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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1107-1110
CORRESPONDENCE
MLL-AF4 Gene Fusions in Normal Newborns
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LETTER |
To the Editor:
Uckun et al1 have reported that bona fide in-frame fusion
MLL-AF4 fusion sequences are detectable, using a sensitive
nested reverse transcription-polymerase chain reaction (RT-PCR) method, in around 25% of normal fetal bone marrow and liver samples and in 1 of 6 infant bone marrows. It was also reported that similar fusion
sequences were present in presumptive normal cells at low levels in
13% of pediatric (noninfant) acute lymphoblastic leukemia (ALL) cases, but were absent in remission bone marrow
samples of similar patients. These are very interesting data and
require careful consideration. Both these authors1 and
editorial articles commenting on the findings2,3 suggested
that the MLL-AF4 fusion gene may be necessary but insufficient
for the clinical development of infant leukemia. As Hunger and
Cleary2 point out in their editorial, it is important that
this result is both substantiated by different methods, eg,
fluorescence in situ hybridization (FISH), genomic sequencing,
and independently confirmed by other investigators.
We have screened a series of unselected normal cord blood samples for
MLL-AF4 fusion sequences as part of a program to evaluate the
frequency of leukemia fusion genes in normal fetal hematopoiesis. We
adapted and standardized a nested RT-PCR assay for maximized sensitivity using primers as previously described for minimal residual
disease detection in infant leukemia4 and could, in standard dilution assays with MLL-AF4 positive leukemic cell
lines, achieve reproducible detection at 10 5 with
variable scores at 106. In screening for other
translocation products, we have in the past had difficulties with
false-positive results due to contamination, and we take appropriate
precautions to prevent this. We have now screened a total of 68 samples
(60 cord blood samples plus 8 fetal liver samples). All samples were
subjected to RT-PCR for an ABL transcript to confirm the
presence of intact mRNA. In the first series of 38 cord blood samples
and 8 fetal liver samples, 5 bone marrows gave an amplified product. In
each case, the product appeared to be slightly different in size from
that anticipated from the cDNA prepared from leukemic cells with an
MLL-AF4 fusion gene.4 Representative products were
cloned and several isolates sequenced, and none were derived from
chimeric MLL-AF4 fusion genes. The sequenced products were
either unrelated to MLL-AF4 or derived from incompletely
spliced AF4 mRNA where the upstream AF4 intron 3 had
some fortuitous homology to the 3' end of the internal MLL primer. Because our RT-PCR primers4 were somewhat different from those used by Uckun et al, we performed screening on an additional 30 cord blood samples with primers identical to those
described,1 but failed to score any positives. We also
rescreened the 8 fetal livers and failed to observe any amplified products.
Other groups have similarly failed to identify MLL-AF4 fusion
sequences in newborn samples5 (J. Trka,
personal communication, September 1998). Intragenic
fusions or partial tandem duplications of AF4 or MLL have been
recorded in normal blood samples,4-7 the significance of
which remains uncertain. Therefore, we are unable to confirm the report
of Uckun et al1 and cannot at present suggest a likely
explanation for the discrepancy. Although there are precedents for the
finding of fusion genes in normal tissue samples,8-10 there
are difficulties in accepting that infant leukemia is "primed" by
widespread or common MLL-AF4 fusions in utero. First, even if
such fusion genes were reproducibly detectable, this would not
necessarily indicate the presence of preleukemic cells awaiting a
second strike. The cellular context is likely to be critical. Such
genes (as with other mutant oncogenes in normal individuals) could
reside in cells that are irrelevant to leukemogenesis. The
MLL-AF4 fusion genes of infant leukemia do arise during fetal
hematopoiesis11,12 and most probably in a stem cell with
B/monocytic lineage potential. The presence of an MLL-AF4 gene
product, albeit in frame, in, say, a T-cell or granulocyte progenitor,
would be of no immediate significance with respect to leukaemogenesis.
