Rare MLL-ELL fusion transcripts in childhood acute myeloid leukemia—association with young age and myeloid sarcomas?
© Panagopoulos et al. 2016
Received: 20 November 2015
Accepted: 1 March 2016
Published: 5 March 2016
The chromosomal translocation t(11;19)(q23;p13) with a breakpoint within subband 19p13.1 is found mainly in acute myeloid leukemia (AML) and results in the MLL-ELL fusion gene. Variations in the structure of MLL-ELL seem to influence the leukemogenic potency of the fusion in vivo and may lie behind differences in clinical features. The number of cases reported so far is very limited and the addition of more information about MLL-ELL variants is essential if the possible clinical significance of rare fusions is to be determined.
Cytogenetic and molecular genetic analyses were done on the bone marrow cells of a 20-month-old boy with an unusual form of myelomonocytic AML with multiple myeloid sarcomas infiltrating bone and soft tissues. The G-banding analysis together with FISH yielded the karyotype 47,XY, +6,t(8;19;11)(q24;p13;q23). FISH analysis also demonstrated that MLL was split. RNA-sequencing showed that the translocation had generated an MLL-ELL chimera in which exon 9 of MLL (nt 4241 in sequence with accession number NM_005933.3) was fused to exon 6 of ELL (nt 817 in sequence with accession number NM_006532.3). RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript.
Based on our findings and information on a few previously reported patients, we speculate that young age, myelomonoblastic AML, and the presence of extramedullary disease may be typical of children with rare MLL-ELL fusion transcripts.
KeywordsAcute myeloid leukemia Myeloid sarcoma RNA-sequencing Chromosomal translocation t(11;19)(q23;p13) MLL ELL Fusion gene
The chromosomal translocation t(11;19)(q23;p13) has been reported in both acute myeloid (AML) and acute lymphoblastic leukemia (ALL) . Breakpoints within subband 19p13.3 are found in both ALL (primarily in infants and children) and AML with the translocation t(11;19)(q23;p13.3) leading to the fusion of MLL with MLLT1 (also known as ENL, LTG19, and YEATS1) generating an MLL-MLLT1 fusion gene . Breakpoints within subband 19p13.1 are found mostly in AML where the translocation t(11;19)(q23;p13.1) results in the MLL-ELL fusion gene . MLL-ELL fusions were recently found also in two biphenotypic leukemias . Two other MLL-fusion genes have also been reported in t(11;19)-positive AML. A recurrent MLL-MYO1F [translocation t(11;19)(q23;p13.2)] fusion gene was seen in infant AML [5, 6], whereas an MLL-SH3GL1 fusion [translocation t(11;19)(q23;p13.3)] was reported in a case of childhood AML .
In the majority of MLL-ELL fusion transcripts, exon 9, 10, 11 or 12 of MLL is fused to exon 2 of ELL [3, 8–13]. A variant form of MLL-ELL fusion transcript has been reported in chronic myelomonocytic leukemia in which MLL exon 9 (exon 10 according to Nilson et al. ) was fused to ELL exon 3 . Furthermore, in a case of congenital acute monoblastic leukemia with a three-way translocation t(1;19;11)(p36;p13.1;q23), De Braekeler et al. showed that the genomic breakpoints in MLL and ELL occurred in introns 9 and 5, respectively [8, 16].
The leukemogenic potency of MLL-ELL fusion genes was demonstrated in murine model systems . Moreover, variant forms of MLL-ELL were shown to impair transforming activities in vitro . These observations suggest that variations in MLL-ELL structure may influence leukemogenic potency of the fusion also in vivo, and they hint that such variability may be behind variation in clinical features. Because so few such cases have been reported, the addition of more cases with MLL-ELL variants is essential if the possible clinical significance of rarer fusions is to be determined. In the present study, we report a childhood leukemia in which a three-way translocation caused the fusion of exon 9 of MLL with exon 6 of ELL. To the best of our knowledge, this is only the second case in which exon 6 of ELL was found to be fused to MLL [8, 16, 18].
