Monitoring of minimal residual leukemia in patients with MLL ... - Nature

Leukemia (1999) 13, 1519–1524  1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu

Monitoring of minimal residual leukemia in patients with MLL-AF9 positive acute myeloid leukemia by RT-PCR G Mitterbauer1, C Zimmer1, C Fonatsch2, OA Haas3, R Thalhammer-Scherrer4, I Schwarzinger4, P Kalhs5, U Jaeger6, K Lechner6 and C Mannhalter1 1

Department of Laboratory Medicine, Division of Molecular Biology, 4Department of Laboratory Medicine, Division of Hematology, Department of Medicine I, Division of Hematology and Hemostaseology, 5Department of Medicine I, Bone Marrow Transplantation Unit, 2 Institute of Medical Biology, University of Vienna; and 3Children’s Cancer Research Institute, St Anna Children’s Hospital, Vienna, Austria 6

Twenty-seven patients with AML and MLL gene rearrangement were analyzed by a reverse transcriptase polymerase chain reaction (RT-PCR) for the MLL-AF9 translocation. The MLL-AF9 fusion transcript was detected in six patients. In five patients, the breakpoint of the AF9 gene was located within the recently described site A; in one patient, a novel breakpoint (AF9 site D) mapped to a position 377 bp 3⬘ of site A. Five patients could be serially monitored for a period of 4–23 months. Two patients became two-step PCR negative in bone marrow and peripheral blood. Molecular remission was achieved rapidly after one cycle of induction chemotherapy. Both patients are in continuous complete remission (CR) at 22 and 15 months, respectively. Two patients who had achieved hematological CR did not become PCR negative and MLL-AF9 fusion transcripts were detectable in all samples after induction and consolidation chemotherapy. One patient relapsed 5 months after achieving CR. The other patient received allogeneic bone marrow transplantation from an HLA-identical sibling 2 months after achieving hematological CR and became PCR negative 4 weeks after transplantation. In the fifth patient, hematological CR could not be achieved with two cycles of intensive induction chemotherapy, and MLL-AF9 transcripts were present in all samples tested. Our data indicate that MLL-AF9 RT-PCR is specific for the t(9;11) translocation. PCR negativity can be achieved in responding patients already 1 month after induction chemotherapy. The fast reduction of MLL-AF9 positive blast cells below the detection limit of RT-PCR seems to be a prerequisite for long-term CR. The results of RT-PCR may be useful for treatment decisions (eg BMT). Keywords: acute myeloid leukemia; t(9;11)(p22;q23); MLL-AF9; RT-PCR; minimal residual disease (MRD)

Introduction In AML and ALL, the human chromosome band 11q23 is frequently involved in chromosomal translocations. As summarized at the European Union Concerted Action Workshop on 11q23, more than 70 chromosome partners had been reported previously. The Workshop described 10 novel chromosome partner sites involved in translocations with chromosome band 11q23.1 Recently, the MLL gene, also known as ALL-1, HTRX-1, or HRX, has been shown to be disrupted by these translocations.2 At least 14 partner genes (on chromosomes 1, 4, 6, 9, 10, 16, 17, 19, 22 and X) have been fully characterized.3–15 The role of these partner genes in leukemogenesis and the possible prognostic impact of distinct 11q23 translocations is largely unexplored. Genomic breakpoints in the MLL gene cluster in a restricted 8.3 kb BamHI fragment spanning exons 5–11.11,16–20 Southern blot analysis with a 0.9 kb cDNA probe can detect almost all Correspondence: G Mitterbauer, Department of Laboratory Medicine, Division of Molecular Biology, University Vienna Medical School, Wa¨hringer Gu¨rtel 18–20, A-1090 Vienna, Austria; Fax: 43 1 40400 2097 Received 29 April 1999; accepted 24 June 1999

