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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 12 (June 15), 1999:
pp. 4079-4085
RAPID COMMUNICATION
Ig Heavy Chain Gene Rearrangements in T-Cell Acute Lymphoblastic
Leukemia Exhibit Predominant DH6-19 and DH7-27 Gene
Usage, Can Result in Complete V-D-J Rearrangements, and Are Rare in
T-Cell Receptor   Lineage
By
Tomasz Szczepanski,
Marja J. Pongers-Willemse,
Anton W. Langerak,
Wietske A. Harts,
Annemarie J.M. Wijkhuijs,
Elisabeth R. van Wering, and
Jacques J.M. van Dongen
From the Department of Immunology, University Hospital
Rotterdam/Erasmus University Rotterdam, Rotterdam, The Netherlands; the
Department of Pediatric Hematology and Chemotherapy, Silesian Medical
Academy, Zabrze, Poland; and the Dutch Childhood Leukemia Study Group,
The Hague, The Netherlands.
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ABSTRACT |
Rearranged IGH genes were detected by Southern blotting in
22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and
31% (8/26) of these cases, respectively. IGH gene
rearrangements were found in 19% (13/69) of CD3 T-ALL
and in 50% of TCR  + T-ALL (12/24), whereas only a
single TCR  + T-ALL (1/25) displayed a monoallelic
IGH gene rearrangement. The association with the T-cell
receptor (TCR) phenotype was further supported by the striking
relationship between IGH and TCR delta (TCRD) gene
rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelic
TCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex
polymerase chain reaction (PCR) analysis with VH and
DH family-specific primers in combination with a JH
consensus primer showed a total of 39 clonal products, representing 7 (18%) VH-(DH-)JH joinings and 32 (82%)
DH-JH rearrangements. Whereas the usage of
VH gene segments was seemingly random, preferential usage of
DH6-19 (45%) and DH7-27 (21%) gene segments was
observed. Although the JH4 and JH6 gene segments were
used most frequently (33% and 21%, respectively), a significant
proportion of joinings (28%) used the most upstream JH1 and
JH2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of
incomplete DH-JH rearrangements, the frequent usage
of the more downstream DH6-19 and DH7-27 gene
segments, and the most upstream JH1 and JH2 gene
segments suggests a predominance of immature IGH rearrangements
in immature (non-TCR+) T-ALL as a result of
continuing V(D)J recombinase activity. More mature -lineage T-ALL
with biallelic TCRD gene deletions apparently have switched off
their recombination machinery and are less prone to cross-lineage
IGH gene rearrangements. The combined results indicate that
IGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes
and completed TCR alpha (TCRA) gene rearrangements.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
WHEN REARRANGED Ig and T-cell receptor
(TCR) genes were found to be useful clonal leukemia-specific markers,
they were initially considered to be lineage-specific.1,2
However, the recognition of abnormal TCR gene rearrangements in
precursor-B-acute lymphoblastic leukemia (ALL) and in some mature
B-cell malignancies together with the finding of rearranged Ig heavy
chain (IGH) genes in some T-ALL raised the question of lineage
infidelity or promiscuity.2,3 TCR gene rearrangements in
precursor-B-ALL were observed in 60% to 80% of patients, indicating
that these recombinations should rather be regarded as an ubiquitously
occurring (cross-lineage) phenomenon in this type of B-cell
malignancy.4-7 In a recent study on 202 precursor-B-ALL
patients, we even found cross-lineage TCR gene rearrangements in 93%
of the patients.8 The occurrence of cross-lineage
rearranged TCR genes is related to the maturation stage, being
significantly lower in immature CD10 precursor-B-ALL
as compared with CD10+ precursor-B-ALL.8,9
Analogous to the hierarchical order during early T-cell development,
rearrangements and/or deletions in the TCR delta (TCRD) locus
occur most frequently (89% of patients), followed by TCR gamma
(TCRG) (59%) and TCR beta (TCRB) (35%) gene rearrangements.4-6,8 Furthermore, most TCRB gene
rearrangements are restricted to the J2 locus, the majority of
TCRG gene rearrangements involve the J1 gene segments, and a
striking predominance of incomplete V2-D3 and D2-D3
rearrangements in the TCRD locus is observed.4-11
In contrast to the high frequency of cross-lineage TCR gene
recombinations in precursor-B-ALL, IGH gene rearrangements in T-ALL are relatively rare. In a meta-analysis of previously reported small patient groups, the prevalence of rearranged IGH genes
was estimated at 10% to 15% of lymphoblastic T-cell
malignancies.11 All early studies were exclusively based on
Southern blotting (SB), and at that time, the configuration of
IGH diversity (D) regions was not completely known. Therefore,
it was not possible to reliably discern between incomplete
DH-JH and complete
VH-(DH-)JH gene rearrangements and to
identify cross-lineage IGH gene rearrangement patterns, which
are characteristic for T-ALL.11,12 The only consistent
finding was that a significant proportion of IGH rearrangements in human leukemic T cells involved the DH7-27 (DQ52) gene
segment, which is located immediately upstream of the JH
region (Fig 1).13,14
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| Fig 1.