Second, MLL-AF4 fusions differ significantly in one particular
respect from other aberrant leukemia- or cancer-associated genes
detectable in normal individuals. Infant ALL/acute myeloblastic
leukemia and secondary acute leukemia13with
MLL fusion genes are associated with uniquely brief latent periods measurable in months as opposed to the norm of
years or decades. Furthermore, the concordance rate of infant ALL in
identical twins derived from an MLL fusion gene in one fetus is
very high somewhere around 50% to 100% of those with a monochorionic
placenta with vascular connections facilitating spread of the
preleukemic clone from one fetus to the other.10,14 One
plausible interpretation of these data is that once an in-frame
MLL fusion gene occurs in an appropriate cell type, evolution
of the leukemic clone to a clinical diagnosis is both highly likely and
rapid. This could occur either if MLL fusion genes are in
themselves sufficient for leukemogenesis or if they very
efficiently provoke other necessary secondary genetic
events.
Either way, if this interpretation is valid, it sits ill at
ease with the presence of MLL-AF4 fusion-positive cells primed
for leukemia in 25% of normal newborns. Back to the drawing board?
M.-H. Kim-Rouille
A. MacGregor
L.M. Wiedemann
Leukaemia Research Fund Centre
Institute of Cancer Research
London, UK
M.F. Greaves
C. Navarrete
National Blood
Service
North London Centre
London, UK
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REFERENCES |
1.
Uckun FM, Herman-Hatten K, Crotty M-L, Sensel MG, Sather HN, Tuel-Ahlgren L, Sarquis MB, Bostrom B, Nachman JB, Steinherz PG, Gaynon PS, Heerema N:
Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation.
Blood
92:810, 1998[Abstract/Free Full Text]
2.
Hunger SP, Cleary ML:
What significance should we attribute to the detection of MLL fusion transcripts?
Blood
92:709, 1998[Free Full Text]
3.
Rowley JD:
Backtracking leukemia to birth.
Nat Med
4:150, 1998[Medline]
[Order article via Infotrieve]
4.
Janssen JWG, Ludwig W-D, Borkhardt A, Spadinger U, Rieder H, Fonatsch C, Hossfeld KK, Harbott J, Schulz AS, Repp R, Sykora K-W, Hoelzer D, Bartram CR:
Pre-pre B acute lymphoblastic leukemia: High frequency of alternatively spliced ALL1-AF4 transcripts and absence of minimal residual disease during complete remission.
Blood
84:3835, 1994[Abstract/Free Full Text]
5.
Yamamoto S, Zaitsu M, Ishii E, Yatsuki H, Mizutani S, Eguchi M, Ihara K, Okamura T, Hara T, Miyazaki S:
High frequency of fusion transcripts of exon 11 and exon 4/5 in AF-4 gene is observed in cord blood, as well as leukemic cells from infant leukemia patients with t(4;11)(q21;q23).
Leukemia
12:1392, 1998[Medline]
[Order article via Infotrieve]
6.
Marcucci G, Strout MP, Bloomfield CD, Caligiuri MA:
Detection of unique ALL1 (MLL) fusion transcripts in normal human bone marrow and blood: Distinct origin of normal versus leukemic ALL1 fusion transcripts.
Cancer Res
58:790, 1998[Abstract/Free Full Text]
7.
Schnittger S, Wormann B, Hiddemann W, Griesinger F:
Partial tandem duplications of the MLL gene are detectable in peripheral blood and bone marrow of nearly all healthy donors.
Blood
92:1728, 1998[Abstract/Free Full Text]
8.
Liu Y, Hernandez AM, Shibata D, Cortopassi GA:
BCL2 translocation frequency rises with age in humans.
Proc Natl Acad Sci USA
91:8910, 1994[Abstract/Free Full Text]
9.
Biernaux C, Loos M, Sels A, Huez G, Stryckmans P:
Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.
Blood
86:3118, 1995[Abstract/Free Full Text]
10.
Muller JR, Mushinski EB, Williams JA, Hausner PF:
Immunoglobulin/Myc recombinations in murine Peyer's patch follicles.
Genes Chromosomes Cancer
20:1, 1997[Medline]
[Order article via Infotrieve]
11.
Ford AM, Ridge SA, Cabrera ME, Mahmoud H, Steel CM, Chan LC, Greaves MF:
In utero origin of rearrangements in the trithorax-related oncogene in infant leukaemias.
Nature
363:358, 1993[Medline]
[Order article via Infotrieve]
12.
Gale KB, Ford AM, Repp R, Borkhardt A, Keller C, Eden OB, Greaves MF:
Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots.
