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Numéro de publicationUS20150037804 A1
Type de publicationDemande
Numéro de demandeUS 14/507,027
Date de publication5 févr. 2015
Date de dépôt6 oct. 2014
Date de priorité18 avr. 2005
Autre référence de publicationCA2700941A1, CN101874119A, CN101874119B, EP2203570A1, EP2203570A4, EP2203570B1, EP2634267A1, EP2634267B1, US20130022979, US20160376660, WO2009039601A1
Numéro de publication14507027, 507027, US 2015/0037804 A1, US 2015/037804 A1, US 20150037804 A1, US 20150037804A1, US 2015037804 A1, US 2015037804A1, US-A1-20150037804, US-A1-2015037804, US2015/0037804A1, US2015/037804A1, US20150037804 A1, US20150037804A1, US2015037804 A1, US2015037804A1
InventeursRyan L. Parr, Robert Thayer, Gabriel Dakubo, Jennifer Creed, Kerry Robinson, Andrea Maggrah, Brian Reguly
Cessionnaire d'origineMitomics Inc.
Exporter la citationBiBTeX, EndNote, RefMan
Liens externes: USPTO, Cession USPTO, Espacenet
3.4 kb mitochondrial dna deletion for use in the detection of cancer
US 20150037804 A1
Résumé
The present invention broadly claims a method for detecting cancer in an individual. The method comprises detecting a deletion in the nucleic acid sequence between residues 10743 and 12125 in mitochondrial DNA. The method comprises obtaining a biological sample from the individual; extracting the mitochondrial DNA (mtDNA) from the sample; quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence between residues 10743 and 14125 of the mtDNA genome; and comparing the amount of mtDNA in the sample having the deletion to at least one known reference sample.
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Revendications(35)
What we claim is:
1. A method of detecting a cancer in an individual comprising;
a) obtaining 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 and 14124 of the mtDNA genome;
c) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA wherein the reference sample is from a known non-cancerous tissue or body fluid or from a known cancerous tissue or body fluid;
wherein if the reference sample is from a known non-cancerous tissue or body fluid, then an elevated level of the deletion in the biological sample compared to the non-cancerous reference sample is indicative of cancer and if the reference sample is from a known cancerous tissue or body fluid sample then an equivalent or elevated level of the deletion in the biological sample compared to the cancerous reference sample is indicative of cancer.
2. The method of claim 1 wherein the deletion has a nucleic acid sequence corresponding to the sequence identified in SEQ ID NO: 1.
3. The method of claim 1 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.
4. The method of claim 3 wherein a 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.
5. The method of claim 1 wherein the step of quantifying is conducted using real-time PCR.
6. The method of claim 1 wherein the cancer is prostate cancer.
7. The method of claim 1 wherein the cancer is breast cancer.
8. The method of claim 1 wherein the biological sample is a body tissue or body fluid.
9. The method of claim 8 wherein the biological sample is breast tissue, prostate tissue, prostate massage fluid, or urine.
10. The method according to claim 3 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.
11. A method of monitoring an individual for the development of a cancer comprising;
a) obtaining a biological sample;
b) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;
c) repeating steps a) to b) over a duration of time; and
d) wherein an increasing level of the deletion over the duration of time is indicative of cancer.
12. The method of claim 11 wherein the deletion has a nucleic acid sequence corresponding to the sequence identified in SEQ ID NO: 1.
13. The method of claim 11 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 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 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.
14. The method of claim 11 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.
15. The method of claim 11 wherein the step of quantifying is conducted using real-time PCR.
16. The method of claim 14 wherein a 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.
17. The method of claim 11 wherein the cancer is prostate cancer.
18. The method of claim 11 wherein the cancer is breast cancer.
19. The method of claim 11 wherein the biological sample is a body tissue or body fluid.
20. The method of claim 19 wherein the biological sample is breast tissue, prostate tissue, prostate massage fluid, or urine.
21. A method of detecting a cancer in an individual comprising;
a) obtaining a biological sample from the individual;
b) quantifying the amount of mtDNA in the sample having a sequence corresponding to the sequence identified in SEQ ID NO: 1;
c) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 to at least one known reference value, wherein the at least one known reference value is the amount of mtDNA corresponding to SEQ ID NO: 1 in a reference sample of mtDNA from known non-cancerous tissue or body fluid or a reference sample of mtDNA from known cancerous tissue or body fluid, and
wherein if the reference value is from a reference sample of mtDNA from known non-cancerous tissue or body fluid then an elevated amount of the mtDNA corresponding to SEQ ID NO: 1 in the biological sample compared to the non-cancerous reference sample is indicative of cancer and wherein if the reference value is from a reference sample of mtDNA from known cancerous tissue or body fluid then an equivalent or elevated amount of the mtDNA corresponding to SEQ ID NO: 1 in the biological sample compared to the cancerous reference sample is indicative of cancer.