Schematic representation of the human IGH locus
on chromosome 14q32.3. The IGH gene complex consists of
numerous (>120) V gene segments, 27 D gene segments, 6 functional J
gene segments, and C gene segments for the constant domains of the
various IgH classes and subclasses, most of which are preceded by
switch sequences (s).23,28,52,53 Pseudogenes are
represented as open bars. The 27 DH gene segments are grouped
in 7 families based on sequence homology. Members of the same
DH family are depicted with the same shading pattern.
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To study cross-lineage IGH gene rearrangements in T-ALL in
detail, we analyzed a large group of 118 T-ALL patients by Southern blotting to determine the precise incidence of rearranged IGH genes. Heteroduplex polymerase chain reaction (PCR) analysis and subsequent sequencing were applied to identify the rearranged IGH gene segments as well as the junctional regions of the
rearrangements. In addition to VH family-specific primers and
a JH consensus primer, we designed a new set of DH
family-specific primers to detect and identify all complete
VH-JH and incomplete DH-JH gene rearrangements.
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MATERIALS AND METHODS |
Cell samples.
Peripheral blood (PB) or bone marrow (BM) samples from 118 T-ALL
patients (84 children and 34 adults) were obtained at initial diagnosis.7,15 Mononuclear cells (MNC) were isolated from PB or BM samples by Ficoll-Paque centrifugation (density, 1.077 g/mL;
Pharmacia, Uppsala, Sweden) and subjected to detailed immunophenotyping according to standard protocols.16,17 To analyze sufficient numbers of CD3+ T-ALL (especially TCR+
T-ALL), we selected T-ALL cell samples based on their CD3/TCR immunophenotype, resulting in 69 CD3 T-ALL (58% of
the total series), 25 TCR + T-ALL (21%), and 24 TCR+ T-ALL (20%). In an entirely random series of
T-ALL, this immunophenotype distribution would approximately be 70%,
20%, and 10%, respectively.18 Although in 2 CD3+ T-ALL cases no information about TCR protein
expression was available, they were included into the
TCR+ T-ALL group based on the Southern blot finding
of biallelic TCRD rearrangements in both cases.
Southern blot analysis.
DNA was isolated from fresh or frozen MNC fractions as described
previously.2 Fifteen micrograms of DNA was digested with the appropriate restriction enzymes (Pharmacia), size-separated in
0.7% agarose gels, and transferred to Nytran-13N nylon membranes (Schleicher and Schuell, Dassel, Germany), as
described.2,19 IGH gene configuration was analyzed
in the 118 T-ALL patients with the IGHJ6 probe (DAKO Corp,
Carpinteria, CA) in Bgl II, BamHI/HindIII, EcoRI, and/or HindIII digests.20 The
configuration of the TCRD genes was analyzed in 101 of 118 patients with the TCRDV2, TCRDD2, TCRDJ1, TCRDJ2, TCRDJ3, TCRDRE, and
TCRAPJ probes (DAKO Corp) in Bgl II, EcoRI, and
HindIII digests.15
Primer design and heteroduplex PCR analysis.
PCR was essentially performed as described previously.7,19
In each 50 µL PCR reaction, 50 ng DNA sample, 6.3 pmol of the 5' and 3' oligonucleotide primers, and 0.5 U AmpliTaq Gold
polymerase (PE Biosystems, Foster City, CA) were used. The sequences of
the oligonucleotides used for amplification of complete
VH-JH gene rearrangements (6 IGH framework-1
VH-family specific primers, and 1 JH consensus
primer) were published before.21,22 Based on recently
published data of germline DNA D-region sequences of the human IGH
locus23 (EMBL accession no. X97051; for the detailed
organization of the IGH D-region, see Fig 1), 7 family-specific DH primers were designed using OLIGO 6.0 software (Dr W. Rychlik, Molecular Biology Insights, Inc, Plymouth, MN) applying
previously described guidelines (Table
1).24 Oligonucleotide primers of 22 to 24 bp were
positioned at least 50 bp upstream of the involved recombination signal
sequence (RSS). Secondary structures such as primer dimers and hairpins
were avoided, and the melting temperature (Tm) was 68°C ± 3°C. All primers were synthesized on an ABI 392 DNA synthesizer (PE
Biosystems) using the solid-phase phosphotriester method.
