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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 810-821
Clinical Significance of MLL-AF4 Fusion Transcript Expression
in the Absence of a Cytogenetically Detectable t(4;11)(q21;q23)
Chromosomal Translocation
By
Fatih M. Uckun,
Kim Herman-Hatten,
Mya-Lisa Crotty,
Martha G. Sensel,
Harland N. Sather,
Lisa Tuel-Ahlgren,
Mireille B. Sarquis,
Bruce Bostrom,
James B. Nachman,
Peter G. Steinherz,
Paul
S. Gaynon, and
Nyla Heerema
From the Children's Cancer Group ALL Biology Reference Laboratory,
Parker Hughes Cancer Center, and the Departments of Biology,
Immunology, and Molecular Genetics, Hughes Institute, St Paul, MN; The
Children's Cancer Group Operations Office, Arcadia, CA; the Department
of Preventive Medicine, University of Southern California School of
Medicine, Los Angeles; the Department of Medical and Molecular
Genetics, Indiana University School of Medicine, Indianapolis; the
Department of Pediatric Hematology-Oncology, Children's Health Care,
Minneapolis, MN; the Section of Pediatric Hematology-Oncology,
University of Chicago Medical Center, Chicago, IL; the Department of
Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY; and
the Department of Pediatric Hematology-Oncology, University of
Wisconsin, Madison.
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ABSTRACT |
Leukemic cells from bone marrow (BM) of 17 infants and 127 children
with newly diagnosed ALL, as well as fetal liver and BM and normal
infant BM samples, were analyzed for presence of a t(4;11)
translocation using standard cytogenetic techniques and expression of
an MLL-AF4 fusion transcript using standard reverse transcriptase-polymerase chain reaction (RT-PCR) assays as well as
nested RT-PCR that is 100-fold more sensitive than standard RT-PCR.
Overall, 9 of 17 infants and 17 of 127 noninfant pediatric ALL patients
were positive for expression of MLL-AF4 fusion transcripts, as
determined by standard and/or nested RT-PCR assays. None of the
MLL-AF4+ cases were positive for E2A-PBX1
or BCR-ABL fusion transcript expression. Although 8 of 9 MLL-AF4+ infants had cytogenetically detectable
t(4;11)(q21;q23), 15 of the 17 MLL-AF4+
noninfants were t(4;11) . Infants with
MLL-AF4+ ALL had poor outcomes, whereas
non-infant MLL-AF4+/t(4;11)
patients had favorable outcomes similar to
MLL-AF4 patients. Notably, MLL-AF4
transcripts also were detected by nested RT-PCR in 4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs, but not in any of the
44 remission BM specimens from pediatric ALL patients. Our results
provide unprecedented evidence that MLL-AF4 fusion transcripts
can be present in normal hematopoietic cells, indicating that their
expression is insufficient for leukemic transformation of normal
lymphocyte precursors.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CHROMOSOMAL TRANSLOCATIONS found in
childhood and adult acute lymphoblastic leukemia (ALL) may result in
the production of chimeric fusion proteins with leukemogenic
potential.1,2 The prototype of such translocations,
t(9;22)(q34;q11), is associated with high risk in B-lineage ALL and
results in the production of BCR-ABL fusion mRNAs and
proteins.3,4 Similarly, the t(4;11)(q21;q23) translocation,
a hallmark of infant ALL, results in the fusion of the MLL gene
on chromosome 11 and the AF4 gene on chromosome 4.5-9 The presence of a t(4;11) translocation is associated
with unfavorable presenting features such as very high white blood cell
(WBC) count,10,11 and the majority of t(4;11)+
infant ALL patients have an extremely poor prognosis.12-16
Recently, the identification of in utero rearrangements have implicated the MLL gene in the leukemogenesis of infant
ALL.17
The MLL gene encodes a protein that contains regions with
homology to the Drosophila trithorax gene,18,19
including A-T hook and transcriptional repression domains located
5 to the MLL-AF4 breakpoint, a zinc finger homology
domain located at the breakpoint, and a transcriptional activation
domain located 3 to the breakpoint.20,21 The A-T
hook domain has been shown to mediate binding of MLL to cruciform DNA,
irrespective of sequence. The AF4 gene encodes a protein with
serine- and proline-rich regions located near the AF4 breakpoint and a
nuclear localization signal domain located 3 to the
breakpoint.19,22 Thus, translocations between MLL
and AF4 that disrupt these functional activities may alter
expression of genes regulated by MLL or AF4, or may endow novel fusion
proteins with transcriptional regulatory activity.
Although fusion transcripts from both reciprocal derivative chromosomes
are found in leukemic cells with t(4;11)(q21;q23) translocations, it is
unclear which of the two products is leukemogenic.19,23 However, clustering of breakpoints found in various 11q23
rearrangements, heterogeneity of breakpoints in 11q23 fusion partners,
and loss of the translocated MLL region telomeric to 11q23 have
lead to the hypothesis that the der(11) fusion product, which is
encoded by the 5 portion of the MLL gene, represents the
biologically more relevant potential oncogenic fusion
protein.23-26
Rubnitz et al,27 as well as Chen et al28 and
Griesinger et al,29 have shown the presence of MLL
gene rearrangements in infant ALL patients with or without
cytogenetically detectable t(4;11) or other structural chromosomal
aberrations involving 11q23. These studies were interpreted to indicate
that the frequency of molecular 11q23 rearrangements and
MLL-AF4 fusion transcripts was greater than that reported using
cytogenetic analyses alone, and suggested the importance of
molecular-based techniques for screening ALL patients.
