Our data show that AFS-PCR is a suitable method for quantification of MRD of tumor cells with ampGR and that it is not limited to neuroblastoma with MYCN-amplification. Despite the almost equal quantification results of AFS-PCR compared to FACS and IgH/TCR-qPCR at days 513 and 533, respectively, the data at day 15 and 29 differ between 5 and 10 fold MRD count. These differences might be explainable at least in part by the different quantification procedures. As described, quantification of MRD by AFS-PCR was calculated in relation to a control gene (Inhibin-Beta-B; INHBB). This is in contrast to quantification of IgH/TCR-qPCR by comparing the PCR values of the subsequent bone marrow samples in relation to a serial dilution of DNA from leukemic blasts from the initial diagnosis into control DNA [6, 7]. INHBB was chosen as control gene in this case of ALL because we have had the information about a numerically diploid karyotype and exclusion of INHBB beeing part of a small copy number variation (CNV) by whole-genome SNP Array in the leukemic blasts. However, although optimisation of qPCR, conditions for the relative quantification of the AFS-PCR against INHBB was performed accurately and although control INHBB-PCR and AFS-PCR both showed good efficiencies between 10-1 and 10-5 dilution steps (Additional file 3: Figure S1), it is not possible to exclude, that minor differences in PCR efficiencies could have influenced the quantification for day 15 and 29, respectively.
From the days 15 and 29 only a very small amount of dried bone marrow was obtainable for DNA isolation for AFS-PCR. Because of the low cell count to these time points, the whole specimens except a few bone marrow smears were used for routine diagnostics before. Indicating the low input DNA into AFS-qPCR, the Ct-values for the INHBB and the AFS-fragment, respectively, were significantly higher compared to those from the day of initial diagnosis or day 513 and day 533 (Additional file 4: Figure S2). The possibility to quantify MRD from such low amounts of input DNA indicates one great advantage for the relative quantification to a control gene, as with AFS-PCR, over the serial dilution method, which depends on an appointed amount of input DNA. However, with respect to differences in PCR efficiancies, the very low amount of input DNA in AFS-qPCR might also have a part in the lower MRD quantification values calculated for day 15 and day 29 as compared to IgH/TCR-qPCR.
Another advantage of AFS-PCR is the absolute specificity for the tumor cells in contrast to IgH/TCR-qPCR. In the latter, the tumor cell specificity varies, dependent on the degree of difference in the rearranged parts of the Ig/TCR genes of the leukemic blasts compared to untransformed B- and T-cells. It is known that the composition of the subpopulations of mononuclear white blood cells change during therapy, influencing the background signal and thereby the quantitative range of IgH/TCR-qPCR, most notably in samples with a low blast count . In contrast, based on the specific sequences to both side of the AFS, it was possible to design AFS-PCR without a significant background for most of the neuroblastoma patients investigated  and the head-to-head junctions of the iAMP21 presented here (Additional file 4: Figure S2a).
Comparable to amplification of MYCN in childhood neuroblastomas, iAMP21 including AML1/RUNX1 is discussed to be an initial event in the development of leukemic blasts comprising this genomic lesion, and no information exists that AFS change over time within these cells . It is well known that in some cases, tumors or leukemias arise out of more than one tumor cell clone. However, in such cases not only AFS-PCR but all specific MRD methods are hampered to monitor the course of the disease exactly, focussing only on one tumor cell clone.
Transferring AFS-PCR to a patient with ALL, we can give additional proof of concept for the usefulness of targeting junction sites within genomic amplicons for MRD quantification. Furthermore, by direct comparison to standard IgH/TCR-qPCR and FACS analysis we can describe AFS-PCR as highly tumor cell-specific and sensitive for MRD quantification.
A major advantage of AFS-PCR over other methods for MRD quantification is the possibility of reliably detecting ampGR in primary tumor or bone marrow specimens, even with low tumor cell content. However, given by the dependency on AFS-sequences, the diagnostic algorithm is limited to patients with malignancies harbouring at least one detectable genomic amplification. Like for all PCR based methods, it might be challenging in some cases to design proper AFS-qPCR in time, to be of use in routine MRD diagnostics. Designing AFS-PCR, it is important to consider all possible orientations of the subsequent ampGR like head-to-tail, as reported for all investigated neuroblastomas or head-to-head and tail-to-tail as shown exemplarily with the ALL-patient described.