Mitochondrial DNA as a Cancer Biomarker
2005; Elsevier BV; Volume: 7; Issue: 2 Linguagem: Inglês
10.1016/s1525-1578(10)60553-3
ISSN1943-7811
AutoresJohn P. Jakupciak, Wendy Wang, Maura E. Markowitz, Delphine S. Ally, Michael D. Coble, Sudhir Srivastava, Anirban Maitra, Peter E. Barker, David Sidransky, Catherine D. O’Connell,
Tópico(s)Genomics and Rare Diseases
ResumoAs part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases. As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases. 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Because mitochondria are the sites of reactive oxygen species production, its DNA is more likely to be damaged than nuclear DNA.37Yakes RM Van Housten B Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress.Proc Natl Acad Sci USA. 1997; 94: 514-519Crossref PubMed Scopus (1424) Google Scholar Mitochondria possess less efficient DNA repair mechanisms than the nucleus,14Fliss MS Usadel H Caballero OL Wu L Buta MR Eleff SM Jen J Sidransky D Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.Science. 2000; 28: 2017-2019Crossref Scopus (698) Google Scholar therefore, mutations are more likely to persist and be representative of clonal expansion. The evolutionary mutation rate is 17-fold higher compared to single copy genes in the nuclear DNA.14Fliss MS Usadel H Caballero OL Wu L Buta MR Eleff SM Jen J Sidransky D Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.Science. 2000; 28: 2017-2019Crossref Scopus (698) Google ScholarAdvantages of using mitochondria DNA (mtDNA) as a potential biomarker for cancer-specific mutation studies are several. The genome is well characterized, with ∼16,568 bp harboring 37 densely packed genes. Secondly, high copy number (hundreds to thousands of mtDNA copies per cell) is a distinct advantage over nuclear DNA for the detection of sequence variants and translates into less tissue required for analysis. Hence, precious clinical samples are conserved. In addition, the mitochondrial genome is more resistant than nuclear DNA to damage caused by isolation and storage due to the small size and covalently closed, circular structure of mtDNA. Repair is also less efficient in the mitochondrial than nuclear genomes therefore, mutations are more quickly identified. Because mitochondria lack introns, mutations that occur will accumulate in the coding regions and are more likely to have biological consequences.26Penta JS Johnson RM Wachsman JT Copeland WC Mitochondrial DNA in human malignancy.Mutat Res. 2001; 488: 119-133Crossref PubMed Scopus (395) Google ScholarDespite the extensive association of mutations with human disease, the role of mitochondrial dysfunction in tumors and the interplay between nuclear and mitochondrial encoded genes remains unclear. Mitochondrial mutations have been observed in colorectal, breast, liver, prostate, pancreatic, and lung cancers. Moreover, sequence variations have been detected in preneoplastic lesions, which suggest mutations occur early in tumor progression.16Ha PK Tong BC Westra WH Sanchez-Cespedes M Parrella P Zahurak M Sidransky D Califano JA Mitochondrial C-tract alteration in premalignant lesions of the head and neck: a marker for progression and clonal proliferation.Clin Cancer Res. 2002; 8: 2260-2265PubMed Google Scholar, 19Parrella P Xiao Y Fliss M Sanchez-Cespedes M Mazzarelli P Rinaldi M Nicol T Gabrielson E Cuomo C Cohen D Pandit S Spencer M Rabitti C Fazio VM Sidransky D Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates.Cancer Res. 2001; 61: 7623-7626PubMed Google Scholar, 22Jeronimo C Nomoto S Caballero OL Usadel H Henrique R Varzim G Oliveira J Lopes C Fliss MS Sidransky D Mitochondrial mutations in early stage prostate cancer and bodily fluids.Oncogene. 2001; 20: 5195-5198Crossref PubMed Scopus (203) Google Scholar For colorectal cancer, 70% of cell lines examined harbored mitochondrial sequence variations in the coding and noncoding regions.21Copeland WC Wachsman JT Johnson FM Penta JS Mitochondrial DNA alterations in cancer.Cancer Invest. 