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Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR).

ABSTRACT: Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cell–specific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets for cancer diagnostics. In this study, we mapped ampGRs around the proto-oncogene MYCN of human neuroblastomas using a high-resolution tiling array (HR-TA). Based on the HR-TA data, we were able to precisely describe the telomeric and centromeric borders of the ampGRs and deduce virtual fusion sites of the joined ampGRs (amplicon fusion sites [AFSs]). These AFSs served as blueprints for the subsequent design of AFS bridging PCR assays (AFS-PCRs). Strikingly, these assays were absolutely tumor cell specific and capable of detecting 1 tumor cell in 1 × 10(6) to 8 × 10(6) control cells. We successfully proved the in vivo practicability of AFS-PCR by detecting and quantifying the specific AFS DNA of human MYCN-amplified neuroblastomas in the patients’ corresponding peripheral blood and bone marrow samples. Thus, we believe AFS-PCR could become a powerful and nevertheless feasible personalized diagnostic tool applicable to a large number of cancer patients, including children with MYCN-amplified neuroblastomas.

SUBMITTER: Weber A

PROVIDER: S-EPMC3026730 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR).

Weber Axel A Taube Sylvia S Starke Sven S Bergmann Eckhard E Christiansen Nina Merete NM Christiansen Holger H

The Journal of clinical investigation 20110201 2

Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cell–specific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets f ...[more]

PMID: 21293059

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Project description:Gene promoter hypermethylation is a vital step in tumorigenesis. This paper set out to explore the use of polymerase chain reaction - surface-enhanced Raman spectroscopy (PCR-SERS) for the detection of gene methylation levels, with a focus on cancer diagnosis. Methods: PCR with methylation independent primers were used on DNA samples to amplify target genes regardless of their methylation states. SERS was used on the obtained PCR products to generate spectra that contained peak changes belonging to CG and AT base pairs. Multiple linear regression (MLR) was then used to deconvolute the SERS spectra so that the CG/AT ratios of the sample could be obtained. These MLR results were used to calculate methylation levels of the target genes. For protocol verification, three sets of seven reference DNA solutions with known methylation levels (0%, 1%, 5%, 25%, 50%, 75%, and 100%) were analysed. Clinically, blood plasma samples were taken from 48 non-small-cell lung cancer (NSCLC) patients and 51 healthy controls. The methylation levels of the genes p16, MGMT, and RASSF1 were determined for each patient using this method. Results: Verification experiment on the mixtures with known methylation levels resulted in an error of less than 6% from the actual levels. When applied to our clinical samples, the frequency of methylation in at least one of the three target genes among the NSCLC patients was 87.5%, but this percentage decreased to 11.8% for the control group. The methylation levels of p16 were found to be significantly higher in NSCLC patients with more pack-years smoked (p=0.04), later cancer stages (p=0.03), and cancer types of squamous cell and large cell versus adenocarcinoma (p=0.03). Prediction accuracy of 88% was achieved from classification and regression trees (CART) based on methylation levels and states, respectively. Conclusion: This research showed that the PCR-SERS protocol could quantitatively measure the methylation levels of genes in plasma. The methylation levels of the genes p16, MGMT, and RASSF1 were higher in NSCLC patients than in controls.

Dataset Information

Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR).

Publications

Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR).

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