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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-10-3252.
NEOPLASIA
From the Children's Cancer Research Institute and St
Anna Kinderspital, Vienna, Austria; Clinic for Blood Group
Serology, University of Vienna, Vienna, Austria; and the
Department of Pediatrics, University of Erlangen, Erlangen,
Germany.
TEL/AML1-positive childhood acute lymphoblastic leukemias (ALLs)
generally have low-risk features, but still about 20% of patients
relapse. Our initial molecular genetic analyses in 2 off-treatment relapses suggested that the initial and relapse clones
represent different subclones that evolved from a common TEL/AML1-positive, treatment-resistant precursor. In order to further elaborate on this hypothesis, we studied 2 patients with late
systemic relapses of their TEL/AML1-positive ALL (41 months and 49 months after initial diagnosis, respectively) who had distinct clonal
antigen receptor gene rearrangements at diagnosis and relapse. These
clone-specific markers enabled us to determine the responsiveness of
the individual clones to treatment. The matching genomic TEL/AML1 breakpoints of the initial and the relapse clones in these patients confirmed their origin from a common progenitor cell. This proof was
especially important in one of these 2 leukemias without a common
antigen receptor gene rearrangement. Our retrospective analysis
revealed that in both cases the relapse clone was already present at
diagnosis. Despite their small sizes (5 × 10 Childhood B-cell precursor (BCP) acute
lymphoblastic leukemia (ALL) is a heterogeneous disease with a variety
of distinct genetic abnormalities. Approximately 25% of cases harbor a
t(12;21) with the molecular equivalent of a TEL/AML1 gene
fusion.1-3 Clinically, patients with this type of leukemia
have low-risk features. Nevertheless, well-treatable relapses occur in
about 20% of cases, typically after a long first remission
period.4-10 However, the relapse incidence varies and
depends on the respective treatment protocol.3-10
The clone-specific TEL/AML1 gene fusion is readily detected in neonatal
blood spots in most children with TEL/AML1-positive leukemia, proving
that it occurred already in utero.11 The long latency
between this first hit and the clinical manifestation suggests that
further events are required to trigger the stepwise progression of the
clone.12,13 One obvious candidate for such a contributing
factor is the concurring deletion of the second TEL allele as seen in
approximately 75% of TEL/AML1-positive cases.14,15
Relapses are assumed to represent the re-emergence of the initial
leukemic clone. This notion is derived from the finding that in the
majority of cases the initial and the relapse clone share clonotypic
antigen receptor gene rearrangements.16-19 Antigen receptor genes of normal lymphoid cells are rearranged during early
development. Each cell and its progeny have their individual immunoglobulin/T-cell receptor (Ig/TCR) rearrangements, and therefore, they can be used for the characterization of a specific lymphoid cell
population, particularly for clonality studies. Thereby, it was seen
that oligoclonality is common in acute lymphoblastic leukemias.20-22
We have recently shown in 2 patients with TEL/AML1-positive ALL that
off-treatment relapses were not derived from the dominant clone at
diagnosis but from a sibling clone as characterized by antigen receptor
gene rearrangements and 12p status. Thus, it was speculated that both
presentations of the leukemia were derived from a TEL/AML1-positive
pre/leukemic clone.23 We now demonstrate that the relapse
and the initial clone are derived from an identical TEL/AML1
preleukemic clone. Further, the pre/leukemic relapse clone is already
present at initial diagnosis, but has a slower response to therapy
compared with the dominant clone. In contrast, this clone responds
rapidly to chemotherapy when it becomes the relapse leukemia.
Patients
Fluorescence in situ hybridization analysis
Determination of leukemia clone-specific antigen receptor gene rearrangements Polymerase chain reaction (PCR) amplification of incomplete and complete IgH rearrangements was performed using family-specific DH and VH primers, respectively, and one JH consensus primer, as previously described.25 For the detection of the IgK-kappa deleting element (KDE) rearrangements we used 4 Vk primers and an intron recombination signal sequence (RSS) primer in combination with a KDE primer. Sequence of the primers and PCR conditions were used as described previously.26 PCR for the detection of incomplete TCRD (D 2-D3, V2-D3) rearrangements and of TCRG rearrangements (V I-J.1/2.1, VII-J1.1/2.1,
VI-J1.3/2.3, VII-J1.3/2.3) was performed as previously
described.26 All primers were purchased from a commercial
supplier (Invitrogen, Carlsbad, CA).
