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Lifelong persistence of AML associated MLL partial tandem duplications (MLL-PTD) in healthy adults
Leukemia Research 30 (2006) 1091–1096
Lifelong persistence of AML associated MLL partial tandem duplications (MLL-PTD) in healthy adults J¨org B¨asecke a,∗ , Martina Podleschny a , Robert Clemens a , Susanne Schnittger b , Volker Viereck c , Lorenz Tr¨umper a , Frank Griesinger a a
Department of Hematology and Oncology, University of Goettingen, Goettingen, Germany Medizinische Klinik III, University of Munich, Klinikum Großhadern, Munich, Germany Department of Gynecology and Obstetrics, University of Goettingen, Goettingen, Germany
b c
Received 28 September 2005; received in revised form 30 January 2006; accepted 2 February 2006 Available online 15 March 2006
Abstract AML-associated MLL-PTD contribute to leukemogenesis by a gain of function and confer an unfavorable prognosis. Like other leukemia associated aberrations they are also present in healthy adults. To delineate the leukemogenic mechanism we tracked down MLL-PTD in normal hematopoiesis and investigated cord blood samples. MLL-PTD were observed in 56/60 (93%) of all cord bloods. In contrast to AML, the transcript frequency in cord blood was four log scales lower as determined by real-time PCR. The CD34+ progenitor cell, CD33+ myeloid, CD19+ B-lymphoid and CD3+ T-lymphoid subfractions were positive. The ubiquitous presence of MLL-PTD in cord blood implicates a lifelong exposure, not an accumulation during lifetime. Since also present in the stem cell subfraction, these factors seem not to be major determinants in MLL-PTD leukemogenesis. © 2006 Elsevier Ltd. All rights reserved. Keywords: MLL-PTD; Cord blood; Healthy newborn; Leukemogenesis
1. Introduction The mixed lineage leukemia protooncogene MLL(11q23) is essential for the development of definitive liver derived hematopoiesis and the generation of hematopoietic stem cells [1,2]. On the other hand MLL is involved in numerous genetic aberrations including reciprocal translocations and duplications which are associated with acute myelogenous or lymphoblastic leukemia, mixed lineage leukemia or myelodysplastic syndrome [3–8]. MLL partial tandem duplications (PTD) are characterized by an in frame repetition of MLL exons in a 5 –3 direction [9], are present in up to 10% of acute myelogenous leukemia (AML) with normal cytogenetics and are also detectable in cases with additional ∗ Corresponding author at: Department of Hematology and Oncology, University of G¨ottingen, Robert-Koch-Straße 40, D-37075 G¨ottingen, Germany. Tel.: +49 551 398535; fax: +49 551 392914. E-mail address: [email protected] (J. B¨asecke).
0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.02.005
chromosomal aberrations [10,11]. MLL-PTD have not been observed in AML with prognostically favorable translocations like t(15; 17), PML/RARalpha and t(8; 21), AML1/ETO [12]. Multiple PTD are detectable in AML but only few (e.g. e9/e3, e10/e3 and e11/e3, numbers indicating the MLL PTD exon fusion points) exhibit a high transcriptional level [13]. Increasing evidence indicates, that MLL-PTD promote or support leukemogenesis. MLL-PTD positive AML exhibit an unfavorable prognosis with a shorter remission duration and a decreased median and overall survival [12,14,15]. Array analysis indicates a distinct mechanism of leukemogenesis [16] and in vitro, MLL exon duplications have a strong transcactivation potential [17]. Recently it has been demonstrated, that MLL-PTD seem to suppress MLL-wild type activity and by restoration of that activity, the leukemic growth is suppressed and apoptosis is promoted [18]. MLL-PTD are also present in adult non-leukemic hematopoiesis and we and others observed MLL-PTD in bone marrow and peripheral blood of adult healthy individ-
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uals [7,19]. Also other leukemia and lymphoma associated aberrations (e.g. the translocations t(9; 22)/BCR-ABL or (t8; 21)/AML1-ETO) have previously been shown to reside in healthy individuals at an incidence of 50% or more [20–22]. We south to delineate the functional role of MLL-PTD in normal and leukemic hematopoiesis with respect to the MLL-PTD transcriptional level, the hematopoietic subfraction in which MLL-PTD are transcribed, and the emergence of MLL-PTD in an individuals lifetime. We investigated cord blood and hematopoietic subfractions and quantified the transcriptional level of MLL-PTD in AML and non-leukemic hematopoiesis.
tive AML sample served as positive control. The sensitivity was 10−5 as determined by a cDNA in cDNA dilution [7]. Amplification products were cycle-sequenced as described [23]. Quantitative PCR was performed using the LightCycler® System (Roche Diagnostics, Mannheim, Germany). Its sensitivity was 10−2 –10−4 (cDNA in cDNA dilution). Primers and hybridisation probes to amplify and quantify the MLL-PTD fusion transcripts were as described [24]. The expression of MLL-PTD was normalized against the expression of the control gene ABL to adjust for variations in RNA quality and efficiencies of cDNA synthesis. 2.2. MACS
2. Materials and methods Cord blood samples of healthy newborns were collected after informed consent according to the convention of Helsinki and approved by the Ethics Committee of the Medical Faculty of the University of Goettingen. Mononuclear cells of 5–10 ml whole blood were freshly isolated. They were either directly subjected to total RNA- or DNAisolation (cord blood mononuclear cells) or subjected to magnetic activated cell sorting for the isolation of hematopoietic subfractions (cord blood subfractions, see below). For NA-isolation the RNAeasy and DNAeasy System (Quiagen, Hilden, Germany) were used as described [7]. The Superscript Preamplification System (GIBCO BRL, Eggenstein, Germany) served for reverse transcription of the RNA samples using random primer. To exclude PCR contaminations, RNA- and DNA-preparation, reverse transcription and PCR were performed in separate laboratories as described earlier [23].
