Minimal residual disease diagnostics in acute lymphoblastic leukaemia: impact of primer characteristics and size of junctional regions

Detection of minimal residual disease (MRD) is a strong prognostic factor in acute lymphoblastic leukaemia (ALL) patients and hence now implemented in many ALL treatment protocols (Schrappe, 2002; Szczepanski, 2007). In most protocols, MRD diagnostics is performed by real-time quantitative polymerase chain reaction (RQ-PCR) analysis employing immunoglobulin (IG) and/or T-cell receptor (TR) gene rearrangements as patient-specific targets (van der Velden et al, 2003, 2007). After detection and characterization of the IG/TR gene rearrangements present in the diagnostic sample, two or more gene rearrangements are selected as MRD-PCR targets based on their expected stability and sensitivity. For these IG/TR gene rearrangements, allele-specific oligonucleotide (ASO) primers encompassing the junctional region are designed and subsequently tested in the RQ-PCR assay (van der Velden & van Dongen, 2009). Most current treatment protocols aim for usage of two MRD-PCR targets: one target with a quantitative range (QR) of ≤10 4 and a second target with a QR of ≤5 9 10 4 and a sensitivity of ≤10 . In order to design an optimal ASO primer, several guidelines have been proposed (Supplementary data) (Verhagen et al, 2000; van der Velden & van Dongen, 2009). In practice, in some cases (5–10%), only a few MRD-PCR targets are available and/or the junctional regions may be small, limiting the possibilities for appropriate positioning of the ASO primers. As a consequence, the designed ASO primers may not fulfil all guidelines. We evaluated how such “nonoptimal” ASO primers performed in RQ-PCR assays. We retrospectively evaluated data from 529 consecutive RQ-PCR assays tested for their applicability for MRD diagnostics. These 529 primers were all ASO-primers designed for a total of 116 consecutive paediatric ALL patients (102 B-cell precursor-ALL and 14 T-ALL). ASO primers were designed for IGH, IGK-Kde, Vj-Jj, Vk-Jk, TRG, TRD (including Vd2-Ja), TRB, and STILTAL1. For about half of rearrangements multiple (up to 5) ASO primers were designed. RQ-PCR data were interpreted according to EuroMRD guidelines (van der Velden et al, 2007). Out of 529 ASO primers tested (68%), 360 fulfilled all the primer guidelines. In 47% of these 360 ASO primers a QR ≤10 4 was obtained. One hundred and sixty-nine primers (32%) could not be designed fully according to the guidelines: 63 (12%) showed poor internal stability (with strong binding at the 3′ end), 15 (3%) had a 3′ dimer, 73 (14%) showed an overall dimer, and 18 (3%) had a 3′ hairpin. The QR of these “non-optimal” ASO primers did not differ significantly from “optimal” ASO primers (QR ≤10 4 in 47% versus 46%; Fig 1A). Also, the sensitivity did not differ between both groups (≤10 4 in 86% versus 84%) and there were no significant differences in the applied Tm (Supplementary data). Finally, there were no significant differences in primer category between the various MRD PCR targets, although “non-optimal” primers were slightly more frequent for incomplete IGH, incomplete TRB, IGK-Kde, IGK and complete TRD gene rearrangements (Fig 1B), which might, at least in part, be due to the high GC content in the 5′ end of IGK-Kde and in the D genes of the TRB and TRD locus. If, for each gene rearrangement, only the best performing ASO primer was used for the evaluation, comparable results were obtained between the “optimal” and “non-optimal” ASO primers, logically with increased percentages of primers reaching a QR ≤10 4 (65% vs. 67%), comparable to published data (Flohr et al, 2008; van der Velden & van Dongen, 2009). The above data indicate that ASO primers that do not fulfil all predefined guidelines generally perform well in RQ-PCR analysis, suggesting that it is the characteristics of the junctional region that determine the QR. No relationship was observed between the QR and the total number of deleted nucleotides (data not shown). However, in line with previous data on TRG rearrangements, (van der Velden et al, 2002) the QR was clearly related to the number of inserted nucleotides: junctions with >5 inserted nucleotides performed comparably well, whereas junctions with 0–5 inserted nucleotides showed variable results with lower QR’s (Fig 1C). Analysis per type of rearrangement showed that the relationship between the number of inserted nucleotides and QR was highest for IGK-Kde (QR of ≤10 4 in 30% of targets with ≤5 inserted nucleotides versus 56% of targets with >5 inserted nucleotides), complete TRB (29% vs. 58%), and TRG targets (16% vs. 48%), but more limited for complete IGH (61% vs. 77%), incomplete IGH (60% vs. 69%) and complete TRD (52% vs. 66%). Furthermore, 48% of targets reached a QR of ≤10 4 if the rearrangement contained only one N-region, whereas this increased to 64% and 83% if two or three N-regions were present, respectively. These data show that the performance of the RQ-PCR is related to the correspondence