Quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) using amplicon-fusion-site polymerase chain reaction (AFS-PCR)

Recently, we described a strategy for developing tumor cell specific PCRs for MYCN amplified neuroblastomas, using junction sites (amplicon fusion sites, AFS) of amplified genomic regions (ampGR) as template (AFS-PCR) [1]. All ampGR and thus, all AFS identified were absolute tumor cell specific and unique for each patient. AFS-PCR was highly sensitive and uncovered one tumor cell out of 106 - 107 control cells. We concluded this method is suitable for MRD quantification of MYCN amplified neuroblastoma. We furthermore hypothesized AFS-PCR might not only be limited to neuroblastoma, but transferable to other cancer types, provided that the individual tumor cells harbour ampGR.The detection and quantification of minimal amounts of residual or recurrent leukemic blasts significantly improved therapy management for adult and childhood acute lymphoblastic leukemia (ALL) patients [2-4]. Routinely, rearrangements in immunoglobulin-chain (Ig) or T-cell receptor genes (TCR) serve as template for the design of tumor cell specific PCRs (Ig/TCR-PCR) [4-7]. Amplification of a part of the long arm of chromosome 21 including the AML1/RUNX1 gene (iAMP21) occurs in 1-2% of ALL [8,9].Bone marrow specimens from initial diagnosis and subsequent time points of an ALL patient with iAMP21 were used to directly compare AFS-PCR to routinely used MRD diagnostic strategies and to give additional proof of concept for AFS-PCR as a method for tumor cell detection, not only for neuroblastoma.Multiple copies of AML1/RUNX1 had been identified by fluorescence-in-situ-hybridization (FISH) in a 9 year and 10 months old patient with precursor-B ALL. We confirmed AML1/RUNX1 to be part of a large amplified genomic region of chromomosome 21 (iAMP21) and excluded coamplified regions or additional ampGR on other chromosomes by whole genome Array (Affymetrix Cytogenetics Whole-Genome 2.7M Array) (Figure 1a). Besides iAMP21, several deletions were identified, with most relevant mapping to chromosomes 7p12.1-2