[Cancer Research 61, 59-63, January 1, 2001]
© 2001 American Association for Cancer Research
The Partial Nontandem Duplication of the MLL (ALL1) Gene Is a Novel Rearrangement That Generates Three Distinct Fusion Transcripts in B-Cell Acute Lymphoblastic Leukemia1
Susan P. Whitman,
Matthew P. Strout,
Guido Marcucci,
Aharon G. Freud,
Lucia L. Culley,
Nancy J. Zeleznik-Le,
Krzysztof Mrózek,
Karl S. Theil,
Ursula R. Kees,
Clara D. Bloomfield and
Michael A. Caligiuri2
Divisions of Hematology and Oncology [S. P. W., G. M., M. A. C.] and Human Cancer Genetics [M. P. S., A. G. F., M. A. C.], Department of Pathology [L. L. C., K. S. T.] and Comprehensive Cancer Center [K. M., C. D. B., M. A. C.], The Ohio State University, Columbus, Ohio 43210; Department of Medicine [N. J. Z.], Loyola University of Chicago, Cardinal Bernadin Cancer Center, Maywood, Illinois 60153; and TVWT Institute for Child Health Research [U. R. K.], University of Western Australia, Perth, Western Australia 6008
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ABSTRACT
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A partial nontandem duplication (PNTD) of mixed lineage leukemia
(MLL) gene is described in B-cell acute lymphoid
leukemia without structural cytogenetic abnormalities at 11q23
and 9p22. A duplicated portion of MLL is interrupted by
the insertion of a region of 9p22 that includes the 3'-end of the
AF9 gene. The PNTD encodes: (a) a PNTD
transcript; (b) a partial tandem duplication of
MLL; and (c) a chimeric transcript fusing
MLL to the 3'-end of AF9, mimicking the
t(9;11)(p22;q23) and expressed 1024-fold higher than the other two. The
MLL PNTD, therefore, contributes toward leukemogenesis
through simultaneous production of fusion transcripts that are
otherwise encoded by three distinct genetic defects.
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Introduction
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The
MLL3
gene (also known as ALL1, HRX, and Htrx-1)
located on chromosome band 11q23 is frequently rearranged in human
acute leukemias usually due to chromosome translocations or by PTDs of
MLL and result in the formation of MLL gene
fusions that are responsible, at least in part, for the leukemic
phenotype (reviewed in Ref. 1
). For example, the
t(9;11)(p22;q23) fuses MLL in-frame with AF9, a
transcription factor, in de novo AML, therapy-related AML
that develops after cytotoxic treatment with a topoisomerase II
inhibitor, and less frequently, in de novo ALL and
therapy-related ALL (2)
. In cases with the MLL
PTD, fusion of a downstream intron of MLL, most commonly
introns 6 or 8, with intron 1 of MLL results in a tandem
duplication of exons 2 through 6 (Fig. 1A)
or 2 through 8 (data not shown; Refs. 3
, 4
).
This rearrangement cannot be detected cytogenetically and is found in a
majority of adult patients with AML and +11 as a sole cytogenetic
abnormality and in 6–10% of adult patients with AML and normal
cytogenetics (3, 4, 5, 6)
. Here, we detail a molecular analysis
characterizing a novel MLL rearrangement in the PER-377 cell
line, a line derived from an infant patient who presented with
biphenotypic acute leukemia (7)
. Southern analysis with
DNA from bone marrow obtained at first and second relapse showed the
same pattern of MLL rearrangement as seen in PER-377 cells,
indicating the MLL rearrangement was present at relapse and
was not a clonal event that occurred during establishment of the cell
line (7)
. Here, we show that the MLL
rearrangement results from a novel duplication of MLL exons
2 through 6 that is nontandem due to insertion of DNA from chromosome
9p22 that includes the 3'-end of the AF9 gene. Three fusion
transcripts are expressed from the MLL PNTD gene
rearrangement, with the most abundant transcript being analogous to the
chimeric transcript that is present in leukemic blasts with a typical
t(9;11)(p22;q23; Ref. 2
).
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Fig. 1. Detection of MLL PTD and MLL
PNTD fusion transcripts. MLL fusion transcripts detected
by RT-PCR using a forward primer from MLL exon 5 (5.3)
and a reverse primer from MLL exon 3 (654c), followed by
PCR using nested primers from MLL exon 6 (6.1) and exon
3 (400c) (Ref. 3
). A, the PTD of
MLL involves MLL exons 2 through 6 and
results in a unique in-frame MLL exon 6-exon 2 fusion,
indicated by the bold vertical line. B,
the PNTD of MLL involves MLL exons 2
through 6, separated by AF9 exon 9, represented by the
shaded box. The PNTD of MLL creates two
unique in-frame exon fusions: MLL exon
6-AF9 exon 9 and AF9 exon
9-MLL exon 2. The vertical lines indicate
all exon junctions. Schematic diagrams are not to scale.
