Fluid from those without Histological Evidence of Prostate Cancer ;[00103] Forty prostate massage fluid samples were collected by urologists from patients who were either subsequently diagnosed with prostate cancer or showed no histological evidence of prostate cancer following a prostate needle biopsy procedure. The sample was deposited on a IsoCode Card™ (Schleicher & Shuell), dried, and then extracted according to the manufacturer's protocol. All DNA extracts were quantified using a NanoDrop™ ND-1000 Spectrophotometer and the DNA concentration normalized to 2ng/ul. Each sample was then amplified according to the following parameters: ;IX iQ SYBR Green Supermix™ (Bio-Rad P/N 170-8880) ;150nmol forward primer ;(5'-TAGACTACGTACATACTAACCCTACTCCTA-3') (SEQ ID NO: 2). 150 nmol reverse primer ;(5'-GAGGTAGGATTGGTGCTGT-3') (SEQ ID NO: 3) ;20 ng template DNA in a 25ul reaction. ;[00104] Reactions were cycled on an Opticon™ 2 DNA Engine (Bio-Rad Canada) according to the following protocol: ;1. 95°C for 3 minutes ;2. 95°C for 30 seconds ;3. 66°C for 30 seconds ;4. 72°C for 30 seconds ;5. Plate Read ;6. Repeat steps 2-5 44 times ;7. 72°C for 10 minutes ;8. Melting Curve from 50°C to 105°C, read every 1°C, hold for 3 ;seconds ;9. 10°C Hold ;21682168.2 ;-19- ;WO 2009/039601 PCT/CA2007/001711 ;1 [00105] Table 9 Results showing the mean Ct Values for Prostate Massage Fluid Test ;Group Sfatisics ;SW. Error firm in ;N ;IVfcan ;DEL3t4 benign ;25 ;37.1869 ;ai8©5 ;.63699 ;malignant ;15 ;33.7712 ;3.98056 ;1.02778 ;2 [00106] Tables 9 and 10 show a significant difference between the mean Ct values ;3 obtained for the benign sample and the malignant sample groups (p=0.005). ;4 [00107] Table 10 Results Showing Difference (p=0.005) for CTvalues of samples. ;W^HiimtS&rpleslest ;Lfivenfe FqriHyn ;Jestfr ;[\AHarpc: ;ttect rrFqi nliK/rf IV/b ;F ;3a. ;t cf ;Sa.Gtaled) ;IVteai □feancB ;3d.Bror □feel® ;95%Q> Irisva nfc ;(icferxB lofthe towar liDff ;CHJJ4 EqjdxaiatES SBBumsd ;Eqjdvaiarcs notasamed ;1251 ;20 ;2980 2825 ;38 21.096 ;.005 .009 ;341570 34*67) 114283 120017 11CE17 52382 572923 59C7SB 5 [00108] Figure 5 is a Receiver Operating Characteristic (ROC) curve illustrating the 6 specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when 7 testing prostate massage fluid. These results were obtained using a cutoff Ct of 37.3683. 8 The sensitivity of the marker at this Ct is 87%, while the specificity is 64%. 9 [00109] The accuracy of the test depends on how well the test separates the group being 10 tested into those with and without the prostate cancer. Accuracy is measured by the area 11 under the ROC curve. Table 11 shows the calculation of the area under the curve for the 12 present example. 13 14 15 21682168.2 -20- WO 2009/039601 PCT/CA2007/001711

[00110] Table 11 Results Showing Area Under the ROC Curve Area Under the Curve Area S td. E rro ra Asymptotic Sia.b Asymptotic 95% Confidence Intprval LowerBound UDDer Bound .768 .074 .005 .622 .914 a- Under the nonparametric assu mption b' Null hypothesis: true area = 0.5

