Mitochondrial DNA mutations in breast cancer tissue and in matched nipple aspirate fluid
Weizhu Zhu*,
Wenyi Qin*,
Paul Bradley,
Amy Wessel,
Charles L. Puckett and
Edward R. Sauter1
Ellis Fischel Cancer Center and Department of Surgery, University of Missouri, Columbia, MO 65212, USA
1 To whom correspondence should be addressed Email: sautere{at}health.missouri.edu
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Abstract
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Unlike nuclear (n)DNA, of which there is one paired copy per cell, there are many copies of mitochondrial (mt)DNA per cell, making PCR amplification of mtDNA easier in samples of limited cellularity. The aims of this study were to (i) determine the mutation patterns of breast cancers through a comprehensive screen of mtDNA mutations, and (ii) assess if mutations in the cancers are also detectable in breast nipple aspirate fluid (NAF), a physiologic fluid which contains shed ductal epithelial cells. Fifteen breast cancers, matched benign tissues and NAF were collected. Nine overlapping primer sets were used to sequence the entire mitochondrial genome from tissue samples. For NAF samples, we focused on the 19 nucleotide positions (np) where mutations were found in a 3701 bp region (np 15331 to 2463), which includes the displacement (D)-loop, a mtDNA mutation hot spot. Fourteen of the fifteen (93%) cancer samples had
1 somatic mtDNA mutation for a total of 45 at 35 np (9 np reported previously, 26 new). Nine of fifteen tumors had
2 mutations. The D-loop contained 17 of 45 (38%) and non-D-loop (coding) regions contained 28 (62%) mutations. Of the 28 mutations in the coding loci, 11 led to an amino acid change. The frequency of mtDNA mutations was higher in the D-loop region (1.5 versus 0.18% of loci). 155 polymorphisms were identified (98 reported previously, 57 new). Sixteen of forty-five (36%) mutations were located at polymorphism sites. Four of nineteen mtDNA mutations in 10 cancers located between np 15331 and 2463 were found in matched NAF (two of eleven mutations in the D-loop and two of eight in non-D-loop regions). No mutations were found in five matched NAF samples from women whose cancers lacked a mutation in the same region. In conclusion, mtDNA mutations in breast cancer occur both within and outside of the D-loop, though the mutation rate in the D-loop is over 7-fold higher than in coding areas. We identified 26 new mutation loci (25 in regions sequenced by others, one in an area not). The high frequency of mtDNA mutations at polymorphic loci requires further investigation. Specific mtDNA mutations can be detected in a subset of NAF samples from women with breast cancer.
Abbreviations: D-loop, displacement loop; MSI, microsatellite instability; mt, mitochondrial; NAF, nipple aspirate fluid; np, nucleotide positions; TTGE, temporal temperature gradient gel electrophoresis
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Introduction
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The human mitochondrial genome, a genetic locus independent of the nuclear genome, is a compactly organized, circular molecule of 16 569 nt. Gene products and functions have been assigned to all mitochondrial genes that include 13 protein coding, two rRNA and 22 tRNA genes that are involved in cellular energy production (1). The displacement (D)-loop is a non-coding region of 1124 bp (nucleotide positions, np 16 024576), which acts as a promoter for both the heavy and light strands of mitochondrial (mt)DNA, and contains essential transcription and replication elements (2). The D-loop is a hot spot for mtDNA mutations, and it contains two hypervariable regions (HV1 at positions 16 02416 383 and HV2 at positions 57372) (2). Cancer development involves the accumulation of various genetic alterations, which are present both in the mitochondrial as well as in the nuclear genomes. mtDNA mutations have been ascribed to high levels of reactive oxygen species produced during oxidative phosphorylation, to a less efficient DNA repair system and to the lack of protective histones as compared with nuclear (n)DNA (3,4). Oxidative mtDNA damage is believed to be associated with cancer. The important roles of mitochondria in energy metabolism, generation of reactive oxygen species, aging and the initiation of apoptosis suggest that the mitochondria may serve as a key switch in shifting the cell from death to abnormal cell growth, thus contributing to the neoplastic process (5).