For the isolation of subfractions mononuclear cells, single cord blood samples were split into five aliquots, each containing 1–2 × 107 cells. One aliquot was subjected either to DNA or RNA isolation and PCR as described above without sorting. The others were subjected to magnetic activated cell-sorting (Miltenyi Biotech Bergisch Glasbach, Germany) using MS-columns for the isolation of CD34+, CD33+, CD19+ and CD3+ cells, respectively, corresponding to early hematopoietic, myeloid, B- and T-lymphoid cells, according to the manufacturer’s instructions. The purity of isolation was investigated by FACS (not shown); subfractions of less than 95% purity were subjected to another MACS-procedure, usually resulting in cell counts of 5 × 104 –5 × 105 purified cells. Purification of subfractions was followed by RNA- or DNA-isolation and RT or genomic MLL-PCR as above.
3. Results 2.1. MLL-PTD RT- and genomic PCR The MLL-PTD RT-PCR primers were as follows (GenBank NM005933): primary: 3.1C 5 -AGGAGAGAGTTTACCTGCTC-3 (bp 863-844), MLLint 5 -CTTCC AGGAAGTCAAGCAAGCAGGT-3 (bp 3892–3916). Nested: 6.1 5 -GTCCAGAGCAGAGCAA ACAG-3 (bp 4036–4055), E3AS 5 -ACACAGATGGATCTGAGAGG-3 (bp609-590). For the genomic MLL-PCR, primers were as follows (Gen Bank AY373585): Primary: i6 5 -GCTGAGATAGAAGGATTGTCTTG-3 (bp 48301–48323), i1.20 5 -TGCTCTGAGATTGCTAAG-3 (bp 32950-32933). Nested: i8 5 GTCCCAATAATTCCTTTATGGC-3 (bp 53261–53282), i1n 5 -ctcctcttcaaagacatctg-3 (bp32895-32876) [7]. An ABL-RT-PCR served as internal positive control, using the primers: 5 -AAAACCTTCTCGCTGGAC-3 and 5 -CTGTTGACTGGCGTGATG-3 . Each PCR was repeated at least two times. If one repetition was positive, the whole sample was scored PTD-positive. The negative control contained no cells and was simultaneously processed starting with DNA-isolation. An MLL-PTD posi-
3.1. MLL-PTD are detectable at low frequency in the majority of all cord blood samples Mononuclear cord blood cells of healthy newborns were subjected to nested MLL-PTD RT-PCR after RNA quality control by an ABL-PCR (Fig. 1a). Only ABL-positive samples were considered informative. A 93% of the samples were MLL-PTD positive in the nested but none in the primary PCR (primary PCR not shown). In >80% (47/56) of all samples more than one MLL-PTD in the initial and repetitive nested RT-PCR cycle was present. Samples which exhibited only one were rare, and in most samples at least three PTD were present. The PTD pattern in an individual sample was not uniform as the pattern differed in repetitive nested RTPCRs. This has also been observed in healthy adults and is most likely due to stochastic effects during PCR amplification of multiple PTD [7]. Specificity of amplification was confirmed by cycle sequencing. The PTD were essentially those, which have been observed in the primary PCR of MLLPTD-positive acute myelogenous leukemias (e9/3, 10/3 and 11/3). Moreover, additional PTD, which have been observed
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Fig. 2. (a) Cord blood mononuclear cells, nested genomic PCR. Due to the localisation of the nested primers in introns 11 and 2 (7), the indicated MLLPTD e11/2 is preferentially amplified. Size of marker lanes is indicated as kb. N; negative control (water). (b) Cord blood subfractions, genomic nested PCR. Two independent PCR cycles have been performed of each sample. Note that the PTD pattern may differ in repetitive PCR cycles of one sample. The PTD e2/11 is indicated. Negative control and size as in (a).
Fig. 1. (a) Cord blood mononuclear cells, nested RT-PCR; This gel shows MLL-PTD in mononuclear cells of cord blood samples as determined by repeated nested RT-PCR. Each sample was submitted twice to PCR and both results are shown in subsequent lanes as indicated. The PTD e9/e3, e10/e3 und e11/e3, which can also be found in AML are indicated. Size of marker lanes is indicated as kb. MW; molecular weight marker. N, P; negative and positive control (water and patient with AML). (b) Cord blood subfractions, nested RT-PCR. Two independent PCR cycles have been performed of each sample and are shown as adjacent lanes for each subfraction. Note that fewer MLL-PTD are present in subfractions than in whole cord blood and that the pattern may differ in repetitive PCR cycles of one sample. AML-associated PTD and marker size (kb) are indicated. CDn; subfraction marker. Controls as in (a). (c) Nested RT-PCR of two cord blood CD34+-progenitor cell subfraction. In two repetitive PCRs of CD34+ samples, including repetition of primary and nested cycles, in sample 1 a PTD e9/3 and e11/3 could be detected, whereas sample 2 was PTD-negative in both PCRs. Controls as in (a).
in peripheral mononuclear cells of healthy adults, were amplified [7]. Both characteristics, numerous MLL-PTD in one sample and an incidence of 93% of all cord blood samples are comparable to what has been observed in healthy adult hematopoiesis [7]. To rule out splicing artifacts which mimic MLL-PTD in RT-PCR, 15 cord blood samples were subjected to nested genomic PCR and sequenced. As shown in Fig. 2a and Table 1, the incidence of positive samples was comparable to that observed by RT-PCR (100% versus 93%). As in adults, MLL-PTD in cord blood therefore derive from a genomic rearrangement and are not RNA splicing artifacts [7]. To investigate the transcription frequency more accurately, we performed a real-time PCR of eight positive cord blood and four AML samples, without any other genetic abnormality. As shown in Table 2, both groups differed markedly. The transcriptional frequency of MLL-PTD in AML samples was much higher than in cord blood. The latter is comparable to the frequency in healthy adult peripheral blood and bone marrow [24].
