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Clinical Chemistry 55: 559-567, 2009. First published January 8, 2009; 10.1373/clinchem.2008.108498
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(Clinical Chemistry. 2009;55:559-567.)
© 2009 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Multimarker Circulating DNA Assay for Assessing Blood of Prostate Cancer Patients

Eiji Sunami1, Masaru Shinozaki1, Celestia S. Higano3, Robert Wollman4, Tanya B. Dorff5, Steven J. Tucker6, Steve R. Martinez1, Frederick R. Singer2 and Dave S.B. Hoon1,a

1 Department of Molecular Oncology and 2 the Breast and Endocrine Program, John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica, CA; 3 Department of Medicine, Division of Oncology, University of Washington and Seattle Cancer Care Alliance, Seattle, WA; 4 Department of Radiation Oncology, Saint John’s Health Center, Santa Monica, CA; 5 The Angeles Clinic and Research Institute, Santa Monica, CA; 6 Pacific Cancer Center, Paragon, Singapore.

aAddress correspondence to this author at: Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404. Fax (310) 449-5282; e-mail hoon{at}jwci.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Prostate cancer (PCa) detection using serum-based prostate specific antigen (PSA) is limited by frequent false-positive and -negative results. Genetic aberrations such as allelic imbalance (AI) and epigenetic changes such as promoter hypermethylation have been detected in circulating DNA of cancer patients. We hypothesized that circulating multimarker DNA assays detecting both genetic and epigenetic markers in serum would be useful in assessing PCa patients.

Methods: We assayed blood from healthy male donors (n = 40) and 83 patients with American Joint Cancer Committee (AJCC) stage I–IV PCa. DNA was assayed for AI of 6 genome microsatellites. We assessed methylation of RASSF1, RARB2, and GSTP1 using a methylation-specific PCR assay and analyzed the sensitivity of each assay for the detection of genetic or epigenetic changes in circulating DNA. The relation between circulating tumor-related DNA detection and prognostic factors was investigated.

Results: The proportion of patients demonstrating AI for ≥1 marker was 47% (38 of 81 patients). Methylation biomarkers were detected in 24 of 83 patients (28%). By combining 2 DNA assays, the number of PCa patients positive for ≥1 methylated or LOH marker increased (52 of 83; 63%). The combined assays detected PCa in 15 of 24 patients (63%) with normal PSA concentrations. The combination of the DNA assays detected the presence of PCa regardless of AJCC stage or PSA concentration. Combination of the DNA and PSA assays gave 89% sensitivity.

Conclusions: This pilot study demonstrates that the combined circulating DNA multimarker assay identifies patients with PCa and may yield information independent of AJCC stage or PSA concentration.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer (PCa)1 is the most frequently diagnosed malignancy in men in the US. The 2007 estimates from the American Cancer Society predicted 218 890 new cases of PCa and 27 050 deaths (1). Although serum concentration of prostate-specific antigen (PSA) remains important for prognosis and diagnosis (2), the PSA assay is not particularly accurate (3)(4)(5)(6): up to 80% of patients with an increased PSA concentration will have negative biopsy results, and up to 25% of patients with a normal serum PSA (<4 µg/L) will ultimately have biopsy evidence of PCa (7). Highly specific, minimally invasive techniques that are cost effective for screening and monitoring would facilitate earlier diagnosis of PCa and may also reduce the number of unnecessary biopsies.

The progression of PCa and other malignancies is characterized by increased genetic and epigenetic aberrations not usually found in normal DNA (8)(9)(10)(11)(12). Of particular interest is allelic imbalance (AI) in a genomic region, which may affect cellular regulatory genes, tumor suppressor genes, or oncogenes (13)(14). An equally important epigenetic change is hypermethylation of tumor-related genes in the promoter region, which can turn off genes and significantly affect the initiation and progression of neoplasia (15)(16). Both genetic and epigenetic changes can be measured in tissue or body fluids, but only the latter specimen type is useful for serial assessments and minimally invasive screening (17)(18)(19). Thus far, however, most investigations of DNA biomarkers in patients with PCa have focused on tissue assessment and have measured a single type of genetic aberration using 1 platform (13)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35).