Recently, however, new questions concerning the clinical significance
of malignancy-associated fusion transcripts have arisen as a result of
several reports that documented their occurrence in normal or
nonleukemic cells. For example, Limpens et al30,31 and
other groups,32-35 using highly sensitive polymerase chain reaction (PCR) techniques, have demonstrated the presence of
lymphoma-associated t(14;18)(q32;q21)36,37 in benign
follicular hyperplasias and normal blood of healthy individuals.
Similarly, Biernaux et al38 recently reported that the
BCR-ABL fusion transcript, which is thought to be derived from
the t(9;22) translocation associated with high-risk ALL, was present in
hematopoietic cells of healthy individuals. Thus, the relationship
between the translocation and transformation in lymphoid malignancies
requires further investigation.
These observations led us to examine primary leukemic cells from ALL
patients, fetal tissues, and normal infant bone marrows (BMs) for the
presence of MLL-AF4 fusion transcripts. Our results show that
MLL-AF4 fusion transcripts, detectable by a sensitive nested
reverse transcriptase (RT)-PCR assay, are frequently generated in
patients whose cells lack cytogenetically detectable t(4;11) and that
expression of MLL-AF4 fusion transcripts is not a significant prognostic factor for these patients. Notably, MLL-AF4 fusion transcripts were also detected by nested PCR in normal hematopoietic cells from fetal tissues and infant BMs. These results suggest that the
presence of MLL-AF4 fusion transcripts may not be sufficient for neoplastic transformation of lymphocyte precursors and add to
recent evidence that leukemia-associated gene rearrangements can occur
in normal hematopoiesis throughout the lifespan of an individual,
thereby providing only a potentially contributory but not final
transforming event.
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MATERIALS AND METHODS |
Patient samples and cell lines.
The patient population included 17 infants and 127 children with newly
diagnosed ALL treated on the Children's Cancer Group (CCG) 1800 series
protocols between December 1993 and December 1994 for whom complete
cytogenetic, immunophenotypic, and clinical data were obtained. Also
included were 44 children with ALL in remission. Most of the children
included in the present analysis had standard-risk ALL. Diagnosis of
ALL was based on morphological, biochemical, and immunological features
of the leukemic cells, including lymphoblast morphology on
Wright-Giemsa-stained BM smears, positive nuclear staining for
terminal deoxynucleotidyl transferase (TdT), and cell-surface
expression of two or more lymphoid differentiation antigens, as
previously described.39 Surplus cells from diagnostic BM
specimens were used for molecular genetic studies. In two cases, 10 sequential BM specimens were examined for MLL-AF4 fusion
transcript expression by nested RT-PCR. Informed consent was obtained
from parents, patients, or both, as deemed appropriate, according to the Department of Health and Human Services guidelines. The RS4;11 cell
line harboring the t(4;11) translocation was obtained from John H. Kersey (University of Minnesota) and served as a positive control in
MLL-AF4 RT-PCR assays.
Fetal tissues and normal infant BMs.
Human fetal livers (N = 13) and fetal BMs (N = 16) from
prostaglandin-induced human abortuses of gestational age 15 to 22 weeks
were used according to the guidelines of the University of Minnesota
Committee on the Use of Human Subjects in research for secondary use of
pathological or surgical tissue. Normal BM specimens were obtained from
infants (N = 6) who were either BM donors in the context of sibling BM
transplantation or had been examined for suspicious cells in the blood
but were found to have a normal BM without any evidence of leukemia.
Immunophenotyping.
Mononuclear cell fractions comprised primarily of leukemic cells were
isolated from diagnostic BM aspirate samples by centrifugation on
Ficoll-Hypaque density gradients, as previously
described.39 Immunophenotyping was performed centrally in
the CCG ALL Biology Reference Laboratory by indirect immunofluorescence
and flow cytometry using monoclonal antibodies (MoAbs) reactive with
the following differentiation antigens: CD2, CD3, CD5, CD7, CD10, CD19,
and CD34, as previously described.39 Patients were
classified as B-cell precursor ALL if  30% of their leukemic cells
were positive for CD19 and <30% of their leukemic cells were
positive for CD2, CD5, or CD7.
Cytogenetic analysis.
Cytogenetic analysis of leukemic cells was performed by local
institutions at diagnosis before initiation of therapy. Banded chromosomes were prepared from unstimulated peripheral blood or direct
and 24-hour cultured preparations of fresh BM, as described previously.12,40 Chromosome abnormalities were designated
using the 1995 International System for Human Cytogenetics
Nomenclature.41 Abnormal clones were defined as two or more
metaphase cells with identical structural chromosomal abnormalities or
extra chromosomes, or three or more metaphase cells with identical
missing chromosomes.
RT-PCR.