[00111] Table 12 Determination of Specificity and Sensitivity Coo id inate s ofth e Curv e T est Re suit V ariable Is): D E L3 .4 Positive if Le ss T h an or E aua I T o Sen stivitv 1 - S d ecificitv 26 .299 2 .00 0 .00 0 27 .378 6 .06 7 .00 0 28 .248 4 .13 3 .00 0 29 .519 3 .20 0 .00 0 30 .175 7 .20 0 .04 0 30 .458 0 .20 0 .08 0 30 .598 0 .26 7 .08 0 31 .570 9 .33 3 .08 0 32 .571 2 .33 3 .12 0 32 .950 0 .33 3 .16 0 33 .331 4 .40 0 .16 0 33 .654 7 .46 7 .16 0 33 .924 7 .53 3 .16 0 34 .355 4 .53 3 .20 0 34 .905 6 .53 3 .24 0 35 .465 0 .53 3 .28 0 35 .917 2 .53 3 .32 0 36 .064 8 .60 0 .32 0 36 .361 6 .66 7 .32 0 36 .642 1 .73 3 .32 0 36 .853 1 .73 3 .36 0 37 .118 8 .80 0 .36 0 37 .368 3 .86 7 .36 0 37 .520 0 .86 7 .40 0 37 .834 1 .86 7 .44 0 38 .253 3 .86 7 .48 0 38 .519 8 .93 3 .48 0 38 .651 9 .93 3 .52 0 38 .855 2 .93 3 .56 0 39 .1 25 8 .93 3 .60 0 39 .273 4 .93 3 .64 0 39 .495 2 .93 3 .68 0 39 .732 3 1.0 00 .68 0 39 .895 6 1.0 00 .72 0 41 .000 0 1.0 00 1.0 00 The smallest cutoff value is the minimum observed test value -1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the average of two consecutive ordered, observed test values. 21682168.2 -21 - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 WO 2009/039601 PCT/CA2007/001711

[00112] The determination of the cutoff Ct of 37.3683 is shown in table 12 above. The results listed in table 12 illustrate that a cutoff Ct of 37.3683 provided the highest sensitivity and specificity.

[00113] Example 6: The 3.4kb Deletion in the Urine of Individuals with Prostate Cancer as Compared to the fluid from those without Histological Evidence of Prostate Cancer

[00114] Urine samples were collected from 5 patients who were diagnosed with prostate cancer and 5 who have had a needle biopsy procedure which was unable to detect prostate malignancy. These samples were collected following a digital rectal exam (DRE) to facilitate the collection of prostate cells.

[00115] Upon receipt of the samples a 5ml aliquot was removed and then 2mls were centrifuged at 14,000 x g to form a pellet. The supernatant was removed and discarded. Pellets were resuspended in 200ul phosphate buffered saline solution. Both the resuspended pellet and the whole urine sample were subjected to a DNA extraction procedure using the QiaAMP™ DNA Mini Kit (Qiagen P/N 51304) according to the manufacturer's directions. The resulting DNA extracts were then quantified using a NanoDrop™ ND-1000 Spectrophotometer and normalized to a concentration of O.lng/ul.

[00116] Samples were analyzed by quantitative real-time PCR with the 3.4kb deletion specific primers according to the following: IX iQ SYBR Green Supermix™ (Bio-Rad P/N 170-8880) 100 nmol forward primer (5' -TAG ACT ACGT AC AT ACT AACCCT ACTCCTA-3') (SEQ ID NO: 2) 100 nmol reverse primer (5'-GAGGTAGGATTGGTGCTGT-3') (SEQ ID NO: 3) 1 ng template DNA in a 25ul reaction.

[00117] Reactions were cycled on an Opticon™ 2 DNA Engine (Bio-Rad Canada) according to the following protocol: 1. 95 °C for 3 minutes 2. 95°C for 30 seconds 3. 69°C for 30 seconds 21682168.2 -22- WO 2009/039601 PCT/CA2007/001711 1 4. 72°C for 30 seconds 2 5. Plate Read 3 6. Repeat steps 2-5 44 times 4 7. 72°C for 10 minutes 5 8. Melting Curve from 50°C to 105°C, read every 1°C, hold for 3 seconds 6 9. 10°C Hold 7 [00118] Table 13 Mean values for Ct scores Group Statistics Std. Error GRPfkiri38 N Mean Ski Dfwistim CTT Benign 5 33.2780 1.10000 .49506 Malignant 5 30.6080 255767 1.14382 8 9 [00119] Tables 13 and 14 show a significant difference between the mean Ct values 10 obtained for benign sample and the malignant sample groups (p=0.005). 11 [00120] Table 14 Results Showing Difference (p=0.005) for C-rvalues of samples. ■ ^ • rapran&rpsea laerg R^riilyn sTeSfcr fUrmR hrfqrilyrfMs F 3a t cf SaQtatef) l\tei □feSTB SfciBcr □ftetE ma nftj defers Icflhe nrre> Lour Hear CIT Eqd\aaiE5 ae&nsi EqMvaaroes notasanBd 122 22 2069 2GS 8 54S (72 -G89 258300 258000 124i72 X2&2 -2sm -54529 54564 57OE30 12 [00121] Figure 6 is a Receiver Operating Characteristic (ROC) curve illustrating the 13 specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when 14 testing urine. These results were obtained using a cutoff Ct of 31.575. The sensitivity of the 15 marker at this Ct is 80%, while the specificity is 100%. 21682168.2 -23 - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 WO 2009/039601 PCT/CA2007/001711

[00122] The determination of the cutoff Ct of 31.575 is shown in table 15. The results listed in table 15 show that a cutoff Ct of 31.575 provided the highest sensitivity and specificity.