Human malignancies have been associated with mtDNA mutations, either deletionsinsertions or base substitutions (68). Studies suggest that the frequency of mtDNA mutations is higher than in nDNA in a variety of human cancers including bladder, head and neck, lung and breast (912). Recently published data indicate that the majority of somatic mtDNA mutations are homoplasmic, indicating that mutant mtDNA had become dominant in the cancer cells, although the mechanism(s) for the development of mtDNA homoplasmy in cancer tissue remains a puzzle (9). It is estimated that each cell contains several hundred to thousands of mitochondria and that each mitochondrion contains 110 mitochondrial genomes (13). mtDNA mutations have been identified in a number of bodily fluids, including urine, saliva and bronchoalveolar lavage (9) and serum (14). Because of the sheer abundance of mtDNA per cell, the tendency for mtDNA mutations to be homoplastic, and the demonstrated ability to detect mtDNA mutations in bodily fluids of limited and mixed cellularity, mtDNA may provide a distinct advantage in terms of feasibility and sensitivity over nDNA-based methods for cancer detection, especially when one is dealing with samples of low cellularity such as nipple aspirate fluid (NAF).
To screen for mtDNA mutations, most reports have used either restriction enzyme digestion (15) or temporal temperature gradient gel electrophoresis (TTGE) (16). Only areas that were abnormal by the screening technique underwent direct sequencing. Although quite sensitive, it appears that these techniques miss a small subset of mutations (17). We elected to directly sequence the entire mitochondrial genome. To the best of our knowledge, this is the first report in breast cancer where the entire mitochondrial genome was sequenced.
For NAF samples, which are generally of limited cellularity, we chose in this proof of principle study to focus on the D-loop and adjacent portions of the cytochrome b, tRNA threonine and 12S ribosomal RNA genes (np 15 3312463), because the mutation frequency appears to be higher in this region (9) of the mitochondrial genome.
The early detection of breast cancer remains inadequate. Our ultimate goal is to use NAF as a diagnostic tool to aid in the early detection of breast cancer. Toward this goal, the aims of this study were to determine the mutation patterns in breast cancers by comprehensively screening the tissues for mtDNA mutations, and to assess if mutations detected in the cancers are also detectable in NAF. Herein we report the results of sequencing the entire mitochondrial genome in 15 matched breast cancer and benign tissues, in which we observed somatic mutations at 35 loci that were present in cancer but not in matched benign breast tissue, and demonstrate the feasibility of identifying identical mtDNA mutations in matched NAF. Our findings suggest that screening NAF for cancer-specific mtDNA mutations, such as those found by ourselves and others, is feasible and can detect mutations in a subset of women with breast cancer.
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Materials and methods
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Subjects
After receiving Institutional Review Board approval, informed consent was obtained from all subjects prior to enrollment. All subjects had biopsy-proven breast cancer that required surgical removal. Fifteen matched breast cancer and benign tissue samples were obtained at the time of surgery. NAF from the ipsilateral breast was collected before or at the time of surgery using a modified breast pump as described previously (18). Malignant and benign frozen tissue provided by the pathologist immediately after removal from the subject was snap frozen and stored at 80°C until use. Serial 10-µm histologic sections were prepared. The first and tenth levels were stained with hematoxylin and eosin for diagnostic purposes. Pathologic diagnosis was verified by light microscopy using levels 1 and 10, and the areas to be microdissected were marked. An average a 2.5 x 2.5-mm area was then microdissected from levels 2 to 9 from matched cancer and benign tissue sections to obtain a cell population sufficient to obtain a detectable band in each PCR. Areas of dissection were chosen to obtain an epithelial cell purity of
80%. The dissection of unstained slides was performed in a laminar flow tissue culture hood.
mtDNA isolation, amplification and sequencing
Isolation. DNA was extracted from malignant and benign tissue samples using proteinase K for digestion followed by heat inactivation at 95°C, then phenolchloroform and ethanol precipitation. The DNA was dissolved in Tris ethylenediamine tetra-acetic acid buffer. A Qiagen Mini-Blood Kit was used to isolate DNA from NAF samples. Briefly, NAF was diluted with 200 µl of PBS and DNA was then extracted following the kit protocol. Isolated DNA was kept at 4°C in a dedicated area that was used only for PCR assembly. No PCR products or equipment used in post-PCR analysis ever entered this area.