All PCR assays were performed centrally in the Children's Cancer Group
ALL Biology Reference Laboratory, as described.42-44 Briefly, total cellular RNA was extracted from cells using the RNeasy
total RNA isolation kit (Qiagen, Santa Clarita, CA), and 20% of the
total RNA sample was used for cDNA synthesis with Moloney murine
leukemia virus (MMLV) reverse transcriptase (GIBCO-BRL, Gathersburg,
MD) in the presence of dNTPs (= reaction mixture 1). For amplification,
cDNA products were denatured, diluted in PCR buffer containing
oligonucleotide primers and Amplitaq DNA polymerase (Perkin Elmer Cetus
Corp, Norwalk, CT; = reaction mixture 2), and subjected to 35 cycles in a DNA thermal cycler as described.42,43 Primers
were as follows: MLL-AF4, 5-AGAGCAGAGCAAACAGAA-3 and 5-GCTGAGAATTTGAGTGAG-3; E2A-PBX1,
5-GCCAGCCAGGCACCCTCCC-3 and
5-GTTGTCCAGCCGCATCAGCT-3; and BCR-ABL,
5-TCCGAGGCCACCATCGTGGGCGTCGGC-3 and
5-TGTGATTATAGCCTAAGACCCGGAG-3. For increased sensitivity, nested PCR11,45,46 was performed using the primers
5-AAGTGGCTCCCCCGCCCAAGTAT-3 and
5-TTGGGTTACAGAACTGACATG-3 for MLL-AF4 and
5-CCAACGATGGCGAGGGCGCCT-3 and
5-CGAGCGGCTTCACTCAGACC-3 for BCR-ABL. PCR
products were separated by electrophoresis in 1.2% agarose,
transferred to nylon membranes, and hybridized with the oligonucleotide
probes specific for internal sequences of E2A-PBX1 or
BCR-ABL as described.44 For the MLL-AF4
fusion transcript, a specific AF4 oligonucleotide probe
(5-TAGGGAAAGGAAACTTGGATG-3) was used. Reactions conducted in the absence of added mRNA substrate served as negative controls. Reactions conducted with RNA isolated from the cell line RS4;11, which
harbors the t(4;11) translocation, and from a patient (unique patient
number [UPN] 100), with t(9;22)+ ALL, were used as
positive controls for MLL-AF4 and BCR-ABL fusion transcripts, respectively. Strict precautions were used to prevent cross-contamination of samples and negative controls were included at
the RNA extraction as well as PCR amplification steps.47 To
prevent carryover of amplified cDNA sequences, we prepared our samples
in a dedicated room separated from the room in which the PCR reactions
were performed. UV germicidal lamps were used in biosafety hoods to
quickly damage any DNA left on exposed surfaces, making it unsuitable
for subsequent amplification. Separate sets of supplies and pipetting
devices were dedicated for sample preparation and for setting up
reactions. Deionized water, buffer solutions, disposable pipette tips,
and microcentrifuge tubes were autoclaved. We divided reagents into
aliquots to minimize the number of repeated samplings necessary. All
reagents used in the PCR reactions were prepared, divided, and stored
in an area that is free of the PCR-amplified products. Similarly,
oligonucleotides used for amplification were synthesized and purified
in a PCR product-free environment. Individuals involved in PCR
reactions were required to wear gloves and change them frequently. The
barrel of pipetting devices may become contaminated with aerosols
containing sample RNA, leading to cross-contamination of samples. To
prevent this, we used pipettes with aerosal-resistant tips. We mixed
reagents before dividing them into aliquots. All PCR reagents were
combined into a "premixture," which was then pipetted into
reaction tubes containing RNA/cDNA. Nonsample components, such as
premixed dNTPs, primers, buffer, and enzyme, were added to
the reaction tubes before sample cDNA (Molecular Bio-Products, San
Diego, CA). Negative controls included PCR products from RNA-free reaction mixture 1 plus reaction mixture 2 (= negative control 1) and
reaction mixture 1 containing RNA from RS4;11 cell line plus DNA
polymerase-free reaction mixture 2 (= negative control 2).
RNA integrity was confirmed by PCR amplification of the cABL mRNA,
which is ubiquitously expressed in human hematopoietic cells using the
primers 5-TTCAGCGGCCAGTAGCATCTGACTT-3 and
5-TGTGATTATAGCCTAAGACCCGGAG-3. In sensitivity assays
of the RT-PCR assay for MLL-AF4 fusion transcript expression,
varying numbers (1 to 107) of RS4;11 cells
were mixed with cells from an Epstein-Barr virus (EBV)-transformed
lymphoblastoid cell line to yield 0.00001% to 100%
MLL-AF4+ cells in a cell population containing a
total of 107 cells. RNA subtracted from those
107 cell samples were analyzed by standard versus nested
RT-PCR.
Molecular characterization of MLL gene rearrangements.