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Materials and Methods
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Chromosomal Analysis.
Cell culture for PER-377 has been described (7)
. SKY was
performed according to the manufacturer’s protocol (Applied Spectral
Imaging, Migdal Haemek, Israel). Spectral analysis and classification
were performed using SkyView 1.2 visualization and analysis software
(Applied Spectral Imaging). FISH was performed using standard
techniques (8)
with the 11q23 bcr probe (Oncor,
Gaithersburg, MD) and the chromosome 9p22 cosmid clone, 213A7
(9)
, which contains the 3'-end of AF9.
Southern Blotting and PCR Analyses.
Southern blotting was performed as described previously using
MLL probes B859 and SAS1 (6
, 10)
and
AF9 genomic DNA probes A403 and Af9e4 (see text below). cDNA
was prepared from total RNA by reverse transcription. To amplify
MLL PTD fusion transcript, first-round PCR and nested PCR
reactions were performed as described previously (3)
and
the PCR products were cloned for sequencing (Sequencing and Genotyping
Unit, Comprehensive Cancer Center, The Ohio State University).
Relative and absolute quantities of fusion transcripts were measured by
real-time RT-PCR, as suggested by the manufacturer’s protocols (ABI
Prism 7700 Sequence Detection System; Perkin-Elmer Corp., Foster City,
CA). Long range DNA PCR was performed according to the manufacturer’s
suggested protocol (Extend Long Template PCR system; Boehringer
Mannheim, GmbH, Germany). DNA analyses were performed using the
basic local alignment search tool, BLAST.
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Results
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MLL Fusion Transcripts Detected by RT-PCR.
Southern analysis of PER-377 with B859 initially showed a
rearrangement of MLL in the absence of a cytogenetic
abnormality at 11q23 (7)
. We noticed that the pattern of
rearrangement in PER-377 was consistent with presence of a
MLL PTD (3
, 4)
and, given the absence of a
cytogenetic abnormality at 11q23, we hypothesized that PER-377 might
carry a MLL PTD. RT-PCR using primers that amplify
MLL PTD fusion transcripts amplified two products of 228 bp
and 300 bp (data not shown). Sequence analysis of the cloned 228-bp PCR
product revealed an in-frame fusion of MLL exon 6 with
MLL exon 2, indicative of the presence of a MLL
tandem duplication of exon 2 through exon 6 (Fig. 1A)
.
Sequence analysis of the cloned 300-bp band revealed a fusion of exon 6
with a 72-bp sequence not present in the normal MLL gene or
its transcript, followed by exon 2. A search of the GenBank database
using the 72-bp DNA sequence indicated that it shared 100% sequence
identity to exon 9 within the 3'-end of the AF9 gene
(9)
. AF9 is a frequent MLL
translocation partner that maps to chromosome 9p22 (1)
. In
this instance, the partial duplication of MLL exons 2
through 6 was nontandem, due to the separation of exons 6 and 2 by the
insertion of AF9 exon 9. Further analysis revealed that the
PNTD fusion transcript of MLL exon 6-AF9 exon
9-MLL exon 2 maintained the open reading frame (Fig. 1B)
.
Southern Analysis of MLL and AF9.
The SAS1 probe can, in many but not all instances, identify a
rearrangement of MLL intron 1 when the breakpoint lies near
the 3'-end (6
, 10)
, but did not in PER-377 (data not
shown), suggesting the break point lies further upstream within the
35-kb MLL intron 1. To assess the genomic status of the
AF9 gene, Southern analysis was performed using A403. This
probe revealed a rearrangement of AF9 in both the
BamHI- (data not shown) and HindIII-digested
PER-377 DNA (Fig. 2A)
. Notably, the rearranged fragments detected by the A403
probe were the same size as fragments detected with the B859
MLL probe (Fig. 2B)
. These data support a genomic
rearrangement of MLL involving AF9 in PER-377. To
determine whether a reciprocal rearrangement was present or absent, the
Af9e4 genomic probe, which lies upstream of the A403 probe and upstream
of the 5' MLL-AF9 genomic fusion (see below), but within the
same AF9 germ-line BamHI fragment, was used in a
Southern analysis. Only the germ-line BamHI fragment was
detected (data not shown), supporting both the absence of a reciprocal
fusion of the 3'-end of MLL to the 5' end of AF9,
as well as the absence of a deletion from this region of
AF9. Finally, in Fig. 2B
, we measured a 2-fold
greater intensity of the germ-line MLL band compared with
the rearranged MLL band using the B859 probe, consistent
with MLL exons 7–11 being intact on both wild-type and
rearranged alleles of PER-377.