22. The method of claim 21 wherein the step of quantifying is conducted using real-time PCR.
23. The method of claim 21 wherein 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.
24. The method of claim 21 wherein one of a pair of primers used in the amplifying of the target region overlaps a rejoining site of the sequence corresponding to SEQ ID NO: 1, after the sequence has re-circularized.
25. The method of claim 23 wherein a primer having a sequence corresponding to SEQ ID NO: 9 is used as part of a pair of amplification primers for amplifying the target region.
26. The method of claim 21 wherein the cancer is prostate cancer.
27. The method of claim 21 wherein the cancer is breast cancer.
28. The method of claim 21 wherein the biological sample is a body tissue or body fluid.
29. The method of claim 28 wherein the biological sample breast tissue, prostate tissue, prostate massage fluid, or urine.
30. The method of claim 22 wherein the reference value is a cycle threshold.
31. A method of detecting a deletion spanning approximately nucleotides 10744 to 14124 of the human mtDNA genome, wherein said deletion is associated with prostate cancer, in a subject having mtDNA, comprising:
a) providing a biological sample from the subject;
b) detecting the presence of the deletion in the mtDNA, wherein presence of the deletion in the mtDNA indicates prostate cancer, a predisposition to prostate cancer or the progression of prostate cancer.
32. The method of claim 31, wherein the step of detecting the presence of the deletion in the mtDNA comprises PCR analysis using a primer that bridges the mtDNA junction formed by the deletion spanning approximately nucleotides 10744 to 14124.
33. A method of detecting a mtDNA deletion spanning approximately nucleotides 10744 to 14124 of the human mtDNA genome as a biomarker for prostate cancer, the method comprising:
a) providing a biological sample from the subject;
b) detecting the presence of the deletion in the mtDNA;
c) comparing the amount of mtDNA in the sample having the deletion to the amount of the deletion in a reference sample of mtDNA wherein the reference sample is from a known non-cancerous tissue or body fluid or from a known cancerous tissue or body fluid;
wherein if the reference sample is from a known non-cancerous tissue or body fluid, then an elevated level of the deletion in the biological sample compared to the non-cancerous reference sample is a biomarker indicative of cancer and if the reference sample is from a known cancerous tissue or body fluid sample then an equivalent or elevated level of the deletion in the biological sample compared to the cancerous reference sample is a biomarker indicative of cancer.
34. The method of claim 33, wherein the deletion is approximately 3379 bp.
35. A method for confirming or refuting a prostate cancer biopsy test from a biopsy sample, comprising:
a) obtaining a normal tissue from a biopsy sample;
b) detecting the absence or presence of a human mtDNA deletion at approximately nucleotides 10744 to 14124 of the human mtDNA genome in the normal tissue; and
c) comparing the amount of the mtDNA deletion in the normal tissue to the amount of the deletion in a reference sample of mtDNA wherein the reference sample is from a known non-cancerous tissue or body fluid or from a known cancerous tissue or body fluid;
wherein if the reference sample is from a known non-cancerous tissue or body fluid, then an elevated level of the deletion in the normal tissue compared to the non-cancerous reference sample is indicative of a cancerous biopsy and if the reference sample is from a known cancerous tissue or body fluid sample then an equivalent or elevated level of the deletion in the biological sample compared to the cancerous reference sample is indicative of a cancerous biopsy.
Description
    CROSS REFERENCE TO RELATED APPLICATIONS
  • [0001]
    This application claims priority under 35 U.S.C. §365 (c) as a continuation of U.S. patent application Ser. No. 12/748,120 filed on Mar. 26, 2010 which application claims the benefit under 35 U.S.C. §120 as a continuation-in-part of U.S. patent application Ser. No. 11/975,390 filed Oct. 18, 2007 which is a continuation of PCT/CA2006/000652, filed Apr. 18, 2006 which application claims the benefit under 35 U.S.C. §119 (e) from U.S. Provisional Patent Application Nos. 60/672,016 filed Apr. 18, 2005, 60/721,522 filed Sep. 29, 2005, and 60/789,872 filed Apr. 7, 2006. In addition, this application claims priority under 35 U.S.C. §365 (c) as a continuation of PCT application PCT/CA2007/001711, filed Sep. 26, 2007, published in English. The entire disclosures of these applications are incorporated by reference herein.
  • REFERENCE TO SEQUENCE IDENTIFICATION LISTING
  • [0002]
    The present application includes a sequence identification listing in .txt format as follows:
  • [0000]
    Filename: Sequence Listing re PCT International Patent Appl. No.
      • PCT_CA2007001711.txt
  • Size: 26.8 KB Date Created: Mar. 22, 2010
  • [0004]
    This sequence identification listing is hereby expressly incorporated by reference in its entirety in the present application.
  • FIELD OF THE INVENTION
  • [0005]
    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.
  • BACKGROUND OF THE INVENTION
  • [0006]
    Mitochondrial DNA (MtDNA) as a Diagnostic Tool
  • [0007]
    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 (Shoffner 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).
  • [0008]
    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; 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).