Table 1 MLL-PTD in cord blood mononuclear cells and subfractions All samples n (Positive/total)
% Positive
RT-PCR n (positive/total)
Genomic PCR n (positive/total)
MNC
56/60
93
41/45
15/15
Subfractions CD34 CD33 CD19 CD3
4/6 4/7 3/5 2/6
67 57 60 33
2/4 3/5 1/3 1/4
2/2 1/2 2/2 1/2
Subfractions of MLL-positive samples have been submitted to nested RT- or nested genomic PCR. Shown is the frequency of positive samples of different individuals. Each of these samples has been investigated as triplicate. CB MNC; cord blood mononuclear cells.
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Table 2 MLL-PTD transcript numbers in AML and cord blood AML
Cord blood
Sample
Normalized ratio
Sample
Normalized ratio
1 2 3 4
112.32 134.78 60.47 455.99
1 2 3 4 5 6 7 8
0.07 0.02 0.02 0.06 0.04 0.05 0.04 0.07
MW S.D.
190.89 179.45
0.05 0.02
MLL-PTD transcripts in cord blood mononuclear cells and PTD-positive AML were determined by real-time PCR. Normalized ratios of MLL-PTD transcripts were calculated against the transcription of the control gene ABL.
3.2. Hematopietic progenitors, myeloid and B- and T-lymphoid subfractions are MLL-PTD positive To investigate the presence of MLL-PTD in hematopoietic subfractions, we isolated subfractions of MLL-PTD cord blood samples by MACS as described above. As shown in Table 1 and pictures 2 and 5 MLL-PTD were present in the CD34+-subfraction which is enriched for hematopoietic progenitor cells as well as in the myeloid (CD33+), B-(CD19+) and T-(CD3+) lymphoid cell subfraction. To rule out splicing artifacts, we also subjected subfractions of MLL-PTD positive cord blood samples to nested genomic PCR and confirmed these results (Fig. 2b). In the RT-PCR of subfractions less amplification products were observed than in cord blood mononuclear cells. If genomic PCR was applied, this effect was also observed (Figs. 1b and 2b).
4. Discussion MLL-PTD, like other leukemia associated genetic aberrations, have been detected in healthy adults at an incidence of 100% [7]. We wanted to further delineate the leukemogenic relevance of MLL-PTD and investigated cord blood mononuclear cells for the presence of PTD. We observed MLL-PTD in mononuclear cells of 93% of all cord blood samples, including exon fusions which were characteristic of MLLPTD positive AML. In none of the cord blood samples were PTD amplified in the primary RT-PCR consistent with findings in healthy adult peripheral blood and bone marrow [7]. This contrasts with findings in MLL-PTD positive AML samples in which amplification was regularly observed in the primary RT-PCR [12]. We confirmed this observation by the application of a real-time PCR. The transcription frequency of MLL-PTD was four log scales lower than in AML and was comparable to the frequency in non-leukemic adult hematopoiesis (see Table 2) [24]. Due to the limited sensitivity of the quantitative PCR (see Section 2) we did not investigate RT-PCR negative cord bloods or subfractions.
Based on these findings, MLL-PTD in healthy individuals seem to emerge in utero or might even be inherited, are not acquired during adulthood and are present in hematopoiesis from the time of birth to adulthood and most likely lifelong. Different exposure times seem therefore not to be a major criterium in MLL-PTD leukemogenesis. MLL-PTD are reminscent of the translocations t(8; 21);AML1/ETO and BCL2/IgH, which are associated with AML and follicular lymphoma but are also present through longer time periods in healthy individuals [23,25]. AML is the result of malignant transformation at the myeloid progenitor cell stage and genetic aberrations in the progenitor cell subfraction seem to be initiating events during leukemogenesis [26]. We therefore investigated the presence of MLL-PTD in hematopoietic subfractions to rule out that MLL-PTD in positive cord blood samples are only present in subfractions that are not relevant for leukemogenesis. We observed MLL-PTD in all studied subfractions, including the CD34+ progenitor cell subfraction. Comparable to cord blood mononuclear cells, in some subfractions more than one PTD was present and the PTD-pattern in one sample differed in repetitive PCR cycles. This was most likely due to stochastic effects during primer binding and amplification since more than one MLL-PTD target sequence exists. Splicing artifacts have been excluded by genomic PCR. Though we used up to two MACS-cycles to achieve highly purified subfractions (see Section 2), a few non-specific cells could still be present in these preparations. Since the usual frequency of PTD in healthy individuals is 1 in 5000 cells [7], it was less likely that PTD results in the subfractions were due to non-specific cells. The overall number of MLL-PTD in the cord blood subfractions was lower than it was in the mononuclear cell samples. This indicates, that the more homogenous a cell population is, the less PTD are transcribed. In AML, which is an example of a highly homogenous population, the MLLPTD positive blasts are known to express only one PTD, due to a monoallelic tandem duplication, whilst the wildtype gene is repressed [18]. Since the MACS-subfractions were discriminated by one lineage specific marker, they were still heterogenous. We could therefore not finally determine if the reduction in the number of PTD in