In this pilot study, we hypothesized that a dual-assay system based on both genetic and epigenetic alterations in multiple microsatellite and methylation markers would be more sensitive for detection of PCa than a single-assay, single-marker approach. We assessed AI and hypermethylation in circulating DNA from the serum of patients with American Joint Cancer Committee (AJCC) stage I, II, III, and IV PCa. AI at loci on chromosomes 6q, 8p, 9p, 10p, and 18q (13)(20)(21)(22)(23)(24)(25)(26) was evaluated by assessing loss of DNA microsatellites. The epigenetic biomarkers evaluated were 3 tumor suppressor genes that are frequently hypermethylated in PCa: GSTP1 (glutathione S-transferase {pi}1),2 RARB2 (retinoic acid receptor β, variant 2), and RASSF1 [Ras association (RalGDS/AF-6) domain family member 1] (28)(29)(30)(31)(32)(33)(34).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
specimen collection and dna isolation
Institutional Review Board approval was obtained from John Wayne Cancer Institute at Saint John’s Health Center, Santa Monica, CA, and the University of Washington, Seattle, WA. All patients provided signed, written informed consent to participate in this study. We enrolled 83 patients receiving treatment for PCa at John Wayne Cancer Institute, The Angeles Clinic and Research Institute, and the University of Washington. Patient characteristics are presented in Table 1 . PCa patients represented AJCC stage I (n = 3), stage II (n = 15), stage III (n = 7), or stage IV (n = 58).


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  		Table 1. Patient characteristics.

We collected 10 mL blood from each patient in a serum separator tube and processed it immediately as follows: separated by centrifugation (1000g, 15 min), filtered through a 13-mm serum filter (Fisher Scientific), separated into aliquots, and cryopreserved at –80 °C. DNA was extracted and processed from serum as previously described (36). Briefly, 500 µL of serum was diluted with 0.9 mol/L NaCl2, SDS, and proteinase K (Qiagen) and incubated at 50 °C for 3 h. An equal volume of phenol–chloroform–isoamyl alcohol (25:24:1) was then added and the sample was vortex-mixed vigorously. After centrifugation at 1000g for 10 min, the aqueous layer was collected. DNA was precipitated using pellet paint NF coprecipitant (Novagen) and isopropanol (36). Extracted DNA from serum was subjected to sodium bisulfite modification (17). Briefly, DNA from 500 µL of serum was supplemented with 1 µg salmon sperm DNA (Sigma) and denatured in 0.3 mol/L NaOH for 3 min at 95 °C. Overall, 550 µL of a 2.5 mol/L sodium bisulfite and 125 mmol/L hydroquinone solution were added. Samples were incubated in the dark for 3 h at 60 °C. Salts were removed using the Wizard DNA Clean-Up System (Promega) and desulfonated in 0.3 mol/L NaOH at 37 °C for 15 min. Modified serum DNA was prepared and stored at –30 °C. We performed DNA quantification on all serum specimens using the PicoGreen quantification assay (Molecular Probes) (17).

Genomic DNA isolated from the peripheral blood lymphocytes of all PCa patients served as internal controls for AI. Additionally, DNA isolated from the serum of 40 healthy donors served as normal controls for the assessment of tumor-related gene hypermethylation. DNA quantification was performed using the PicoGreen assay (Molecular Probes) according to manufacturer directions. Samples were run in triplicate, and the results are expressed as the mean concentration for each patient.

microsatellite analysis
The following microsatellite markers and their corresponding chromosomal loci were evaluated for AI: D6S286 at 6q14, D8S261 at 8p22, D8S262 at 8p23, D9S171 at 9p21, D10S591 at 10p15, and D18S70 at 18q23. Forward primer sets were labeled with WellRed phosphoramidite-linked dye (Research Genetics, Inc.). PCR reactions were performed in 10-µL volumes with 1 µL template for 40 cycles of 30 s at 94 °C, 30 s at 55 °C, and 30 s at 72 °C, followed by a final extension of 7 min at 72 °C, as described (18). We performed post-PCR product separation using capillary array electrophoresis (CAE) (CEQ 8000XL; Beckman Coulter Inc.). AI was defined when a ≥40% reduction in serum DNA allele peak intensity was noted compared to the respective patient’s normal lymphocyte DNA allele peak using CEQ 8000XL software.

bisulfite modification and methylation-specific pcr
Extracted DNA from serum was subjected to sodium bisulfite modification (17). DNA from 500 µL serum was supplemented with 1 µg salmon sperm DNA (Sigma) and denatured in 0.3 mol/L NaOH for 3 min at 95 °C. Overall, 550 µL of a 2.5 mol/L sodium bisulfite and 125 mmol/L hydroquinone solution were added. Samples were incubated in the dark for 3 h at 60 °C. Salts were removed using the Wizard DNA Clean-Up System (Promega) and desulfonated in 0.3 mol/L NaOH at 37 °C for 15 min. Modified serum DNA was prepared and stored at –30 °C.