PCR amplification. Nine pairs of published overlapping primer sets (19) were used to amplify the entire 16.569 kb mitochondrial genome. The generated fragments were 18862075 bp in length. DNA prepared from human mtDNA-less cells (143b rho 0 cells, kind gift of Dr Timothy Johns, McGill University) were used as a negative control to exclude the possibility of pseudogene amplification. Briefly, total DNA was subjected to the following PCR protocol: initial DNA denaturization at 95°C for 15 min, followed by 35 cycles at 95°C for 52 s, 57°C for 50 s, 70°C for 2 min, and a final extension at 72°C for 10 min. The annealing temperature for the fragment, which covered np 41556220 was set at 54°C and the other parameters were kept the same as the other eight fragments for PCR amplification. PCR products were purified with a Qiagen gel extraction kit (Qiaquick columns; Qiagen, Chatsworth, CA) and stored at 4°C until use for sequencing.
DNA sequencing. DNA sequencing was executed using a dye terminator cycle sequencing kit (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ) and an ABI 377 (Applied Biosystems, Foster City, CA) automated sequencer. Briefly, 30 ng of purified PCR products were used as a template with a primer concentration of 2 µM in the sequencing mixture. Each fragment was sequenced with four overlapping (
100 bp overlap) primers (primer sequences are not shown but are available upon request) that walked the entire length of each fragment. All mutations were sequenced from the opposite direction to ensure reading accuracy. The results of the DNA sequence analysis were compared with the published Cambridge Sequence (1) using Mutation Surveyor Version 1.4 DNA mutation analysis software (Softgenetics, State College, PA). Independent sequence readings were performed by three different individuals (W.Z., W.Q. and P.B.). Sequence differences between malignant and matched benign tissue were recorded as somatic mtDNA mutations. Sequence variations found in both malignant and matched benign mtDNA were scored as germline polymorphisms. Each polymorphism was then checked against the Mitomap database (http://www.mitomap.org/) and further classified as novel or reported, depending on whether or not it is recorded in the database. Ten cancer specimens with an mtDNA mutation located within the 15 3312463 bp area, as well as five cancer specimens lacking an mtDNA mutation in the same region, had a matched NAF sample sequenced from both directions to assure sequence reading precision.
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Results
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Somatic mtDNA mutations
Our first step was to ensure that the mtDNA primers amplified only mtDNA sequences. We evaluated each of our nine mtDNA primers in two cell lines (Figure 1), one normal (143b) and the other lacking mtDNA (143b rho 0). The odd lanes represent the amplification of 143b rho 0 cells with our mtDNA primers, whereas the even lanes represent the amplification of 143b cells. The absence of a band in the lanes where the mtDNA-less cells were amplified, and a single band in the lanes where cells contain mtDNA, suggests that the primers are mtDNA specific.

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Fig. 1. mtDNA primers amplify only mtDNA sequences. Nine mtDNA primers designed to amplify the entire mtDNA genome were PCR amplified in one normal (143b) cell line and in one line lacking mtDNA (143b rho 0). The predicted sizes of each of the products range from 1886 to 2075 bp. Odd lanes represent the amplification of 143b rho 0 cells with our mtDNA primers, whereas even lanes represent the amplification of 143b cells. M = 1 kb DNA ladder.