The study was approved by the regional ethics committee (Regional komité for medisinsk forskningsetikk Sør-Øst, Norge, http://helseforskning.etikkom.no), and written informed consent was obtained from the patient’s parents to publication of the case details. The ethics committee’s approval included a review of the consent procedure. All patient information has been anonymized.
Interphase FISH analyses of bone marrow cells using the Cytocell multiprobe ALL panel (Cytocell, http://www.cytocell.co.uk/) showed a split signal of the MLL locus in 146 out of 201 investigated interphase nuclei (data not shown). FISH analysis of metaphase spreads using the MLL breakpoint probe (Cytocell, http://www.cytocell.co.uk/) showed that the red signal (distal) had moved not to chromosome 19 but to the q arm of chromosome 8 (Fig. 4b). Thus, the modified karyotype after G-banding analysis and FISH was 47,XY, +6,t(8;19;11)(q24;p13;q23)  (Fig. 4a, b).
Mainly on the basis of the detected MLL-rearrangement, we interpreted the boy’s disease as an unusual form of myelomonocytic AML with multiple myeloid sarcomas infiltrating bone and soft tissues.
During the investigation, the patient’s extensive skeletal lesions increased causing therapy-resistant pain and his general condition deteriorated. After 3 weeks, AML-directed therapy was begun according to the NOPHO-DBH AML 2012 protocol . He received five courses (MEC, ADxE, HAM, HA3E, FLA) at 5–6 weeks intervals. For details, see Additional file 1: Figure: S1.
Clinically, the boy recovered rather quickly. Evaluation before course two showed no remaining MLL-rearranged cells by FISH in the bone marrow. MRI controls of his bony lesions demonstrated continuous, but slow, regression. At 9 months after cessation of treatment, the boy is clinically healthy. As expected, MRI still shows several small residual bone lesions undergoing regression. Unexpectedly, however, the patient has developed a new, small (about 5 %) clone in the bone marrow with a solitary 7q deletion. This may or may not represent an emerging secondary malignancy [20–23], and the situation is being monitored closely to see whether the clone expands and gives rise to hematologically recognizable disease.
Initial RT-PCR experiments
Primers used for PCR amplification and Sanger sequencing analyses
Sequence (5´– >3´)
Because of the negative RT-PCR results, the less than typical cytogenetic findings, and the clinical picture, three µg of the total RNA extracted from the patients’ bone marrow at the time of diagnosis were subjected to high-throughput paired-end RNA-sequencing at the Norwegian Sequencing Centre, Oslo University Hospital (http://www.sequencing.uio.no/) as described elsewhere [24, 26]. The raw sequencing data were subsequently analyzed using FusionCatcher which is a program designed to detect fusion genes from high throughput sequencing data . More than 100 potential fusion transcripts were found (Additional file 2: Table: S1), among them an MLL-ELL in which exon 9 of MLL (nt 4241 in sequence with accession number NM_005933.3) was fused to exon 6 of ELL (nt 817 in sequence with accession number NM_006532.3). No reciprocal ELL-MLL fusion transcript was found.
Sequences retrieved with the «grep» command using the expression “GACTTTAAGGTGGCCAACAT”
Molecular genetic confirmation of the fusion
PCR with the MLL-3878F and ELL-1044R1 primer combination (Table 1) amplified a fragment from the patient’s bone marrow cDNA (Fig. 4d). Sanger sequencing of the amplified product showed that it was a chimeric MLL-ELL cDNA fragment in which exon 9 of MLL was fused to exon 6 of ELL, i.e., the same MLL-ELL fusion transcript found by RNA-sequencing (Fig. 4e; Table 2).
