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 4

Identification of AFS sequences in the type 2 amplicon of the IMR-32 cell line.

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Identification of AFS sequences in the type 2 amplicon of the IMR-32 cel...
(A) HR-TA data indicate the virtual telomeric and centromeric borders of 4 ampGRs that appear to be separated by nonamplified genomic regions. (B) ampGR order within a single building block. ampGR-2 is fused between ampGR-1 and ampGR-4 inversely, as identified by different combinations of PCR primers and subsequent sequencing. No combination of primers for the known ampGRs successfully integrated ampGR-3 in the amplicon. Most likely, additional ampGRs that were not covered by the HR-TA are adjacent to both sides of ampGR-3, avoiding a successful PCR. (C) Sequencing of the AFS-PCR products from both sides resulted in the exact AFS. In both AFSs, additional short sequences were found between the ampGR borders. In the AFS between ampGR-4 and ampGR-2 (inverse), an additional ampGR, 20 bp long, was identified. For shorter sequences (lower case) of both AFSs, it was not possible to identify the definite chromosomal origin. (D) Simplified model of the architecture of a complex type 2 amplicon. Different ampGRs are joined together in head-to-tail, head-to-head, or tail-to-tail orientation, resulting in different tumor cell–specific AFSs. The different ampGRs build single building blocks that are subsequently joined together. The entirety of all ampGRs corresponds to the whole, individual amplicon.