Molecular characterization and clinical course of MLL-ACTN4 rearrangement in therapy-related hematologic malignancies

Rearrangement of the MLL gene located at 11q23 is found with over 70 different partner genes in approximately 5–10% of acute leukemia cases. While the most common is the MLL-AF4 fusion resulting from the translocation t(4;11)(q21;q23), several rare and novel partner genes have been detected and characterized throughout the last decade.1 A novel MLL-ACTN4 was first reported by our group in a case of CD10- B-cell precursor (BCP) acute lymphoblastic leukemia (ALL).2 The ACTN4 gene located at chromosome 19q13.2 encodes an ubiquitously expressed actin-bundling protein associated with cell motility and cancer invasion.3 Its mutation or deficiency is mainly associated with focal segmental glomerulosclerosis (FSGS);4 however, its association with leukemogenesis is not clear.

To focus on the identification of MLL-ACTN4 as a rare but recurrent MLL rearrangement, we present 2 cases of MLL-ACTN4 rearrangement, and compare these patients with regard to diagnostic findings and clinical courses. To our knowledge, these are the only reported cases of MLL-ACTN4 rearrangement so far, and thus a comprehensive analysis of these 2 MLL-ACTN4 cases possibly provides an insight into this rare MLL rearrangement.

All analyses were carried out using bone marrow (BM) specimens obtained at the time of diagnosis. Both patients were analyzed by BM cytology and immunophenotyping. Fluorescence in situ hybridization (FISH) analysis using MLL probe (LSI dual color, break-apart probes) and a conventional karyogram were available in the second case. Long-distance inverse-polymerase chain reaction (LDI-PCR) at the Diagnostic Center of Acute Leukemia (DCAL, Frankfurt, Germany) was conducted in both cases. Microarray analysis was performed using Cytoscan 750K array (Affymetrix, Santa Clara, CA, USA) for the second case.

The first MLL-ACTN4 case was a 69-year old woman who developed a secondary BCP-ALL after radio-chemotherapy treatment of a marginal zone lymphoma. Four cycles of chemotherapy under the FCM regimen (fludarabine, cyclophosphamide and mitoxantrone) and local radiotherapy of 30 Gy were used as treatment. This case was presented as a part of previous study.2 Both LDI-PCR and reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed reciprocal fusions of MLL and ACTN4, resulting from fusion of MLL intron 9 and ACTN4 intron 1. The patient died one month after induction chemotherapy due to myocardial infarction. The second MLL-ACTN4 case was found in a 5-year old male diagnosed with therapy-related myelodysplastic syndrome (t-MDS) after chemotherapy for previously diagnosed rhabdomyosarcoma. Treatment for rhabdomyosarcoma was chemotherapy using cisplastin, vincristin and etoposide. Karyotype was 46,XY,der(6)t(6;15)(p22;q21),t(11;19)(q23;q13.1) in all 20 cells analyzed (Figure 1). MLL FISH result was nuc ish (nuclear in situ hybridization) (MLL×2)(5′MLL sep 3′MLL×1) in 191 out of 200 cells observed (95.5%) (Figure 1). The LDI-PCR identified genomic fusions of both the MLL-ACTN4 and its reciprocal ACTN4-MLL allele, resulting from fusion of MLL intron 11 and ACTN4 intron 1 (Figure 1). No RT-PCR analysis was possible. Microarray result was arr 6p25.2p22.2(381, 135–28, 638, 046)×1, 15q21.3q26.3(59, 048, 632–102, 429, 040)×3, showing about 28Mb loss at 6p25.2p22.2 and about 43Mb gain at 15q21.3q26.3. The patient is alive three years after diagnosis, and remains in remission. A detailed description of the laboratory and clinical features of these 2 cases is shown in Table 1.

Figure 1.A. (a) Breakpoint cluster regions (BCRs) of the ACTN4 gene. (b) Size and location of functional domains of the MLL wt, ACTN4 wt, and of the MLL-ACTN4 fusion protein. (B~E; case 2). B. Identification of genomic fusion breakpoint between intron 11 of MLL and intron 1 of ACTN4. C. Bone marrow mophology showing myelodysplastic features. D. Giemsa banded karyogram, abnormal chromosomes indicated by arrows. E. Flourescence in situ hybridization (FISH) image showing break-apart MLL signals. AT: AT hook; SNL: subnuclear localization; MT: methyltransferase; BD: binding domain; TAD: transcriptional activation domain; PHD: plant homeo domain; SET: Su(var)3e9, Enhancer-of-zeste, Trithorax; CH: calponin-homology domain; S: spectrin-like repeats; EF: EF-hand motif.

Table 1.Laboratory and clinical features of the 2 patients with MLL-ACTN4 rearrangement.

Based on our findings, MLL-ACTN4 rearrangements appear to be therapy-related. Use of topoisomerase-II inhibitors, such as mitoxantrone and etoposide, is likely to be responsible for therapy-related hematologic malignancies with MLL rearrangements, especially involving chromosome bands 11q23 or 21q22.5 Clinical and laboratory data from both cases suggest the likelihood of cytotoxic (radio-)chemotherapy being responsible for the occurrence of these leukemias. The ACTN4 gene is composed of 21 exons and has a coding capacity of 911 amino acids (Figure 1). In both patients, interestingly, the breakpoints were localized in ACTN4 intron 1 which leads to an in-frame fusion of nearly the entire ACTN4 open reading frame (except the first 54 amino acids encoded by exon 1) to the N-terminal portion of MLL gene (Figure 1). Since the ACTN4 as well as ENL and ELL genes are located on chromosome 19, albeit on different arms, confirmatory tests should be conducted to unambigously distinguish between either of these three possibilities in patients with a t(11;19) translocation. In circumstances of t(11;19) with a negative result for MLL-ENL or MLL-ELL, a genomic breakpoint analysis using LDI-PCR should be able to readily identify MLL-ACTN4 or other rare MLL rearrangements (MLL-ACER1, MLL-MYO1F).61 As MLL-ACTN4 rearrangements are rarely found, little is known of its response to therapy and prognosis. Additional reports in the future will shed light on the understanding of leukemogenesis of this rare MLL-related fusion gene.