We assessed the methylation status of GSTP1, RARB2, and RASSF1 using methylation-specific PCR (MSP) using 2 sets of fluorescent-labeled primers specifically designed to amplify methylated and unmethylated DNA sequences, respectively. In total, 100 ng bisulfite-modified DNA was subjected to PCR amplification in a final reaction volume of 10 µL containing PCR buffer, 2.5–4.5 mmol/L MgCl2, dNTPs, 0.3 µmol/L primers, 0.5 U AmpliTaq gold polymerase (Applied Biosystems), and 50 pmol of each forward (F) and reverse (R) primer set for methylated (M) and unmethylated (U) primers as follows: RARB2, (M) F-GAACGCGAGCGATTCGAGT and R-GACCAATCCAACCGAAACG, (U) F-GGATTGGGATGTTGAGAATGT and R-CAACCAATCCAACCAAAACAA; RASSF1, (M) F-GTGTTAACGCGTTGCGTATC and R-AACCCCGCGAACTAAAAACGA, (U) F-TTTGGTTGGAGTGTGTTAATGTG and R-CAAACCCCACAAACTAAAAACAA; GSTP1, (M) F-TTCGGGGTGTAGCGGTCGTC and R-GCCCCAATACTAAATCACGACG, (U) F-GATGTTTGGGGTGTAGTGGTTGTT and R-CCACCCCAATACTAAATCACAACA. We carried out PCR after optimizing annealing temperatures for each primer set (60 °C for RASSF1 and 59 °C for RARB2 and GSTP1). An initial 10-min incubation at 95 °C was followed by 40 cycles of denaturation at 95 °C for 30 s, annealing for 30 s, and extension at 72 °C for 30 s, and a final hold for 7 min at 72 °C. Sodium bisulfite–modified DNA from healthy donors’ lymphocytes and {phi}-29–treated genomic DNA served as unmethylated negative control DNA for each PCR reaction, whereas lymphocytes treated with SssI Methylase (New England Biolabs) followed by sodium bisulfite modification served as positive methylated control DNA (37). Molecular biology–grade water without DNA served as a reagent control. Post-PCR products were separated and assessed using CAE. CAE was performed in a 96-well microplate format, combining 1 µL of each methylated and unmethylated PCR reaction product with 40 µL loading buffer and 0.5 µL dye-labeled size standard (Beckman Coulter) in a single well. Forward MSP primers were labeled with D4pa dye, whereas forward unmethylated specific primers were labeled with D2pa dye. This permitted discrimination of the 2 peaks for direct comparison of post-PCR methylated and unmethylated products in a single run. CEQ 8000XL software was used to determine PCR product signal intensity.

statistical analysis
Descriptive statistics, such as mean, SD, median, frequency, and percentage, were used to summarize patient characteristics, AI status, and gene hypermethylation status. We used ANOVA with the Dunnett method for posthoc testing (for continuous variables) and the {chi}2 test (for categorical variables) for comparing clinical factors among tumors demonstrating AI vs heterozygous alleles or hypermethylation vs nonmethylation. Statistical analysis was by JMP software (SAS). All statistical tests were 2-sided, with significant P values <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
loss of heterozygosity in circulating dna
We evaluated the presence of AI in serum circulating tumor-associated DNA from PCa patients using 6 microsatellite markers known to demonstrate frequent loss of heterozygosity (LOH) in PCa. The number of informative patient samples for any 1 microsatellite marker ranged from 33% (27 informative patient samples) for D8S261 to 70% (58 informative patient samples) for D18S70. The most common microsatellite for AI was D6S286, which was detected in 14 of 45 (31%) informative patient samples. This was followed by D8S262 (11 of 41, 27%), D8S261 (7 of 27, 26%), D10S591 (11 of 45, 24%), D9S171 (9 of 46, 20%), and D18S70 (11 of 58, 19%). Of patient samples with at least 1 informative marker (81 cases), 20 (25%) had only 1 LOH marker detected in the serum sample, 13 samples (16%) had 2 LOH markers detected, and 5 samples (6%) had 3 or more LOH markers detected. The proportion of informative patient samples demonstrating AI for ≥1 marker was 47% (38 of 81 patient samples; Fig. 1A ). Healthy male donors (n = 40) did not have these specific circulating LOH markers in their sera. Detection of AI correlated with AJCC PCa stage overall. There were no significant differences in AI status between individual stages.


Figure 1
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  		Figure 1. Positivity ratio of LOH and methylation markers.