An aliquot of 500 ng DNA was used for each amplification reaction in a 50 µL volume of 1× buffer (Applied Biosystems, Foster City, CA) containing 200 nM of each deoxynucleotide triphosphate (dNTP), 30 pmol of each primer, and 0.5 U AmpliTaq Gold (Applied Biosystems). Amplifications were performed on a Thermocycler (PTC-200; Techne, Cambridge, United Kingdom). PCR products were then size-fractionated on 4% to 12% nondenaturing polyacrylamide gels (Invitrogen) and analyzed for heteroduplex formation in order to see if more than one clonal product of identical size was present and to separate clonal bands from polyclonal background.26 After elution in water, PCR products were directly sequenced (VBC Genomics, Vienna, Austria). All sequences were confirmed in different independent PCR reactions. The involved gene segments were identified by the Imunogenetics Database (IMGT; http://www.ebi.ac.uk/imgt/), and all Ig rearrangements by comparison with sequences of all known human Ig genes obtained from the IGBlast (http://www.ncbi.nlm.nih.goc/blast/) or VBASE directory (http://www.mrc-cpe.cam.ac.uk/imt-doc/). Determination of genomic TEL/AML1 breakpoints Breakpoint-spanning DNA fragments were amplified by nested long-range PCR. Primer sequences were deduced from gene bank sequence data covering the breakpoint cluster regions of both genes involved, TEL (U61375) and AML1 (AP001721), respectively, and designed with the help of OMIGA software (Oxford Molecular, Oxford, England). PCR was performed on an Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany). We used 2 nested primer sets located within exon 5 and intron 5 of TEL, respectively, together with 16 primer sets located within intron 1 of AML1 in all combinations possible. PCR was performed with a conventional kit (Gene Amp XL, Perkin Elmer, Foster City, CA) following the manufacturer's recommendations using 50 ng template DNA. Cycling conditions consisted of an initial denaturation step at 92°C for 2 minutes, followed by 30 cycles (92°C for 10 seconds, 68°C for 10 minutes and 30 seconds). The first-round PCR product (1 µL) was used as template for the nested reaction. Amplified products were excised from agarose gels, eluted, and directly sequenced. By using this approach, a breakpoint-spanning DNA sequence was obtained from patient 2 only. In patient 1, we assumed that the negative result was due to the breakpoint located within intron 2 of AML1, as is the case in about 20% of t(12;21) translocations. Intron 2 of AML1, however, is very rich in G/C residues. For this reason, a different set of 8 nested PCR primers was used to keep the size of possible PCR products lower. We combined 4 nested PCR primers binding within intron 2 of AML1 with 4 TEL primers. The PCR reaction mix was also modified to destabilize G/C bonds. PCR was performed in a total volume of 50 µL containing 100 ng template DNA, 1× buffer (Invitrogen), 12.5 µL enhancer-solution, 50 mM magnesium chloride, 10 mM of each dNTP (Biozyme, Heidelberg, Germany), 20 µM of each primer, and 1 U Taq polymerase (Taq DNA Polymerase; Invitrogen). Initial denaturation for 2 minutes at 94°C was followed by 35 cycles (94°C for 10 seconds, 68°C for 3 minutes) and a final annealing/extension step for 7 minutes. PCR primers giving rise to a product in both approaches are listed in Table 1. Primers for amplification of the derivative 21 breakpoint in patient 1 were selected on basis of the derivative 12 sequence.
Clone-specific PCR Clone-specific oligonucleotide primers were designed homologous to the junctional regions of the Ig/TCR rearrangements and used for quantitative PCR, as previously described.24 Optimal thermocycling conditions were established for each patient-specific primer pair to detect the clone-specific sequence. The specificity and sensitivity of the primer combinations was determined for each clonal marker on DNA from either initial or relapse leukemia according to their presence in the screening PCR. Leukemic DNA was diluted (10 1 to 107) in DNA from peripheral blood
MNCs from healthy donors.
DNA for Ig/TCR and TEL/AML gene fusions (500 ng and 100 ng, respectively) was used as a template in a 50-µL reaction volume. Negative controls were included in each experiment: one sample containing the reaction mix without DNA, and another sample containing DNA from peripheral blood MNCs from healthy donors as a polyclonal control. DNA obtained at diagnosis from each patient was used as a positive control. Samples and controls were always analyzed in triplicate. Precautions to avoid contamination were followed as previously described.27
The 2 children had a late systemic relapse of their
TEL/AML1-positive ALL. The clinical data as well as the duration of
first remission are listed in Table 2.