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We sequenced the entire mitochondrial genome in 15 malignant and matched benign breast tissues. We were able to read 98.5% of the resulting sequences. We identified one or more somatic mutations in 93% (14/15) of breast cancers. Overall, 45 somatic mutations (six specimens had one mutation, five specimens had two mutations, one had three, one had four, one had six and one had 16 mutations) at 35 unique np were identified (Table I and Figure 2). Of the 35 unique np, nine were reported previously and 26 are new (Table II). Fifty-three percent (24/45) of the alterations were homoplasmic and 47% (21/45) heteroplasmic (Figure 3). Seventeen of the 45 (38%) were located in the D-loop (non-coding) and 28 (62%) in non-D-loop (coding) regions. The relative mutation frequency in the D-loop was 7.3-fold higher than in other areas (17 mutations/1121 np, 1.5%, versus 29/15 448 np, 0.18%). The highest mutation frequency was in the 310 C-repeat area in the D-loop, where we found seven mutations (one deletion, four insertions and two substitutions).

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Fig. 2. mtDNA mutations found by ourselves and others. Every tick represents a mutation locus detected in this study. Outer rods indicate mutations that were also found by others (see Table II for details). Abbreviations: 12S, 12S ribosomal RNA; 16S, 16S ribosomal RNA; ND1, 2, 4, 5, 6, NADH dehydrogenase subunit 1, 2, 4, 5, 6; ATPase, ATP synthase F0 subunit 8; COI, II, III, cytochrome c oxidase subunit I, II, and III; Cyt B, cytochrome b.
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Fig. 3. Heteroplasmic mtDNA mutations in breast cancer tissue compared with normal tissue. (A) T > CT mutation in the 310 region (np 303315); (B) A > AG mutation at the 15924 locus.
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Eleven of the 37 genes encoded in the mitochondrial genome (four encoding RNA: 12S, 16S, TI and TT, and seven encoding proteins: ND1, ND2, ND4, ND5, ATPase, COIII and CYTB) had at least one mutation (Table I and Figure 2). Of the 45 mutations, 11 resulted in an amino acid change at 10 unique loci. Of the 26 new mutations that were identified, 25 were in regions of the mtDNA genome that had been sequenced previously. One mutation, located at np 8498, to the best of our knowledge is in a region for which sequence results in breast cancer have not been reported by other investigators.
Germline sequence polymorphisms
When the sequences of both malignant and benign tissue were compared with that of the published Cambridge sequence, 155 polymorphisms were found (Table III). Fifty-five of the polymorphisms (35%) were in the D-loop and 65% in the coding regions of the mitochondrial genome. Fifty-seven of these variations are novel, and 98 of them have been recorded in the Mitomap database. The frequency of novel polymorphisms was 10% (6/55) in the D-loop and 49% (49/100) in the coding regions.
We observed a high frequency of mutations at polymorphic sites (Table IV). We identified 16 somatic mutations at 155 polymorphic sites as opposed to 29 mutations at 16 414 sites (relative frequency of 10 versus 0.18%) where we did not find a polymorphism.
Detection of mtDNA mutations in NAF
We were able to amplify all NAF samples and obtained a usable mtDNA sequence. Because of the variable cellularity in NAF, in this proof of principle study we elected to evaluate NAF mutations in a known mtDNA hot spot, the D-loop. We used two of our primers to amplify the region that covered the D-loop and part of the adjacent cytochrome b, tRNA threonine and 12S ribosomal RNA genes. In the region amplified in NAF samples, 10 of the 15 cancers contained mutations at 19 unique np, while five cancers lacked mutations. Of the 19 np found to harbor an mtDNA mutation in breast cancer tissue from this area (the 310 region, which includes a series of cytosines from 303 to 309 and from 311 to 315, with a thymidine at 310, was considered one mutation site), four NAF samples had mutations (Table V). Two of eleven (18%) mutations detected in the D-loop were found in matched NAF samples, compared with two or eight (25%) in coding regions (Figure 4). An example of a homoplasmic mutation found in matched tumor and NAF is illustrated in Figure 5. Of the five cancers that lacked an mtDNA mutation in the sequenced region, there were no false positive mtDNA mutations in matched NAF.

