CHARACTERIZATION OF KMT2A (MLL) CHROMOSOMAL BREAKPOINTS

Background

The KMT2A gene (MLL, mixed lineage leukemia) is located at chromosome region 11q23.  Chromosomal rearrangements involving the human MLL gene are associated with development of childhood, adult and therapy-related acute leukemia. The presence of certain MLL rearrangements is an independent prognostic factor and patients are usually treated according to high-risk protocols. Minimal residual disease (MRD) detection provides an objective assessment of treatment response and enables risk stratification of patients. However, MRD assays using MLL fusion transcripts as molecular markers usually do not provide sufficiently sensitive detection of residual leukemic cells. 

Aims

Identification of the unique chromosomal breakpoints of the MLL gene, which is necessary for the design of a quantitative real-time PCR (qPCR) DNA-based MRD assay.

Methods

For the identification of patient-specific MLL breakpoint sequences we used two different technical approaches. The first approach was long-range PCR followed by sequencing of the PCR products. The second approach included a combination of conventional chromosome microdissection, amplification of the microdissected material, next-generation sequencing, long-range PCR and sequencing of the final PCR products. Patient-specific sequences of the chromosomal breakpoint were used to develop MRD assays and enabled us to perform sensitive monitoring of MRD using qPCR in six acute leukemia patients.

Results

We identified unique breakpoint sequences in five patients with acute myeloid leukemia and one patient with acute lymphoblastic leukemia at the time of diagnosis. Using first approach (i.e. long-range PCR followed by sequencing of the PCR products) we detected MLL/AF6, MLL/AF9 (two patients) and MLL/ELL. A combination of conventional chromosome microdissection, amplification of the microdissected material, next-generation sequencing, long-range PCR and sequencing of the PCR products was used for identification of MLL/AF10 and MLL/AF4 breakpoints. Identification of unique breakpoint sequences was followed by the design of sufficiently sensitive qPCR MRD assays. The MRD levels of residual leukemic cells correlated with clinical outcome. 

Summary

MLL breakpoints could be identified by various methods (e.g. inverse PCR, panhandle PCR). Our results show other approaches for identification of unique MLL breakpoint sequences, which can be utilized for the design of the leukemia-specific assay for DNA-based MRD monitoring in patients with acute leukemia.

Keyword(s): Acute leukemia, MLL, MRD

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