The children are still in second complete continuous remission (CCR).
The children received chemotherapy for non-high-risk leukemias of the
Berlin-Frankfurt-Münster (BFM) ALL 90 protocol in
Austria.28 Remission induction chemotherapy consisted of
one week prednisone (60 mg/m2 per day) plus one
intrathecal methotrexate application on day 1, followed by
weekly administrations of intravenous vincristine (1.5 mg/m2) and daunorubicine per infusion (30 mg/m2). Asparaginase infusions (10.000 IE/m2)
were given twice a week for 4 weeks. On day 33 the morphologic remission status was evaluated in the bone marrow. Relapses were treated according to the BFM Rez ALL 95 study.29 The first
treatment block consisted of dexamethasone 20 mg/m2 on days
1 to 5, triple intrathecal administration on day 1, vincristine 1.5 mg/m2 on days 1 and 6, methotrexate 1000 mg/m2
per infusion (36 hours) on day 1 and E coli-asparaginase
10.000 IE/m2 on day 4 (infused over 24 hours).
Both patients achieved a morphologic remission after the initial and
relapse induction chemotherapy, respectively.
In the first patient, we found a total of 6 rearrangements, 4 at diagnosis and 2 different, completely unrelated ones at relapse. In the second patient, we identified 5 distinct rearrangements at diagnosis and 6 at relapse, one of which was found in both instances. Based on our results that in both leukemias a maximum of 2 different rearrangements per antigen receptor gene were detected, we concluded that the initial diseases and relapses were monoclonal. Thus, with regard to the antigen gene receptor gene rearrangements, the diagnostic and relapse samples of the first patient appeared as having arisen from 2 completely unrelated clones. This is in contrast to the second patient, in whom a clonal relationship between diagnostic and relapse leukemia was evident. TEL/AML1 FISH reveals clonal diversification The first patient had a TEL/AML1-positive as well as TEL/AML1-negative cell population, but both with very similar secondary changes. At diagnosis, a deletion of one TEL allele was seen in 9 of 13 TEL/AML1-negative metaphases. A TEL/AML1 fusion was present in 81% of the 80 analyzable interphase cells. The following percentages always refer to the whole number of analyzed interphase cells. The 19% TEL/AML1-negative cells comprised some with a TEL deletion together with an additional AML1 signal (3.8%, indicative of the presence of an additional chromosome 21), whereas 7.6% had none of these changes. Another 7.6% had 2 additional AML1 signals, but no TEL deletion. The TEL/AML1-positive cell population consisted of 31% with and 50% without a TEL deletion. The former comprised 23% with 2 and 7.6% with 3 and the latter 3.8% with one, 31% with 2, and 15.3% with 3 extra AML1 signals. In the relapse sample we were able to screen 130 cell nuclei, of which 48% were TEL/AML1-negative and 15% TEL/AML1-negative, but with a TEL deletion. The 37% TEL/AML1-positive cells included 7% with only a TEL deletion, 10% with 2 extra AML1 signals, and 20% without a TEL deletion, but with one extra AML1 signal.In summary, these FISH data suggest that in the first patient all TEL/AML1-negative and -positive clones were also present in the relapse sample, albeit with a different distribution. This clonal heterogeneity most likely indicates that the TEL/AML1 gene fusion and the subsequent clonal evolution have occurred in a transformed clone before the immune receptor rearrangements. In the second patient, a colocalization of the TEL and AML1 signals was found in 62% of interphase cells from the diagnostic sample. Moreover, in line with the presence of an additional derivative chromosome 21, der(21), 7% of nuclei showed a second colocalization, whereas the absence of a noncolocalized TEL signal indicated that the residual TEL allele was deleted in this cell population. In the relapse sample, on the other hand, 74% of nuclei contained a TEL/AML1 gene fusion and 48% of them had 2 colocalizations, whereas the signal for the second TEL allele was always present. In this patient the relapse clone without TEL deletion could not have evolved from the diagnostic clone with a deleted TEL. Initial leukemia and relapse are derived from a patient-specific TEL/AML1 leukemic progenitor cell To test our