We report a case of AML genetically characterized by a three-way translocation, t(8;19;11)(q24;p13;q23), leading to rearrangement of the MLL gene and the generation of a chimeric MLL-ELL transcript with fusion of MLL exon 9 to ELL exon 6. The initial RT-PCR amplifications relied on forward primers located in exon 7 of MLL and reverse primers located in exon 4 of ELL; this choice was based on findings in previous studies in which MLL was shown to fuse with exon 2 or 3 of ELL [3, 10, 11, 15, 29, 30]. The PCRs with these primer sets (first PCR with MLL-3878F/ELL-498R1, then nested PCR with the primers MLL-3947F1/ELL-415R) failed to amplify any cDNA fragments. It was a combination of three methods—banding cytogenetics, FISH, and RNA-sequencing—that helped us identify the present MLL exon 9-ELL exon 6 fusion. G-banding analysis showed what appeared to be a regular chromosomal translocation t(11;19)(q23;p13) (Fig. 4a), a well-known change in acute leukemia. FISH showed that although MLL was split, the distal part of the gene was moved not to the derivative 19 but, surprisingly, to the long arm of chromosome 8 (Fig. 4b). Finally, RNA-sequencing showed that exon 9 of MLL was fused to exon 6 of ELL (Table 2). RT-PCR using a new reverse primer located in exon 8 of ELL (primer ELL-1044R1, MLL-3878F and ELL-1044R1 primer combination) then confirmed the fusion transcript (Fig. 4d, e).
In 2009, De Braekeleer and coworkers reported a case of congenital acute monoblastic leukemia with a three-way translocation, t(1;19;11)(p36;p13.11;q23), which involved the MLL gene and generated an MLL-ELL fusion identical to that of the present case [8, 16, 18]. To the best of our knowledge, these are the only two cases hitherto reported in which exon 6 of ELL is fused to MLL. It is certainly intriguing that three-way translocations, an unusual phenomenon behind MLL-rearrangements, had occurred in both cases; it may hint at some currently hidden mechanism behind the generation of the genomic change. The number of AML cases with MLL exon 9-ELL exon 6 fusions might actually be underestimated when assessed by means of RT-PCR amplifications using primer sets based on hitherto published studies [3, 10, 11, 15, 29, 30]. This situation may be remedied by use of a new RT-PCR method that includes primers to detect fusion of MLL also with exon 6 of ELL .
The case we describe presented unusual clinical features. Most conspicuous were the widespread, very painful bone lesions with soft tissue involvement which were interpreted as multiple myeloid sarcomas and extensive bone infarcts, not mere marrow infiltration. The validity of this diagnosis is supported by the gradual, protracted resolution of these lesions taking place during and after therapy. To the best of our knowledge, only four cases, including the present one, have been reported of very young AML patients displaying myelomonocytic features, myeloid sarcomas, and involvement of the MLL-ELL fusion gene [8, 16, 32, 33]. The four patients have similar cytogenetic and genetic (MLL-ELL fusion) features. Three of them had a three-way translocation generating the MLL-ELL fusion: the present case with t(8;19;11)(q24;p13;q23), a female newborn with t(1;19;11)(p36;p13.11;q23) [16, 32], and a three-month-old boy with t(6;19;11)(p22;p13;q23) . None of the patients had the type 1 MLL-ELL fusion (see above). In the patient with t(1;19;11)(p36;p13.11;q23), the data suggest an MLL exon 9-ELL exon 6 fusion transcript, similar to our case [16, 32]. In a two-month-old child reported by De Braekeler et al, the genomic breakpoints in MLL and ELL indicated an MLL exon 9-ELL exon 3 fusion transcript [8, 32]. In the three-month-old boy with t(6;19;11)(p22;p13;q23), the translocation resulted in an MLL exon 8-ELL exon 3 fusion transcript .
Due to the small number of patients described, it is impossible to make definite statements about prognosis. Nevertheless, treatment results so far on patients carrying rare MLL-ELL fusion genes seem to have been encouraging. The newborn patient did not receive antileukemic therapy and died 24 h after birth , but the three treated patients, including the present case, went into remission [32, 33]. Two of them seem to be long-term survivors [32, 33], and our patient is in complete clinical remission 1 year after diagnosis. The clinical importance of the small clone with a 7q deletion that has emerged in remission is unclear. Recurrent cytogenetic abnormalities are sometimes seen in AML and ALL patients who are in complete clinical remission and may persist for years in the bone marrow even in the absence of progression to leukemia [20–23]. A wait-and-see approach is therefore prudent.