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Fig. 2. Southern analysis of MLL and
AF9 gene rearrangement in PER-377 cells. Genomic DNA was
isolated and digested with the indicated restriction enzymes.
A, Southern analyses were performed with A403, which
detects rearrangements of the 3'-end of AF9.
B, Southern analyses were performed using B859, which
detects rearrangements of the MLL bcr. Note that the
rearranged HindIII fragment in A is the
same size as the rearranged HindIII fragment in
B. PER, PER-377 DNA; PTD,
AML case positive for the MLL PTD defect;
C, normal peripheral blood mononuclear cell control. The
arrows indicate germ-line fragments.
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Cytogenetic Analysis of PER-377.
We analyzed PER-377 chromosomes by SKY analysis to perhaps detect
a cryptic translocation involving bands 9p22 and 11q23 (Fig. 3A)
because chromosomes 9 and 11 appeared normal by G-banding
(7)
. The results led to two revisions to the originally
described karyotype: the t(2;13)(p12;q34) to t(2;13)(p12;q32) and a
loss of one copy of chromosome 14, indicating that a clonal evolution
had occurred during maintenance of this cell line. SKY did not reveal
any microscopically detectable chromosome aberrations involving 11q23
and 9p22 (Fig. 3A)
. Next, dual-color FISH was performed with
the 9p22 cosmid probe 213A7, which contains the 3'-end of
AF9, and the 11q23 bcr probe. The results shown in Fig. 3B
confirmed that MLL was not involved in a
balanced translocation as indicated by fluorescence signals detected on
chromosome bands 11q23 and not on chromosomes 9p. The 9p22 probe,
213A7, identified two normal 9p22 signals (Fig. 3B)
, and
this was confirmed in a second experiment using the 9p22 cosmid probe
c48, which also contains AF9 exon 9 (data not shown). Fig. 3B
also shows that two 11q23 bcr probe signals are
separated by the yellow/green signal of
AF9 probe 213A7 on one chromosome 11. As suggested by the
RT-PCR data above (Fig. 1B)
, this FISH pattern is consistent
with the insertion of part of AF9 between the partially
duplicated portions of MLL on chromosome 11q23. An
alternative explanation for the above observation is that a region of
9p22 inserts into a normal MLL allele. However, Southern
analysis (Fig. 2B)
does not detect the predicted second
rearranged BamHI fragment with B859 involving the 3'-end of
the MLL bcr (exons 7 through 11), arguing against such a
gene rearrangement.
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Fig. 3. SKY and FISH analyses of PER-377. A,
representative karyotype of a PER-377 cell subjected to SKY analysis.
Two images of each chromosome are displayed. On the
left, the chromosomes are shown in spectra-based
classification color; on the right, inverted and
contrast-enhanced 4',6-diamidino-2-phenylindole image of the same
chromosome. No visible rearrangement of chromosomes 9 and 11 is seen.
See text for description of clonal chromosome aberrations in PER-377.
B, dual-color FISH using biotin-labeled 11q23
MLL bcr probe (red signals) and
digoxigenin-labeled 213A7 from 9p22 (green signals).
Normal green 213A7 signals are detected on each
chromosome 9p22, and a normal red MLL bcr
signal is present on one chromosome, 11q23. The yellow
arrow indicates the abnormal chromosome 11q23 with an
interstitial insertion of a portion of 9p22
(green/yellow) flanked by 11q23
MLL bcr signals (red).
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Characterization of the Genomic Breakpoints in PER-377.
To identify the 5'-genomic fusion of MLL with
AF9, PCR was performed on PER-377 DNA and DNA extracted from
the patient’s leukemic blasts at second relapse (Fig. 4A
, Lanes 2 and 3, respectively).