[00123] Table 15: Determination of Crcutoff Coordinates of the Curve Tfist Rfisnlt Van ahlfi/sV CTf Positive if LessThan nr Ffilial To Rfinativitv 1 - Snfirifir.itv 26.2900 .000 .000 28.4950 .200 .000 30.3850 .400 .000 31.0800 .600 .000 31.5750 .800 .000 32.1400 .800 .200 32.8150 .800 .400 33.8700 .800 .600 34.3350 .800 .800 34.3550 1.000 .800 35.3700 1.000 1.000 a- The smallest cutoff value isthe minimum observed test value minus 1, and the largest cutoff value isthe maximum observed test value plus 1. All the other cutoff values a re the averages of two consecutive ordered observed test values.

[00124] Example 7: Detection of Re-circularized 3.4kb Deleted Sequence in Prostate Malignant and Benign Tissue In this example, the amount of re-circularized 3.4 kb deleted mtDNA molecules in samples was tested as an indicator for prostate cancer. As mentioned above, the 3.4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. Amplification of a target region from the deleted 3.4 kb mtDNA sublimon was conducted using a primer pair (SEQ ID NOS: 9 and 10). The forward primer (SEQ ID NO: 9), overlaps the rejoining site of the ends of the 3.4 kb sequence. 21682168.2 -24- WO 2009/039601 PCT/CA2007/001711 1 [00125] Prostate tissue was formalin-fixed paraffin embedded prostate tissue needle 2 biopsies. 3

[00126] The reagent setup used for this example was as follows: 4 250nmol each primer 5 12.5ul of 2X reaction mix, 6 20ng (lOul of 2ng/ul) template in 25 ul reaction volume. 7

[00127] The cycling parameters were as follows: 8 1. 95 degrees Celsius for 3 minutes 9 2. 95 degrees Celsius for 30 seconds 10 3. 62 degrees Celsius for 30 seconds 11 4. 72 degrees Celsius for 30 seconds 12 5. Plate Read 13 6. Repeat steps 2-5 44 times 14 7. 72 degrees for 10 minutes 15 8. Melting Curve from 50-100 degrees, reading every 1 degree for 3 seconds 16 9 4 degrees HOLD. 17

[00128] Amplification of a target region from the deleted 3.4 kb mtDNA sublimon was 18 conducted using a primer pair (SEQ ID NOS: 9 and 10). 19 [00129] Table 16 below provides a summary of testing conducted for the detection of the 20 actual 3.4 kb deleted in mtDNA obtained from malignant and benign prostate tissue. Using a 21 Ct score of 30.0, a clear identification of malignant and benign tissue was possible. As such, 22 an increase in the amount of the 3.4 kb molecule present in a sample was indicative of cancer. 23 [00130] Table 16: Ct scores for Detection of Cancer in Prostate Tissue Description CT Benign sample 1 33.75 Malignant sample 1 28.79 Benign sample 2 30.96 Malignant sample 2 28.4 Benign sample 3 32.19 Malignant sample 3 27.38 21682168.2 -25 - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 WO 2009/039601 PCT/CA2007/001711

[00131] Although the invention has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art without departing from the spirit and scope of the invention as outlined in the claims appended hereto.

[00132] REFERENCES

[00133] Birch-Machin MA, Online Conference Report (Sunburnt DNA), International Congress of Biochemistry and Molecular Biology, New Scientist, 2000(a)

[00134] Birch-Machin MA, Taylor RW, Cochran B, Ackrell BAC, Tumbull DM. Ann Neurol 48: 330-335, 2000(b)

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[00139] Huoponen, Kirsi, Leber hereditary optic neuropathy: clinical and molecular genetic findings, Neurogenetics (2001) 3: 119-125.

[00140] Hayward SW, Grossfeld GD, Tlsty TD, Cunha GR., IntJOncol 13:35-47, 1998

[00141] Huang GM, Ng WL, Farkas J, He L, Liang HA, Gordon D, Hood R., Genomics 59(2): 178-86,1999

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[00144] Lee HC, Lu CY, Fahn HJ, Wei YHu. Federation of European Biochemical Societies, 441:292-296,1998

[00145] Mitochondrial Research Society http://www.mitoresearch.org/diseases.html.