Total cellular DNA extracted from BM leukemic cells, fetal liver/BM, or
normal infant BM (= test samples) and unrearranged control DNA isolated
from normal peripheral blood lymphocytes were analyzed by Southern
blotting using standard techniques. DNA was digested using the
restriction endonucleases BamI-II, EcoRI, and
HindIII. Fragments were electrophoretically separated on 0.8%
agarose gels, transferred to nylon membranes, and hybridized with the
radiolabeled MLL-specific probes PS/4, 98.40, and
4.2E.16,28 Membranes were washed under high stringency
conditions and exposed to Kodak XAR-5 film (Eastman Kodak, Rochester,
NY).
MLL-AF4 sequence analysis.
MLL-AF4 cDNA from RT-PCR reaction mixtures was purified with
the QIAquick PCR purification kit (Qiagen). Purified PCR products were
cloned into the pCR II sequencing vector using the TA Cloning kit
(Invitrogen, San Diego, CA). Clones (24/patient) were sized by
EcoRI restriction analysis and two to four clones were randomly chosen for sequencing. The cloned PCR products were purified with a
Qiagen plasmid isolation kit and sequenced automatically with the
Thermosequenase sequencing kit (Amersham, Arlington Heights, IL) and
the ALF Sequencer (Pharmacia, LKB Biotech, Piscataway, NJ). The
sequences were compared with the published MLL-AF4 and the
t(4;11) translocation breakpoint region sequences obtained through
Bionet (Bionet Accession codes HUMMLLAF44F, HSMLLAF4F, and S67825).
Statistical analysis.
Event-free survival (EFS) for ALL patients with or without
MLL-AF4 fusion transcripts was analyzed using life table
methods and associated statistics. Events included induction failure
(nonresponse to therapy or death during induction), leukemic relapse at
any site, death during remission, or second malignant neoplasm,
whichever occurred first. Patients not experiencing an event at the
time of EFS analysis were censored at the time of their last contact. Life table estimates were calculated by the Kaplan-Meier (KM) procedure, and the standard deviation of the life table estimate was
obtained using Greenwood's formula.48 An approximate 95% confidence interval can be obtained from the life table estimate ± 1.96 SDs. Life table comparisons of EFS outcome pattern for patient
groups generally used the log rank statistic,49,50 and
P values for life table comparisons are based on the pattern of
outcome across the entire period of patient
follow-up.51
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RESULTS |
Detection of MLL-AF4 in infants and children with ALL by standard as
well as nested RT-PCR.
A balanced t(4;11)(q21;q23) translocation is frequently observed in
infant ALL.9,11,14,15 Of 56 infants with newly diagnosed ALL entered on the most recent CCG infant ALL protocol, 21 (37.5%) had
a t(4;11).13 By comparison, t(4;11) is a very rare
cytogenetic abnormality in childhood ALL. There were only 8 t(4;11)+ patients (0.6%) among 1,322 children with newly
diagnosed ALL entered on all CCG-1800 series protocols and only 2 of
653 (0.3%) children with standard-risk ALL entered on CCG-1881 and
CCG-1891 risk-adjusted protocols had a t(4;11) (N.H., F.M.U.,
unpublished data, June 1998). In a recent study we used
standard RT-PCR assays to examine primary leukemic cells from 642 children with ALL for the expression of MLL-AF4 fusion
transcript and found MLL-AF4 expression only in 0.7% of the
patient population, which excluded infants.44 Since Downing
et al11 reported that standard PCR was not sensitive enough
and nested PCR analyses were required for the detection of
MLL-AF4 fusion transcripts in 26% of t(4;11) ALL cases, we
decided to examine primary leukemic cells for the expression of
t(4;11)-specific MLL-AF4 fusion transcripts using both standard
as well as nested RT-PCR assays. When we compared the detection levels
achieved by standard versus nested PCR assays, we found that standard
PCR was capable of detecting 1% MLL-AF4+ cells
among 107 cells, whereas nested PCR was 100-fold more
sensitive, allowing the detection of 0.01%
MLL-AF4+ cell contamination among 107
cells (Fig 1).
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| Fig 1.
Sensitivity of standard and nested RT-PCR assays for
detection of MLL-AF4 fusion transcript-positive cells. Varying
numbers (1 to 107) of MLL-AF4+ RS4;11 cells
were mixed with cells from an EBV-transformed lymphoblastoid B-cell
line to yield 0.00001% to 100%
MLL-AF4+ cells in a test cell
population containing a total of 107 cells. RNA was
subtracted from these 107 cell samples and analyzed by
standard RT-PCR as well as nested RT-PCR for MLL-AF4
positivity, as described in Materials and Methods. CON, control.
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We first examined leukemic cells from 17 infants with ALL for
MLL-AF4 expression. For comparison, cells were also examined for expression of t(1;19)-specific E2A-PBX1, and
t(9;22)-specific BCR-ABL fusion transcripts. While 6 cases
(35%) were determined to be MLL-AF4+ by
standard PCR, 9 cases (53%) were found to be
MLL-AF4+ by nested PCR
(Table 1). Data for all 17 infants is summarized in
Table 2. Cytogenetic analysis showed
balanced t(4;11)(q21;q23) in 8 infants, normal diploid karyotypes in 2 infants, as well as t(1;19)(q23;p13), t(11;15)(q23;q26), and
t(7;11)(q22;q23) in 1 infant each. Three additional infants had complex
rearrangements and 1 infant was hyperdiploid without structural
abnormalities. Both standard and nested RT-PCR results generally were
in accord with the cytogenetics data. Specifically, all 6 standard
PCR+ infants also had a cytogenetically detectable t(4;11).