hypothesis that the clones from initial disease and relapse are derived from a common TEL/AML1-positive progenitor cell, we cloned the patient-specific TEL/AML1 and the reciprocal AML1/TEL fusion sites on the DNA level. In the first patient, the primer combinations P1/P3 and P2/P4 revealed a breakpoint-spanning PCR product of the AML1/TEL fusion on the derivative chromosome 12, der(12), in intron 5 of the TEL and in intron 2 of the AML1 gene. Sequencing was performed with primer P13. Sequence data were deposited in public databases (accession number AJ494735). TaqMan real-time PCR was performed with primers P16/P17 and the probe P18. In this patient, also a TEL/AML1 breakpoint-spanning DNA fragment on the der(21) was amplified with primers P5/P7 and P6/P8 and sequenced using primer P14. In the second patient, the der(12) genomic breakpoint was detected in the intron 5 of TEL and in intron 1 of AML1 with the primer combinations P9/P11 and P10/P12. Sequencing was performed with primer P15 (database accession number AJ494736). Real-time PCR was performed with P19/P20 and the probe P21. Primers P22/P23 and probe P24 were used to quantifiy the single copy gene APM-1 as a basis for calculating the number of TEL/AML1-positive cells in each sample. The breakpoint sequences, identified in the diagnostic and relapse samples, were clonotypic for each patient demonstrating unambiguously the common clonal origin of the leukemia.Relapse originates from a small subclone present at diagnosis The individual configurations of the immune receptor gene rearrangements enabled us to retrospectively confirm that the relapse clones were actually already present at diagnosis in both patients (Table 3). In the first patient, the relapse clone with its specific TCRGc and TCRGd gene rearrangements constituted only approximately 1 in 104 cells at the time of diagnosis. The relapse clone of the second patient, which was defined by unique IgHd, TCRDb, and TCRGb gene rearrangements, comprised 0.5% of the initial leukemic cell load. However, despite a high sensitivity (104) of the
allele-specific PCR, we were unable to detect 2 of the relapse-specific
rearrangements (IgHc and TCRGc) in this patient (Table 3).
These rearrangements were also not found in samples that were obtained
during the complete treatment phase (data not shown). We therefore
presume that the relapse clone had evolved from a cell with ongoing
recombinatorial potential that was related to the smaller subclone at
diagnosis. We also used clone-specific PCR to find out whether the
initial clone might have persisted (Table 3). However, in neither case
were the respective clonotypic rearrangements detectable.
Subclones show a different response to initial and relapse chemotherapy In both children the response of the different subclones to treatment was evaluated by quantitative PCR of the unique rearrangements from the initial and relapse leukemias, respectively (Figure 1). In patient 1, both leukemic subclones had an initial slow response after 2 weeks of treatment, although it was even slower in the minor subclone. One cell of the dominant clone was still detectable among 100 normal cells at day 15 as analyzed by the IgH and TCRD rearrangements, whereas the relapse clone, detected through its 2 TCRG rearrangements, persisted at similar low levels (104) as before treatment. These clones remained
undetectable at all time points including 2 months after the end of
chemotherapy. No further samples were obtained until relapse. The
leukemia at relapse responded very promptly to chemotherapy as shown in
Figure 1.
In patient 2, the dominant clone at initial diagnosis was not detected
(< 10 To test whether quantification of the TEL/AML1 breakpoints would confirm the results obtained by immune receptor gene analysis, or detect a higher copy number of the TEL/AML1 fusion gene, we applied patient-specific TEL/AML1 real-time TaqMan PCRs using primers 16/17 and 19/20 and probes 18 and 21, for patient 1 and 2, respectively (Table 1). We detected similar amounts of PCR products in all diagnostic and follow-up samples (indicated by an asterisk in Figure 1), thereby confirming the slower response and the persistence of the relapse clone, as well as the lack of amplification during later phases of the chemotherapy.