  • [0009]
    Specifically, these mutations or alterations include point mutations (transitions, transversions), deletions (one base to thousands of bases), inversions, duplications, (one base to thousands of bases), recombinations and insertions (one base to thousands of bases). In addition, specific base pair alterations, deletions, or combinations thereof have been found to be associated with early onset of prostate, skin, and lung cancer, as well as aging (e.g. Polyak et al., 1998), premature aging, exposure to carcinogens (Lee et al., 1998), etc.
  • [0010]
    Prostate Cancer
  • [0011]
    Prostate cancer is a frequently diagnosed solid tumour that most likely originates in the prostate epithelium (Huang et al. 1999). In 1997, nearly 10 million American men were screened for prostate specific antigen (PSA), the presence of which suggests prostate cancer (Woodwell, 1999). Indeed, this indicates an even higher number of men screened by an initial digital rectal exam (DRE). In the same year, 31 million men had a DRE (Woodwell, 1999). Moreover, the annual number of newly diagnosed cases of prostate cancer in the United States is estimated at 179,000 (Landis et al., 1999). It is the second most commonly diagnosed cancer and second leading cause of cancer mortality in Canadian men. In 1997 prostate cancer accounted for 19,800 of newly diagnosed cancers in Canadian men (28%) (National Cancer Institute of Canada). It is estimated that 30% to 40% of all men over the age of forty-nine (49) have some cancerous prostate cells, yet only 20% to 25% of these men have a clinically significant form of prostate cancer (SpringNet—CE Connection, internet, www.springnet.com/ce/j803a.htm). Prostate cancer exhibits a wide variety of histological behaviour involving both endogenous and exogenous factors, i.e. socio-economic situations, diet, geography, hormonal imbalance, family history and genetic constitution (Konishi et al. 1997; Hayward et al. 1998). Although certain mtDNA alterations have been previously associated with prostate cancer, the need exists for further markers for the detection of prostate cancer.
  • [0012]
    3.4 Kb mtDNA Deletion and the Detection of Prostate Cancer.
  • [0013]
    In the applicant's pending PCT application bearing publication no. WO/06/111029 (the entire contents of which are incorporated herein by reference) a deletion of a 3379 bp segment of mtDNA was identified through full mitochondrial genome amplification of prostate tissue. The 3379 bp deletion (referred to as 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 the diagnosis of prostrate cancer when tissue samples are tested.
  • [0014]
    The 3.4 kb deletion removes all or part of the following genes from the mtDNA genome: (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.
  • [0015]
    Breast Cancer
  • [0016]
    Breast cancer is a cancer of the glandular breast tissue and is the fifth most common cause of cancer death. In 2005, breast cancer 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 Cancer 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.
  • BRIEF SUMMARY OF THE INVENTION
  • [0017]
    In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;
  • [0018]
    a) obtaining a biological sample from the individual;
  • [0019]
    b) extracting mitochondrial DNA, mtDNA, from the sample;
  • [0020]
    c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;
  • [0021]
    d) comparing the amount of mtDNA in the sample having the deletion to at least one known reference value.
  • [0022]
    In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;
  • [0023]
    a) obtaining a biological sample from the individual;
  • [0024]
    b) extracting mitochondrial DNA, mtDNA, from the sample;
  • [0025]
    c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;
  • [0026]
    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;
  • [0027]
    wherein an elevated amount of the deletion in the biological sample compared to the reference sample is indicative of cancer.
  • [0028]
    In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;
  • [0029]
    a) obtaining a biological sample from the individual;
  • [0030]
    b) extracting mitochondrial DNA, mtDNA, from the sample;
  • [0031]
    c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;
  • [0032]
    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;
  • [0033]
    wherein a similar level of the deletion in the biological sample compared to the reference sample is indicative of cancer.
  • [0034]
    In one embodiment, the present invention provides a method of monitoring an individual for the development of a cancer comprising;
  • [0035]
    a) obtaining a biological sample;
  • [0036]
    b) extracting mtDNA from the sample;
  • [0037]
    c) quantifying the amount of mtDNA in the sample having a deletion in the nucleic acid sequence spanning approximately residues 10744 and 14124 of the mtDNA genome;
  • [0038]
    d) repeating steps a) to c) over a duration of time;
  • [0039]
    e) wherein an increasing level of the deletion over the duration of time is indicative of cancer.
  • [0040]
    In one embodiment, the present invention provides a method of detecting a cancer in an individual comprising;
  • [0041]
    a) obtaining a biological sample from the individual;
  • [0042]
    b) extracting mitochondrial DNA, mtDNA, from the sample;
  • [0043]
    c) quantifying the amount of mtDNA in the sample having a sequence corresponding to the sequence identified in SEQ ID NO: 1;
  • [0044]
    d) comparing the amount of mtDNA in the sample corresponding to SEQ ID NO: 1 to at least one known reference value.
  • BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
  • [0045]
    An embodiment of the invention will now be described by way of example only with reference to the appended drawings wherein:
  • [0046]
    FIG. 1 is a schematic diagram showing the design and sequence of a primer useful for the detection of the 3.4 kb deletion. The primer (SEQ ID NO: 2) binds to bases 10729-10743/14125-14139 of the mtDNA genome, wherein the portion of the primer that binds to bases 10729-10743 is depicted in gray in the upper left corner of the figure (nucleotides 1-15 of SEQ ID NO: 2) and the portion of the primer that binds to bases 14125-14139 is depicted in white in the lower right corner of the figure (nucleotides 16-30 of SEQ ID NO: 2).
  • [0047]
    FIG. 2 is a graph showing a comparison of cycle threshold between malignant and symptomatic benign participants in the 3.4 kb study.
  • [0048]
    FIG. 3 is a graph showing cycle threshold as related to Example 1.
  • [0049]
    FIG. 4 shows a ROC curve illustrating the specificity and sensitivity of one embodiment of the present invention.
  • [0050]
    FIG. 5 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.
  • [0051]
    FIG. 6 shows real-time PCR data relating to 3.4 kb mtDNA deletion levels associated with breast cancer.
  • [0052]
    FIG. 7 shows a ROC curve illustrating the specificity and sensitivity of another embodiment of the present invention.
  • DETAILED DESCRIPTION OF THE INVENTION
  • [0053]
    As used herein, “cycle threshold” (CT) is the point at 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.
  • [0054]
    As defined herein, “sensitivity” refers to the fraction of true positives (true positive rate) results obtained using the method of the present invention.
  • [0055]
    As defined herein, “specificity” refers to the fraction of false positives (false positive rate) results obtained using the method of the present invention.
  • [0056]
    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 3.4 kb mtDNA deletion (SEQ ID NO:1) 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.
  • [0057]
    As disclosed in PCT WO/06/111029, the 3.4 kb 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.
  • [0058]
    The deletion results in the creation of two deletion monomers, one of 3.4 kb in size (small sublimon) and one of approximately 12.6 kb 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.
  • [0059]
    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.
  • [0060]
    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 (eg. years) in order to monitor the genesis or progression of cancer. Increasing levels of the 3.4 kb deletion over time could be indicative of the beginning or progression of cancer.
  • [0061]
    Age related accumulation of the 3.4 kb mtDNA deletion may predispose an individual to, for example, prostate cancer or breast cancer, which is prevalent in middle aged and older men, and middle aged and older women, respectively. According to one aspect of the invention, a method is provided wherein regular cancer screening may take place by monitoring over time the amount of the 3.4 kb deletion in body tissues such as breast tissue or body fluids such as prostate massage fluid, or urine.
  • [0062]
    The system and method of the present invention may be used to detect cancer at an early stage, and before any histological abnormalities. For example, the system and method of the present invention may be used to detect pre-neoplasia in breast tissue.
  • [0063]
    The following primer sequences are preferred for the detection of the 3.4 kb deletion:
  • [0000]
    3.4 forward (binds to bases 10729-10743/14125-
    14139 of the mtDNA genome)
    (SEQ ID NO: 2)
    5′-TAGACTACGTACATACTAACCCTACTCCTA-3′;
    3.4 reverse (binds to bases 14361-14379 of the  
    mtDNA genome)
    (SEQ ID NO: 3)
    5′-GAGGTAGGATTGGTGCTGT-3′. 
  • [0064]
    In one embodiment of the present invention, a pair of amplification primers are used to amplify a target region indicative of the presence of the 3.4 kb deletion. In this embodiment, one of the pair of amplification primers overlaps a spliced region of mtDNA after deletion of the 3.4 kb sequence has occurred (ie. a splice at a position spanning approximately residues 10744 and 14124 of the mtDNA genome). Therefore, extension of the overlapping primer can only occur if the 3.4 kb section is deleted.
  • [0065]
    In another embodiment of the present invention, a pair of amplification primers are used to amplify a target region associated with the deleted 3.4 kb sequence. The deleted 3.4 kb sequence, upon deletion, may reform as a circular mtDNA molecule. In this embodiment, one of the pair of amplification primers overlaps the rejoining site of the ends of the 3.4 kb sequence. Thus, an increase in the amount of the 3.4 kb molecule detected in a sample is indicative of cancer. The below primer pair is preferred for the detection of the deleted 3.4 kb nucleic acid.
  • [0000]
    (SEQ ID NO: 9)
    Forward 14115/10755 5′-CCCACTCATCACCTAAACCTAC-3′
    (SEQ ID NO: 10)
    Reverse 10980R 5′-GGTAGGAGTCAGGTAGTTAG-3′.
  • [0066]
    In one aspect of the invention, a kit for diagnosing cancer, for example prostate or breast cancer, comprising means for extraction of mtDNA, primers having the nucleic acid sequences recited in SEQ ID NOS: 2 and 3, or SEQ ID NOS: 9 and 10, reagents and instructions, is provided.
  • [0067]
    Another aspect of the invention provides methods for confirming or refuting the presence of a cancer biopsy test from a biopsy sample (eg. prostate or breast cancer), comprising: obtaining non-cancerous tissue from a biopsy sample; and detecting and quantifying the amount of the 3.4 kb mtDNA deletion in the non-diseased tissue.