IP designed the research, performed the molecular genetic analyses, interpreted the data, and wrote the manuscript. LG and KA performed the cytogenetics and FISH experiments and interpreted the data. GK, SS, and AT did the hematopathological and immunological evaluations. LTNO made flow cytometry and immunophenotyping, evaluation of the flowcytometric data, and immunologic evaluation. L-SOM did the MRI examinations. MH treated the patient. BZ treated the patient, supervised the project, and wrote the manuscript. SH supervised the project, designed the research, evaluated the cytogenetics and FISH data, and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by Grants from the Norwegian Radium Hospital Foundation.
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Heim S, Mitelman F. Cancer cytogenetics: chromosomal and molecular genetic aberrations of tumor cells. 4th ed. Hoboken: Wiley-Blackwell; 2015.View ArticleGoogle Scholar
- Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell. 1992;71(4):691–700.View ArticlePubMedGoogle Scholar
- Thirman MJ, Levitan DA, Kobayashi H, Simon MC, Rowley JD. Cloning of ELL, a gene that fuses to MLL in a t(11;19)(q23;p13.1) in acute myeloid leukemia. Proc Natl Acad Sci USA. 1994;91(25):12110–4.View ArticlePubMedPubMed CentralGoogle Scholar
- Turkmen S, Timmermann B, Bartels G, Groger D, Meyer C, Schwartz S, et al. Involvement of the MLL gene in adult T-lymphoblastic leukemia. Genes Chromosom Cancer. 2012;51(12):1114–24. doi:https://doi.org/10.1002/gcc.21996.View ArticlePubMedGoogle Scholar
- Duhoux FP, Ameye G, Libouton JM, Bahloula K, Iossifidis S, Chantrain CF, et al. The t(11;19)(q23;p13) fusing MLL with MYO1F is recurrent in infant acute myeloid leukemias. Leuk Res. 2011;35(9):e171–2. doi:https://doi.org/10.1016/j.leukres.2011.04.022.View ArticlePubMedGoogle Scholar
- Taki T, Akiyama M, Saito S, Ono R, Taniwaki M, Kato Y, et al. The MYO1F, unconventional myosin type 1F, gene is fused to MLL in infant acute monocytic leukemia with a complex translocation involving chromosomes 7, 11, 19 and 22. Oncogene. 2005;24(33):5191–7. doi:https://doi.org/10.1038/sj.onc.1208711.View ArticlePubMedGoogle Scholar
- So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ, et al. EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia. Proc Natl Acad Sci USA. 1997;94(6):2563–8.View ArticlePubMedPubMed CentralGoogle Scholar
- De Braekeleer E, Meyer C, Douet-Guilbert N, Basinko A, Le Bris MJ, Morel F, et al. Identification of MLL partner genes in 27 patients with acute leukemia from a single cytogenetic laboratory. Mol Oncol. 2011;5(6):555–63. doi:https://doi.org/10.1016/j.molonc.2011.08.003.View ArticlePubMedGoogle Scholar
- Megonigal MD, Rappaport EF, Wilson RB, Jones DH, Whitlock JA, Ortega JA, et al. Panhandle PCR for cDNA: a rapid method for isolation of MLL fusion transcripts involving unknown partner genes. Proc Natl Acad Sci USA. 2000;97(17):9597–602. doi:https://doi.org/10.1073/pnas.150241797.View ArticlePubMedPubMed CentralGoogle Scholar
- Mitani K, Kanda Y, Ogawa S, Tanaka T, Inazawa J, Yazaki Y, et al. Cloning of several species of MLL/MEN chimeric cDNAs in myeloid leukemia with t(11;19)(q23;p13.1) translocation. Blood. 1995;85(8):2017–24.PubMedGoogle Scholar
