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 1

Identification and diagnostic use of AFSs in newly diagnosed malignomas.

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Identification and diagnostic use of AFSs in newly diagnosed malignomas....
The diagnostic algorithm combines the possibility of screening for ampGRs by whole-genome CGH analysis, the precision of HR-TA as a platform for subsequent base-exact AFS mapping, the absolute specificity of the AFSs for the respective malignoma cells, and the high sensitivity of real-time PCR. For malignomas with already described ampGRs, isolated genomic DNA can be hybridized directly on the corresponding HR-TA, covering the region of interest. If no ampGRs are described, or common ampGRs are not found in preliminary investigations, whole-genome CGH analysis could be used to screen for regions with ampGRs for further fine mapping on a HR-TA. Once the borders of the ampGRs are mapped, AFS-PCR, and subsequently AFS-RQ-PCR, can be designed. AFS-PCR can be used as a feasible tool in the initial staging of malignoma diseases and, furthermore, for highly sensitive and absolutely specific tracking of remaining or recurrent malignoma cells during the course of the diseases.