MLL gene rearrangements in which the 5⬘ region of the MLL gene is juxtaposed to the 3⬘ part of translocation partner genes. The MLL translocations usually lead to the generation of fusion genes that are transcribed into fusion mRNA. The translocation t(9;11)(p22;q23) is the most common of the 11q23 translocations in both de novo and therapy-related AML and results in the fusion of parts of the MLL gene and the AF9 gene at 9p22.4,21 In most cases, the subtle reciprocal translocation exchanging the terminal short and long arm segments of chromosomes 9 and 11, respectively, is associated with FAB subtypes with monocytic features, M4 and M5.22,23 The MLL-AF9 fusion gene leads to the generation of a fusion protein that appears to play a crucial role in leukemogenesis.18 At the molecular level, most of the breaks within the AF9 gene occurred in the central breakpoint cluster region, called site A (at nucleotide 1321 according to the mRNA numbering reported by Nakamura et al).4,21,24–27 In some patients, the breakpoints mapped to a more 3⬘ region, site B (nucleotide 1627).4,26,27 In a recently described patient, a new fusion site C was located at nucleotide 616, upstream of the previously identified breakpoints A and B.26 Typically, AF9 mRNA is fused with exons 5, 6, 7, or 8 of the MLL gene. A study of 125 t(9;11) positive patients with AML showed that white cell count (WBC) and age are significant predictors of event-free survival. Central nervous system (CNS) involvement and karyotype class (sole, with trisomy 8, or with other chromosomal abnormalities) also contribute to prognosis.28 Previous studies in childhood and adult AML have shown that patients with t(9;11)(p22;q23) may have a significantly better prognosis than those with translocations between 11q23 and other partner chromosomes.29–31 Preliminary findings suggest that intensive postremission therapy containing high-dose cytarabine in first complete remission (CR) may be especially beneficial in adult patients with t(9;11).31 For this reason, reliable diagnostic techniques for detection of t(9;11) are required, but conventional karyotyping does not always provide sufficient metaphases in all bone marrow (BM) specimen. During the last few years, sensitive PCR-based techniques for detection of 11q23 translocations have become available. We studied the efficacy and clinical applicability of RT-PCR for the detection and monitoring of minimal residual disease (MRD) in MLL-AF9 positive AML patients. Materials and methods

Patients Two hundred and nine patients with AML, who were diagnosed at the Department of Medicine I, Division of Hematology, between 1992 and 1998, were included in the study. Diagnosis of AML was made according to the French–American–British (FAB) classification. All patients but one were

Minimal residual leukemia in MLL-AF9 positive AML patients G Mitterbauer et al

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classified as de novo AML. The only exception was an MLLAF9 positive patient (case 4) who developed AML (FAB M1) 5 years after immunosuppression with azathioprine following renal transplantation for end-stage renal disease secondary to diabetes mellitus. Four MLL-AF9 positive patients (cases 1–3, 5) received induction chemotherapy containing daunorubicin, etoposide and cytarabine.32 The patient with biphenotypic leukemia (case 5) was treated with an AML protocol because pretreatment with prednisone had no cytoreductive effect on blast cells. Patient 4 received induction chemotherapy with intermediate-dose cytarabine. Consolidation chemotherapy consisted of four cycles of intermittent high-dose cytarabine in patient 1,33 one cycle of cytarabine-mitoxantrone and three cycles of intermittent high-dose cytarabine in patient 2, one cycle of cytarabine-mitoxantrone in patient 3, and one cycle of daunorubicin, etoposide and cytarabine in patient 4. One patient (case 3) received allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling 2 months after achieving hematological CR (Table 1).

Cytogenetics Bone marrow (BM) for cytogenetic analysis was obtained at initial diagnosis or relapse. Unstimulated isolated BM cells were cultured for 24–72 h. Chromosomes were prepared, and G-banding after trypsin or 2 × SSC pretreatment was performed according to standard techniques. If possible, 20 metaphases were analyzed. Karyotyping was performed on a PSI chromosome analysis system (PSI, Halladale, UK) or after microphotography of metaphases and the results were described according to ‘An International System for Human Cytogenetic Nomenclature (ISCN)’.34

Southern blot analysis All 209 patients were screened for translocations involving the MLL gene by Southern blot analysis with a cDNA probe for the MLL-breakpoint cluster region. High-molecular weight DNA was prepared from BM and/or peripheral blood (PB) using proteinase K (Boehringer Mannheim, Mannheim, Germany) digestion, phenol extraction and ethanol precipitation. Genomic DNA was digested with BamHI, electrophoresed through 0.8% agarose gels, transferred to nylon membranes (GeneScreen; NEN Life Science Products, Boston, MA, Table 1

Patient

USA) and hybridized with a random primed 32P-dCTP labeled cDNA probe (provided by ML Cleary, Department of Pathology, Stanford University Medical Center, Stanford, CA, USA). Membranes were washed at a final stringency of 0.1 × saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 65°C before autoradiography.