cord blood subfractions were due to a unique PTD expression pattern in highly purified cells and if the PTD in these cells would derive from mono- or biallelic tandem duplication. Notably, MLL-PTD were present in the CD34+ progenitor cell subfraction. This subfraction is still heterogenous and included multipotent stem cells as well as early hematopoietic cells, capable of dividing and differentiation, which have entered early lineage specific differentiation [27]. The majority of mononuclear cells either in adults or newborn, which have initially been found to be MLL-PTD positive, are non-dividing cells. Though we can not delineate the origin of the MLL-PTD in this subfraction any further, e.g. to multipotent stem cells, the existence of MLL-PTD in a proliferating and differentiating subfraction indicates,
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that PTD in healthy individuals may derive from a positive progenitor cell subfraction instead of emerging spontaneously in further matured cells. Furthermore, the presence of MLL-PTD in a subfraction which is sensitive to leukemic transformation, limits its inherent leukemogenic potential. The transcription level of MLL-PTD in AML was much higher than in normal hematopoiesis. This may indicate that either PTD-positive cells in normal hematopoietic are less frequent than in AML but have identical transcription levels. On the other hand, the intracellular transcription level in AML blasts may be higher than in normal PTD positive cells, due to e.g. additional alterations in regulatory elements. As recently observed in samples of patients with AML, highly transcribed MLL-PTD seem to repress the MLL-wildtype gene expression. Inappropriate dosage of the MLL-wildtype gene, known to impair stem cell development and definitive hematopoiesis [1] and a gain of function mechanism by MLLPTD, as suggested by the strong transctivation potential of MLL-PTD in vitro [28], may therefore represent a pivotal mechanism leading to malignant transformation. In conclusion the lifelong exposure of the hematopoietic system to MLL-PTD and their presence in the progenitor cell subfraction are not key determinants in their contribution to leukemogenesis. MLL-PTD are somewhat reminiscent of the translocation t(8; 21), characteristic of AML FAB M2, which also present at low levels in healthy newborn and adults but, if transcribed at high levels, induce proleukemogenic alterations in mice [7,23]. MLL-PTD seem to have an inherent low leukemogenic potential and their role in the process of transformation may be dependent on a high transcription rate and/or cooperating alterations. In vivo systems will be helpful to further delineate the function of MLL-PTD in the process of leukemogenesis. Acknowledgment This work is supported by the German Hector-Stiftung (grant M18) to J.B. and F.G. References [1] Ernst P, Fisher JK, Avery W, Wade S, Foy D, Korsmeyer SJ. Definitive hematopoiesis requires the mixed-lineage leukemia gene. Dev Cell 2004;6(3):437–43. [2] Dorshkind K, Witte O. Got MLL? Definitive hematopoiesis requires MLL gene expression. Mol Cell 2004;13(6):765–6. [3] Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa III R, Patel Y, Harden A, et al. Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 1991;88(23):10735–9. [4] Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, et al. Ten novel 11q23 chromosomal partner sites. European 11q23 Workshop participants. Leukemia 1998;12(5):811–22. [5] Rowley JD. Rearrangements involving chromosome band 11Q23 in acute leukaemia. Semin Cancer Biol 1993;4(6):377–85. [6] So CW, Cleary ML. Dimerization: a versatile switch for oncogenesis. Blood 2004;104(2):919–22.
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[7] Schnittger S, Wormann B, Hiddemann W, Griesinger F. Partial tandem duplications of the MLL gene are detectable in peripheral blood and bone marrow of nearly all healthy donors. Blood 1998;92(5):1728–34. [8] So CW, Ma ZG, Price CM, Dong S, Chen SJ, Gu LJ, et al. MLL self fusion mediated by Alu repeat homologous recombination and prognosis of AML-M4/M5 subtypes. Cancer Res 1997;57(1): 117–22. [9] Schichman SA, Caligiuri MA, Strout MP, Carter SL, Gu Y, Canaani E, et al. ALL-1 tandem duplication in acute myeloid leukemia with a normal karyotype involves homologous recombination between Alu elements. Cancer Res 1994;54(16):4277–80. [10] Yu M, Honoki K, Andersen J, Paietta E, Nam DK, Yunis JJ. MLL tandem duplication and multiple splicing in adult acute myeloid leukemia with normal karyotype. Leukemia 1996;10(5):774–80. [11] Caligiuri MA, Strout MP, Lawrence D, Arthur DC, Baer MR, Yu F, et al. Rearrangement of ALL1 (MLL) in acute myeloid leukemia with normal cytogenetics. Cancer Res 1998;58(1):55–9. [12] Schnittger S, Kinkelin U, Schoch C, Heinecke A, Haase D, Haferlach T, et al. Screening for MLL tandem duplication in 387 unselected patients with AML identify a prognostically unfavorable subset of AML. Leukemia 2000;14(5):796–804. [13] Steudel C, Wermke M, Schaich M, Schakel U, Illmer T, Ehninger G, et al. Comparative analysis of MLL partial tandem duplication and FLT3 internal tandem duplication mutations in 956 adult patients with acute myeloid leukemia. Genes Chromosomes Cancer 2003;37(3):237–51. [14] Shiah HS, Kuo YY, Tang JL, Huang SY, Yao M, Tsay W, et al. Clinical and biological implications of partial tandem duplication of the MLL gene in acute myeloid leukemia without chromosomal abnormalities at 11q23. Leukemia 2002;16(2):196–202. [15] Dohner K, Tobis K, Ulrich R, Frohling S, Benner A, Schlenk RF, et al. Prognostic significance of partial tandem duplications of the MLL gene in adult patients 16–60 years old with acute myeloid leukemia and normal cytogenetics: a study of the Acute Myeloid Leukemia Study Group Ulm. J Clin Oncol 2002;20(15):3254–61. [16] Ross ME, Mahfouz R, Onciu M, Liu HC, Zhou X, Song G, et al. Gene expression profiling of pediatric acute myelogenous leukemia. Blood 2004. [17] Martin ME, Milne TA, Bloyer S, Galoian K, Shen W, Gibbs D, et al. Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell 2003;4(3):197–207. [18] Whitman SP, Liu S, Vukosavljevic T, Rush LJ, Yu L, Liu C, et al. The MLL partial tandem duplication: evidence for recessive gainof-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy. Blood 2005;106(1):345–52. [19] Marcucci G, Strout MP, Bloomfield CD, Caligiuri MA. Detection of unique ALL1 (MLL) fusion transcripts in normal human bone marrow and blood: distinct origin of normal versus leukemic ALL1 fusion transcripts. Cancer Res 1998;58(4):790–3. [20] Basecke J, Feuring-Buske M, Brittinger G, Schaefer UW, Hiddemann W, Griesinger F. Transcription of AML1 in hematopoietic subfractions of normal adults. Ann Hematol 2002;81(5):254–7. [21] Bose S, Deininger M, Gora-Tybor J, Goldman JM, Melo JV. The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. Blood 1998;92(9):3362–7. [22] Biernaux C, Sels A, Huez G, Stryckmans P. Very low level of major BCR-ABL expression in blood of some healthy individuals. Bone Marrow Transplant 1996;17(Suppl. 3):S45–7. [23] Basecke J, Cepek L, Mannhalter C, Krauter J, Hildenhagen S, Brittinger G, et al. Transcription of AML1/ETO in bone marrow and cord blood of individuals without acute myelogenous leukemia. Blood 2002;100(6):2267–8. [24] Weisser M, Kern W, Schoch C, Hiddemann W, Haferlach T, Schnittger S. RT-PCR based monitoring of MLL-PTD expression
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levels enables risk assessment in AML patients with normal karyotypes. Haematologica 2005;90(7):881–9. [25] Basecke J, Griesinger F, Trumper L, Brittinger G. Leukemia- and lymphoma-associated genetic aberrations in healthy individuals. Ann Hematol 2002;81(2):64–75. [26] Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol 2004;5(7):738–43.
[27] Terstappen LW, Huang S, Safford M, Lansdorp PM, Loken MR. Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells. Blood 1991;77(6):1218–27. [28] Martin ME, Milne TA, Bloyer S, Galoian K, Shen W, Gibbs D, et al. Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell 2003;4(3):197–207.
Lifelong persistence of AML associated MLL partial tandem duplications (MLL-PTD) in healthy adults J¨org B¨asecke a,∗ , Martina Podleschny a , Robert Clemens a , Susanne Schnittger b , Volker Viereck c , Lorenz Tr¨umper a , Frank Griesinger a a
Department of Hematology and Oncology, University of Goettingen, Goettingen, Germany Medizinische Klinik III, University of Munich, Klinikum Großhadern, Munich, Germany Department of Gynecology and Obstetrics, University of Goettingen, Goettingen, Germany
b c
Received 28 September 2005; received in revised form 30 January 2006; accepted 2 February 2006 Available online 15 March 2006
Abstract AML-associated MLL-PTD contribute to leukemogenesis by a gain of function and confer an unfavorable prognosis. Like other leukemia associated aberrations they are also present in healthy adults. To delineate the leukemogenic mechanism we tracked down MLL-PTD in normal hematopoiesis and investigated cord blood samples. MLL-PTD were observed in 56/60 (93%) of all cord bloods. In contrast to AML, the transcript frequency in cord blood was four log scales lower as determined by real-time PCR. The CD34+ progenitor cell, CD33+ myeloid, CD19+ B-lymphoid and CD3+ T-lymphoid subfractions were positive. The ubiquitous presence of MLL-PTD in cord blood implicates a lifelong exposure, not an accumulation during lifetime. Since also present in the stem cell subfraction, these factors seem not to be major determinants in MLL-PTD leukemogenesis. © 2006 Elsevier Ltd. All rights reserved. Keywords: MLL-PTD; Cord blood; Healthy newborn; Leukemogenesis
1. Introduction The mixed lineage leukemia protooncogene MLL(11q23) is essential for the development of definitive liver derived hematopoiesis and the generation of hematopoietic stem cells [1,2]. On the other hand MLL is involved in numerous genetic aberrations including reciprocal translocations and duplications which are associated with acute myelogenous or lymphoblastic leukemia, mixed lineage leukemia or myelodysplastic syndrome [3–8]. MLL partial tandem duplications (PTD) are characterized by an in frame repetition of MLL exons in a 5 –3 direction [9], are present in up to 10% of acute myelogenous leukemia (AML) with normal cytogenetics and are also detectable in cases with additional ∗ Corresponding author at: Department of Hematology and Oncology, University of G¨ottingen, Robert-Koch-Straße 40, D-37075 G¨ottingen, Germany. Tel.: +49 551 398535; fax: +49 551 392914. E-mail address: [email protected] (J. B¨asecke).