(A), AI positive (%). (B), methylation positive (%).

hypermethylation of tumor-related genes
We assessed serum circulating DNA from PCa patients for hypermethylation of the tumor-related genes RASSF1, GSTP1, and RARB2 (Supplemental Fig. 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol55/issue3), which have shown to be hypermethylated in primary prostate tumors (28)(29)(30)(31)(32)(33)(34). The most frequently detected methylated gene circulating in serum was RASSF1, detected in 20 of 83 (24%) patient samples. RARB2 and GSTP1 were detected in 10 of 83 (12%) and 11 of 83 (13%) patient samples, respectively. The proportion of patient samples demonstrating hypermethylated circulating DNA of only 1 tumor-related biomarker was 17% (14 of 83), whereas 4% (3 of 83) and 8% (7 of 83) demonstrated 2 and 3 hypermethylated DNA markers, respectively. The combination of all 3 methylation markers increased the detection rate to 24 of 83 (29%) PCa patients (Fig. 1BUp ). Healthy male donors did not have these specific circulating methylated DNA markers in their sera under optimal assay conditions.

circulating loh and methylated dna correlation with serum psa
To assess the clinical relevance of AI and methylation, we correlated LOH and hypermethylation with serum PSA. We assessed blood drawn for serum LOH and for serum PSA in 81 of 83 (98%) patients. No significant differences were seen in the mean serum PSA concentrations among patients with AI of D9S171, D10S591, D8S261, D6S286, D8S262, and D18S70 and heterozygous patients. The mean serum PSA concentration in patients with hypermethylated tumor-related genes (RASSF1, RARB2, and GSTP1) was significantly higher than in patients without hypermethylated genes (P = 0.002, 0.017, and <0.0001, respectively). Similarly, patients with ≥1 hypermethylated tumor-related gene had serum PSA concentrations that were significantly higher than patients without circulating hypermethylated DNA (P = 0.002; Fig. 2 ).


Figure 2
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  		Figure 2. Methylation status and serum PSA concentrations (ng/mL).

(A), RASSF1. (B), RARB2. (C), GSTP1. (D), Any methylation marker.

correlation of gleason score with circulating dna loh and methylation
Primary PCa tumor Gleason score also correlated with AI and methylation status. The Gleason score was available in 70 (84%) of 83 patients. There were no significant differences between the mean Gleason score in patients with AI of D9S171, D8S261, D6S286, D8S262, and D18S70 and that of heterozygous patients. The mean PCa Gleason score in patients with hypermethylated RASSF1, RARB2, and GSTP1 was significantly higher (P = 0.017, 0.042, and 0.016, respectively) than in patients without hypermethylated DNA. Patients with ≥1 hypermethylated circulating tumor-related gene had tumors with significantly higher Gleason scores than patients without hypermethylated DNA (P = 0.019; Table 2 ).


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  		Table 2. Serum methylation and AI biomarkers vs Gleason score.

correlation of ajcc stage with circulating dna methylation
Detection of circulating methylated tumor-related DNA also correlated with AJCC PCa stage. Circulating methylated DNA of RARB2 and GSTP1 in serum was more common with increasing AJCC stage. Stage IV patients showed significantly more frequent hypermethylation of RARB2 and GSTP1 than stage I, II, and III patients (P = 0.027 and 0.019, respectively). Circulating methylated DNA of RARB2 and GSTP1 was not seen in stage I, II, or III patients (Table 3 ).


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  		Table 3. Methylation status and AJCC stage.

combination of circulating loh and methylated dna
Neither LOH nor hypermethylated DNA markers alone were able to identify all patients with PCa. By combining the 2 circulating DNA assays, the number of PCa patients positive for ≥1 methylation or LOH marker increased to 52 of 83 (63%). Of the 81 patients with available serum PSA (closest analysis to blood draw for DNA assays), 24 (30%) had concentrations considered to be at low risk for a PCa diagnosis (<4 µg/L). By combining the MSP and LOH assays, we identified 15 of 24 (63%) PCa cases not detected by PSA concentration alone (Fig. 3A ). The dual platform multimarker assays detected circulating tumor-related DNA with similar sensitivity, regardless of AJCC stage. Methylation or LOH of circulating tumor-related DNA was detected in 18 of 25 (72%) stage I/II/III and 34 of 58 (59%) stage IV PCa patients (Fig. 3B ). In 57 (70%) of the 81 PCa patients, PSA >4 µg/L was detected. Overall, the combination of the DNA assays and PSA assay positivity in the analysis of 81 PCa patients gave a sensitivity of 89%.