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Fig. 4. mtDNA mutations identified in matched NAF samples. Two of the positive NAF samples were in the D-loop, one in the cytochrome b gene and one in the interval space between the two where the tRNA threonine gene is located.
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Fig. 5. Homoplasmic mtDNA mutation in breast cancer tissue and matched NAF. Compared with normal tissue, A > G mutation at the 16 293 locus.
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We next determined if the ability to detect mtDNA mutations in NAF was related to tumor stage, grade or to NAF cytology. Tumor stage in the NAF samples that were positive for mtDNA mutations ranged from 1 to 3, grade from moderate to severe, and cytology from scant cells to atypia. This range was not different from NAF samples in which an mtDNA mutation was not found.
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Discussion
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Using single-strand conformation polymorphism (SSCP), two-dimensional (2-D) gene scanning, or TTGE, investigators have published reports in which they screened between 7 and 100% of the mitochondrial genome of a variety of human cancers (9,11,12,2025). The screening approaches used are limited in three ways. First, in most cases only a portion of the mitochondrial genome was screened. Secondly, screening techniques such as SSCP, 2-D gene scanning and TTGE may miss mutations that are detected by direct sequencing (17). Finally, abnormalities found by screening techniques require confirmation by direct sequencing.
The report with the greatest mtDNA coverage to date analyzing breast cancer tissues (13) that employed direct sequencing to screen the mitochondrial genome used a combination of manual sequencing (55% of the mitochondrial genome) and automated sequencing (29% of mitochondrial genome) to identify mtDNA mutations in 18 breast cancers. Twelve mutations were detected, five (42%) in the D310 region and seven elsewhere, similar to our findings of 38% being located in the D-loop. We identified a greater number of mtDNA mutations both in the D-loop [17 (at 11 loci) versus 7, for a mutation frequency of 1.5 versus 0.62%] and in coding regions [28 mutations (at 24 loci) versus five, for a mutation frequency of 0.18 versus 0.039%]. We believe that this is due to the greater mtDNA sequence coverage and the use of automated versus manual sequencing, which allowed us to use software designed to aid in mutation detection.
Of the 45 somatic mutations that we found, 35 were at unique loci. Mutations at nine of the loci (seven in the D-loop and two in non-D loop areas) have been reported in other cancer types (Table II) suggesting that these sites are susceptible to mutation in a variety of human malignancies. Our observations reinforce the findings of others (9,13,26) that somatic mtDNA mutations in cancers are concentrated in the D-loop and that the D310 region is a mutation hot spot. The sequence between np 303 and 315 is a conserved sequence of mtDNA. Length variation or mutations in this region, therefore, may play an important role in regulating mtDNA replication. The seven mutations that we and others detected in this conserved region in cancer specimens may therefore reflect neoplastic initiation. The D-loop mutations at np 204, 207 and 16 293 were reported previously (25) in breast cancer cells, suggesting that mutations at np 204, 207 and 16 293 may indicate the presence of breast cancer.
We observed 11 mutations at 10 loci that led to changes in amino acids. These mutations occurred in NADH dehydrogenase subunits 2, 4 and 5; ATP synthase F0 subunit 8; cytochrome c oxidase subunit III; and cytochrome b. Each of these mutations may be of functional significance, but more extensive biochemical and molecular studies will be necessary to determine their effects on energy metabolism in malignant cells.
Most tumors had more then one mutation. Specifically, of the 14 tumors with mutations, nine (64%) had two or more, four had three or more (29%) and two had six or more (14%). Multiple mtDNA mutations are common in human tumors, including glioblastomas and ovarian, prostate and thyroid carcinomas (Table II) (27). This high mutation rate has been termed mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, which leads to a burst of multiple mtDNA mutations (27).