Sequence analyses of the resulting 2.2-kb PCR products revealed a
genomic fusion point between MLL intron 6 and AF9
intron 8 in both sources of DNA (Fig. 4B)
. This is
consistent with the original Southern data showing the defect was
present in the patient’s leukemic cells at relapse and did not arise
during establishment of the cell line (7)
. Further
analysis of the break points revealed a VDJ-like recombination sequence
at the junction between MLL intron 6 and the AF9
intron 8 (Fig. 4B
, underlined). Cloning of the
3'-genomic fusion between AF9 exon 9 and MLL
intron 1 was attempted by long range DNA PCR. Long range DNA PCR was
successful up to 
16 kb of 9p22 sequence downstream of the
MLL intron 6-AF9 intron 8 junction and
demonstrated the inclusion of the last AF9 exon, exon 10,
and the 3'-UTR of AF9, herein termed AF9 exon 10
3'-UTR (Fig. 4C)
. Examination of published DNA sequences
(see GenBank accession numbers AC002050 and AF036405) revealed an
abundance of Alu repeat elements present in 9p22, downstream
of the AF9 gene as well as throughout MLL intron
1, respectively. These Alu repeat elements likely
contributed to the difficulty in cloning the 3'
AF9-MLL intron 1 genomic fusion.
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Fig. 4. Genomic analysis of the MLL PNTD. Long
range DNA PCR was carried out using a forward primer from
MLL exon 6 and a reverse primer from the
AF9 exon 9. A, ethidium bromide staining
of electrophoresed PCR products shows the same 2.2-kb PCR product is
amplified in both PER-377 (Lane 2) and in the patient
relapse sample (Lane 3). Lane 1 is a
water control. B, sequencing of the cloned products
revealed a fusion of MLL intron 6 with
AF9 intron 8. A VDJ heptamer-like recombination sequence
(underlined) was identified at the genomic fusion.
C, diagram of the PNTD of MLL in PER-377
cells. The vertical arrow indicates the known upstream
break point. The open boxes represent MLL
exons. The shaded boxes represent the AF9
exons. Introns are represented by horizontal lines (not
drawn to scale). The black and white box represents a
region of Alu elements present within 9p22, downstream
of the AF9 3'-UTR and within the upstream region of
MLL intron 1 (flanked by slanted lines,
unknown distance). The downstream break point is predicted to occur
within this Alu-rich region. B,
BamHI; H, HindIII.
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Analysis of the Fusion Transcripts Generated by the
MLL PNTD Gene Rearrangement.
As described above, our initial RT-PCR analysis of PER-377 using
primers that amplify MLL PTD fusion transcripts detected two
fusion transcripts: an MLL exon 6-MLL exon 2
fusion and an MLL exon 6-AF9 exon
9-MLL exon 2 fusion. The presence of the 3'-UTR
of AF9 in the PNTD at the genomic level suggested that a
transcript similar to that expressed from a t(9;11)(p22;q23)
chromosomal translocation (i.e,, a MLL exon
6-AF9 exons 9–10-3'-UTR) might be detected using a
forward primer from MLL exon 5 and a reverse primer from the
3'-UTR of AF9. Indeed, RT-PCR amplified a single product
(data not shown), the sequence analysis of which revealed the
additional transcript, an MLL exon 6-AF9 exons
9–10-3'-UTR, which, while similar to that generated by the
t(9;11)(p22;q23) (Ref. 2
), resulted in this instance from
the PNTD gene rearrangement. These three transcripts are likely the
result of various splicing events. Because we and others have
previously detected MLL PTD fusions in total RNA from normal
donors using nested RT-PCR (11, 12, 13)
, fluorescence-based
real-time RT-PCR assays were performed (Fig. 5)
. With this method, the fewer the number of PCR cycles required to
reach fluorescent threshold (i.e., the cycle threshold or
CT), the more abundant is the transcript in the
original sample. The MLL exon 6-AF9 exon 9 fusion
detected by primers/probe I (Fig. 5A)
, is present in the
PNTD transcript (Fig. 5B)
and the MLL exon
6-AF9 exons 9–10-3'-UTR fusion transcript (Fig. 5D)
. The CT was 25.6 (Fig. 5E
, curve I). The CT for
MLL exon 6-AF9 exons 9–10-3'-UTR
transcript (Fig. 5D)
, uniquely detected by primers/probe IV
(Fig. 5A)
, was also 25.6 (Fig. 5E
, curve
IV). In contrast, the CT for both the
MLL PTD transcript (Fig. 5C)
and the
CT for the MLL PNTD (Fig. 5B)
, detected by primers/probe sets II and III (Fig. 5A)
, respectively, was 35.8 (Fig. 5E
, curves
II and III). A linear phase, followed by a
plateau phase, in real-time curves is predicted by PCR amplification
kinetics (14)
, hence, the results shown in Fig. 5E
represent a true exponential amplification of the target
sequences during first-round PCR, and not nonspecific amplification.