Figure 3
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  		Figure 3. Combination of circulating LOH and methylated DNA biomarkers.

Comparison of all patients and patients with PSA ≤4 µg/L (A) and AJCC stages I–III and stage IV (B).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The evaluation of serum for PCa-associated genetic and epigenetic changes has several advantages. Unlike tissue biopsy, blood sampling is minimally invasive, has little or no morbidity, and can be repeated to monitor changes in disease or detect recurrence. Additionally, the use of multiple molecular markers based on appropriate tumor-related genes minimizes the false-positive rate (19). Currently, the only blood-based PCa screening method is serum PSA measurement, which is limited in its utility because of the high rate of false positives and false negatives (3)(4)(5)(6).

Our study of circulating DNA from patients with PCa examined 6 AI markers associated with chromosomes 6q, 8p, 9p, 10p, and 18q. In primary PCa tissue, AI has been reported on these chromosomes (13)(20)(21)(22)(23)(24)(25)(26) at frequencies that vary with tumor stage/Gleason score and with assay parameters; however, very few studies have examined AI in body fluid specimens from patients with PCa. A recent report demonstrated 0%–18% rates of LOH for various chromosomal loci in plasma DNA; 44% of specimens tested positive for at least 1 of 15 LOH markers (27). By comparison, 47% of analyzed serum specimens in our study tested positive for at least 1 of 6 LOH markers. The similar rates of positive markers in these 2 independent multimarker studies suggest that LOH may be detecting patients having a specific clinical status.

Our study also examined hypermethylation of the promoter region for 3 tumor-related genes. GSTP1, RASSF1, and RARB2 are reportedly hypermethylated in 36%–99%, 53%–96%, and 53%–75% of PCa tissue specimens, respectively (28)(29)(30)(31)(32)(33)(34). GSTP1 is hypermethylated in 15%–52% of serum specimens from patients with PCa (38)(39), but there are no published reports on hypermethylation of RASSF1A and RARB2 in circulating DNA. We found that GSTP1, RASSF1, and RARB2 were hypermethylated in 13% (11 of 83), 24% (20 of 83), and 12% (10 of 83) of serum specimens from PCa patients, respectively. In combination, the 3 markers identified 29% of patients with PCa.

A DNA marker’s half-life and clearance from the bloodstream may impact its clinical utility. Similar to other malignancies, PCa is characterized by genetic and epigenetic heterogeneity, particularly during tumor progression (13)(23)(24)(29)(30). Hoque et al. (40) used a 9-marker panel to detect promoter region hypermethylation in DNA obtained from the urine sediment of PCa patients. Although 5 of these markers were also positive in the urine of normal controls, the authors proposed that a combination of CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); alias, p16], ARF (ADP-ribosylation factor), MGMT (O-6-methylguanine-DNA methyltransferase), and GSTP1 could theoretically identify 87% of patients with PCa. While these results are impressive, their diagnostic and prognostic utility remains to be assessed.

The average DNA amount extracted from serum of healthy donors was about 260 µg/L (17) using our method. The amount of DNA extracted from the sera of prostate cancer patients was 2- to 3-fold higher than the amount from healthy donors. Therefore, for methylation analysis, lack of hypermethylation meant that not only could we not detect any methylation peak, but also that an unmethylated peak could be detected by CAE; the latter is a built-in control for the presence of DNA. In LOH analysis of circulating DNA, the detection of LOH is more difficult to assess because circulating DNA from cancer patients contains DNA from both normal (no LOH) and cancer cells (with LOH). In our study, neither hypermethylation nor LOH was detected in circulating DNA from normal donors, confirming the high specificity and positive predictive value of our dual-platform assay. To be precise, the number of normal donors assessed was 40, so at a 95% CI the assay has specificity between 91% and 100%. We attempted to bring clinical relevance to our assay by correlating genetic and epigenetic changes to known diagnostic and prognostic factors, such as Gleason score and AJCC stage, which are independent of serum. We could not detect a significant correlation between Gleason score and any AI; however, each methylation-positive group (RASSF1, RARB2, GSTP1, and any 1 methylated marker) showed significantly higher mean Gleason scores than each methylation-negative group (P = 0.017, P = 0.042, P = 0.016, and P = 0.02, respectively). The correlation of higher mean Gleason scores to methylation of tumor-related genes indicates that more advanced or aggressive tumors were likely to harbor these epigenetic aberrations.