2002; 20: 557-569Crossref PubMed Scopus (195) Google Scholar DNA alterations were also found in 45% of colorectal cancer specimens. Mitochondrial genome instabilities were found in 48% of breast cancer tissues38Richard SM Bailliet G Paez GL Bianchi MS Peltomaki P Bianchi NO Nuclear and mitochondrial genome instability in human breast cancer.Cancer Res. 2000; 60: 4231-4237PubMed Google Scholar and 42% of breast cancer specimens contained mitochondrial DNA sequence variants in the D-loop.19Parrella P Xiao Y Fliss M Sanchez-Cespedes M Mazzarelli P Rinaldi M Nicol T Gabrielson E Cuomo C Cohen D Pandit S Spencer M Rabitti C Fazio VM Sidransky D Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates.Cancer Res. 2001; 61: 7623-7626PubMed Google Scholar The mononucleotide repeat (C-stretch) at D303 to 310 was identified as a candidate hotspot in these breast primary tumors as well as cervical cancer. However, a rapid polymerase chain reaction (PCR) analysis of the C-stretch region from 56 cervical, bladder, and breast tumors, and endometrial neoplasia resulted in the detection of only 13 sequence variants (23%).39Parrella P Seripa D Matera MG Rabitti C Rinaldi M Mazzarelli P Gravina C Gallucci M Altomare V Flammia G Mutations of the D310 mitochondrial mononucleotide repeat in primary tumors and cytological specimens.Cancer Lett. 2003; 190: 73-77Abstract Full Text Full Text PDF PubMed Scopus (73) Google ScholarSequence variants in the D-loop region of the mitochondrial genome are also present in lung cancer. In one study of the noncoding region from 28 cell lines and 55 non-small cell lung cancer samples, sequence alterations were detected in 61% of the cell lines and 20% of the tumors.40Suzuki M Toyooka S Miyajima K Iizasa T Fujisawa T Bekele NB Gazdar AF Alterations in the mitochondrial displacement loop in lung cancers.Clin Cancer Res. 2003; 9: 5636-5641PubMed Google Scholar In further studies, lung cancer cell lines and tumor tissues were demonstrated to contain sequence variants at 70% and 43%, respectively.13Polyak K Li Y Zhu H Lengauer C Willson JKV Markowitz SD Trush MA Kinzler KW Vogelstein B Somatic mutations of the mitochondrial genome in human colorectal tumors.Nat Genet. 1998; 20: 291-293Crossref PubMed Scopus (731) Google Scholar, 14Fliss MS Usadel H Caballero OL Wu L Buta MR Eleff SM Jen J Sidransky D Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.Science. 2000; 28: 2017-2019Crossref Scopus (698) Google Scholar Sequence variations in mitochondrial DNA may serve as early indicators of lung cancer, thus enabling detection of cancer in early stages when treatment is most effective.The DNA Technologies Group at National Institute of Standards and Technology (NIST), an Early Detection Research Network Biomarker Validation Laboratory, conducts measurements to validate the utility of genes and gene products as biomarkers after their initial characterization by the Biomarker Discovery Laboratories. The purpose of this study was to evaluate mitochondrial sequence variants as biomarkers of early lung cancer. Lung cancer is the second most common cancer among men and women and is the leading cause of death among the most common cancers.41Hirsch FR Franklin WA Gazdar AF Bunn Jr, PA Early detection of lung cancer; clinical perspectives of recent advances in biology and radiology.Clin Cancer Res. 2001; 7: 5-22PubMed Google Scholar The incidence is as great as 117 per 100,000 individuals.42Miller BA, Kolonel LN, Bernstein L, et al (eds): Racial/Ethnic Patterns of Cancer in the United States. 1988–1992, National Cancer Institute: US Cancer Patterns. Bethesda, National Institutes of Health pub 96–4104Google Scholar Reductions in cancer can be expected if improved diagnostics and population screening technologies are implemented. Improvement in lung cancer prognosis can be primarily achieved via the development and validation of accurate early detection assays, which include strategies for noninvasively collected samples coupled with automation and sensitive detection limits. Currently, no clinically applied DNA biomarker exists for lung cancer. Initial pathological diagnosis is based on small bronchoscopic biopsy.43Garber ME Troyanskaya OG Schluens K Petersen S Thaesler Z Pacyna-Gengelbach M van de Rijn Matt Rosen GD Perou CM Whyte RI Diversity of gene expression in adenocarcinoma of the lung.Proc Natl Acad Sci USA. 2001; 98: 13784-13789Crossref PubMed Scopus (1097) Google Scholar Thus, reliable, high-throughput assays are needed to determine the role of sequence variation in the mitochondrial genome and the manifestation of cancer in clinical samples and bodily fluids.In this study, 22 mitochondrial genomes were fully sequenced from blood and tumor DNAs obtained from 11 individuals with lung cancer. Using a nested protocol and M13 primers for sequencing, an automated capillary electrophoresis protocol obtained 97 to 100% (average, 99.8%) sequence coverage for the forward strand and 90 to 100% (average, 98.4%) sequence coverage for the reverse strand. Sequence variants were identified in the tumor samples with respect to patient blood in 5 of 11 individuals (45%). Overall, eight sequence variants were identified. Twenty-two percent of the sequence variants identified were found in the D-loop region, which