- Rubnitz JE, Behm FG, Curcio-Brint AM, Pinheiro RP, Carroll AJ, Raimondi SC, et al. Molecular analysis of t(11;19) breakpoints in childhood acute leukemias. Blood. 1996;87(11):4804–8.PubMedGoogle Scholar
- Takeuchi M, Nakaseko C, Miyagi S, Takeda Y, Ozawa S, Ohwada C, et al. Clonal expansion of non-leukemic cells expressing two novel MLL-ELL variants differing in transforming activity. Leukemia. 2008;22(4):861–4. doi:https://doi.org/10.1038/sj.leu.2404954.View ArticlePubMedGoogle Scholar
- Yamamoto K, Nagata K, Tsurukubo Y, Inagaki K, Ono R, Taki T, et al. Translocation (8;12)(q13;p13) during disease progression in acute myelomonocytic leukemia with t(11;19)(q23;p13.1). Cancer Genet Cytogenet. 2002;137(1):64–7.View ArticlePubMedGoogle Scholar
- Nilson I, Lochner K, Siegler G, Greil J, Beck JD, Fey GH, et al. Exon/intron structure of the human ALL-1 (MLL) gene involved in translocations to chromosomal region 11q23 and acute leukaemias. Br J Haematol. 1996;93(4):966–72.View ArticlePubMedGoogle Scholar
- Kakihana K, Kubo F, Wakabayashi S, Kurosu T, Miki T, Murakami N, et al. A novel variant form of MLL-ELL fusion transcript with t(11;19)(q23;p13.1) in chronic myelomonocytic leukemia transforming to acute myeloid leukemia. Cancer Genet Cytogenet. 2008;184(2):109–12. doi:https://doi.org/10.1016/j.cancergencyto.2008.04.001.View ArticlePubMedGoogle Scholar
- De Braekeleer E, Meyer C, Douet-Guilbert N, Morel F, Le Bris MJ, Marschalek R, et al. A complex 1;19;11 translocation involving the MLL gene in a patient with congenital acute monoblastic leukemia identified by molecular and cytogenetic techniques. Ann Hematol. 2009;88(8):795–7. doi:https://doi.org/10.1007/s00277-008-0656-8.View ArticlePubMedGoogle Scholar
- Lavau C, Luo RT, Du C, Thirman MJ. Retrovirus-mediated gene transfer of MLL-ELL transforms primary myeloid progenitors and causes acute myeloid leukemias in mice. Proc Natl Acad Sci USA. 2000;97(20):10984–9. doi:https://doi.org/10.1073/pnas.190167297.View ArticlePubMedPubMed CentralGoogle Scholar
- Burmeister T, Meyer C, Groger D, Hofmann J, Marschalek R. Evidence-based RT-PCR methods for the detection of the 8 most common MLL aberrations in acute leukemias. Leuk Res. 2015;39(2):242–7. doi:https://doi.org/10.1016/j.leukres.2014.11.017.View ArticlePubMedGoogle Scholar
- Zwaan CM, Kolb EA, Reinhardt D, Abrahamsson J, Adachi S, Aplenc R, et al. Collaborative efforts driving progress in pediatric acute myeloid leukemia. J Clin Oncol. 2015;33(27):2949–62. doi:https://doi.org/10.1200/JCO.2015.62.8289.View ArticlePubMedGoogle Scholar
- Beach DF, Barnoski BL, Aviv H, Patel V, Schwarting R, Strair R, et al. Duplication of chromosome 1 dup(1)(q21q32) as the sole cytogenetic abnormality in a patient previously treated for AML. Cancer Genetics. 2012;205(12):665–8. doi:https://doi.org/10.1016/j.cancergen.2012.09.004.View ArticlePubMedGoogle Scholar
- Farina M, Rossi G, Bellotti D, Marchina E, Gale RP. Is having clonal cytogenetic abnormalities the same as having leukaemia? Acta Haematol. 2016;135(1):39–42. doi:https://doi.org/10.1159/000437202.View ArticlePubMedGoogle Scholar
- Raynaud SD, Brunet B, Chischportich M, Bayle J, Gratecos N, Pesce A, et al. Recurrent cytogenetic abnormalities observed in complete remission of acute myeloid leukemia do not necessarily mark preleukemic cells. Leukemia. 1994;8(2):245–9.PubMedGoogle Scholar