We could not detect a significant correlation between PSA concentration and any AI; however, each methylation-positive group (RASSF1, RARB2, GSTP1, and any 1 methylated marker) showed significantly higher mean PSA concentrations (P = 0.002, P = 0.017, P < 0.001, and P = 0.002, respectively). These patients represent the portion of PCa cases that may be missed if diagnosis is based on serum PSA in the absence of a palpable mass on digital rectal examination. Among the 30% of patients with a normal serum PSA concentration (<4 µg/L), 54% had at least 1 positive microsatellite marker, 17% had at least 1 positive methylation marker, and 63% tested positive for PCa based on dual-platform assay criteria.

AI was independent of AJCC stage of PCa, possibly because AJCC staging considers nodal and distant metastasis as well as the primary tumor. Thus the primary tumor of a patient with AJCC stage I/II PCa may have a high Gleason score. Also, tissue studies show that AI is often independent of AJCC T-stage but may be inversely linked to invasive potential (23)(29). In our study, no single LOH marker identified all patients with PCa, and no combination of LOH markers identified more than 47% of cases. In contrast, the frequency of hypermethylation increased directly with AJCC stage of disease. The observation that RARB2 and GSTP1 were methylated only in patients with stage IV disease is consistent with a putative role for these genes in invasion and metastasis.

As hypothesized, the combination of LOH and methylation assays was more informative than either assay alone. At least 1 marker of LOH or methylation was detected in circulating DNA from 63% of patients (52 of 83) with PCa, compared with 16% for LOH analysis alone and 34% for methylation analysis alone. As expected, the range of our dual-platform, multimarker assay compensated not only for the expected heterogeneity of tumor cells (13)(23)(24)(29)(30) but also for varying rates of clearance for free circulating DNA. Our approach may represent a useful complement to serum PSA for the diagnosis or surveillance of PCa. In our study, PSA alone detected 71% of PCa cases, whereas the combination of PSA, LOH, and methylation detected 89% of PCa cases without increasing the rate of false-positive results.

Several factors may improve the overall sensitivity and specificity of our dual-platform molecular assay. Serial assessment of patient blood may provide additional sensitivity and may be particularly helpful for early-stage disease and detection of recurrence. Different/additional methylation and LOH markers may increase the assay’s ability to accommodate tumor heterogeneity and differences in the clearance rates of various lengths of circulating tumor-related DNA. Although the assay had limited correlation with known diagnostic and prognostic factors of PCa, its sensitivity in patients with early-stage disease merits its incorporation into prospective treatment protocols, where it can be investigated as a monitoring tool. The assays may be of clinical utility in monitoring patients receiving treatment or those with potentially aggressive disease.


   Acknowledgments
 
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors’ Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: None declared.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: US Department of Defense prostate grant award number DAMD17-03-1-0261 and the Associates for Breast and Prostate Cancer Studies.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played a direct role in the design of study, choice of enrolled patients, review and interpretation of data, and preparation and approval of manuscript.

Acknowledgments: We thank the Molecular Core and Database/specimen coordinator personnel of the Department of Molecular Oncology.


   Footnotes
 
1 Nonstandard abbreviations: PCa, prostate cancer; PSA, prostate-specific antigen; AI, allelic imbalance; AJCC, American Joint Cancer Committee; CAE, capillary array electrophoresis; MSP, methylation-specific PCR; LOH, loss of heterozygosity.

