Multiple clonal MLL fusions in a patient receiving CHOP-based chemotherapy

Abstract

MLL rearrangements were analysed in the blood of a patient receiving chemotherapy for diffuse large B-cell lymphoma using inverse polymerase chain reaction targeting exon 12, parallel sequencing and a custom algorithm design. Of thirteen MLL rearrangements detected, five were capable of generating MLL fusion genes, including MLL-MLLT3, the most common fusion in acute myeloid leukaemia (AML). Other fusions, all previously clinically unobserved, included MLL-NKD1, a fusion to the negative regulator of Wnt/β-catenin signaling, a pathway linked to leukaemic cell proliferation. The majority of the fusions exhibited clonal persistence from before treatment until 6 months post-chemotherapy, suggesting the fusions may confer a survival advantage to the mutant clone. MLL breakpoints were partly clustered at a specific location, indicating commonality in the process of their formation. Further, the same MLL breakpoint location exhibited a 50-100-fold increase in C to T transitions, consistent with attack by activation-induced cytidine deaminase (AICDA). As is also observed in AML and acute lymphoblastic leukaemia, in this single patient setting, MLL is capable of interacting with multiple fusion partners. This finding defines a discrete site of MLL susceptibility to fragmentation, linked to possible deregulation of AICDA function.

Fig. 1. Putative 101bp Stem-loop structure within MLL exon 12

Red arrows identify palindromic regions drawn approximately to scale. Green shaded region shows location of previously identified consensus binding site for Topoisomerase II (Broeker, et al 1996). Panel indicates site of MLL breakpoints identified using both in vitro (top) and patient (bottom) based data, numbering corresponds to the MLL break cluster region notation of Gu (Gu, et al 1992, Le, et al 2009). Inset shows the base of the predicted stem loop structure with an AICDA binding site (AGC) linked to a cytosine duplex at the base of the stem.

Fig. 2. Analysis of MLL breakpoint location from multiple rearrangements in a single patient

(A) Polymerase chain reaction signal of specific MLL-MLLT3 genomic rearrangement identified in this patient from before treatment until 6 months later. The same MLL-MLLT3 clone persists for the entire period, though it was not detected at the 1-month time point. B, baseline (B) Histogram of all 13 MLL breakpoint locations referenced to sequence at the 5′ base of the putative stem-loop structure pictured in Fig 1. The cytosine within (AGC) is designated position “0”. All fusions shown in Table I are referenced from this point using the 3′ edge of the fusion microhomology as the marker – the region of microhomology means it is not possible to determine the exact site of MLL fragmentation. (C) MLL-MLLT3 breakpoint sequence from the identified clone indicating the region of microhomology in bold. Also shown are all other breakpoint locations identified in this single patient, which occur outside of the stem loop structure, and which were captured by the IPCR screen. Though each pair is at a very similar location, they represent unique rearrangements that occur at approximate 180 bp multiples of the inter-nucleosomal repeat distance, when referenced to the stem-loop base.

Fig. 3. Cytosine to Thymine transition mutation within MLL exon 3 of a single patient undergoing chemotherapy

Data extracted from parallel sequencing analysis of total blood DNA following IPCR amplification of MLL exon 12 region to identify rearrangements. Data therefore collected from cells that both did, or did not, contain an MLL rearrangement. ■ -Before treatment or ● - 1 month ○ - 3 months ▼ - and 6 months after treatment initiation. The % mutation rate was calculated from comparison to all sequenced material within the sample analysed.

Fig. 4. Schematic showing possible role of transcription in generating site-restricted MLL translocations

Transcriptional progress may be stalled at the identified stem loop through binding of Topoisomerase II to the terminal tip of the loop. Adoption of a single stranded structure promoting the stem loop structure may be assisted by the presence of the stalled transcriptional complex and associated single stranded DNA bubble. AICDA action, requiring the transcriptional component Spt 5, single stranded DNA, target sequence (AGC) and promoted by supercoil stress facilitates cytosine attack, generating both mutations and DNA breaks (Pavri, et al 2010, Shen, et al 2009, Yamane, et al 2011).