Proc Natl Acad Sci USA
94:13950, 1997[Abstract/Free Full Text]
13.
Smith MA, McCaffrey RP, Karp JE:
The secondary leukemias: Challenges and research directions.
J Natl Cancer Inst
88:407, 1996[Abstract/Free Full Text]
14.
Greaves M:
A natural history for pediatric acute leukemia.
Blood
82:1043, 1993[Free Full Text]
Response to Trka et al and Greaves et al
MLL-AF4 Fusion Transcripts in Normal and Leukemic
Hematopoietic Cells
In a recent study,1 we used nested polymerase
chain reaction (PCR) assays to determine the expression frequency of
MLL-AF4 in infant and childhood acute lymphoblastic leukemia
(ALL). In infants, nested PCR assays were in agreement
with the standard cytogenetics data. Of 17 infants, 9 were MLL-AF4
positive by nested PCR and 8 of these 9 infants had a cytogenetically
detectable t(4;11).2 In contrast, nested PCR
assays showed MLL-AF4 positivity in 17 of 127 children with ALL,
including children without a cytogenetically detectable t(4;11),
although standard PCR showed MLL-AF4 positivity only in one
case.1 Surprisingly, nested PCR assays (but not standard
PCR) detected MLL-AF4 fusion transcript expression in some normal fetal
liver or bone marrow cells as well. We1 and others3 commented that our results suggest that the t(4;11) translocation may be a common occurrence in utero, and may be a
critical but not sufficient step for leukemogenesis.
In their Letter to Editor,4 Trka et al provide several
suggestions and criticisms regarding our study. Their statements require further clarification and comment. The authors state that they
have no data to support our hypothesis that MLL-AF4 expression is not
sufficient for leukemogenesis, which was based upon detection of
MLL-AF4 fusion transcripts in fetal hematopoietic tissues. This should
not be surprising when one considers the fact that the authors have not
examined MLL-AF4 expression in any fetal tissue. Furthermore, we
studied only a very limited number of fetal tissue specimens and
therefore cannot comment on the frequency of MLL-AF4 expression. Fresh
samples need to be analyzed since MLL-AF4 transcripts in normal cells
may be unstable. We were unable to detect MLL-AF4 transcripts
by nested RT-PCR in any of 21 cryopreserved fetal liver specimens. The
authors based their statement on their failure to detect MLL-AF4
positivity in cord blood samples from 103 full-term healthy newborns.
Unfortunately, the authors did not include any positive controls in
their study to show that their failure to detect MLL-AF4 fusion
transcripts was not a false-negative result due to technical
difficulties. Specifically, it is prudent that the authors show the
presence of MLL-AF4 positive leukemic cells added to their cord blood
samples at a 0.01% level. Furthermore, the authors do not comment on
the cellular composition of the cord blood samples. The nucleated cell
content as well as freshness of cord blood samples will undoubtedly
affect the ability of the authors to detect MLL-AF4 positivity, which
required even for highly hematopoietic cell-enriched mononuclear cell
preparations from fetal tissues the use of a highly sensitive nested
PCR assay.1 It should also be noted that the authors used a
different PCR assay with different primers for nested PCR.