RT-PCR Mononuclear cells (MNC) were separated from BM and/or PB by density gradient centrifugation using Ficoll (Pharmacia Biotech, Uppsala, Sweden). Aliquots of 107 cells were stored in RNAzol B (Biotecx, Houston, TX, USA) at −80°C. Total RNA was extracted according to the RNAzol B protocol supplied by the manufacturer (modified acid guanidinium thiocyanate-phenol-chloroform method).35 The integrity of RNA was controlled by electrophoresis through 1% agarose gels containing formaldehyde. cDNA synthesis was performed with 2 ␮g total RNA in a reaction volume of 20 ␮l as previously described.36 Aliquots of 3 ␮l cDNA were amplified with GeneAmp PCR Core Reagents (Perkin Elmer Cetus, Norwalk, CT, USA) using 10 pmol of primer MLL6S (5⬘-GCAAACAGAAAAAAGTGGCTCCCCG-3⬘) and AF9AS3 (5⬘-TCACGeneAmp GATCTGCTGCAGAATGTGTCT-3⬘), 200 ␮M dNTPs, 2.5 mM MgCl2 and 1 U AmpliTaq Gold DNA polymerase in a total reaction volume of 50 ␮l. After an initial denaturation of 10 min at 95°C, 35 cycles of 1 min at 95°C, 1 min at 64°C and 1 min at 72°C, followed by a final extension at 72°C for 10 min were performed in a Perkin Elmer Cetus DNA Thermal Cycler (Emeryville, CA, USA). The PCR products were analyzed on 6%-polyacrylamide gels (Novex, San Diego, CA, USA) and visualized by SYBR Green I (Molecular Probes, Leiden, The Netherlands) staining. The specificity of PCR products was proved by a second PCR amplification of 3 ␮l 1:1000 diluted first round PCR products with inner primers MLL7S (5⬘-CCTCCGGTCAATAAGCAGGAGAATG-3⬘) and AF9AS1 (5⬘-CAGAGTCATTGTCGTTATCCTCCAC-3⬘). PCR conditions were as above, but only 30 cycles were carried out. Quality of cDNA was assessed by concurrent amplification of MLL transcripts from the same cDNA using primer MLL7S and MLL9AS (5⬘-TTGTAGCCTGATGTTGCCTTCCACA-3⬘). Precautions to prevent crosscontamination of the amplified material were taken according to published recommendations.37,38 Nucleotide sequences were determined by cycle sequen-

Patients’ characteristics, treatment and clinical outcome

Age/Sex

FAB subtype

1 2 3

30/M 58/M 22/F

M5a M5b M5a

4

74/M

M1

5 6

68/M 67/M

Karyotype

47,XY,t(9;11)(p22;q23),+8 46,XY,t(9;11)(p22;q23) 46,XX,t(9;11)(p21苲22;q23), der(10)t(1;10)(q25;q22) 46,XY,t(9;11)(p21;q23)

bipheno 46,XY,t(9;11)(p21苲22;q23) M5a 47,XY,inv(4)(p16q12)c, inv(9)(p12q13),ins(11;9)(q23; p22p23),+20.ish ins(11;9)(wcp11+;wcp9+)

Southern blot

MLL fusion AF9 fusion point point

Therapy

R R R

Exon 7 + 8 Exon 7 + 8 Exon 7 + 8

Site A Site A Site A

R

Exon 7 + 8

Site A

Ind, C1–C4 Ind, C1–C4 Ind, C1, allo BMT Ind, C1

R R

Exon 7 + 8 Exon 6

Site A Site D

Ind, Reind none

Current status

alive in CR 22 months+ alive in CR 15 months+ alive in CR 11 months+ died (10 months) in first relapse died (4 months)/NR early death

bipheno, biphenotypic AML; R, MLL gene rearrangement; Ind, induction chemotherapy; C1–C4, consolidation chemotherapy 1–4; NR, no remission.