Of the additional 3 infants determined to be
MLL-AF4+ by nested PCR only, 2 had a t(4;11) and 1 was normal diploid (Table 1 and Table 2). The E2A-PBX1 and
BCR-ABL fusion transcripts were observed only in cells from
infants who were t(1;19)+ and t(9;22)+,
respectively. Infants with other translocations, including t(7;11) and
t(11;15), did not express MLL-AF-4 fusion transcripts in their leukemic cells (Table 2).
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Table 1.
Expression of MLL-AF4 Fusion Transcript in
Infants (N = 17) and Children (N = 127) With Cytogenetically
Classified Acute Lymphoblastic Leukemia
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In contrast to the high expression frequency of MLL-AF4 in
infant ALL, standard PCR detected MLL-AF4 positivity in only 1 of 127 (0.8%) pediatric ALL cases. However, MLL-AF4 fusion
transcripts were found in leukemic cells from 17 (13%) patients when
nested PCR assays were applied (Table 1).
Figure 2 depicts representative experiments
illustrating the detection of MLL-AF4 fusion transcripts in
t(4;11) ALL by standard and nested PCR as well as detection of
MLL-AF4 expression in non-t(4;11) ALL patients by nested PCR. RNA was reextracted from cryopreserved cells in 10 of the 15 MLL-AF4+ non-t(4;11) ALL cases and reanalyzed by nested
RT-PCR to confirm the initial results shown in Table 1 and Fig 2. As
shown in Fig 3, each of these 10 non-t(4;11) cases were MLL-AF4+ when retested by
nested PCR.
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| Fig 2.
Detection of MLL-AF4 fusion transcripts in
pediatric ALL BM specimens by standard and nested RT-PCR. RNA samples
from primary leukemic cells of 32 (A, 14 patients; B, 18 patients)
newly diagnosed pediatric ALL patients were examined for
MLL-AF4 fusion transcript expression by standard RT-PCR (A1 and
B1) and nested RT-PCR (A2 and B2). PEDS 76 shown in A.2 was a normal
diploid case (see Tables 3 and 6). PEDS 87 shown in B.1. and B.2 was a
hyperdiploid t(4;11) ALL case and PEDS 58 shown in B.2. was a
hyperdiploid non-t(4;11) case with del(11)(q23) (see Tables 3 and 6).
Amplified mRNA from the RS4;11 cell line was used as a positive control
(POS CON). Negative controls were PCR products from RNA-free reaction mixture 1 plus reaction mixture 2 (NEG CON 1) and reaction mixture 1 plus DNA polymerase-free reaction mixture 2 (NEG CON 2).
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| Fig 3.
Detection of MLL-AF4 fusion transcripts in non-t(4;11)
leukemic cells by PCR. Ethidium bromide-stained gel (A) and Southern blot (B) of the PCR reaction products from ALL patients who lack cytogenetically detectable t(4;11). First and last lanes contain molecular size markers. UPN-PEDS 69, 76, 86, and 106 had normal diploid
karyotypes. UPN-PEDS 79 had a pseudodiploid karyotype. UPN-PEDS 43, 44, 57, and 59 had hyperdiploid karyotypes with structural abnormalities.
UPN-PEDS 111 had hyperdiploid karyotypes without structural
abnormalities. UPN-PEDS 87, a patient with t(4;11), was included as a
positive control (see Tables 3 and 6).
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Of the 1,322 children treated on the CCG-1800 series protocols for whom
centrally acceptable cytogenetic data were available, 399 (30.2%) were
normal diploid, 74 (5.6%) were hypodiploid, 368 (27.8%) were
pseudodiploid, and 481 (36.4%) were hyperdiploid (N.H., F.M.U.,
unpublished data, June 1998). Similarly, of the 127 children included in the present study, 49 (38.6%) were normal diploid, 3 (2.4%) were hypodiploid, 33 (26.0%) were pseudodiploid, and 42 (33.1%) were hyperdiploid. There were only 2 patients with t(4;11) (1 pseudodiploid and 1 hyperdiploid case); standard PCR detected MLL-AF4 expression in 1 of these cases, whereas both cases were positive by nested PCR (Table 1). Notably, 15 of the remaining 125 non-t(4;11) patients (12%), including 3 pseudodiploid, 6 normal diploid, and 6 hyperdiploid cases, were found to be nested PCR
positive for MLL-AF4 fusion transcript expression even though standard PCR was negative in each of these 15 cases. Cytogenetic and
PCR data as well as the age and WBC count at presentation for the 17 pediatric ALL patients who expressed MLL-AF4 fusion transcripts are detailed in Table 3. None
of these 17 MLL-AF4 positive cases were positive for
E2A-PBX1 (standard PCR) or BCR-ABL expression (standard
and nested PCR). In summary, 17 of 127 noninfant pediatric ALL patients
were positive for expression of MLL-AF4 fusion transcripts, yet
only 2 of these patients had cytogenetically detectable
t(4;11)(q21;q23). Thus, MLL-AF4 fusion transcripts were found
in leukemic cells from patients lacking t(4;11) translocations.