Specifically, the authors report that they used primers in MLL exon 5 for both standard and nested PCR even though our sequencing results
provide conclusive evidence that exon 6 of the MLL gene is frequently
fused to AF4a in normal hematopoietic cells as well as non-t(4;11) ALL
cells.1 Another interesting aspect of the letter by Trka et
al is their statement that the authors usually find up to six bands in
their gel analysis of their nested PCR products and therefore find the results shown in Fig 3 of our report1 surprising. While
most investigators, including our group, frequently find multiple bands in the post-PCR gels of the MLL-AF4 standard PCR product of a given
case, this is not to be expected after a nested PCR reaction. Therefore, the question arises of how the nested PCR reactions were
performed and how the band profiles differed from those of standard PCR
products. The post-PCR gels reported in our paper are indeed very
similar to those reported by Downing et al,2 who used the
same nested PCR methodology. Nevertheless, the results reported by Trka
et al,4 if confirmed by others and validated after
inclusion of appropriate positive controls, would certainly expand our
knowledge of MLL-AF4 expression in hematopoietic tissues. I certainly
agree with the authors that MLL-AF4 positive cord blood specimens
cannot be considered free of leukemic cells and find nothing in our
report to suggest this rather unusual interpretation. To the contrary,
the detection of in utero rearrangements reported to
date5-7 indicates that extreme caution is needed when
autologous or syngeneic cord blood stem cells are to be used for
transplantation, as recently discussed by Rowley.3 For
example, Greaves reported the development of a T-cell leukemia with
identical T-cell receptor rearrangements at the ages of 9 and 10 years
in twins, indicating that the transforming events occurring in utero
may lead to leukemia after a very long latency period.3
Regarding the failure of the authors to detect MLL-AF4 positive cells
in remission bone marrow samples, I would like to point out that this
is consistent with our own experience as well.1 Specifically, in Table 5 of our paper, we reported that none of the 44 remission bone marrow samples expressed MLL-AF4 by standard or nested
PCR.1 We were interested to know if patients with t(4;11)
ALL would show persistent MLL-AF4 fusion transcript expression after
chemotherapy. As shown in Fig 1A, nested
PCR analysis of sequential bone marrow specimens from a
t(4;11)+ infant ALL case (INF 1 from Table 2 of ref 2)
showed disappearance of MLL-AF4 positivity 5 months after initiation of
chemotherapy. More than 2 years after the first disappearance of
MLL-AF4 positivity and in complete remission off therapy for more than
a year, nested PCR continues to show no evidence of MLL-AF4 expression
in this patient. Therefore, we postulate that all MLL-AF4 positive
cells in this case were involved in the original leukemic
transformation. Similarly, we were interested to know if the MLL-AF4
positivity detectable only by nested PCR in a non-t(4;11) ALL case
represents low-level fusion transcript expression in leukemic cells or
detection of MLL-AF4 fusion transcript expression in nonleukemic cells. As shown in Fig 1B, the MLL-AF4 positivity of bone marrow samples from
a normal diploid ALL patient (PEDS 5) disappeared after remission induction, was transiently detectable again towards the end of the
maintenance chemotherapy (ie, 1/95 bone marrow sample),
and has been undetectable by nested PCR for more than 2 years as the patient continues to be in complete remission off chemotherapy. These
results suggest that the MLL-AF4 positivity in this case was likely
caused by low-level fusion transcript expression in leukemic cells. In
view of the disappearance of MLL-AF4 positivity in nested PCR positive
cases after remission induction and the nested PCR negativity of
remission bone marrow specimens from 44 children with standard-risk
ALL, I do not believe that bone marrow specimens from healthy children
(excluding infants) would be nested PCR positive for MLL-AF4
expression.
View larger version (45K):
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| Fig 1.
Chemotherapy-induced elimination of MLL-AF4 positive
cells in bone marrow specimens from ALL patients. (A) INF1 is an infant
t(4;11) ALL case diagnosed in March 1995. (B). PEDS5 is a normal
diploid ALL case diagnosed in December 1993. Sequential
bone marrow specimens were analyzed for the presence of MLL-AF4 fusion
transcripts using nested RT-PCR, as described. Amplified mRNA from the
RS4;11 cell line was used as a positive control (POS CON). Negative
controls (NEG CON) were PCR products from RNA-free and
DNA-polymerase-free reaction mixtures.2
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We have not performed Southern blot analyses on a routine basis to
detect MLL gene rearrangements. Since the nested PCR assay did not
appear to be a useful diagnostic test because it failed to distinguish
patients with t(4;11) ALL and those who might have MLL-AF4 in a minor
population of cells, we used Southern blotting in only 9 patients, 2 fetal livers, and 1 infant bone marrow to highlight these distinctions.
Interestingly, cells from all t(4;11) ALL patients, including a
4.7-year-old child (PEDS87) whose leukemic cells had MLL-AF4 positivity
detectable by nested PCR only, showed 11q23 gene rearrangements by
Southern blot analysis. Thus, in some cases with 11q23 gene
rearrangements and MLL-AF4 positivity, the level of expression for
MLL-AF4 may be so low that a nested PCR is required for detection. This
conclusion is also supported by findings of Hilden et
al.8 Do patients with no 11q23 gene rearrangement
detectable by Southern blotting and MLL-AF4 positivity detectable only
by nested PCR have a small subpopulation of MLL-AF4 positive cells or a
cryptic MLL gene rearrangement in all leukemic cells with a very
low-level fusion transcript expression detectable only by nested PCR?
