Asymmetric multiplex‐polymerase chain reaction – a high throughput method for detection and sequencing genomic fusion sites in t(4;11)

Abstract

SummaryChromosomal translocations are a characteristic feature of leukaemia and other malignant diseases. As clonal markers, they can be applied to identify and quantify the number of malignant cells by polymerase chain reaction (PCR) methods. The translocation t(4;11) is present in >60% of infant leukaemia. In order to facilitate the sequencing of chromosomal breakpoints, we developed an optimized set of 30 PCR primers and a new approach, designated as asymmetric multiplex PCR (am-PCR). Due to the high number of primers, small breakpoint-spanning DNA fragments are obtained in one nested multiplex PCR reaction. All PCR products contain an identical binding site for the initiation of direct sequencing. By using am-PCR, the translocation t(4;11) was examined in bone marrow and blood samples from children with acute leukaemia. Compared with previously described methods for the determination of genomic breakpoints, am-PCR may be advantageous with regard to its simplicity and rapidity. Breakpoint-spanning sequences were also evaluated with regard to their applicability as unique clonal markers to design primers and probes for minimal residual disease quantification by real-time PCR. This approach can easily be adapted to other chromosomal translocations in malignant diseases for the detection and analysis of clonespecific DNA markers.

Prognostic significance of minimal residual disease in infants with acute lymphoblastic leukemia treated within the Interfant-99 protocol

“…The recent development of new methods to characterize MLL break points at the DNA level allows the routine application of MLL rearrangements as MRD-PCR targets. 17,18,[28][29][30] In fact, MLL rearrangements are preferred over Ig/TCR rearrangements as MRD-PCR targets, as MLL rearrangements are assumed to be present in the total leukemic clone, which is in contrast to the frequently oligoclonal Ig/TCR rearrangements in infant ALL. 3 Consequently, MRD data obtained by analysis of MLL rearrangements may provide a more accurate estimation of the level of MRD.…”

“…3 For patients with MLL rearrangements, the break point at the DNA level was analyzed as described previously. [16][17][18][28][29][30] Patientspecific primers were designed and used in combination with MLL probes for real-time quantitative-PCR analysis. Due to the large variation in break point locations, most patients required the design of specific oligonucleotides.…”

“…Out of 46 patients with available data at day 15 (TP1), the vast majority had very high (29) or high (6) MRD levels, resulting in 18 relapses overall. Of the 11 remaining patients, two patients were MRD-negative and remained disease-free, whereas out of the nine patients with low-to-moderate MRD levels, one suffered an off-therapy relapse.…”

The MLL recombinome of acute leukemias

“…They have successfully analyzed 33 t(4;11), 20 t(9;11) and 16 t(11;19) leukemia patients in parallel to our study (see Table 1b, marked by asterisk) by using different genomic PCR approaches. 14,15 Upon taking these data together, the five different chromosomal translocations mentioned above account for about 90% of all MLL-positive leukemias. Routine diagnostic methods based on RT-PCR might be restricted to these few TPGs in order to identify the vast majority of MLL gene rearrangements.…”

Mixed Lineage Leukemia–Rearranged Childhood Pro-B and CD10-Negative Pre-B Acute Lymphoblastic Leukemia Constitute a Distinct Clinical Entity

“…Detailed protocols used for the assessment of the E2A/PBX1, BCR/ABL, BCR1/ABL, and MLL/AF4 fusion transcripts by RT-PCR and multiplex PCR to detect different MLL abnormalities have been described elsewhere (15,16,18,24,25). In addition, a newly established long-distance inverse-PCR method, allowing to detect any kind of MLL rearrangements if located within the breakpoint cluster region, was applied in cases of MLL abnormalities other than those detectable by FISH or RT-PCR (26).…”