cing of PCR products using the Ampli Cycle Sequencng Kit (Perkin Elmer, Branchburg, NJ, USA). Results

Sensitivity of MLL-AF9 RT-PCR Mononuclear cells of patient 5 at diagnosis (⬎90% blast cells in BM) were serially diluted with MNC from a healthy volunteer. RNA samples extracted from these mixtures were amplified by RT-PCR. In patient 5, two alternatively spliced fusion mRNAs (MLL exon 8–AF9 site A and MLL exon 7–AF9 site A) could be demonstrated. After one-step PCR, both fusion transcripts were detected up to the 10−3-fold dilution; in the 10−4-fold dilution only the PCR product generated by fusion between MLL exon 8 and the AF9 gene could be amplified. After two-step PCR, both fusion transcripts could be detected reliably up to the 10−4-fold dilution. In the 10−5- and 10−6-fold dilution, only one fusion transcript could be amplified in part of the tested material (Figure 1).

Detection of MLL-AF9 mRNA in AML patients with rearranged MLL gene The MLL-AF9 fusion transcript was detected in six of 27 patients with rearranged MLL gene: four patients with FAB M5, one patient with M1 and one patient with biphenotypic acute leukemia (Table 1). The blast cells of the patient with biphenotypic leukemia expressed CD33 (40%), CD15 (90%), CDw65 (80%), CD19 (90%) and intracytoplasmic CD79a (40%). The median age of these patients was 62 years (range 22–74 years). In patients 1–5, the t(9;11) was detectable by cytogenetic analysis. Additional chromosomal abnormalities

were found in patients 1 (trisomy 8) and in patient 3 (der(10)t(1;10)(q25;q22). In patient 6, conventional cytogenetic analysis did not exhibit a t(9;11). FISH analysis with whole chromosome painting (wcp) probes showed an inversion of chromosome 9 and an insertion of a small segment of chromosome 9 into the long arm of chromosome 11 (47,XY,inv(4)(p16q12)c,inv(9)(p12q13), ins(11;9)(q23; p22p23),+20.ish ins(11;9)(wcp11+;wcp9+). No t(9;11) was detected by cytogenetic analysis of MLL-AF9 negative patients with rearranged MLL gene. In patients 1–5, PCR products with two different sizes of 765 bp and 651 bp were obtained with primers MLL6S and AF9AS3 (Figure 2). Sequence analysis of the larger band showed fusion between MLL exon 8 and AF9 fusion site A; the smaller band contained sequences of MLL exon 7 fused to AF9 site A. In patient 6, RT-PCR amplification gave a 142 bp product (Figure 2). Sequence analysis showed that this PCR product was generated by fusion of MLL exon 6 to a novel breakpoint in the AF9 gene. This AF9 ‘D’ fusion site is located at nucleotide 1698 (numbering according to the mRNA sequence reported by Nakamura et al4), downstream of the previously identified breakpoints. In some samples (Figure 2: patients 1, 4 and 6), additional faint bands were amplified during RT-PCR. These bands showed variable size, could not be reliably reproduced in repeated experiments, and could not be amplified with a set of inner primers in a nested PCR. Therefore, these bands appear to be the result of non-specific amplification.

Serial monitoring of MLL-AF9 positive AML patients by RT-PCR Five t(9;11) positive patients (cases 1–5) could be serially monitored for a period of 4–23 months for detection of minimal residual disease by a two-step ‘nested primer’ RT-PCR

Figure 1 Sensitivity of MLL-AF9 RT-PCR. Both alternatively transcribed fusion transcripts (MLL exon 8–AF9 and MLL exon 7–AF9) can be reliably amplified up to the 104-fold dilution by two-step PCR. M, 100 base pair ladder size marker; neg, negative control.