0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.02.005
chromosomal aberrations [10,11]. MLL-PTD have not been observed in AML with prognostically favorable translocations like t(15; 17), PML/RARalpha and t(8; 21), AML1/ETO [12]. Multiple PTD are detectable in AML but only few (e.g. e9/e3, e10/e3 and e11/e3, numbers indicating the MLL PTD exon fusion points) exhibit a high transcriptional level [13]. Increasing evidence indicates, that MLL-PTD promote or support leukemogenesis. MLL-PTD positive AML exhibit an unfavorable prognosis with a shorter remission duration and a decreased median and overall survival [12,14,15]. Array analysis indicates a distinct mechanism of leukemogenesis [16] and in vitro, MLL exon duplications have a strong transcactivation potential [17]. Recently it has been demonstrated, that MLL-PTD seem to suppress MLL-wild type activity and by restoration of that activity, the leukemic growth is suppressed and apoptosis is promoted [18]. MLL-PTD are also present in adult non-leukemic hematopoiesis and we and others observed MLL-PTD in bone marrow and peripheral blood of adult healthy individ-
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uals [7,19]. Also other leukemia and lymphoma associated aberrations (e.g. the translocations t(9; 22)/BCR-ABL or (t8; 21)/AML1-ETO) have previously been shown to reside in healthy individuals at an incidence of 50% or more [20–22]. We south to delineate the functional role of MLL-PTD in normal and leukemic hematopoiesis with respect to the MLL-PTD transcriptional level, the hematopoietic subfraction in which MLL-PTD are transcribed, and the emergence of MLL-PTD in an individuals lifetime. We investigated cord blood and hematopoietic subfractions and quantified the transcriptional level of MLL-PTD in AML and non-leukemic hematopoiesis.
tive AML sample served as positive control. The sensitivity was 10−5 as determined by a cDNA in cDNA dilution [7]. Amplification products were cycle-sequenced as described [23]. Quantitative PCR was performed using the LightCycler® System (Roche Diagnostics, Mannheim, Germany). Its sensitivity was 10−2 –10−4 (cDNA in cDNA dilution). Primers and hybridisation probes to amplify and quantify the MLL-PTD fusion transcripts were as described [24]. The expression of MLL-PTD was normalized against the expression of the control gene ABL to adjust for variations in RNA quality and efficiencies of cDNA synthesis. 2.2. MACS
2. Materials and methods Cord blood samples of healthy newborns were collected after informed consent according to the convention of Helsinki and approved by the Ethics Committee of the Medical Faculty of the University of Goettingen. Mononuclear cells of 5–10 ml whole blood were freshly isolated. They were either directly subjected to total RNA- or DNAisolation (cord blood mononuclear cells) or subjected to magnetic activated cell sorting for the isolation of hematopoietic subfractions (cord blood subfractions, see below). For NA-isolation the RNAeasy and DNAeasy System (Quiagen, Hilden, Germany) were used as described [7]. The Superscript Preamplification System (GIBCO BRL, Eggenstein, Germany) served for reverse transcription of the RNA samples using random primer. To exclude PCR contaminations, RNA- and DNA-preparation, reverse transcription and PCR were performed in separate laboratories as described earlier [23].
For the isolation of subfractions mononuclear cells, single cord blood samples were split into five aliquots, each containing 1–2 × 107 cells. One aliquot was subjected either to DNA or RNA isolation and PCR as described above without sorting. The others were subjected to magnetic activated cell-sorting (Miltenyi Biotech Bergisch Glasbach, Germany) using MS-columns for the isolation of CD34+, CD33+, CD19+ and CD3+ cells, respectively, corresponding to early hematopoietic, myeloid, B- and T-lymphoid cells, according to the manufacturer’s instructions. The purity of isolation was investigated by FACS (not shown); subfractions of less than 95% purity were subjected to another MACS-procedure, usually resulting in cell counts of 5 × 104 –5 × 105 purified cells. Purification of subfractions was followed by RNA- or DNA-isolation and RT or genomic MLL-PCR as above.
3. Results 2.1. MLL-PTD RT- and genomic PCR The MLL-PTD RT-PCR primers were as follows (GenBank NM005933): primary: 3.1C 5 -AGGAGAGAGTTTACCTGCTC-3 (bp 863-844), MLLint 5 -CTTCC AGGAAGTCAAGCAAGCAGGT-3 (bp 3892–3916). Nested: 6.1 5 -GTCCAGAGCAGAGCAA ACAG-3 (bp 4036–4055), E3AS 5 -ACACAGATGGATCTGAGAGG-3 (bp609-590). For the genomic MLL-PCR, primers were as follows (Gen Bank AY373585): Primary: i6 5 -GCTGAGATAGAAGGATTGTCTTG-3 (bp 48301–48323), i1.20 5 -TGCTCTGAGATTGCTAAG-3 (bp 32950-32933). Nested: i8 5 GTCCCAATAATTCCTTTATGGC-3 (bp 53261–53282), i1n 5 -ctcctcttcaaagacatctg-3 (bp32895-32876) [7]. An ABL-RT-PCR served as internal positive control, using the primers: 5 -AAAACCTTCTCGCTGGAC-3 and 5 -CTGTTGACTGGCGTGATG-3 . Each PCR was repeated at least two times. If one repetition was positive, the whole sample was scored PTD-positive. The negative control contained no cells and was simultaneously processed starting with DNA-isolation. An MLL-PTD posi-
3.1. MLL-PTD are detectable at low frequency in the majority of all cord blood samples Mononuclear cord blood cells of healthy newborns were subjected to nested MLL-PTD RT-PCR after RNA quality control by an ABL-PCR (Fig. 1a). Only ABL-positive samples were considered informative. A 93% of the samples were MLL-PTD positive in the nested but none in the primary PCR (primary PCR not shown). In >80% (47/56) of all samples more than one MLL-PTD in the initial and repetitive nested RT-PCR cycle was present. Samples which exhibited only one were rare, and in most samples at least three PTD were present. The PTD pattern in an individual sample was not uniform as the pattern differed in repetitive nested RTPCRs. This has also been observed in healthy adults and is most likely due to stochastic effects during PCR amplification of multiple PTD [7]. Specificity of amplification was confirmed by cycle sequencing. The PTD were essentially those, which have been observed in the primary PCR of MLLPTD-positive acute myelogenous leukemias (e9/3, 10/3 and 11/3). Moreover, additional PTD, which have been observed
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Fig. 2. (a) Cord blood mononuclear cells, nested genomic PCR. Due to the localisation of the nested primers in introns 11 and 2 (7), the indicated MLLPTD e11/2 is preferentially amplified. Size of marker lanes is indicated as kb. N; negative control (water). (b) Cord blood subfractions, genomic nested PCR. Two independent PCR cycles have been performed of each sample. Note that the PTD pattern may differ in repetitive PCR cycles of one sample. The PTD e2/11 is indicated. Negative control and size as in (a).