We next used Southern blot analyses to examine HindIII-,
EcoRI-, or BamHI-digested genomic DNA of leukemic cells
from a subset of standard PCR, nested
PCR+ patients with MLL-AF4 fusion transcripts
for the presence of MLL gene rearrangements. As shown in
Table 4 and
Fig 4, cells from INF-6 and PEDS-87, two
t(4;11)+, standard PCR, nested
PCR+ ALL patients, had MLL gene rearrangements
occurring in the HindIII, EcoRI, and BamHI
fragments as detected by the PS/4, 98.4, or 4.2E probes. Similarly,
cells from PEDS-27, PEDS-44, and PEDS-5, three of the five
t(4;11), standard PCR, nested
PCR+ ALL patients, had MLL gene rearrangements
(Table 4, Fig 5). These results are
consistent with the notion that in some ALL cases with MLL
gene rearrangements and MLL-AF4 positivity, the level of
expression for MLL-AF4 fusion transcripts in leukemic cells
may be so low that a nested PCR is required for their detection. By
comparison, the absence of MLL gene rearrangements in some patients with MLL-AF4 positivity (eg, PEDS-59 and PEDS-106
in Table 4) indicates the existence of a very small subpopulation (detectable by nested PCR only) of leukemic cells expressing the MLL-AF4 fusion transcript.
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| Fig 4.
MLL rearrangement in leukemic cells from patients
with ALL. Genomic DNA was digested with BamHI or EcoRI
restriction enzymes, separated by electrophoresis, blotted to nylon
membranes, and hybridized with the radiolabeled P/S4 probe. (A) Probes
for analysis of MLL gene rearrangement. Partial restriction map
of the 11q23 region containing the MLL gene. Approximate
positions of MLL exons 4 through 13 (of 21 exons) are shown as gray
boxes. Areas mapped by probes P/S4, 98.40, and 4.2E are indicated.
Abbreviations: H, HindIII; E, EcoRI; B, BamHI;
S, Sac I. (B) BamHI digest. (C) EcoRI digest.
RS4;11 cells were used as a positive control and normal peripheral
lymphocytes served as a negative control. bp, base pairs.
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| Fig 5.
MLL rearrangement in leukemic cells, normal BM,
and fetal liver. Genomic DNA was digested with EcoRI, separated
by electrophoresis, blotted to nylon membranes, and hybridized with the
radiolabeled probes. (A) P/S4 probe. (B) 98.40 probe. RS4;11 cells were
used as a positive control and normal peripheral lymphocytes served as
a negative control. FL7 was isolated from a fetus age 19 gestational weeks. NBM4 was a BM sample from a normal infant BM donor age 6 months.
UPN-PEDS no. 91 was a male age 17.7 years with newly diagnosed t(4;11)
ALL. UPN-PEDS no. 5 was a female age 2.9 years with newly diagnosed
normal diploid ALL. bp, base pairs.
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Prognostic significance of MLL-AF4 fusion transcripts in
t(4;11) pediatric ALL patients.
Eight of the 9 infants with MLL-AF4+ ALL had
cytogenetically detectable t(4;11) and presented with very high WBC;
all 9 MLL-AF4+ ALL infants were
CD10 (Table 2). In accordance with previous reports,
outcome among the MLL-AF4+ infants was poor, with
only 2 of 9 patients surviving disease-free longer than 12 months after
diagnosis despite intensive chemotherapy and/or BM
transplantation (data not shown). Unlike MLL-AF4+
infants, who were CD10, all but 1 of the
MLL-AF4+ children were CD10+, and the
majority of these children (13 of 15) were ages 2 to 9.9 years (Table
3).
The 15 MLL-AF4+/t(4;11) patients
were similar to fusion transcript
(MLL-AF4/BCR-ABL/E2A-PBX1 = PCR) patients with respect to important presenting
clinical characteristics, including age, WBC count, organomegaly, risk
classification, and modal chromosome number.
The majority of patients in both groups were less than 10 years of age,
had WBC counts <50,000/µL, and were assigned to CCG protocols for
low- or intermediate-risk ALL, which corresponds to standard risk
according to NCI classification.52
All MLL-AF4+/t(4;11) patients
and the majority (96%) of PCR patients achieved
remission after induction chemotherapy. Outcome for
MLL-AF4+/t(4;11) patients also
was similar to that of the 97 MLL-AF4,
BCR-ABL, E2A-PBX1 patients
(Fig 6), with both groups achieving
excellent EFS at 2 years of follow-up (86.7%, SD = 8.8% v
88.4%, SD = 3.3%, respectively). Thus, among this subset of primarily
standard-risk pediatric ALL patients, presence of an MLL-AF4
fusion transcript in the absence of a cytogenetically detectable
t(4;11) translocation was not associated with poor outcome.
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| Fig 6.
EFS for patients with t(4;11)
negative/MLL-AF4+ ALL. Percentage of patients surviving
event-free was compared for 15 MLL-AF4+ patients who lacked
cytogenetically detectable t(4;11) and 97 patients who showed no
evidence for MLL-AF4, BCR-ABL, or E2A-PBX1 fusion transcripts. (Inset) Number of patients in follow-up at indicated times.
|
|
Expression of MLL-AF-4 fusion transcripts in normal infant BM
and fetal hematopoietic cells.