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Figure 2

MLL-AF9 fusion transcripts in patients with AML. M, 100 base pair ladder size marker; neg, negative control.

Figure 3 Monitoring of MLL-AF9 positive AML patients by RT-PCR. Closed bar, more than 5% blast cells in bone marrow; open bar, hematological complete remission; circles, peripheral blood; squares, bone marrow; closed symbols, MLL-AF9 positive PCR; open symbols, MLL-AF9 negative PCR; Ind, induction chemotherapy; Reind, reinduction chemotherapy; C1–C4, consolidation chemotherapy 1–4; allo BMT, allogeneic bone marrow transplantation.

(Figure 3). Four to 16 samples per patient (median, 15 samples) were analyzed (BM: 2–6 samples; median, four samples; PB: 2–12 samples; median, nine samples). Patient 6 could not be monitored, because he died before the start of chemotherapy. Patients 1–4 achieved hematological CR after one cycle induction chemotherapy. Patients 1 and 2 became two-step PCR negative in BM and PB. Molecular remission was achieved within 1 month after the start of induction chemotherapy. Both PCR negative patients are in continuous hematological and molecular CR at 22 and 15 months after diagnosis, respectively. Patients 3 and 4 achieved a hemato-

logical CR but no molecular CR. MLL-AF9 fusion transcripts were detectable in all BM and PB samples after induction and consolidation therapy. Patient 3 received an allogeneic BMT from an HLA-identical sibling 2 months after achieving hematological CR. She became PCR negative 4 weeks after BMT and is still in molecular remission at 8 months after BMT. Patient 4 relapsed 5 months after achieving hematological CR. In patient 5, hematological CR could not be induced with two cycles of intensive induction chemotherapy, and MLL-AF9 transcripts could be amplified in all BM and PB samples tested.


Discussion The demonstration of MLL gene rearrangements alone, without simultaneous identification of the partner chromosome, is not sufficient for the prediction of the clinical outcome in patients with 11q23 abnormalities.31 RT-PCR is a useful approach for the detection of the involved partner gene and can also be used to monitor minimal residual disease. The good agreement between cytogenetic and molecular analyses of our study indicates that MLL-AF9 RT-PCR is specific for the t(9;11) translocation. Recent studies have shown that the detection of fusion transcripts by RT-PCR allows the most precise and sensitive diagnosis of some genetic subtypes of AML. Preliminary molecular monitoring studies of minimal residual disease suggest that this technique also allows the identification of patients at high risk for relapse who require further therapy. The clinical significance of detection of minimal residual disease in CR depends on the underlying genetic abnormality. The elimination of PML-RAR␣ positive cells below the detection limit of 1 in 104 is a prerequisite for stable remissions in acute promyelocytic leukemia,39 while the AML1-ETO fusion transcript is consistently detected in patients with t(8;21) positive AML after attainment of complete morphologic and cytogenetic remission and even after BMT.40 To date, molecular studies of AML patients with other translocations are limited and the clinical significance of PCR monitoring is still a controversial issue. Larger studies using new technologies (eg real-time PCR) may allow even more precise detection and the quantitation of residual leukemic cells. We have used a two-step MLL-AF9 RT-PCR assay with a sensitivity of 1 in 104 to 1 in 106 to monitor minimal residual disease in five AML patients with t(9;11). In all cases, we found corresponding results in BM and PB samples collected on the same day (Figure 3). Because of the small number of comparisons that could be performed, we cannot exclude that patients may be found who exhibit PCR negativity in PB and PCR positivity in BM at certain time points. Four patients responded to standard induction chemotherapy with hematological CR, but only two patients achieved a molecular CR. PCR negativity in responding patients was observed already 1 month after the start of induction chemotherapy. The fast reduction of MLL-AF9 positive blast cells below the detection limit of RT-PCR seems to be a prerequisite for long-term CR, as one patient who did not reach PCR negativity relapsed already 5 months after achieving CR despite high-dose consolidation chemotherapy. Another patient who was still MLLAF9 positive after conventional chemotherapy received allogeneic BMT. She became PCR negative and is in continuous hematological and molecular remission at 8 months after BMT. The continuous hematological and molecular CR of patients 1 and 2 indicates that consolidation chemotherapy with high-dose cytarabine may be effective in patients with t(9;11) as it has been observed in patients with core binding factor (CBF) type abnormalities; ie t(8;21), inv(16), t(16;16), and del(16).41 Our findings show that RT-PCR provides a rapid, accurate, and sensitive tool for diagnosing leukemia with t(9;11)(p22;q23) and monitoring response to treatment in these patients. Even though our data allow only limited conclusions because of the small number of patients, they indicate that PCR negativity should be the primary therapeutic goal in AML with t(9;11). Without molecular CR, no long-term remissions could be achieved. The results of RT-PCR may be useful for treatment decisions (eg BMT in patients who remain