  • [0068]
    In one embodiment the present invention provides a method for screening individuals for prostate or breast cancer from a body fluid sample comprising; obtaining a body fluid sample, and detecting and quantifying the level of the 3.4 kb mtDNA deletion in the body fluid.
  • [0069]
    Although real-time quantitative PCR methods, as described in the examples below, represent the preferred means for detecting and quantifying the presence or absence of the 3.4 kb deletion, other methods that would be well known to an individual of skill in the art could also be utilized. For example quantification of the deletion could be made using Bio-Rad's Bioplex™ System and Suspension Array technology. Generally, the method requires amplification and quantification of sequences using any known methods.
  • [0070]
    The examples provided below illustrate that not only can this deletion be used for the detection of prostate cancer in prostate tissue, but can also be used to detect the presence of cancer in other biological samples, for example prostate massage fluid, urine, and breast tissue. Based on the findings in these examples, the 3.4 kb mtDNA deletion may be used as a biomarker for cancer.
  • [0071]
    The various examples provided illustrate a difference in the amount of mtDNA having the 3.4 kb deletion between samples obtained from subjects having cancer, and 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.
  • Example 1 3.4 Kb Deletion in the mtDNA of Prostate Tissue
  • [0072]
    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. FIG. 1 is a schematic diagram showing the design and sequence of the primer (ie. SEQ ID 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.
  • [0073]
    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.
  • [0074]
    The 3.4 kb 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.
  • [0075]
    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. 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 2 ng/ul. Each sample was amplified using 20 ng 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).
  • [0076]
    As shown in FIG. 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.
  • Example 2 3.4 Kb Deletion Blinded Study—Comparison of Cycle Threshold
  • [0077]
    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%.
  • Example 3 3.4 Kb Deletion Study—Methods (n=76)
  • [0078]
    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 had hyperplasia. All specimens were needle biopsies taken from the investigators' tissue archives.
  • [0079]
    Prostate Specimens
  • [0080]
    A tapelift was performed on each slide using Prep-Strips (Catalogue Number LCMO207) 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 2 ng/μl were prepared for the purpose of Quantitative Polymerase Chain Reaction (qPCR).
  • [0081]
    Primers (Oligonucleotides)
  • [0082]
    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.
  • [0000]
    TABLE 1
    Amplification Primers.
    Length of
    amplified 
    Position product
    Primer Amplified  (base
    Pair 5′-3′ pairs)
    3.4 Deletion Real- 10729-14379 273
    Time (less 3379 bp
     at 10744-14124)
    12s mtDNA 708-945 238
    TNF 3756-3886 131
    3.4 forward (10729-10743-14125-14139) 5′TAGACTACGTACATACTAACCCTACTCCTA-3′ SEQ ID NO: 2
    3.4 reverse (14361-14379) 5′-GAGGTAGGATTGGTGCTGT-3′ SEQ ID NO: 3
    12s forward (708-728) 5′-CGTTCCAGTGAGTTCA CCCTC-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′-GGTTTCGAAGTGGTGGTC TTG-3′ SEQ ID NO: 7
  • [0083]
    Real-Time Polymerase Chain Reaction
  • [0084]
    Three separate PCRs were performed on each sample. Each reaction was 25 μl total volume and included template DNA, one pair of primers (12s or 3.4 Deletion or TNF), an iQ™ SYBR Green Supermix™ kit (Catalogue Number 170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water (ddH2O). The TNF (tumor necrosis factor) comprised single copy nuclear gene primers, and 12s comprised total mitochondrial genome primers. The volume and concentrations for template DNA, primers, and reaction buffer are listed below.
  • [0000]
    TABLE 2
    qPCR Components.
    Concentration per Volume per
    Reagent Reaction Reaction
    Reaction Buffer 1X 12.5 μl
    Primer (forward 250 nM 0.0625 μl of each
    and reverse) 100 umole stock
    ddH2O N/A 2.375. μl
    Template DNA 20 ng 10.0 μl
    Total 25 μl
  • [0085]
    The cycling parameters for each amplicon are listed in Table 3.
  • [0000]
    TABLE 3
    Cycling Parameters.
    Step Temperature (° C.) Duration
    1 95 3 min
    2 95 30 sec
    3 66 (3.4 deletion primers) or 30 sec
    61.5 (12s primers) or
    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.
  • [0086]
    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 CT 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.
  • [0087]
    The determination of malignancy is based upon the quantity of the 3.4 kb 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 3.4 kb 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.
  • [0088]
    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.
  • [0089]
    Cluster Analysis
  • [0090]
    The clustering was not normalized nor were logarithmic functions used due to the similar and small range of data.
  • [0091]
    FIG. 3 shows the actual movement and trends of the data. The x-axis is the patient number and the y-axis is the cycle threshold obtained from real time PCR.
  • [0092]
    It is important to note that the higher the cycle threshold is, the lower amount of the deletion is present.