[00146] MITOMAP: A human mt genome database (www.gen.emory.edu/mitomap.html) 21682168.2 -26- 1 2 WO 2009/039601 PCT/CA2007/001711

[00147] Naviaux, RK., Mitochondrial Disease- Primary Care Physican's Guide. Psy-Ed. Corp D/B/A Exceptional Parents Guide: 3-10, 1997 3 [00148] Parrella P, Xiao Y, Fliss M, Sanchez-Cespedes M, Mazzarelli P, Rinaldi M, Nicol 4 T, Gabrielson E, Cuomo C, Cohen D, Pandit S, Spencer M, Rabitti C, Fazio VM, Sidransky 5 D: Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle 6 aspirates. Cancer Res 2001, 61:7623-7626 7 [00149] Polyak Y, et al., Nature Genet. 20 (3):291-293, 1998 8 [00150] Seidman, M.D. et al., Arch. Otolaryngol Head Neck Surg., 123: 1039-1045, 1997 9 [00151] Sherrat EJ, Thomas AW, Alcolado JC., Clin. Sci. 92:225-235,1997 10 [00152] Shoffner JM, Brown MD, Torroni A, Lott MT, Cabell MF, Mirra SS, Beal MF, 11 Yang C, Gearing M, Salvo R, Watts RL, Juncos JL, Hansen LA, Crain BJ, Fayad M, 12 Reckford CL, and Wallace DC., Genomics 17: 171-184, 1993 13 [00153] SpringNet - CE Connection: Screening, Diagnosis: Improving Primary Care 14 Outcomes. Website: http://www.springnet.com/ce/i803a.htm 15 [00154] Taniike, M. et al., BioChem BioPhys Res Comun, 186: 47-53, 1992 16 [00155] Valnot, Isabelle, et al., A mitochondrial cytochrome b mutation but no mutations 17 of nuclearly encoded subunits in ubiquinol cytochrome c reductase (complex III) deficiency, 18 Human Genetics (1999) 104: 460-466 19 [00156] von Wurmb, N, Oehmichen, M, Meissner, C., Mutat Res. 422:247-254, 1998 20 [00157] Wallace et al., Mitochondiral DNA MUtatio Assoicated with Leber's Hereditary 21 Optic Neuropathy, Science, 1427-1429 22 [00158] Wei YH. Proceedings of the Nat. Sci. Council of the Republic of China April 23 22(2):5567, 1998 24 [00159] Woodwell DA. National Ambulatory Medical Care Survey: 1997 Summary. 25 Advance data from vital and health statistics; no. 305. Hyattsville, Maryland: National 26 Center for Health Statistics. 1999 21682168.2 -27- WO 2009/039601 PCT/CA2007/001711 1 [00160] Yeh, J.J., et al., Oncogene Journal, 19: 2060-2066, 2000 2 [00161] Zhang et al., Multiple mitochondiral DNA deletions in an elderly human 3 individual, FEBS Lett, 297, 34-38 1992 4 [00162] Zhang, C., et al., BioChem. BioPhys. Res. Comun., 195: 1104-1110, 1993 5 21682168.2 -28- From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 23 of 35 Received mail@iponz 12 June 2012 A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that tire information it contains was part of the common general knowledge as al the priority date of any of the claims 5 Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they arc to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. - 28a - </div>

Claims

From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 28 of 35 Received mail@iponz 12 June 2012 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of detecting a cancel' in an individual comprising; a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; 5 b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 to 14324 of the mtDNA genome; c) comparing the amount, of mtDNA in the sample having the deletion to at least one known reference value, wherein the known reference value is: (i) the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and/or (ii) the 10 amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid; - wherein, if the reference value is (i), an elevated amount of the deletion in the biological sample compared to the reference value is indicative of cancer; - and wherein, if the reference value is (ii), a similar level of the deletion in the biological 15 sample compared to the reference value is indicative of cancer; - with the proviso that the cancer is not prostate cancer.

2. The method of claim 1, wherein the deletion has a nucleic acid sequence corresponding to the sequence identified in SKQ ID NO: 1. 20

3. The method of claim ! or 2, wherein the step of comparing comprises the use of reference value (i) and reference value (ii).