Fig. 1. (a) Cord blood mononuclear cells, nested RT-PCR; This gel shows MLL-PTD in mononuclear cells of cord blood samples as determined by repeated nested RT-PCR. Each sample was submitted twice to PCR and both results are shown in subsequent lanes as indicated. The PTD e9/e3, e10/e3 und e11/e3, which can also be found in AML are indicated. Size of marker lanes is indicated as kb. MW; molecular weight marker. N, P; negative and positive control (water and patient with AML). (b) Cord blood subfractions, nested RT-PCR. Two independent PCR cycles have been performed of each sample and are shown as adjacent lanes for each subfraction. Note that fewer MLL-PTD are present in subfractions than in whole cord blood and that the pattern may differ in repetitive PCR cycles of one sample. AML-associated PTD and marker size (kb) are indicated. CDn; subfraction marker. Controls as in (a). (c) Nested RT-PCR of two cord blood CD34+-progenitor cell subfraction. In two repetitive PCRs of CD34+ samples, including repetition of primary and nested cycles, in sample 1 a PTD e9/3 and e11/3 could be detected, whereas sample 2 was PTD-negative in both PCRs. Controls as in (a).
in peripheral mononuclear cells of healthy adults, were amplified [7]. Both characteristics, numerous MLL-PTD in one sample and an incidence of 93% of all cord blood samples are comparable to what has been observed in healthy adult hematopoiesis [7]. To rule out splicing artifacts which mimic MLL-PTD in RT-PCR, 15 cord blood samples were subjected to nested genomic PCR and sequenced. As shown in Fig. 2a and Table 1, the incidence of positive samples was comparable to that observed by RT-PCR (100% versus 93%). As in adults, MLL-PTD in cord blood therefore derive from a genomic rearrangement and are not RNA splicing artifacts [7]. To investigate the transcription frequency more accurately, we performed a real-time PCR of eight positive cord blood and four AML samples, without any other genetic abnormality. As shown in Table 2, both groups differed markedly. The transcriptional frequency of MLL-PTD in AML samples was much higher than in cord blood. The latter is comparable to the frequency in healthy adult peripheral blood and bone marrow [24].
Table 1 MLL-PTD in cord blood mononuclear cells and subfractions All samples n (Positive/total)
% Positive
RT-PCR n (positive/total)
Genomic PCR n (positive/total)
MNC
56/60
93
41/45
15/15
Subfractions CD34 CD33 CD19 CD3
4/6 4/7 3/5 2/6
67 57 60 33
2/4 3/5 1/3 1/4
2/2 1/2 2/2 1/2
Subfractions of MLL-positive samples have been submitted to nested RT- or nested genomic PCR. Shown is the frequency of positive samples of different individuals. Each of these samples has been investigated as triplicate. CB MNC; cord blood mononuclear cells.
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Table 2 MLL-PTD transcript numbers in AML and cord blood AML
Cord blood
Sample
Normalized ratio
Sample
Normalized ratio
1 2 3 4
112.32 134.78 60.47 455.99
1 2 3 4 5 6 7 8
0.07 0.02 0.02 0.06 0.04 0.05 0.04 0.07
MW S.D.
190.89 179.45
0.05 0.02
MLL-PTD transcripts in cord blood mononuclear cells and PTD-positive AML were determined by real-time PCR. Normalized ratios of MLL-PTD transcripts were calculated against the transcription of the control gene ABL.
3.2. Hematopietic progenitors, myeloid and B- and T-lymphoid subfractions are MLL-PTD positive To investigate the presence of MLL-PTD in hematopoietic subfractions, we isolated subfractions of MLL-PTD cord blood samples by MACS as described above. As shown in Table 1 and pictures 2 and 5 MLL-PTD were present in the CD34+-subfraction which is enriched for hematopoietic progenitor cells as well as in the myeloid (CD33+), B-(CD19+) and T-(CD3+) lymphoid cell subfraction. To rule out splicing artifacts, we also subjected subfractions of MLL-PTD positive cord blood samples to nested genomic PCR and confirmed these results (Fig. 2b). In the RT-PCR of subfractions less amplification products were observed than in cord blood mononuclear cells. If genomic PCR was applied, this effect was also observed (Figs. 1b and 2b).
4. Discussion MLL-PTD, like other leukemia associated genetic aberrations, have been detected in healthy adults at an incidence of 100% [7]. We wanted to further delineate the leukemogenic relevance of MLL-PTD and investigated cord blood mononuclear cells for the presence of PTD. We observed MLL-PTD in mononuclear cells of 93% of all cord blood samples, including exon fusions which were characteristic of MLLPTD positive AML. In none of the cord blood samples were PTD amplified in the primary RT-PCR consistent with findings in healthy adult peripheral blood and bone marrow [7]. This contrasts with findings in MLL-PTD positive AML samples in which amplification was regularly observed in the primary RT-PCR [12]. We confirmed this observation by the application of a real-time PCR. The transcription frequency of MLL-PTD was four log scales lower than in AML and was comparable to the frequency in non-leukemic adult hematopoiesis (see Table 2) [24]. Due to the limited sensitivity of the quantitative PCR (see Section 2) we did not investigate RT-PCR negative cord bloods or subfractions.