This question will be addressed using in situ nested PCR assays in our
future studies. The authors refer to two patients (INF-6, PEDS-5) with
normal diploid ALL and MLL-AF4 positivity with nested PCR.
The detection of MLL gene rearrangements by Southern blot analysis has
been extensively reported by others who examined normal diploid ALL
cases.8-10 We do not believe that the nested PCR positivity
in INF-6 or PEDS-5 has much to do with the Southern blot evidence of
MLL gene rearrangement.
Finally, I would like to mention that recent results from our
laboratory indicate the presence of additional molecular abnormalities in t(4;11) infant leukemia cells, which further supports our hypothesis that the expression of MLL-AF4 may be critical but not sufficient for
leukemic transformation.11 However, our published
findings1 that were obtained using nested RT-PCR still need
to be validated using genomic sequencing, which we have not yet
performed. I would like to thank the authors for their interest in our
work and this opportunity to clarify a number of issues regarding
MLL-AF4 fusion transcript expression in human lymphocyte ontogeny.
Fatih M. Uckun
Parker Hughes Cancer Center and
the
CCG ALL Biology Reference Laboratory
Hughes Institute
Roseville,
MN
| |
REFERENCES |
1.
Uckun FM, Herman-Hatten K, Crotty M-L, Sensel MG, Sather HN, Tuel-Ahlgren L, Sarquis MB, Bostrom B, Nachman JB, Steinherz PG, Gaynon PS, Heerema N:
Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation.
Blood
92:810, 1998
2.
Downing JR, Head DR, Raimondi SC, Carroll AJ, Curcio-Brint AM, Motroni TA, Hulshof MG, Pullen J, Domer PH:
The der(11)-encoded MLL/AF4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia.
Blood
83:330, 1994[Abstract/Free Full Text]
3.
Rowley JD:
Backtracking leukemia to birth.
Nat Med
4:150, 1998
4.
Trka J, Zuna J, Hru ák O, Michalová K, Mu íková K, Kalinová M, Starý J:
No evidence for MLL/AF4 expression in normal cord blood samples.
Blood
93:1106, 1999[Free Full Text]
5.
Gale KB, et al:
Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots.
Proc Natl Acad Sci USA
94:13950, 1997
6.
Ford AM, Bennett CA, Price CM, Bruin MC, Van Werring ER, Greaves M:
Fetal origins of the TEL-AML1 fusion gene in identical twins with leukemia.
Proc Natl Acad Sci USA
95:4584, 1998[Abstract/Free Full Text]
7.
Ford AM, Ridge SA, Cabrera ME, Mahmoud H, Steel CM, Chan LC, Greaves M:
In utero rearrangements in the trithorax-related oncogene in infant leukemias.
Nature
363:358, 1993
8.
Hilden JM, Frestedt JL, Moore RO, Heerema NA, Arthur DC, Reaman GH, Kersey JH:
Molecular analysis of infant acute lymphoblastic leukemia: MLL gene rearrangement and reverse transcriptase-polymerase chain reaction for t(4;11)(q21;q23).
Blood
86:3876, 1995[Abstract/Free Full Text]
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Rubnitz JE, Link MP, Shuster JJ, Carroll AJ, Hakami N, Frankel LS, Pullen DJ, Cleary ML:
Frequency and prognostic significance of HRX rearrangements in infant acute lymphoblastic leukemia: A Pediatric Oncology Group Study.
Blood
84:570, 1994[Abstract/Free Full Text]
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Chen CS, Sorensen PH, Domer PH, Reaman GH, Korsmeyer SJ, Heerema NA, Hammond GD, Kersey JH:
Molecular rearrangements on chromosome 11q23 predominate in infant acute lymphoblastic leukemia and are associated with specific biologic variables and poor outcome.
Blood
81:2386, 1993[Abstract/Free Full Text]
11. Sun L, Heerema N, Crotty L, Wu X, Navara C, Vassilev A,
Sensel M, Reaman GH, Uckun FM: Expression of dominant negative and
mutant isoforms of the antileukemic transcription factor Ikaros in
infant acute lymphoblastic leukemia. Proc Natl Acad Sci USA 1999 (in
press)
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