4. The method according to any one of claims ] to 3, wherein the biological sample is a 25 body (issue or body fluid.

5. The method of claim 4, wherein the biological sample is breast tissue or urine.

6. The method according to any one of claims 1 to 5, wherein the quantifying of the deletion 30 includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount' of the amplified target region. -29- From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 29 of 35 Received mail@iponz 12 June 2012

7. The method of claim 6, wherein a PCR primer having a sequence corresponding to SEQ ID NO: 2 is used as part of a pair of amplification primers for amplifying the target region,

8. The method of claim 6, wherein the amplifying of the target region is conducted using a 5 pair of amplification primers, one of the pair of amplification primers overlapping a splice joining regions on opposite ends of the deletion.

9. The method according to any one of claims 1 to 8, wherein the step of quantifying is conducted using real-time PCR, 10

10. The method according to any one of claims 1 to 9, wherein the cancer is breast canccr.

11. A method of detecting a cancer in an individual comprising; a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; 15 b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 to 14124 of the mtDNA genome; e) comparing the amount of mtDNA in the sample having the deletion I.o a known refcrcncc value; •• wherein the step of quantifying is conducted using real-time PCR, the quantifying of the 20 deletion includes first amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region; - and wherein the known refcrcncc value is a cycle threshold; - with the proviso that the canccr is not prostate cancer. 25

12. , The method of claim 11, wherein the deletion has a miclcic acid sequence corresponding to the sequence identified in SEQ ll> NO: 1.

13. The method of claim 11 or 12, wherein the amplifying of the target region is conducted using a pair of amplification primers, one of the pair of amplification primers overlapping a 30 splice joining regions on opposite ends of the deletion.

14. , The method according t.o any one of claims 11 to 13, wherein the biological sample is a body tissue or body fluid, -30- From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 30 of 35 Received mail@iponz 12 June 2012

15. The method of claim 14, wherein the biological sample is breast tissue or urine.

16. The: method according to any one of claims 11 to 15, wherein the canccr is breast cancer. 5

17. A method of monitoring an individual for the development of a canccr comprising; a) extracting tnt.DNA. from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid :sequence spanning approximately residues 10744 to 14124 of the mtDNA genome; 10 c) repeating steps a) and b) over a duration of time; d) wherein an increasing level of the deletion over the duration of time is indicative of cancer; - with the proviso that the cancer is not prostate cancer. 15

18. The method of claim 17, wherein the deletion has a nucleic acid sequence corresponding to the sequence identi (led in SRQ ID NO: 1,

19. The method (if claim 17 or 18, further comprising at least one step selected from the group consisting of: (a) comparing the amount of mtDNA in the .sample having the deletion to 20 the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and (b) comparing the amount of mlDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body fluid. 25 2,0.

20. The method according to any one of claims 17 to 19, wherein the quantifying of the deletion includes amplifying a target region of mtDNA that is indicative of the deletion, and quantifying the amount of the amplified target region.

21. , The method of claim 20, wherein a PCR. primer having a sequence corresponding to SEQ 30 ID NO: 2 is used as part of a pair of amplification primers for amplifying the target region. -31 - From Phillips Ormonde Fitzpatrick Tue 12 Dun 2012 16:53:11 EST Page 31 of 35 Received mail@iponz 12 June 2012

22. The method of claim 20, wherein the amplifying of the target region is conducted using a pair of amplification primers, one of the pair of amplification primers overlapping a splice joining regions on opposite ends of the deletion. 5

23. The method according to any one of claims 17 to 22, wherein the step of quantifying is conducted using real-time PCR.

24. The method of claim 17 to 23, wherein the biological sample is a body tissue or body fluid. 10

25. The method of chum 24, wherein the biological sample is breast tissue or urine,

26. The method according to any one of claims 17 to 25, wherein the cancer is breast cancer. 1 5 27, A method of detecting breast cancer in an individual comprising; a) extracting mitochondrial DNA, mtDNA, from a biological sample from the individual; b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid scqucncc spanning approximately residues 10744 to 14124 of the mtDNA genome, and wherein the deletion has a nucleic acid sequence corresponding to the sequence identified in SHQ ID NO: 20 1; c) comparing the amount of mtDNA in the sample having the deletion to al least one known reference value, wherein the known reference value is; (i) the amount of the deletion in a reference sample of mtDNA from known non-cancerous tissue or body fluid; and/or (ii) the amount of the deletion in a reference sample of mtDNA from known cancerous tissue or body 25 fluid; - wherein, if the reference value is (i), an elevated amount of the deletion in the biological sample compared to the reference value is indicative of breast canccr; - and wherein, if the reference value is (ii), a similar level of the deletion in the biological sample compared to the reference value is indicative of breast cancer. 30 28, The method of claim 1, 11, 17 or 27, substantially as hereinbefore described with reference to any of the examples and/or figures. -32-