  • [0093]
    The general trend shown in FIG. 3 is based upon the differences/ratios between the variables of Deletion, Total, and TNF. The deletion is low to absent for the benign/normal samples (right side) and increases (toward the left) with abnormal benign and malignant samples. The abnormal benign and malignant samples begin to differentiate themselves from each other based on the cycle threshold ratio of Deletion to TNF.
  • [0094]
    Supervised Learning
  • [0095]
    Supervised learning is based on the system trying to predict outcomes for known samples. Half of the data 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.
  • [0096]
    Data SET: 5% to 35%—Benign
      • 35% to 65%—Hyperplasia
      • 65% to 95%—Malignant
  • [0099]
    Artificial Neural Network (ANN) Algorithm (shown schematically below):
      • Half of Data set used for Training ANN
      • Other half used to compare the accuracyAccuracy=Compare expected data set with obtained data set→86.6%
  • [0000]
  • [0102]
    Supervised Learning of Deletion Data using Artificial Neural Network (ANN)
  • [0103]
    Three Classifications:
      • Benign
      • Hyperplasia
      • Malignant
  • [0107]
    Three variables for each classification were used based on Real Time PCR Cycle Threshold CT:
  • [0108]
    Tumour Necrosis Factor (TNF)—Nuclear copy control.
  • [0109]
    Total Mitochondria—Mitochondria copy control
  • [0110]
    Deletion—Mitochondria in the deleted state.
  • [0111]
    Results:
  • [0112]
    Half of data set is used to train the ANN, and the remaining half is used to compare the accuracy.
  • [0113]
    Three Classification Accuracy=86.6%
  • [0114]
    Positive Predictive Value (PPV);
  • [0115]
    Benign to Malignant=88.2%
  • [0116]
    Negative Predictive Value (NPV)
  • [0117]
    Benign to Malignant=76.5%
  • Example 4 3.4 Kb Deletion in mtDNA Associated with Breast Cancer
  • [0118]
    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.
  • [0119]
    DNA was isolated and purified from the samples using a QIAamp DNA Mini Kit (Qiagen, Cat. #51304) according to manufacturer's specifications.
  • [0120]
    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.
  • [0121]
    Real-Time Polymerase Chain Reaction
  • [0122]
    Three separate PCRs were performed on each sample. Each reaction was 25 μl total volume and included template DNA, one pair of primers (12s or 3.4 Deletion or TNF), an iQ™ SYBR Green Supermix kit (Catalogue Number 170-8882, Bio-Rad Laboratories Inc.) and distilled deionized water (ddH2O). The TNF (tumor necrosis factor) comprised single copy nuclear gene primers, and 12s comprised total mitochondrial genome primers. The volume and concentrations for template DNA, primers, and reaction buffer are listed below:
  • [0000]
    TABLE 4
    qPCR Components.
    Concentration per Volume per
    Reagent Reaction Reaction
    Reaction Buffer 1X 12.5 μl
    Primer (forward 250 nM 0.0625 μl of each
    and reverse) 100 μmole stock
    ddH2O N/A 2.375. μl
    Template DNA 20 ng 10.0 μl
    Total 25 μl
  • [0123]
    The cycling parameters for each amplicon are listed in Table 5.
  • [0000]
    TABLE 5
    Cycling Parameters.
    Step Temperature (° C.) Duration
    1 95 3 min
    2 95 30 sec
    3 66 (3.4 deletion primers) or 30 sec
    61.5 (12s primers) or
    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.
  • [0124]
    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 CT to that of the standards.
  • [0125]
    The determination of malignancy was based upon the quantity of the 3.4 kb 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 3.4 kb deletion. This may be expressed as a percent of the total mitochondrial DNA.
  • [0126]
    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.
  • [0127]
    Table 6 and FIG. 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 CT for 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.
  • [0000]
    TABLE 6
    Mean values for CT scores
    Group Statistics
    Std. Std.
    GRP N Mean Deviation Error Mean
    del3.4 normal 9 30.5889 2.53897 .84632
    malignant 9 27.8533 2.52253 .84084
  • [0128]
    FIG. 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%.
  • [0129]
    Table 7 shows the calculation of the area under the curve for the present example. As a measure of the accuracy of the test.
  • [0000]
    TABLE 7
    Results Showing Area Under the Curve
    Area Under the Curve
    Test Result Variable(s): del3.4
    Asymptotic 95%
    Std. Asymptotic Confidence Interval
    Area Errora Sig.b Lower Bound Upper Bound
    .790 .112 .038 .570 1.010
    aUnder the nonparametric assumption
    bNull hypothesis: true area = 0.5
  • [0130]
    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.
  • [0000]
    TABLE 8
    Determination of CT cutoff.
    Coordinates of the Curve
    Test Result Variable(s): del3.4
    Positive if
    Less Than
    or Equal To a Sensitivity 1 - Specificity
    24.6000 .000 .000
    25.6800 .111 .000
    25.7700 .222 .000
    25.9250 .333 .000
    26.2050 .444 .000
    26.8400 .556 .000
    27.4800 .556 .111
    28.1600 .556 .222
    28.8800 .667 .222
    29.1900 .778 .222
    29.4600 .778 .333
    29.8750 .778 .444
    30.5850 .778 .556
    31.2200 .778 .667
    31.5000 .889 .667
    31.7650 .889 .778
    32.9900 1.000 .778
    34.3350 1.000 .889
    35.6400 1.000 1.000
    a The smallest cutoff value is the minimum observed test value minus 1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.