- Sandoval C, Mayer SP, Giamelli J, Farley T, Ozkaynak MF, Tugal O, et al. Cytogenetic abnormalities during clinical, immunophenotypic, and molecular remission in pediatric acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2000;118(1):9–13.View ArticlePubMedGoogle Scholar
- Panagopoulos I, Micci F, Thorsen J, Haugom L, Buechner J, Kerndrup G, et al. Fusion of ZMYND8 and RELA genes in acute erythroid leukemia. PLoS One. 2013;8(5):e63663. doi:https://doi.org/10.1371/journal.pone.0063663.View ArticlePubMedPubMed CentralGoogle Scholar
- Panagopoulos I, Torkildsen S, Gorunova L, Tierens A, Tjonnfjord GE, Heim S. Comparison between karyotyping-FISH-reverse transcription PCR and RNA-sequencing-fusion gene identification programs in the detection of KAT6A-CREBBP in acute myeloid leukemia. PLoS One. 2014;9(5):e96570. doi:https://doi.org/10.1371/journal.pone.0096570.View ArticlePubMedPubMed CentralGoogle Scholar
- Panagopoulos I, Gorunova L, Zeller B, Tierens A, Heim S. Cryptic FUS-ERG fusion identified by RNA-sequencing in childhood acute myeloid leukemia. Oncol Rep. 2013;30(6):2587–92. doi:https://doi.org/10.3892/or.2013.2751.PubMedPubMed CentralGoogle Scholar
- Nicorici D, Satalan H, Edgren H, Kangaspeska S, Murumagi A, Kallioniemi O, et al. FusionCatcher—a tool for finding somatic fusion genes in paired-end RNA-sequencing data. bioRxiv. 2014. doi:https://doi.org/10.1101/011650.Google Scholar
- Panagopoulos I, Gorunova L, Bjerkehagen B, Heim S. The “grep” command but not FusionMap, FusionFinder or ChimeraScan captures the CIC-DUX4 fusion gene from whole transcriptome sequencing data on a small round cell tumor with t(4;19)(q35;q13). PLoS One. 2014;9(6):e99439. doi:https://doi.org/10.1371/journal.pone.0099439.View ArticlePubMedPubMed CentralGoogle Scholar
- Yamamoto K, Seto M, Iida S, Komatsu H, Kamada N, Kojima S, et al. A reverse transcriptase-polymerase chain reaction detects heterogeneous chimeric mRNAs in leukemias with 11q23 abnormalities. Blood. 1994;83(10):2912–21.PubMedGoogle Scholar
- Andersson A, Hoglund M, Johansson B, Lassen C, Billstrom R, Garwicz S, et al. Paired multiplex reverse-transcriptase polymerase chain reaction (PMRT-PCR) analysis as a rapid and accurate diagnostic tool for the detection of MLL fusion genes in hematologic malignancies. Leukemia. 2001;15(8):1293–300.View ArticlePubMedGoogle Scholar
- Muntean AG. Mechanisms of mixed-lineage leukemia. Int J Hematol Oncol. 2013;2(3):207–17. doi:https://doi.org/10.2217/ijh.13.20.View ArticlePubMedPubMed CentralGoogle Scholar
- De Braekeleer E, Douet-Guilbert N, Meyer C, Morel F, Marschalek R, De Braekeleer M. MLL-ELL fusion gene in two infants with acute monoblastic leukemia and myeloid sarcoma. Leuk Lymphoma. 2012;53(6):1222–4. doi:https://doi.org/10.3109/10428194.2011.648632.View ArticlePubMedGoogle Scholar
- Tuborgh A, Meyer C, Marschalek R, Preiss B, Hasle H, Kjeldsen E. Complex three-way translocation involving MLL, ELL, RREB1, and CMAHP genes in an infant with acute myeloid leukemia and t(6;19;11)(p22.2;p13.1;q23.3). Cytogenet Genome Res. 2013;141(1):7–15. doi:https://doi.org/10.1159/000351224.View ArticlePubMedGoogle Scholar