2 Human genes: GSTP1, glutathione S-transferase {pi}1; RARB2, retinoic acid receptor β, variant 2; RASSF1, Ras association (RalGDS/AF-6) domain family member 1; CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) (alias, p16); ARF, ADP-ribosylation factor; MGMT, O-6-methylguanine-DNA methyltransferase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43-66.[Abstract/Free Full Text]
  2. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, Morrow M. AJCC cancer staging manual 6th ed. 2002:421 p. Springer-Verlag New York. .
  3. Beatty PA. Prostate cancer with low PSA levels. N Engl J Med 2004;351:1802-1803.[Free Full Text]
  4. Bock JL, Klee GG. How sensitive is a prostate-specific antigen measurement? How sensitive does it need to be?. Arch Pathol Lab Med 2004;128:341-343.[Web of Science][Medline] [Order article via Infotrieve]
  5. Pfister C, Basuyau JP. Current usefulness of free/total PSA ratio in the diagnosis of prostate cancer at an early stage. World J Urol 2005;23:236-242.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  6. Thompson IM, Pauler DK, Goodman PJ, Tangen CM, Lucia MS, Parnes HL, et al. Prevalence of prostate cancer among men with a prostate-specific antigen level < or =4.0 ng per milliliter. N Engl J Med 2004;350:2239-2246.[Abstract/Free Full Text]
  7. American Cancer Society. Prostate cancer: early detection. http://www.cancer.org/docroot/CRI/content/CRI_2_6x_Prostate_Cancer_Early_Detection.asp?sitearea=&level (Accessed January 2009)..
  8. Dahiya R, Lee C, McCarville J, Hu W, Kaur G, Deng G. High frequency of genetic instability of microsatellites in human prostatic adenocarcinoma. Int J Cancer 1997;72:762-767.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. Dong JT, Boyd JC, Frierson HF, Jr. Loss of heterozygosity at 13q14 and 13q21 in high grade, high stage prostate cancer. Prostate 2001;49:166-171.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  10. Karan D, Schmied BM, Dave BJ, Wittel UA, Lin MF, Batra SK. Decreased androgen-responsive growth of human prostate cancer is associated with increased genetic alterations. Clin Cancer Res 2001;7:3472-3480.[Abstract/Free Full Text]
  11. Karan D, Lin MF, Johansson SL, Batra SK. Current status of the molecular genetics of human prostatic adenocarcinomas. Int J Cancer 2003;103:285-293.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  12. Canto EI, Shariat SF, Slawin KM. Molecular diagnosis of prostate cancer. Curr Urol Rep 2004;5:203-211.[CrossRef][Medline] [Order article via Infotrieve]
  13. von Knobloch R, Konrad L, Barth PJ, Brandt H, Wille S, Heidenreich A, et al. Genetic pathways and new progression markers for prostate cancer suggested by microsatellite allelotyping. Clin Cancer Res 2004;10:1064-1073.[Abstract/Free Full Text]
  14. Konishi N, Nakamura M, Kishi M, Ishida E, Shimada K, Matsuyoshi S, et al. Genetic mapping of allelic loss on chromosome 6q within heterogeneous prostate carcinoma. Cancer Sci 2003;94:764-768.[CrossRef][Medline] [Order article via Infotrieve]
  15. Harden SV, Sanderson H, Goodman SN, Partin AA, Walsh PC, Epstein JI, Sidransky D. Quantitative GSTP1 methylation and the detection of prostate adenocarcinoma in sextant biopsies. J Natl Cancer Inst 2003;95:1634-1637.[Abstract/Free Full Text]
  16. Singal R, Ferdinand L, Reis IM, Schlesselman JJ. Methylation of multiple genes in prostate cancer and the relationship with clinicopathological features of disease. Oncol Rep 2004;12:631-637.[Web of Science][Medline] [Order article via Infotrieve]
  17. Mori T, O'Day SJ, Umetani N, Martinez SR, Kitago M, Koyanagi K, et al. Predictive utility of circulating methylated DNA in serum of melanoma patients receiving biochemotherapy. J Clin Oncol 2005;23:9351-9358.[Abstract/Free Full Text]
  18. Fujimoto A, O'Day SJ, Taback B, Elashoff D, Hoon DS. Allelic imbalance on 12q22–23 in serum circulating DNA of melanoma patients predicts disease outcome. Cancer Res 2004;64:4085-4088.[Abstract/Free Full Text]
  19. Mori T, Martinez SR, O'Day SJ, Morton DL, Umetani N, Kitago M, et al. Estrogen receptor-alpha methylation predicts melanoma progression. Cancer Res 2006;66:6692-6698.[Abstract/Free Full Text]
  20. Cooney KA, Wetzel JC, Consolino CM, Wojno KJ. Identification and characterization of proximal 6q deletions in prostate cancer. Cancer Res 1996;56:4150-4153.[Abstract/Free Full Text]
  21. Verhagen PC, Hermans KG, Brok MO, van Weerden WM, Tilanus MG, de Weger RA, et al. Deletion of chromosomal region 6q14–16 in prostate cancer. Int J Cancer 2002;102:142-147.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  22. Lu W, Takahashi H, Furusato B, Maekawa S, Ikegami M, Sudo A, et al. Allelotyping analysis at chromosome arm 8p of high-grade prostatic intraepithelial neoplasia and incidental, latent, and clinical prostate cancers. Genes Chromosomes Cancer 2006;45:509-515.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  23. Matsuyama H, Pan Y, Oba K, Yoshihiro S, Matsuda K, Hagarth L, et al. Deletions on chromosome 8p22 may predict disease progression as well as pathological staging in prostate cancer. Clin Cancer Res 2001;7:3139-3143.[Abstract/Free Full Text]
