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Next Article 
Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 709-711
COMMENTARY
What Significance Should We Attribute to the Detection of
MLL Fusion Transcripts?
By
Stephen P. Hunger and
Michael L. Cleary
From the Section of Pediatric Hematology/Oncology, the Department of
Pediatrics, University of Colorado School of Medicine, Denver; and the
Department of Pathology, Stanford University School of Medicine,
Stanford, CA.
TRANSLOCATIONS involving the mixed
lineage leukemia (MLL) gene (also termed ALL-1, HRX,
HTRX1) at chromosome band 11q23 occur in 5% to 10% of acute
lymphoblastic and acute myeloid leukemias (ALLs and AMLs) and serve as
particularly illustrative examples of the clinical, biological, and
epidemiological implications of contemporary molecular oncology
investigations.1,2 Remarkably, various translocations in
ALL and AML fuse MLL to more than two dozen different partner
genes, one dozen or more of which have been cloned and identified at
the current time.3,4 MLL also undergoes partial
tandem duplication in some patients with AML.5 In ALL, the
t(4;11)(q21;q23), which fuses MLL to AF4
(FEL), occurs most frequently and accounts for about half of
all MLL translocations. Cytogenetic data, supported by gene
transfer studies and knock-in mouse models, indicate that the critical
product is the der(11)-encoded fusion protein that consists of amino
terminal ~1,200 amino acids of MLL fused to carboxy
terminal polypeptides of various sizes specified by the
different partner genes.6-8
Epidemiologically, MLL translocations occur at greatly
increased frequency in two clinical settings: infant leukemias and secondary leukemias that arise after treatment with chemotherapeutic agents classified as inhibitors of DNA topoisomerase II.1,2 Molecular studies have shown that MLL translocations are
present in 70% to 80% of ALLs and 50% to 60% of AMLs that occur in
infants less than 1 year of age. The occasional diagnosis of leukemias with MLL translocations in the immediate neonatal period, twin studies, and retrospective analysis of cord blood specimens indicate that MLL translocations can occur in utero.9,10
Clinically, MLL translocations have aroused particular interest
because of their prognostic significance. Infants with ALL and
MLL gene rearrangements have an extremely poor outcome when treated with various different chemotherapy regimens.9
Older children and adults with ALL carrying MLL translocations
also tend to fare poorly.11
How does this information affect clinical practice? Many centers and
cooperative groups are beginning to routinely screen patients with ALL,
particularly infants, for MLL abnormalities, and consider
altering therapy if these are detected. For example, the Children's
Cancer Group (CCG) and the Pediatric Oncology Group (POG), which
collectively treat almost all infants with ALL in the United States,
are currently conducting trials that explore the use of allogeneic bone
marrow (BM) transplant, using either matched sibling or unrelated
donors, in first remission for infants with ALL bearing an MLL
translocation.
Against this backdrop, the report by Uckun et al12 in this
issue of Blood presents fascinating, and occasionally
perplexing, information that has significant implications for
understanding the molecular details of leukemogenesis, and for
interpreting molecular analyses that clinicians must now consider in
treating patients with leukemia. These investigators used single-step
and nested reverse transcriptase-polymerase chain reaction (RT-PCR) to
amplify MLL-AF4 fusion transcripts from infants and children with ALL. The salient points of this manuscript include the following:
(1) MLL-AF4 fusion transcripts were amplified by single-step
RT-PCR from only 6 of 8 infants and 1 of 2 older children with a
cytogenetically evident t(4;11) (we will refer to these as standard PCR+). This is quite similar to results previously
published in Blood by Downing et al,13 who, using
the same primers and reaction conditions, found that MLL-AF4
fusion transcripts were amplified by single-step RT-PCR from 17 of 23 children with t(4;11)+ ALL. In both studies,
MLL-AF4 transcripts were amplified from each of the remaining
t(4;11)+ patients when a second round of PCR was performed
(nested PCR+).
(2) Fifteen of 125 children (12%) with ALL who lacked cytogenetic
evidence of a t(4;11) were found to have an MLL-AF4 fusion transcript by nested PCR. Overall, these children did not have the
clinical features and poor treatment outcome typically associated with
the t(4;11), but rather were similar to the larger group of
t(4;11) /nested PCR children with
ALL. However, for some of these nested PCR+
leukemias (3 of 5 tested), Southern blot studies showed MLL
gene rearrangements, indicating that a cryptic t(4;11) was present in
the leukemic cells. Conversely, 2 of 5 nested PCR+
leukemias had no evidence of MLL gene rearrangements,
suggesting that the t(4;11) was present in only a minor (<5%)
subpopulation of the cells.
(3) The investigators detected MLL-AF4 fusion transcripts by
nested PCR in a substantial proportion of samples from nonleukemic sources (4 of 16 fetal BMs, 5 of 13 fetal livers, and 1 of 6 normal infant BMs). All of the nested PCR+ normal samples that
were tested by Southern blot lacked MLL gene rearrangements.
One important message of this study is that standard PCR, as used by
the investigators, lacks sufficient sensitivity to identify all
patients with a t(4;11). Furthermore, nested PCR, while identifying all
patients with a t(4;11), lacks acceptable specificity since it was
positive in some children with ALL that lacked cytogenetic evidence of
a t(4;11) and an adverse treatment outcome. In addition, nested PCR was
positive in a subset of samples obtained from normal children and
apparently healthy fetuses. Although the investigators found that none
of the children with standard-risk ALL who were in remission at the end
of induction were nested PCR+, the study also raises
concerns about the use of RT-PCR for detection of minimal residual
disease in patients with t(4;11)+ ALL. Taken together, the
observations indicate that RT-PCR cannot be used alone as a screening
assay.