Over a third (55/155, 35%) of the polymorphisms that we detected were in the D-loop, an area that acts as a promoter for both the heavy and light strands of mtDNA, and contains essential transcription and replication elements, suggesting that polymorphisms may play a role in carcinogenesis. There was a much higher rate (49 versus 10%) of newly discovered polymorphisms in coding regions than in the D-loop. Although we cannot be sure, this may simply reflect the fact that the D-loop has been more intensively investigated than the coding regions. Additionally, we found that 36% (16/45) of mtDNA mutations were located at polymorphic sites (Table IV), even though these sites made up <1% of the bases in the mitochondrial genome. This raises the question of whether polymorphic loci are prone to develop mutations (25,28).
Although not a primary aim of our investigation, we also determined if there was evidence of mtDNA microsatellite instability (MSI). Contradictory observations regarding the frequency of MSI in the mitochondrial genome have been reported. CAn repeats were found at a low frequency in various types of cancer, including endometrial [2% (29)], ovarian [8% (20)], glioblastoma [6% (23)] and gastric [19% (30)] cancers, but was much higher [42% (15)] in one but not in a second (25) analysis of breast cancer, where MSI was found in 0 of 19 tumors at a CAn repeat site. Methods used to detect MSI were either fragment size or RFLP analysis, which may not be the most accurate means to detect mitochondrial MSI because of the interference of shadow bands and the possibility of incomplete digestion. We detected no changes in the three CAn repeat loci of the mitochondrial genome that we tested, including np 514523, 13 93213 937 and 16 23216 237 areas. Differences in the frequency of MSI may be related to differences in methodology used, as well as the loci evaluated.
Our previous studies demonstrate that NAF provides a promising non-invasive approach to obtain breast epithelial cells to detect pre- and invasive breast cancer (18). We have also observed that multiple DNA markers, including loss of heterozygosity, MSI and DNA methylation, are detectable from the epithelial cells that are present in NAF (3133). A previous report (34) evaluated six NAF samples from four women without breast cancer, two with a BRCA1 mutation and two without. The D310 microsatellite marker, spanning
300 bp, was PCR amplified. Presumed mutations were identified by running the PCR products on a gel, to determine if a mutation was present. One of the six NAF samples was suspected of having an mtDNA mutation, although this was not confirmed by direct sequencing. We evaluated NAF samples from 15 breast cancers, 10 with 19 unique (24 total) mutations and five lacking a mutation in matched tumor tissue in a 3701-bp region, which included the D-loop region. The 3701-bp region was sequenced directly. From tumors with one or more mutations in the region, we found an identical mutation in four of ten matched NAF specimens, whereas zero of five NAF specimens contained a mutation when the matched tumor lacked a mutation.
Given that NAF is likely to contain both normal and malignant cells, we were concerned that we might encounter ambiguous sequencing results, with two or more bases at the same np, but we did not. All 15 NAF samples provided clear, unambiguous sequence results using both mutation analysis software and manual reading. An example of this is illustrated in Figure 5. On the other hand, the fact that only a subset of mutations present in the tumor were also identified in NAF suggests that not all NAF samples had sufficient tumor cells to detect the mutation identified in the tissue. Of the 19 unique mutations found in breast cancer tissue using the primer set that included the D-loop, 4 (21%) identical mutations were found in matched NAF. At least one mutation was identified in four of ten tumors which contained a mutation in the D-loop region, and in four of fifteen tumors overall. Since 14 of 15 tumors contained at least one mtDNA mutation, wider sequencing coverage of the mitochondrial genome using NAF should allow us to identify a significant subset of breast cancers. We do not feel, based on our experience with sequencing
22% (3701/16 569) of the mitochondrial genome using NAF, that the amount of DNA in NAF will be limiting. Additional NAF biomarkers such as cytology, in which suspicious cells were detected in two women with breast cancer when an mtDNA mutation was not observed in NAF, should increase the sensitivity of NAF as a non-invasive breast cancer early detection tool.
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Notes
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* The first two authors contributed equally to this work. 
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Acknowledgments
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Supported in part by Department of Defense grant DAMD17-01-1-0426.
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Received June 26, 2004;
revised August 9, 2004;
accepted September 5, 2004.