| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
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
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
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
).
|
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
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.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
.
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.
|
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.
|
, 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.
|
. 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.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
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.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Miremadi, M. Z. Oestergaard, P. D.P. Pharoah, and C. Caldas Cancer genetics of epigenetic genes Hum. Mol. Genet., April 15, 2007; 16(R1): R28 - R49. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Libura, D. J. Slater, C. A. Felix, and C. Richardson Therapy-related acute myeloid leukemia-like MLL rearrangements are induced by etoposide in primary human CD34+ cells and remain stable after clonal expansion Blood, March 1, 2005; 105(5): 2124 - 2131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-K. So, Y. Nie, Y. Song, G.-Y. Yang, S. Chen, C. Wei, L.-D. Wang, N. A. Doggett, and C. S. Yang Loss of Heterozygosity and Internal Tandem Duplication Mutations of the CBP Gene Are Frequent Events in Human Esophageal Squamous Cell Carcinoma Clin. Cancer Res., January 1, 2004; 10(1): 19 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. J. de Mollerat, F. Gurrieri, C. T. Morgan, E. Sangiorgi, D. B. Everman, P. Gaspari, J. Amiel, M. J. Bamshad, R. Lyle, J.-L. Blouin, et al. A genomic rearrangement resulting in a tandem duplication is associated with split hand-split foot malformation 3 (SHFM3) at 10q24 Hum. Mol. Genet., August 15, 2003; 12(16): 1959 - 1971. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. R. Kees, J. Ford, M. Watson, A. Murch, M. Ringner, R. L. Walker, and P. Meltzer Gene Expression Profiles in a Panel of Childhood Leukemia Cell Lines Mirror Critical Features of the Disease Mol. Cancer Ther., July 1, 2003; 2(7): 671 - 677. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Blanco, T. Dervieux, M. J. Edick, P. K. Mehta, J. E. Rubnitz, S. Shurtleff, S. C. Raimondi, F. G. Behm, C.-H. Pui, and M. V. Relling Molecular emergence of acute myeloid leukemia during treatment for acute lymphoblastic leukemia PNAS, August 28, 2001; 98(18): 10338 - 10343. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
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).