accounts for only 6.8% of the genome. The other sequence variants were spread throughout the coding region. Despite the reasonable assumption that heteroplasmies should comprise multiple subpopulations of mutated mtDNA molecules, the majority of the tumors were homoplasmic for sequence variants. Our work describes sequence variant detection by fluorescent capillary electrophoresis and compares these results to previous studies using radiolabeled sequencing. Further, limited comparison between these results and those obtained using DNA resequencing microarrays are described. In summary, a robust protocol for the identification of both heteroplasmic and homoplasmic sequence variants in clinical samples (human tissues and bodily fluids) is described.Materials and MethodsNucleic Acid IsolationDNA was extracted from microdissected tissue obtained from cryostat-embedded snap-frozen sections. DNA from primary tumors and bodily fluids was evaluated. Paired normal and tumor specimens were collected after surgical resection with prior consent from patients in the Johns Hopkins University Hospital. DNA from tumor sections was digested with 1% sodium dodecyl sulfate/Proteinase K, extracted by phenol chloroform, and ethanol precipitated. Tumor samples were obtained from males and females and represented broncholoalveolar, squamous cell, and adenocarcinomas. Samples were collected from stage I and IV tumors (Table 1).Table 1Pathology and Staging of Clinical SamplesSampleAgePathologyStageJHU sequenceSequence variants159BronchioloalveolarIComplete1274AdenocarcinomaID-loop0362Squamous cellID-loop0484AdenocarcinomaID-loop0547AdenocarcinomaIVComplete1678AdenocarcinomaIComplete0767AdenocarcinomaID-loop0864BronchioloalveolarIVD-loop1982Squamous cellIComplete41041AdenocarcinomaID-loop11166Large cell undifferentiatedIVD-loop0 Open table in a new tab PCR AmplificationPCR amplification primers selected were reported previously44Taylor RW Taylor GA Durham SE Turnbull DM The determination of complete mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations.Nucleic Acids Res. 1999; 29: e74-e84Crossref Scopus (148) Google Scholar to specifically amplify mitochondrial encoded DNA sequences. PCR amplification conditions were modified to optimize these primers and to use PCR-ready amplification microtiter plates. Briefly, primers were synthesized by Operon/Qiagen (Alameda, CA) and were shipped frozen at 100 μmol/L. Amplification was performed in two steps: a primary amplification followed by a nested, secondary amplification. For the primary amplification, nine overlapping primer sets were used to amplify the entire mtDNA genome from 20 ng of DNA template (Table 2). These PCR products ranged in size from 1886 to 2075 bp. The DNA, primers (0.6 μmol/L of each), and deionized H2O were added to a Clontech BD Sprint Advantage 96-well plate (Clontech Laboratories, Inc., Palo Alto, CA) containing lyophilized BD Sprint Advantage polymerase mix, dNTPs, optimized PCR buffer, and a mix of cryoprotectants for a total reaction volume of 25 μl. Thermal cycling conditions were as follows: preamplification denaturation: (1 cycle), 95°C for 1 minute; amplification (30 cycles): 95°C for 30 seconds; annealing, 58°C for 1 minute; elongation, 68°C for 3 minutes; final elongation (1 cycle), 68°C for 3 minutes; 4°C hold.Table 2Primary and Nested Primers for Full Mitochondrial Genome Sequence AnalysisPrimer Set 1NT PositionProduct LengthPrimer Set 2NT PositionProduct LengthPrimer Set 3NT PositionProduct LengthTay-1503–24841982Tay-22384–42491886Tay-34155–52202066tay2-1*Nested primers contain M13 sequence (forward or reverse) for fluorescent sequencing.44516–1190711tay2-42395–3074716tay2-74184–4869722tay2-21138–1801700tay2-52995–3648687tay2-84832–775775tay2-31754–2444725tay2-63536–4239740tay2-95526–6188699Primer Set 4NT PositionProduct LengthPrimer Set 5NT PositionProduct LengthPrimer Set 6NT PositionProduct LengthTay-46113–80171905Tay-57925–98841980Tay-69167–117481982tay2-106115–6781703tay2-137960–8641718tay2-169821–10516732tay2-116730–7398705tay2-148563–9231705tay2-1710394–11032675tay2-127349–8012697tay2-159181–9867723tay2-1810985–11708760Primer Set 7NT PositionProduct LengthPrimer Set 8NT PositionProduct LengthPrimer Set 9NT PositionProduct LengthTay-711614–136382025Tay-813539–154311893Tay-915331–8362075tay2-1911634–12361765tay2-2213568–14275745tay2-2515372–16067732tay2-2012284–13007758tay2-23142527–14928738tay2-D115879–16545703tay2-2112951–13614700tay2-2414732–15420724tay2-D216495–390482tay2-D3213–803525* Nested primers contain M13 sequence (forward or reverse) for fluorescent sequencing.44Taylor RW Taylor GA Durham SE Turnbull DM