Based on these findings, MLL-PTD in healthy individuals seem to emerge in utero or might even be inherited, are not acquired during adulthood and are present in hematopoiesis from the time of birth to adulthood and most likely lifelong. Different exposure times seem therefore not to be a major criterium in MLL-PTD leukemogenesis. MLL-PTD are reminscent of the translocations t(8; 21);AML1/ETO and BCL2/IgH, which are associated with AML and follicular lymphoma but are also present through longer time periods in healthy individuals [23,25]. AML is the result of malignant transformation at the myeloid progenitor cell stage and genetic aberrations in the progenitor cell subfraction seem to be initiating events during leukemogenesis [26]. We therefore investigated the presence of MLL-PTD in hematopoietic subfractions to rule out that MLL-PTD in positive cord blood samples are only present in subfractions that are not relevant for leukemogenesis. We observed MLL-PTD in all studied subfractions, including the CD34+ progenitor cell subfraction. Comparable to cord blood mononuclear cells, in some subfractions more than one PTD was present and the PTD-pattern in one sample differed in repetitive PCR cycles. This was most likely due to stochastic effects during primer binding and amplification since more than one MLL-PTD target sequence exists. Splicing artifacts have been excluded by genomic PCR. Though we used up to two MACS-cycles to achieve highly purified subfractions (see Section 2), a few non-specific cells could still be present in these preparations. Since the usual frequency of PTD in healthy individuals is 1 in 5000 cells [7], it was less likely that PTD results in the subfractions were due to non-specific cells. The overall number of MLL-PTD in the cord blood subfractions was lower than it was in the mononuclear cell samples. This indicates, that the more homogenous a cell population is, the less PTD are transcribed. In AML, which is an example of a highly homogenous population, the MLLPTD positive blasts are known to express only one PTD, due to a monoallelic tandem duplication, whilst the wildtype gene is repressed [18]. Since the MACS-subfractions were discriminated by one lineage specific marker, they were still heterogenous. We could therefore not finally determine if the reduction in the number of PTD in cord blood subfractions were due to a unique PTD expression pattern in highly purified cells and if the PTD in these cells would derive from mono- or biallelic tandem duplication. Notably, MLL-PTD were present in the CD34+ progenitor cell subfraction. This subfraction is still heterogenous and included multipotent stem cells as well as early hematopoietic cells, capable of dividing and differentiation, which have entered early lineage specific differentiation [27]. The majority of mononuclear cells either in adults or newborn, which have initially been found to be MLL-PTD positive, are non-dividing cells. Though we can not delineate the origin of the MLL-PTD in this subfraction any further, e.g. to multipotent stem cells, the existence of MLL-PTD in a proliferating and differentiating subfraction indicates,
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that PTD in healthy individuals may derive from a positive progenitor cell subfraction instead of emerging spontaneously in further matured cells. Furthermore, the presence of MLL-PTD in a subfraction which is sensitive to leukemic transformation, limits its inherent leukemogenic potential. The transcription level of MLL-PTD in AML was much higher than in normal hematopoiesis. This may indicate that either PTD-positive cells in normal hematopoietic are less frequent than in AML but have identical transcription levels. On the other hand, the intracellular transcription level in AML blasts may be higher than in normal PTD positive cells, due to e.g. additional alterations in regulatory elements. As recently observed in samples of patients with AML, highly transcribed MLL-PTD seem to repress the MLL-wildtype gene expression. Inappropriate dosage of the MLL-wildtype gene, known to impair stem cell development and definitive hematopoiesis [1] and a gain of function mechanism by MLLPTD, as suggested by the strong transctivation potential of MLL-PTD in vitro [28], may therefore represent a pivotal mechanism leading to malignant transformation. In conclusion the lifelong exposure of the hematopoietic system to MLL-PTD and their presence in the progenitor cell subfraction are not key determinants in their contribution to leukemogenesis. MLL-PTD are somewhat reminiscent of the translocation t(8; 21), characteristic of AML FAB M2, which also present at low levels in healthy newborn and adults but, if transcribed at high levels, induce proleukemogenic alterations in mice [7,23]. MLL-PTD seem to have an inherent low leukemogenic potential and their role in the process of transformation may be dependent on a high transcription rate and/or cooperating alterations. In vivo systems will be helpful to further delineate the function of MLL-PTD in the process of leukemogenesis. Acknowledgment This work is supported by the German Hector-Stiftung (grant M18) to J.B. and F.G. References [1] Ernst P, Fisher JK, Avery W, Wade S, Foy D, Korsmeyer SJ. Definitive hematopoiesis requires the mixed-lineage leukemia gene. Dev Cell 2004;6(3):437–43. [2] Dorshkind K, Witte O. Got MLL? Definitive hematopoiesis requires MLL gene expression. Mol Cell 2004;13(6):765–6. [3] Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa III R, Patel Y, Harden A, et al. Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 1991;88(23):10735–9. [4] Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, et al. Ten novel 11q23 chromosomal partner sites. European 11q23 Workshop participants. Leukemia 1998;12(5):811–22. [5] Rowley JD. Rearrangements involving chromosome band 11Q23 in acute leukaemia. Semin Cancer Biol 1993;4(6):377–85. [6] So CW, Cleary ML. Dimerization: a versatile switch for oncogenesis. Blood 2004;104(2):919–22.
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