The study raises several vexing questions. For instance, why do some
t(4;11)+ ALLs display discrepant PCR results (standard
PCR/nested PCR+) when Southern blot data
indicate that the t(4;11) is present in most, and presumably all, cells
within the leukemic clone? One possibility is problems with the PCR
reaction itself. Mutations may be present in the regions to which the
first-round primers anneal, but not affecting the second-round primers.
However, this seems highly unlikely. It is important to note that no
mention is made in this report of efforts to optimize the PCR assay, by using different amplification primers and/or varying the
reaction conditions, so that products would be obtained from all
t(4;11)+ patients after a single round of amplification.
The experience of several other groups is that MLL-AF4 fusion
transcripts can be amplified by nested PCR from most/all
t(4;11)+ ALLs.13-18 Interestingly, all of these
reports used nested PCR assays for routine diagnosis of
t(4;11)+ patients. Although details were not provided, this
suggests that others may also have encountered problems with the
sensitivity of single-step PCR reactions in t(4;11)+ ALL. A
more likely explanation for the low sensitivity of the MLL-AF4
PCR assays is that some leukemias may express fusion transcripts (and
proteins) at very low levels, perhaps because high-level expression is
deleterious and only tolerated in the presence of specific compensatory
mutations. This notion is consistent with experimental data, eg, the
inability to stably express exogenous MLL fusion genes after
transfections of cultured cells in vitro. Notably, experimental
immortalization of primary murine BM cells by MLL-ENL was associated
with exceedingly low levels of fusion transcript and
protein.8
Conversely, why do leukemias that lack evidence of MLL gene
rearrangements contain MLL-AF4 transcripts detected by RT-PCR? Given the precautions used by Uckun et al to exclude PCR contamination, and the reproducibility of their results, it seems very unlikely that
these findings can be explained by false-positive PCR results. In the
future it will be important for other investigators to attempt to
confirm these results. It would be particularly convincing if the
presence of t(4;11)+ cells could be documented by other
techniques, such as FISH (fluorescence in situ hybridization) or PCR
using genomic DNA. Although it is possible that the Southern blot data
are incorrect and these patients really have a cryptic t(4;11) present
in most/all cells, the nature of the fusion transcripts indicates that
the strategy used should have detected gene rearrangements if they were
present in greater than 1% to 5% of the cell population (the
threshold of detection for Southern blot studies). Therefore, we must
hypothesize that the t(4;11) is present in only a small percentage of
cells in some children with ALL. The fact that the
t(4;11)/nested PCR+ leukemias in this study
did not have the clinical features and poor treatment outcome typically
associated with the t(4;11) seems to support this hypothesis. If
correct, then these patients might represent the counterparts of the
healthy fetuses/infants with rare t(4;11)+ cells (see
below). However, this would not explain why MLL-AF4 transcripts
are not detected once patients enter remission unless the t(4;11), in
the absence of other mutations, renders cells more sensitive to
chemotherapy.
The detection of MLL-AF4 transcripts by RT-PCR in BM from one
presumably healthy infant and normal fetal liver and BM adds the
t(4;11) to the list of molecular abnormalities t(14;18), t(9;22), t(8;14) and MLL tandem duplicationthat are present, at low
levels, in hematopoietic cells of normal individuals, and apparently
tolerated without adverse consequences.19-22 Perhaps these
results should no longer be surprising. All available data (statistical
models, transgenic animal studies, latency following in utero
mutations) indicate that development of leukemia, like solid tumors, is
a multistep process that requires cooperative mutations in more than
one oncogene and/or tumor suppressor gene. Thus, it is likely that cells with mutations typically found in leukemias and lymphomas frequently arise in normal individuals. However, additional mutations necessary for progression to clinical malignancy must ostensibly occur
in only a subset of individuals. In others, "single-hit" mutations might provide the cell with a survival advantage that allows
long-term persistence, ie, Bcl-2 overexpression in a
t(14;18)+ cell. In other cases, it might render the cell
more susceptible to spontaneous or exogenously induced cell death, as
postulated above for MLL-AF4.
What then should we conclude from this and similar reports? First,
molecular analyses, like all other clinical information, must be
interpreted cautiously and in the context of other available data. At
the present time, it seems prudent to use tests other than RT-PCR, such
as Southern blot analysis, to screen for MLL abnormalities, or
to insist that MLL abnormalities be confirmed by another
technique in those patients that are nested PCR+ but not
standard PCR+. Nested PCR assays must be rigorously tested
to ensure that they reliably distinguish between leukemia/lymphoma
patients that have low levels of disease, and healthy individuals that
carry rare nonmalignant cells that possess a specific molecular defect.
Biologically, we must move beyond focusing exclusively on
translocations and other "sentinel" molecular defects toward a
more comprehensive characterization of the spectrum of mutations
present in malignant cells.
 |
FOOTNOTES |
Submitted May 26, 1998;
accepted May 26, 1998.
S.P.H. is supported by a Professional Development Award from The
Children's Hospital Research Institute, Denver, CO.
Address reprint requests to Stephen P. Hunger, MD, UCHSC Campus Box
C229, 4200 E Ninth Ave, Denver, CO 80262; e-mail:
Stephen.Hunger{at}UCHSC.edu.
| |
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References