The determination of complete mitochondrial DNA sequences in single cells: implications for the study of somatic mitochondrial DNA point mutations.Nucleic Acids Res. 1999; 29: e74-e84Crossref Scopus (148) Google Scholar Open table in a new tab PCR amplification products were subsequently analyzed for both quality and quantity of DNA using the Caliper AMS 90 SE electrophoresis system (Caliper Technologies Corp., Mountain View, CA). To facilitate sequencing, the nine amplicons were reamplified using M13-tagged primers. The secondary amplification uses 28 primer pairs to amplify smaller fragments of the primary amplicons (482 bp and 775 bp, Table 2). Secondary amplification reactions were conducted using 2 μl (0.6 to 45 ng) of the primary PCR product and the Clontech 96-well plates and reagents used in the primary PCR step. Thermal cycling conditions were as follows: preamplification denaturation: (1 cycle), 95°C for 1 minute; amplification (30 cycles): 95°C for 30 seconds; annealing, 58°C for 1 minute; elongation, 68°C for 1 minute; final elongation (1 cycle), 68°C for 3 minutes; 4°C hold.PCR Clean-UpPCR clean-up protocols were optimized for high-throughput sequencing using both Exo l-SAP and filtration. The first method, Exo I-SAP, was used for partial plates and individual reactions. The following reagents were added to a final 25-μl reaction volume: 1.0 unit of SAP (USB Corp., Cleveland, OH), 5.0 units of Exo I (USB), and 4.85 μl of deionized H2O. For multiple reactions, a master mix of these reagents was made and dispensed in 6.25-μl aliquots. Samples were then placed on the 9700 thermocycler (Applied Biosystems, Foster City, CA) for 15 minutes at 37°C, then 30 minutes at 72°C. The samples were then diluted with 40 μl of deionized H2O for analysis on the AMS 90 SE.The second method used for PCR clean-up was the Montage PCRμ96 plate (Millipore Corp., Billerica, MA). This method was used for full 96-well plates containing the secondary PCR reactions. Seventy-five μl of deionized H2O were added to each well of the secondary amplification plate to a final volume of 100 μl/well. The contents of each well were then transferred to the Millipore filtration plate and placed on the vacuum manifold for 30 to 45 minutes or until all of the wells were dry. Once dry, 65 μl of deionized H2O were added to each well. The plate was then placed on a shaker at high speed for 10 minutes. The samples were then transferred to a 96-well plate compatible with the Caliper AMS 90 SE for quantification.DNA QuantificationAfter clean-up, samples were separated on the AMS 90 SE to size and quantify the DNA. Fifty ng of each sample were then transferred to a new plate and dried in a vacuum centrifuge. The samples were resuspended in 50 μl of deionized water to a final concentration of 1 ng/μL for sequencing.DNA SequencingSecondary products tagged with M13 forward and reverse primers, (tgtaaaacgacggccagt and ggaaacagctatgaccat, respectively) were sequenced using the Big Dye Terminator (BDT) version 3.1 cycle sequencing kit (Applied Biosystems). A one-eighth cycle-sequencing reaction was used for all sequencing. The 28 secondary PCR amplicons for each sample were sequenced on both strands with the M13 primers, for a total of 56 sequencing reactions for each mitochondrial genome. Each reaction contained 1 μl of each of the following reagents: BigDye Terminator, DNA (1 ng/μl), M13 primer (forward or reverse; 5 pmol/μl), 5× dilution buffer (Applied Biosystems), and dH2O to a final volume of 5 μl. Cycling sequencing conditions were as follows: (40 cycles): 96°C for 10 seconds; annealing, 50°C for 5 seconds; elongation, 60°C for 4 minutes; 4°C hold. All separations were performed using the ABI 3100 genetic analyzer using an 80-cm capillary and POP4 polymer system. Samples were electrokinetically injected (30 seconds, 1 kV) and separated at 14.6 kV. Sequences were aligned using the DNA Star SeqMan II (5.05) program (DNASTAR, Inc, Madison, WI) and scanned for polymorphisms and sequence variants. Both homoplasmic (identical mitochondrial genomes) and heteroplasmic (differences in mitochondrial DNA genomes present) sequence variants were expected.13Polyak K Li Y Zhu H Lengauer C Willson JKV Markowitz SD Trush MA Kinzler KW Vogelstein B Somatic mutations of the mitochondrial genome in human colorectal tumors.Nat Genet. 1998; 20: 291-293Crossref PubMed Scopus (731) Google Scholar, 14Fliss MS Usadel H Caballero OL Wu L Buta MR Eleff SM Jen J Sidransky D Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.Science. 2000; 28: 2017-2019Crossref Scopus (698) Google Scholar The
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