In 2 children with late systemic relapses of their TEL/AML1-positive leukemias we show that the initial and the relapse leukemias arise independently from a common TEL/AML1 precursor cell. At initial diagnosis, the dominant clone was sensitive to therapy, whereas the subclone, which later developed into the relapse leukemia, responded poorly. At the time of relapse, however, this clone was sensitive to treatment. The differentiation stage of a transformed lymphoid cell can be
characterized by its immune receptor rearrangements. These rearrangements occur independently from the oncogenic process. Thus,
they can be used as markers for the timing of molecular events during
leukemogenesis as well as for determining the time point of an early
event during ontogeny (fetal versus postnatal development).30-34 We propose an order of molecular events
during leukemogenesis, which is based on a current model by M. Greaves.35,36 Accordingly, at least 2 "hits" are
required for the manifestation of leukemia. The TEL/AML1 translocation
might be such a "first hit" in a precursor cell, either before the
onset of somatic recombination, or, at a later stage, when immune
receptor genes rearrange. Thus, continuing rearrangement processes lead
to a variety of immune receptor rearrangements in individual cells of
the TEL/AML1-positive preleukemic clone, resulting in a heterogeneous
population. A second crucial molecular hit, probably a deletion of the
nonrearranged TEL allele, drives one of these cells to overt leukemia.
The following chemotherapy eradicates that leukemia, while the
preleukemic clone may survive. If one of these preleukemic cells
acquires a second mutation another leukemia develops, which harbors an
identical TEL/AML1 gene fusion, but may carry a different TEL deletion
and also different Ig/TCR gene rearrangements. In both patients the identical TEL/AML1 gene fusion was found at initial manifestation of
the leukemia and at relapse. This gene fusion presumably transformed a
cell with its immune receptor genes in germ line configuration or with
an IgK rearrangement in patients 1 and 2, respectively, which may, on
some occasions, precede IgH rearrangements.37 The
difference in the TEL deletion status between initial presentation and
relapse in patient 2 concords with the proposed model, whereas the
clonal heterogeneity in the first patient makes an interpretation and
valid conclusion extremely difficult. At initial presentation of the
leukemia, the relapse clones ranged from 10 Patient 2 from our study had a long persistence of the minor pre/leukemic subclone. Likewise, the persistence of a preleukemic clone carrying the leukemia-specific fusion gene during morphologic remission has been reported in patients with acute myeloid leukemia with the t(8;21) chromosomal translocation. Thereby, the AML/ETO fusion gene resides in a preleukemic multipotent stem cell, which is resistant to treatment and develops to overt leukemia due to secondary genetic events.39-42 A rapid and sustained disappearance of the dominant leukemia clones and
a slow response of the smaller clones to first chemotherapy were
observed in both children. According to current concepts, the slow
response of the minor clones results from a primary drug resistance.
In-line, slow-responding subclones were described previously in
children with TEL/AML1-negative ALL.43 These patients experienced an early disease recurrence, whereas the 2 patients from
this report had long first remission durations of 48 months and 40 months, respectively. Typically, TEL/AML1-positive leukemias have late
relapses as exemplified by the cases from this report. These relapses
respond very well to relapse chemotherapy with persistent or
long-lasting second remissions.8 Such biologic differences between TEL/AML1-positive and -negative leukemias allow an alternative interpretation, that is, that these
minor The observation from the 2 cases reported here should, however, not
suggest the exclusive use of breakpoint-specific sequences for MRD
analysis In conclusion, we have shown that a minor subclone of TEL/AML1-positive ALL responds slowly to chemotherapy, whereas the major clone has a rapid response. This biologic behavior of the smaller subclone during first chemotherapy can be interpreted as a feature of the preleukemic stage, which contrasts its very rapid response when it has evolved to the relapse leukemia.
This study was performed using the network of the Biology Group within the I-BFM Group. We would like to thank Uli Monschein and Marion Zavadil for help in preparing the manuscript.
Submitted October 28, 2002; accepted December 10, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-10-3252.
Supported in part by a grant from the FWF 13757-MED (E.R.P.-G.) and by the "Österreichische Kinderkrebshilfe."
M.K. and M.M. contributed equally to the content of this paper.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: E. Renate Panzer-Grümayer, CCRI, St Anna Kinderspital, Kinderspitalg.6, A-1090 Vienna, Austria; e-mail: renate.panzer{at}ccri.univie.ac.at.
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Abstract
Introduction
indicates the dominant leukemic clone
at diagnosis as detected by immune receptor rearrangements (patient 1 [pt 1]: IgH, TCRD; pt 2: IgHb, TCRDa, TCRGa); 
shows the relapse
clone detected by immune receptor rearrangements (pt 1: TCRGa and b; pt
2: IgHd, TCRDb, TCRGb); filled symbols indicate a quantifiable amount
of the clone (more than 5 × 10
presumably preleukemic