Detection of human tumor cells by amplicon fusion site polymerase chain reaction (AFS-PCR)
Axel Weber, … , Nina Merete Christiansen, Holger Christiansen
Axel Weber, … , Nina Merete Christiansen, Holger Christiansen
Published January 10, 2011
Citation Information: J Clin Invest. 2011;121(2):545-553. https://doi.org/10.1172/JCI44415.
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Technical Advance Oncology

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 × 106 to 8 × 106 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.

Authors

Axel Weber, Sylvia Taube, Sven Starke, Eckhard Bergmann, Nina Merete Christiansen, Holger Christiansen

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Figure 3

Identification of the AFS sequence in the type 1 amplicon of the KELLY cell line.

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Identification of the AFS sequence in the type 1 amplicon of the KELLY c...
(A) HR-TA data indicate the virtual telomeric and centromeric borders of the ampGR of the KELLY cell line by referring to the first and last amplified oligonucleotide. The relative copy number for each printed oligonucleotide is presented as fluorescent intensity of the Cy5-labeled test DNA (cell lines) normalized to the Cy3-labeled reference DNA (healthy human female) (log2 ratios). The mean signal intensity values of the continuous genomic regions calculated are indicated by red lines. The ampGR is highlighted green. (B) The last (red) and first (blue) 1,000 amplified bps are fused together, resulting in a virtual AFS. (C) Primers for a first AFS-PCR are designed up- and downstream of the virtual AFS. (D) Sequencing of the excised AFS fragment from both sides, using the previously designed primers, results in the exact AFS. (E) Simplified model of the architecture of a simple type 1 amplicon. ampGRs are subsequently joined together in head-to-tail orientation, resulting in a tumor cell–specific AFS.