After equalization for starting transcript, the relative fold
difference in CTs between fusion transcripts
detected by primers/probe I and IV (i.e., 25.6) and those
detected by primers/probe II and III (i.e., 35.8) is 10.2
cycles. Each cycle that passes is an approximate 2-fold change in
expression. Therefore, the 10.2 cycle difference represents over a
1024-fold higher level of the MLL fusion transcript detected
by primers/probe I and IV [i.e., the MLL exon
6-AF9 exons 9–10-3'-UTR transcript (Fig. 5D)
,
compared with the MLL PNTD and MLL PTD
transcripts]. Furthermore, transcript copy numbers derived from
standard curves supported the above conclusion. Thus, it is the
MLL exon 6-AF9 exons 9–10-3'-UTR transcript,
analogous to the chimeric transcript generated by a t(9;11)(p22;q23),
that is the predominant transcript arising from the PNTD gene
rearrangement in the PER 377 cell line.
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Discussion
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The discovery of the PNTD as a novel rearrangement of
MLL is supported by several lines of evidence. First, was
the identification by dual-color FISH of a 3' portion of AF9
between two MLL bcr signals. Second, was the Southern blot
analysis with the MLL B859 probe and the AF9
probe each identifying a single rearranged BamHI fragment of
identical size in the PER-377 cell line. Third, was the identification
of the MLL intron 6-AF9 intron 8 genomic fusion,
and the MLL exon 6-AF9 exon 9-MLL exon
2 fusion transcript. Fourth, was the absence of evidence, by standard
cytogenetic analysis, FISH, SKY, or Southern analysis, of a reciprocal
or nonreciprocal translocation between chromosomes 11q23 and 9p22, or
of a deletion of either MLL or AF9. Furthermore,
the 2-fold greater intensity of the germ-line MLL band
compared with the rearranged MLL band using the B859 probe
in Fig. 2B
is consistent with MLL exons 7–11
being intact on both wild-type and rearranged alleles in the PER-377
cell line. MLL exons 7–11 can be intact in the rearranged
allele that fuses MLL exon 6 to AF9 exon 9 at the
transcript level and MLL intron 6 to AF9 intron 8
at the genomic level if a partial duplication of the MLL bcr
follows the MLL-AF9 fusion (Fig. 4C)
. The
identical MLL gene rearrangement present in the PER-377 cell
line was present in blasts from the first and second relapse, in the
absence of a structural cytogenetic abnormality at 11q23 (Ref.
7
and our study). Furthermore, we demonstrated that the
identical MLL intron 6-AF9 intron 8 fusion was
found in PER-377 and in the second relapse sample. Cytogenetic and
molecular analyses of 11q23 and MLL, respectively, could not
be performed on the diagnostic sample due to lack of material.
The detection of a six of seven nucleotide match for a VDJ
recombinase recognition sequence (consensus, CAC-A/T-GTG) at the
junction between MLL intron 6 and AF9 intron 8
(Fig. 4B
, underlined) in this case of acute
leukemia with a known immunoglobulin heavy-chain gene rearrangement
(7)
suggests that the 5'-fusion may have involved an
illegitimate VDJ recombination event. Illegitimate VDJ recombination
has been implicated in a case of infant de novo AML with the
t(9;11)(p22;q23) after treatment with etoposide, a topoisomerase II
inhibitor, and in cases of infant ALL with the t(4;11)(q21;q23) (Refs.
15
and 16
). Aside from the association of VDJ
recombination in therapy-related acute leukemias, topoisomerase II
cleavage sites have been identified within the 3'-end of the
MLL bcr region and are associated with treatment-related AML
(17)
. Moreover, a de novo topoisomerase II
cleavage site has been identified in AF9 intron 7
(9)
. Finally, the MLL PTD has been shown to
occur via homologous recombination between Alu elements, and
such elements are present within MLL intron 1 and downstream
of the AF9 3'-UTR. Similarities in DNA structural properties
in the breakpoint cluster regions of AF9 and MLL
have been suggested to facilitate recombination and lead to
MLL-AF9 fusions (9)
. Thus, the
MLL PNTD may have arisen as a consequence of multiple
recombination events involving different mechanisms, a process, as far
as we are aware, not previously described for MLL gene
rearrangements, but none of our molecular data allows us to determine
whether it was related to prior therapy.