The presence of MLL-AF-4 fusion transcripts in leukemia cells
without a cytogenetically detectable t(4;11) translocation prompted the
hypothesis that MLL-AF4 fusion transcripts may be similarly expressed in rare populations of normal lymphohematopoietic cells. To
test this hypothesis, we used RT-PCR to determine if
MLL-AF4 fusion transcripts were present in fetal BMs and fetal
livers (gestational age, 15 to 22 weeks), as well as BM samples from normal infants.
As summarized in Table 5 and illustrated in
Fig 7, an MLL-AF4 fusion
transcript was detected by nested (but not standard) PCR in 1 (NBM 4)
of 6 (17%) normal infant BM samples. MLL-AF4 transcripts were
detected by nested (but not standard) PCR in 4 of 16 (25%) fetal BMs:
FBM 4, 19 weeks; FBM 6, 20 weeks; FBM 7, 21 weeks; FBM 11, 18 weeks.
Representative samples are shown in Fig 7A. MLL-AF4 also was
detected in 5 of 13 fetal livers: FL 1, 16 weeks gestational age; FL
14, 19 weeks gestational age; FL 10, 20 weeks gestational age; FL
12, 21 weeks gestational age; FL 13, 22 weeks gestational age; but not
in any of the 44 remission BM specimens from pediatric ALL patients
(Table 5). Nested PCR data from representative fetal liver
samples are shown in Fig 7B. Unlike MLL-AF4 fusion
transcripts, BCR-ABL fusion transcripts were not expressed in
any of the normal infant BM, fetal BM, or fetal liver samples by
standard or nested PCR (Fig 7).
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| Fig 7.
Detection of MLL-AF4 fusion transcripts in normal
hematopoietic cells by Southern blot analysis. (A) PCR products from
normal infant and fetal BM. (B) PCR products from fetal liver. PCR
products were transferred to nylon membranes and hybridized as
described in Materials and Methods with 32P-labeled
oligonucleotide detection probes for MLL-AF4 and
BCR-ABL. Amplified mRNA from the RS4;11 and patient UPN100,
with t(9;22)+ ALL, were used as positive controls (Pos
Con) for MLL-AF4 and BCR-ABL fusion transcripts, respectively. Negative
controls (Neg Con) were PCR products from RNA-free reaction mixture 1 plus reaction mixture 2 (negative control 1) and reaction mixture 1 plus DNA polymerase-free reaction mixture 2 (negative control 2).
Gest., gestational.
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|
As expected, MLL gene rearrangements were detected in
leukemic cells from both of the t(4;11)+, standard
PCR+, nested PCR+ cases (ie, INF-12 and
PEDS-91) examined. In contrast, no MLL gene rearrangements were
detected by Southern blot analysis in any of the DNA samples from
normal cells with nested PCR positivity for MLL-AF4 fusion
transcript expression (Table 4, Fig 5). The nested PCR positivity of
these standard PCR samples is likely caused by the
presence of a small subpopulation of normal cells expressing
MLL-AF4 fusion transcripts rather than very low level
MLL-AF4 expression in the majority of the cells.
Molecular characterization of MLL-AF4 fusion transcripts in
leukemic and normal cells.
The predominant amplification products of the MLL-AF4 nested
RT-PCR assays from 2 t(4;11)+ infants, 1 t(4;11) infant, 2 t(4;11)+ children, and
13 t(4;11) children with ALL, as well as 5 fetal
livers and 2 fetal BMs, were characterized by sequence analyses. These
analyses confirmed that the PCR-generated products in each of the 25 cases were MLL-AF4 fusion transcripts and showed common
splicing variants in the various samples assayed. MLL-AF4
sequence analysis data for these 25 samples are summarized in
Table 6. In 3 of 4 t(4;11) leukemia cases,
sequence analysis indicated that the PCR products resulted from fusions
between MLL exon 6 and AF4 exon a, whereas the PCR product in the remaining case was the result of an MLL exon
7/AF4 exon b fusion. Among the 14 MLL-AF4+
non-t(4;11) cases, 4 had MLL exon 6/AF4 exon a fusion
transcripts, 7 had MLL exon 7/AF4 exon a fusion
transcripts, and 3 had MLL exon 7/AF4 exon b fusion
transcripts. By comparison, the nested PCR products from 7 fetal liver
(N = 5)/fetal BM (N = 2) specimens resulted from fusions between
MLL exon 6 and AF4 exon a (N = 4) or MLL exon 7 and AF4 exon b (N = 3).
| |
DISCUSSION |
We have examined the expression of MLL-AF4 fusion transcripts
in pediatric and infant ALL patients with or without cytogenetically detectable t(4;11)(q21;q23), as well as in normal BM cells and fetal
tissues using standard and nested RT-PCR assays. Overall, MLL-AF4 transcripts were detected by nested PCR in 9 of 17 infant ALL patients and 17 of 127 childhood ALL patients.