  • Example 5 The 3.4 Kb Deletion in the Prostate Massage Fluid of Individuals with Prostate Cancer as Compared to the Fluid from Those without Histological Evidence of Prostate Cancer
  • [0131]
    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 2 ng/ul. Each sample was then amplified according to the following parameters:
  • [0000]
    1X iQ SYBR Green Supermix ™ (Bio-Rad P/N 170-8880)
    150 nmol forward primer
    (SEQ ID NO: 2)
    (5′-TAGACTACGTACATACTAACCCTACTCCTA-3′).
    150 nmol reverse primer
    (SEQ ID NO: 3)
    (5′-GAGGTAGGATTGGTGCTGT-3′)
    20 ng template DNA
    in a 25 ul reaction.
  • [0132]
    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
  • [0000]
    TABLE 9
    Results showing the mean CT Values for Prostate Massage Fluid Test
    Group Statistics
    Std. Std.
    Group N Mean Deviation Error Mean
    DEL3.4 benign 25 37.1869 3.18495 .63699
    malignant 15 33.7712 3.98056 1.02778
  • [0142]
    Tables 9 and 10 show a significant difference between the mean CT values obtained for the benign sample and the malignant sample groups (p=0.005).
  • [0000]
    TABLE 10
    Results Showing Difference (p = 0.005)for CT values of samples.
    Independent Samples Test
    t-test for Equality of Means
    Levere's Test for 95% Confidence Interval
    Equality of Variances Sig. Mean Std. Error of the Difference
    F Sig. t df (2-tailed) Difference Difference Lower Upper
    DEL34 Equal variances 1.251 .270 2989 38 .005 341570 1.14283 1.10217 5.72923
    assumed
    Equal variances 2825 24.696 .009 341570 1.20917 .92382 5.90758
    not assumed
  • [0143]
    FIG. 5 is a Receiver Operating Characteristic (ROC) curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when testing prostate massage fluid. These results were obtained using a cutoff CT of 37.3683. The sensitivity of the marker at this CT is 87%, while the specificity is 64%.
  • [0144]
    The accuracy of the test depends on how well the test separates the group being tested into those with and without the prostate cancer. Accuracy is measured by the area under the ROC curve. Table 11 shows the calculation of the area under the curve for the present example.
  • [0000]
    TABLE 11
    Results Showing Area Under the ROC Curve
    Area Under the Curve
    Test Result Variable(s): DEL3.4
    Asymptotic 95%
    Std. Asymptotic Confidence Interval
    Area Errora Sig.b Lower Bound Upper Bound
    .768 .074 .005 .622 .914
    aUnder the nonparametric assumption
    bNull hypothesis: true area = 0.5
  • [0000]
    TABLE 12
    Determination of Specificity and Sensitivity
    Coordinates of the Curve
    Test Result Variable(s): DEL3.4
    Positive if
    Less Than
    or Equal To a Sensitivity 1-Specificity
    26.2992 .000 .000
    27.3786 .067 .000
    28.2484 .133 .000
    29.5193 .200 .000
    30.1757 .200 .040
    30.4580 .200 .080
    30.5980 .267 .080
    31.5709 .333 .080
    32.5712 .333 .120
    32.9500 .333 .160
    33.3314 .400 .160
    33.6547 .467 .160
    33.9247 .533 .160
    34.3554 .533 .200
    34.9056 .533 .240
    35.4650 .533 .280
    35.9172 .533 .320
    36.0648 .600 .320
    36.3616 .667 .320
    36.6421 .733 .320
    36.8531 .733 .360
    37.1188 .800 .360
    37.3683 .867 .360
    37.5200 .867 .400
    37.8341 .867 .440
    38.2533 .867 .480
    38.5198 .933 .480
    38.6519 .933 .520
    38.8552 .933 .560
    39.1258 .933 .600
    39.2734 .933 .640
    39.4952 .933 .660
    39.7323 1.000 .680
    39.8956 1.000 .720
    41.0000 1.000 1.000
    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.
  • [0145]
    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.
  • Example 6 The 3.4 Kb Deletion in the Urine of Individuals with Prostate Cancer as Compared to the Fluid from Those without Histological Evidence of Prostate Cancer
  • [0146]
    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.
  • [0147]
    Upon receipt of the samples a 5 ml aliquot was removed and then 2 mls were centrifuged at 14,000×g to form a pellet. The supernatant was removed and discarded. Pellets were resuspended in 200 ul 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 0.1 ng/ul.