MLL-AF9 positive after conventional chemotherapy). The good results in some of our patients confirm the findings of Mro´zek et al,31 that the outcome of adults with t(9;11) positive AML is more favorable than that of patients with other 11q23 abnormalities, especially if these patients receive intensive postremission therapy. Nevertheless, a significant proportion of t(9;11) positive AML patients with more resistant disease might profit from MRD-based treatment modification. Acknowledgements This work was supported by a grant from the Kommission Onkologie of the University of Vienna. References 1 Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM. Ten novel 11q23 chromosomal partner sites. Leukemia 1998; 12: 811–822. 2 Ziemin-van der Poel S, McCabe NR, Gill HJ, Espinosa R III, Patel Y, Harden A, Rubinelli P, Smith SD, Le Beau MM, Rowley JD, Diaz MO. Identification of a gene, MLL, that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc Natl Acad Sci USA 1991; 88: 10735–10739. 3 Bernard OA, Mauchauffe M, Mecucci C, Van-den Berghe H, Berger R. A novel gene, AF-1p, fused to HRX in t(1;11)(p32;q23), is not related to AF-4, AF-9 nor ENL. Oncogene 1994; 9: 1039– 1045. 4 Nakamura T, Alder H, Gu Y, Prasad R, Canaani O, Kamada N, Gale RP, Lange B, Crist WM, Nowell PC, Croce CM, Canaani E. Genes on chromosomes 4, 9, and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs. Proc Natl Acad Sci USA 1993; 90: 4631–4635. 5 Prasad R, Gu Y, Alder H, Nakamura T, Canaani O, Saito H, Huebner K, Gale RP, Nowell PC, Kuriyama K, Miyazaki Y, Croce CM, Canaani E. Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation. Cancer Res 1993; 53: 5624–5628. 6 Hillion J, Le Coniat M, Jonveaux P, Berger R, Bernard OA. AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. Blood 1997; 90: 3714–3719. 7 Chaplin D, Ayton P, Bernard OA, Saha V, Della Valle V, Hillion J, Gregorini A, Lillington D, Berger R, Young BD. A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia. Blood 1995; 85: 1435–1441. 8 Taki T, Shibuya N, Taniwaki M, Hanada R, Morishita K, Bessho F, Yanagisawa M, Hayashi Y. ABI-1, a human homolog to mouse Abl-interactor 1, fuses the MLL gene in acute myeloid leukemia with t(10;11)(p11.2;q23). Blood 1998; 92: 1125–1130. 9 Sobulo OM, Borrow J, Tomek R, Reshmi S, Harden A, Schlegelberger B, Housman D, Doggett NA, Rowley JD, Zeleznik-Le NJ. MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3). Proc Natl Acad Sci USA 1997; 94: 8732–8737. 10 Prasad R, Leshkowitz D, Gu Y, Alder H, Nakamura T, Saito H, Huebner K, Berger R, Croce CM, Canaani E. Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia. Proc Natl Acad Sci USA 1994; 91: 8107–8111. 11 Tkachuk DC, Kohler S, Cleary ML. Involvement of a homolog of Drosophila trithorax by 11q23 chromosomal translocations in acute leukemias. Cell 1992; 71: 691–700. 12 Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway JW. An RNA polymerase II elongation factor encoded by the human ELL gene. Science 1996; 271: 1873–1876. 13 So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ, Sham MH, Wiedemann LM, Chan LC. 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

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