Cytogenetically detectable t(4;11) translocations were found in 8 of
the 9 MLL-AF4+ infants, but in only 2 of 17 MLL-AF4+ children with ALL. Sequence analysis confirmed
that the PCR-generated products were MLL-AF4 fusion transcripts
and revealed common splicing variants in the various samples assayed.
Notably, MLL-AF4 transcripts were found in 9 of 29 fetal
tissues and 1 of 6 normal infant BM samples.
Although previous investigators15,27-29 have shown that
MLL-AF4 fusion transcripts were present in infant and adult ALL
patients who were cytogenetically t(4;11), to our
knowledge this is the first report to document the expression of
MLL-AF4 fusion transcripts in noninfant pediatric ALL patients lacking cytogenetic evidence of t(4;11). Interestingly, the expression of MLL-AF4 fusion transcripts in these patients was not
associated with high-risk features or poor treatment outcome. Behm et
al16 recently reported that MLL rearrangements
confer poor outcome in pediatric ALL patients regardless of age.
However, all patients in the study by Behm et al had MLL
rearrangements in concert with a cytogenetically detectable 11q23
abnormality. Numerous investigators have documented the poor prognosis
of ALL patients with t(4;11),12-16 although in one study
the subset of patients 1 to 9 years of age with t(4;11) was distinct in
having a favorable outcome.53
The observation that MLL-AF4 fusion transcripts are present in
normal hematopoietic tissues raises crucial questions regarding the
significance of MLL-AF4 rearrangements in leukemogenesis of ALL. Rearrangements between the 11q23 and 4q21 loci may occur in normal
cells where their biological impact may be dependent on additional
leukemogenic events. Altered expression or loss of expression of genes
other than MLL and AF4 could be involved in both
leukemogenesis and the poor prognosis of t(4;11)+ patients.
A crucial role for MLL in mammalian development has been shown
by disruption of 11q23 in transgenic mice.54
MLL+/ mice exhibited retarded growth,
hematologic abnormalities, and disrupted segmental development of the
axial skeleton, and MLL/ was lethal
to embryonic development. Although these studies clearly indicate that
disruption of normal MLL expression throughout the organism can
have deleterious effects, the effects of MLL-AF4 gene fusions
restricted to blood-forming tissues in normal individuals or
t(4;11) leukemia patients may be much less dire.
Further studies of MLL-AF4+ ALL patients and normal
individuals will be required to confirm this hypothesis.
Consistent with our finding of MLL-AF4 fusion transcripts in
normal tissues, recent reports by others have documented similar findings for other lymphoid malignancy-associated gene fusion products.
BCL2-JH fusions, which were thought to arise via
t(14;18)(q32;q21) in follicular cell lymphoma and diffuse large-cell
lymphoma,36,37 have now been detected in peripheral blood
cells from healthy individuals.31,33-35
BCL2-JH fusions also were reported to occur in
benign hyperplastic lymphoid tissues,30,32 as well as in
all hematopoietic lineages of patients with B-cell non-Hodgkin's
lymphoma (NHL).55 In addition, the BCR-ABL fusion
transcript, which is thought to occur as a result of the t(9;22)
translocation that represents a significant adverse risk factor in
pediatric ALL,3,4,56 was recently identified in the blood
of normal individuals38 as well as in a
Ph case of chronic myelogenous
leukemia.57 In our current analysis, BCR-ABL
fusions were detected in two ALL patients lacking classic t(9;22)
translocations, but not in any of the normal tissues examined. It also
is noteworthy that the gene fusion partners AF9 and
ENL, which are translocated in the 11q23 rearrangements t(9;11)
and t(11;19), like AF4, encode proteins with serine- and
proline-rich regions and nuclear localization
signals.19,22,58,59 In light of our current findings, it
would be of interest to determine if MLL-AF9 and
MLL-ENL fusion transcripts are present in normal cells or
leukemic cells lacking cytogenetically detectable t(9;11) and t(11;19).
In view of the findings described above, the contribution of
MLL-AF4 and other fusion transcripts toward leukemic
transformation of lymphopoietic precursors requires further
investigation. Furthermore, the utility of sensitive PCR-based
detection of these fusion transcripts as sole indicators of minimal
residual disease60 or for potential screening of cord blood
before transplant should be reconsidered given their presence in
nonleukemic cells.
| |
FOOTNOTES |
Submitted December 23, 1997;
accepted April 24, 1998.
Supported in part by Department of Health and Human
Services grants, including CCG Chairman's Grant Nos.
CA-13539 and CA-60437 from the National Cancer Institute. F.M.U. is a
Stohlman Scholar of the Leukemia Society of America and Parker Hughes
Chair in Oncology.
Presented in part at the 39th Annual Meeting of the American Society of
Hematology, December 5-9, 1997, San Diego, CA.
Address reprint requests to Fatih M. Uckun, MD, PhD, Hughes Institute,
2665 Long Lake Rd, Suite 330, St Paul, MN 55113.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr James Downing (St Jude Children's
Research Hospital, Memphis, TN) for his generous and comprehensive assistance in establishing the RT-PCR assays in the CCG ALL Biology Reference Laboratory.
| |
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Abstract
Introduction
Abstract