PURPOSE: To advance the characterization of tumor-associated cell-free DNA in blood and bone marrow (BM), a rapid profiling method using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was established. MS-MLPA detects genetic and epigenetic aberrations of 37 tumor suppressor genes (TSG) in a single reaction and might, therefore, avoid the cumbersome single gene analyses. METHODS: The validity of MS-MLPA for using cell-free plasma DNA was assessed by analyzing blood and BM samples of 91 patients with prostate cancer. As reference analyses, the methylation patterns of 4 genes (CD44, E-cadherin, CDKN2A and PTEN) chosen from the TSG set of the MS-MLPA kit were investigated in single reactions by sodium bisulfite DNA sequencing. RESULTS: Copy number changes and aberrant DNA methylation of 37 circulating TSG could be analyzed in BM and blood of 30 and 13 of the 91 patients, respectively, whereas the DNA content in the remaining samples was too low (<50 ng/μl of eluted DNA). The copy number of 28 of the 37 TSG was altered, and most changes were found for APC, CHFR, TP73 and GSTP1 genes in BM plasma. Statistical evaluations showed an association between copy number changes of TP73 and a positive resection margin of the prostate (p = 0.05). Both MS-MLPA and sodium bisulfite sequencing techniques showed that all genes were unmethylated. CONCLUSIONS: Our results demonstrate the potential and limitation of MS-MLPA for multiplex characterization of TSG in cell-free plasma DNA as a new non-invasive approach to obtain information on the molecular tumor biology of individual cancer patients.
PURPOSE: To advance the characterization of tumor-associated cell-free DNA in blood and bone marrow (BM), a rapid profiling method using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was established. MS-MLPA detects genetic and epigenetic aberrations of 37 tumor suppressor genes (TSG) in a single reaction and might, therefore, avoid the cumbersome single gene analyses. METHODS: The validity of MS-MLPA for using cell-free plasma DNA was assessed by analyzing blood and BM samples of 91 patients with prostate cancer. As reference analyses, the methylation patterns of 4 genes (CD44, E-cadherin, CDKN2A and PTEN) chosen from the TSG set of the MS-MLPA kit were investigated in single reactions by sodium bisulfite DNA sequencing. RESULTS: Copy number changes and aberrant DNA methylation of 37 circulating TSG could be analyzed in BM and blood of 30 and 13 of the 91 patients, respectively, whereas the DNA content in the remaining samples was too low (<50 ng/μl of eluted DNA). The copy number of 28 of the 37 TSG was altered, and most changes were found for APC, CHFR, TP73 and GSTP1 genes in BM plasma. Statistical evaluations showed an association between copy number changes of TP73 and a positive resection margin of the prostate (p = 0.05). Both MS-MLPA and sodium bisulfite sequencing techniques showed that all genes were unmethylated. CONCLUSIONS: Our results demonstrate the potential and limitation of MS-MLPA for multiplex characterization of TSG in cell-free plasma DNA as a new non-invasive approach to obtain information on the molecular tumor biology of individual cancerpatients.
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