  24. Perinchery G, Bukurov N, Nakajima K, Chang J, Li LC, Dahiya R. High frequency of deletion on chromosome 9p21 may harbor several tumor-suppressor genes in human prostate cancer. Int J Cancer 1999;83:610-614.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  25. Trybus TM, Burgess AC, Wojno KJ, Glover TW, Macoska JA. Distinct areas of allelic loss on chromosomal regions 10p and 10q in human prostate cancer. Cancer Res 1996;56:2263-2267.[Abstract/Free Full Text]
  26. Jenkins R, Takahashi S, DeLacey K, Bergstralh E, Lieber M. Prognostic significance of allelic imbalance of chromosome arms 7q, 8p, 16q, and 18q in stage T3N0M0 prostate cancer. Genes Chromosomes Cancer 1998;21:131-143.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  27. Schwarzenbach H, Chun FK, Lange I, Carpenter S, Gottberg M, Erbersdobler A, et al. Detection of tumor-specific DNA in blood and bone marrow plasma from patients with prostate cancer. Int J Cancer 2007;120:1465-1471.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  28. Liu L, Yoon JH, Dammann R, Pfeifer GP. Frequent hypermethylation of the RASSF1A gene in prostate cancer. Oncogene 2002;21:6835-6840.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Woodson K, Gillespie J, Hanson J, Emmert-Buck M, Phillips JM, Linehan WM, Tangrea JA. Heterogeneous gene methylation patterns among pre-invasive and cancerous lesions of the prostate: a histopathologic study of whole mount prostate specimens. Prostate 2004;60:25-31.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  30. Maruyama R, Toyooka S, Toyooka KO, Virmani AK, Zochbauer-Muller S, Farinas AJ, et al. Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clin Cancer Res 2002;8:514-519.[Abstract/Free Full Text]
  31. Kang GH, Lee S, Lee HJ, Hwang KS. Aberrant CpG island hypermethylation of multiple genes in prostate cancer and prostatic intraepithelial neoplasia. J Pathol 2004;202:233-240.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  32. Nakayama T, Watanabe M, Yamanaka M, Hirokawa Y, Suzuki H, Ito H, et al. The role of epigenetic modifications in retinoic acid receptor beta2 gene expression in human prostate cancers. Lab Invest 2001;81:1049-1057.[Web of Science][Medline] [Order article via Infotrieve]
  33. Enokida H, Shiina H, Urakami S, Igawa M, Ogishima T, Li LC, et al. Multigene methylation analysis for detection and staging of prostate cancer. Clin Cancer Res 2005;11:6582-6588.[Abstract/Free Full Text]
  34. Bernardini S, Miano R, Iori R, Finazzi-Agro E, Palmieri G, Ballerini S, et al. Hypermethylation of the CpG islands in the promoter region of the GSTP1 gene in prostate cancer: a useful diagnostic and prognostic marker?. Clin Chim Acta 2004;350:181-188.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  35. Muller I, Beeger C, Alix-Panabieres C, Rebillard X, Pantel K, Schwarzenbach H. Identification of loss of heterozygosity on circulating free DNA in peripheral blood of prostate cancer patients: potential and technical improvements. Clin Chem 2008;54:688-696.[Abstract/Free Full Text]
  36. Hoon DS, Spugnardi M, Kuo C, Huang SK, Morton DL, Taback B. Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients. Oncogene 2004;23:4014-4022.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  37. Umetani N, de Maat MF, Mori T, Takeuchi H, Hoon DS. Synthesis of universal unmethylated control DNA by nested whole genome amplification with phi29 DNA polymerase. Biochem Biophys Res Commun 2005;329:219-223.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  38. Jeronimo C, Usadel H, Henrique R, Silva C, Oliveira J, Lopes C, Sidransky D. Quantitative GSTP1 hypermethylation in bodily fluids of patients with prostate cancer. Urology 2002;60:1131-1135.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  39. Ellinger J, Haan K, Heukamp LC, Kahl P, Buttner R, Muller SC, et al. CpG Island hypermethylation in cell-free serum DNA identifies patients with localized prostate cancer. Prostate 2008;68:42-49.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  40. Hoque MO, Topaloglu O, Begum S, Henrique R, Rosenbaum E, Van Criekinge W, et al. Quantitative methylation-specific polymerase chain reaction gene patterns in urine sediment distinguish prostate cancer patients from control subjects. J Clin Oncol 2005;23:6569-6575.[Abstract/Free Full Text]



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