The MLL-AF9 fusion transcripts generated from the
MLL PNTD in this B-ALL cell line involved AF9
exon 9, located downstream of the ALL-associated fusion site B
(2
, 18)
. This is consistent with the notion put forth by
others that MLL fusion with the more 3'-AF9
region may influence the phenotype of the acute leukemia or,
alternatively, that the lineage of the cell may determine or influence
which AF9 exon is fused to MLL (2
, 18
, 19) . Thus, in functional terms, the MLL PNTD gene
rearrangement, expressing predominately the MLL-AF9 fusion
transcript, may represent a typical MLL-AF9
translocation. Cases of B-ALL having the MLL-AF9
fusion and data from the mouse MLL-AF9 knock-in
model (20)
suggest that MLL-AF9
alone is sufficient to promote B-ALL. The MLL PNTD may be
another example of the complex mechanisms that facilitate the
production of oncogenic MLL fusion proteins, with this B-ALL
case having MLL-AF9 as the critical transforming
MLL transcript. However, the potential roles for the PTD and
PNTD in promoting B-ALL in this case have not yet been addressed.
Notably, cases of pediatric pre-B-ALL have been described having the
MLL PTD gene defect (21)
. Although the presence
of a t(9;11)(p22;q23) generally confers a better prognosis in adult
de novo AML patients in comparison to other cytogenetically
detected 11q23 abnormalities (1)
, 11q23 abnormalities and,
more recently, a 9p abnormality in ALL, especially of B-lineage, were
shown to be adverse risk factors in childhood leukemias
(22)
. The patient, from whom PER-377 cells were
established, had an aggressive disease, having relapsed a second time
with B-ALL at 18 months of age, 12 months from diagnosis.
Unfortunately, by 25 months of age, the patient died from refractory
leukemia. The PER-377 cell line established from this case lacked
cytogenetic evidence of 9p22 and 11q23 abnormalities, and the
t(9;11)(p22;q23) chromosomal defect is found infrequently in pediatric
ALL. The data from our report suggest that molecular analysis for
evidence of 9p22 and 11q23 abnormalities may be warranted when such
cytogenetic evidence is lacking.
In summary, this report characterizes a novel molecular defect in a
cell line derived from a bilineage leukemia and provides what we
believe to be the first genomic evidence for a PNTD of the
MLL gene in B-ALL. We demonstrated that this abnormality
generates three distinct fusion transcripts, each of which was
quantitated, with the most abundant fusion transcript analogous to that
derived from a common t(9;11)(p22;q23) chromosomal aberration. It
appears that the MLL PNTD present in PER-377 at the genomic
level was also present in the patient’s relapsed samples, however, we
cannot say if the novel gene rearrangement of MLL was
present at diagnosis or was associated with the use of topoisomerase II
inhibitor therapy during induction. The MLL PNTD, therefore,
represents a novel mechanism of MLL gene rearrangement in
acute leukemia.
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ACKNOWLEDGMENTS
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We thank Dr. Bedrich Mosinger for thoughtful discussions.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grant P30-CA16058 from
the National Cancer Institute (Bethesda, MD) and The Coleman
Leukemia Research Fund. S. P. W. is supported by a fellowship from
the Ladies’ Auxiliary to Veterans of Foreign Wars and Oncology
Training Grant CA09338 from the National Cancer Institute. 
2 To whom requests for reprints should be
addressed, at Division of Hematology and Oncology, The Ohio State
University Medical Center, A458 Starling-Loving Hall, 320 West 10th
Avenue, Columbus, OH 43210. Phone: (614) 293-7521; Fax: (614) 293-7522;
E-mail: caligiuri-1{at}medctr.osu.edu
3 The abbreviations used are:
MLL, mixed lineage leukemia; AML, acute myeloid
leukemia; ALL, acute lymphoid leukemia; B-ALL, B-cell ALL; bcr,
breakpoint cluster region; SKY, spectral karyotyping; FISH,
fluorescence in situ hybridization; PTD, partial tandem
duplication; PNTD, partial nontandem duplication; UTR, untranslated
region.
Received 7/18/00.
Accepted 11/14/00.
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Top
ABSTRACT
Introduction
Rn)
versus PCR cycle number (i.e., time). The
vertical lines indicate the point at which fluorescence
crosses the predetermined threshold (horizontal line).
The most abundant transcript derived from the MLL PNTD
gene rearrangement is analogous to a t(9;11)(p22;q23) chimeric
transcript (see results).