  • [0148]
    Samples were analyzed by quantitative real-time PCR with the 3.4 kb deletion specific primers according to the following:
  • [0000]
    1X iQ SYBR Green Supermix ™
    (Bio-Rad P/N 170-8880)
    100 nmol forward primer
    (SEQ ID NO: 2)
    (5′-TAGACTACGTACATACTAACCCTACTCCTA-3′)
    100 nmol reverse primer
    (SEQ ID NO: 3)
    (5′-GAGGTAGGATTGGTGCTGT-3′)
    1 ng template DNA
    in a 25 ul reaction.
  • [0149]
    Reactions were cycled on an Opticon™ 2 DNA Engine (Bio-Rad Canada) according to the following protocol:
  • [0150]
    1. 95° C. for 3 minutes
  • [0151]
    2. 95° C. for 30 seconds
  • [0152]
    3. 69° C. for 30 seconds
  • [0153]
    4. 72° C. for 30 seconds
  • [0154]
    5. Plate Read
  • [0155]
    6. Repeat steps 2-5 44 times
  • [0156]
    7. 72° C. for 10 minutes
  • [0157]
    8. Melting Curve from 50° C. to 105° C., read every 1° C., hold for 3 seconds
  • [0158]
    9. 10° C. Hold
  • [0000]
    TABLE 13
    Mean values for CT scores
    Group Statistics
    Std. Std.
    GRPfluid38 N Mean Deviation Error Mean
    CTf Benign 5 33.2780 1.10900 .49596
    Malignant 5 30.6980 2.55767 1.14382
  • [0159]
    Tables 13 and 14 show a significant difference between the mean CT values obtained for benign sample and the malignant sample groups (p=0.005).
  • [0000]
    TABLE 14
    Results Showing Difference (p = 0.005) for CT values of samples.
    Independent Samples Test
    t-test for Equality of Means
    Levere's Test for 95% Confidence Interval
    Equality of Variances Sig. Mean Std. Error of the Difference
    F Sig. t df (2-tailed) Difference Difference Lower Upper
    CTf Equal variances 1.272 .292 2069 8 .072 258000 124672 −.29494 5.45494
    assumed
    Equal variances 2069 5453 .089 258000 124672 −.54639 5.70639
    not assumed
  • [0160]
    FIG. 6 is a Receiver Operating Characteristic (ROC) curve illustrating the specificity and sensitivity of the 3.4 kb mtDNA deletion as a marker for prostate cancer when testing urine. These results were obtained using a cutoff CT of 31.575. The sensitivity of the marker at this CT is 80%, while the specificity is 100%.
  • [0161]
    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.
  • [0000]
    TABLE 15
    Determination of CT cutoff.
    Coordinates of the Curve
    Test Result Variable(s): CTf
    Positive if
    Less Than
    or Equal To a Sensitivity 1 - Specificity
    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 is the minimum observed test value minus 1, and the largest cutoff value is the maximum observed test value plus 1. All the other cutoff values are the averages of two consecutive ordered observed test values.
  • Example 7 Detection of Re-Circularized 3.4 Kb Deleted Sequence in Prostate Malignant and Benign Tissue
  • [0162]
    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.
  • [0163]
    Prostate tissue was formalin-fixed paraffin embedded prostate tissue needle biopsies.
  • [0164]
    The reagent setup used for this example was as follows:
  • [0165]
    250 nmol each primer
  • [0166]
    12.5 ul of 2λ reaction mix,
  • [0167]
    20 ng (10 ul of 2 ng/ul) template in 25 ul reaction volume.
  • [0168]
    The cycling parameters were as follows:
  • [0169]
    1. 95 degrees Celsius for 3 minutes
  • [0170]
    2. 95 degrees Celsius for 30 seconds
  • [0171]
    3. 62 degrees Celsius for 30 seconds
  • [0172]
    4. 72 degrees Celsius for 30 seconds
  • [0173]
    5. Plate Read
  • [0174]
    6. Repeat steps 2-5 44 times
  • [0175]
    7. 72 degrees for 10 minutes
  • [0176]
    8. Melting Curve from 50-100 degrees, reading every 1 degree for 3 seconds
  • [0177]
    9 4 degrees HOLD.
  • [0178]
    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).
  • [0179]
    Table 16 below provides a summary of testing conducted for the detection of the actual 3.4 kb deleted in mtDNA obtained from malignant and benign prostate tissue. Using a CT score of 30.0, a clear identification of malignant and benign tissue was possible. As such, an increase in the amount of the 3.4 kb molecule present in a sample was indicative of cancer.
  • [0000]
    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
  • [0180]
    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.
  • REFERENCES
  • [0000]
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Citations de brevets
Brevet cité Date de dépôt Date de publication Déposant Titre
US8008008 *18 oct. 200730 août 2011Mitomics Inc.Mitochondrial mutations and rearrangements as a diagnostic tool for the detection of sun exposure, prostate cancer and other cancers
Classifications
Classification aux États-Unis435/6.12, 435/6.14
Classification internationaleC12Q1/68, C12N15/11, C12N15/10
Classification coopérativeC12Q2600/156, C12Q1/6886, C12Q2600/112, C12Q2600/118, G01N33/57434, G01N33/57415