PCR conditions were initial denaturation for 3 minutes at 92°C,
followed by 35 cycles of 45 seconds at 92°C, 90 seconds at 60°C, and 2 minutes at 72°C using a Perkin-Elmer 480 thermal
cycler (PE Biosystems). After the last cycle, an additional extension step of 10 minutes at 72°C was performed. Appropriate positive and
negative controls were included in all experiments.24
Heteroduplex analysis of PCR products included denaturation at 94°C
for 5 minutes after the final cycle of amplification and subsequent
renaturation at 4°C for 60 minutes to induce duplex formation.25 Afterwards, the duplexes were immediately
loaded on 6% nondenaturing polyacrylamide gels in 0.5×
Tris-boric acid-EDTA (TBE) buffer, run at room temperature, and
visualized by ethidium bromide staining.25 A 100-bp DNA
ladder (Promega Corp, Madison, WI) was used as size marker.
Sequence analysis of IGH gene rearrangements.
PCR products found to be clonal by heteroduplex analysis were directly
sequenced except for cases in which heteroduplex PCR analysis showed
more than two clonal bands, ie, either two homoduplexes, or an
additional upper band resulting from extension to downstream JH segments, or a DH7-27-JH1 germline band
accompanying a DH7-27-JH rearrangement. In such
cases, homoduplexes were excised from the polyacrylamide gel and eluted
as described before.26,27 The eluted PCR products were
either directly sequenced or subjected to second-step PCR with the same
primer pair to increase the amount of template for sequence analysis.
Sequencing was performed using the dye-terminator cycle sequencing kit
with AmpliTaq DNA polymerase FS on an ABI 377 sequencer (PE
Biosystems). Briefly, 50 to 200 ng of PCR product and 3.2 pmol primer
were used in a 15 µL reaction volume. The cycling protocol was
96°C for 30 seconds, followed by 60°C for 4 minutes for a total
of 25 cycles. Each PCR product was sequenced in two directions.
Interpretation of sequence data.
VH, DH, and JH segments were identified
using DNAPLOT software (W. Müller, H-H. Althaus, University of
Cologne, Cologne, Germany) by searching for homology with all known
human germline VH, DH, and JH sequences
obtained from the VBASE directory of human Ig genes
(http://www.mrc-cpe.cam.ac.uk/imt-doc/).28 For alignments
of D segments in VH-DH-JH or
DH-DH-JH rearrangements, it was necessary to
have at least 10 consecutive matching nucleotides.23 Palindromic (P-region) nucleotides (maximally 2) generated during the
joining process were recognized as being palindromic to the juxtaposed
nucleotides of an untrimmed rearranged gene segment.29 Extensive N-regions (nucleotides that cannot be assigned to V, D, J
gene segments, or P-regions) were analyzed in more detail by comparing
them to the most recent update of GenBank using the BLAST sequence
similarity-searching tool (National Center for Biotechnology
Information; http://www.ncbi.nlm.nih.gov/BLAST/).30
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RESULTS |
Southern blot analysis of IGH gene rearrangements in T-ALL.
IGH gene rearrangements were found in 22% (26/118) of T-ALL
patients and were equally distributed between the different age groups,
ie, 23% (19/84) of children and 21% (7/34) of adults. In the majority
of cases, this concerned monoallelic rearrangements (69% [18/26]),
and in 31% (8/26) biallelic rearrangements were observed. In 2 of
these patients, SB analysis showed weak bands, most probably derived
from subclones. Cross-lineage IGH gene rearrangements were
found in 19% (13/69) of CD3 T-ALL and in 50% of
TCR+ T-ALL (12/24), whereas only a single
TCR+ T-ALL (4% [1/25]) displayed a rearranged
IGH gene on one allele.
IGH gene rearrangements coincide with TCRD gene rearrangements.
Because CD3 T-ALL theoretically represent precursor
stages of both TCR+ and TCR+ T-ALL,
we analyzed the configuration of the TCRD genes in 57 CD3 T-ALL cases and used it as an additional marker
for further subdivision of this group.15,18 The
TCRD gene configuration of each allele can potentially pass
three consecutive stages: germline, rearrangement, and
deletion.15,18 Except for 1 case, all IGH gene
rearrangements were found in patients with at least one TCRD
gene rearrangement; in this single CD3 T-ALL, a
monoallelic IGH gene rearrangement was observed in combination with deletion of both TCRD alleles.
Based on the above-described results, we analyzed the association of
cross-lineage IGH gene rearrangements and TCRD gene
configuration for 101 of the 118 T-ALLs. Rearrangements in the IGH
locus appeared to be almost exclusively associated with TCRD
gene rearrangements. They were evenly distributed between cases
with 1 rearranged and 1 germline TCRD allele (1 of 3 cases
[33%]), cases with biallelic TCRD rearrangements (15 of 47 cases [32%]), and cases with 1 rearranged and 1 deleted TCRD
allele (7 of 22 cases [32%]). Remarkably, only 1 of 26 cases
with biallelically deleted TCRD genes (4%) displayed a
cross-lineage IGH gene rearrangement.
Taking these data together, cross-lineage IGH gene
rearrangements occurred in only 5% (2/38) of -lineage T-ALL, ie,
either TCR+ T-ALL (n = 25) or CD3
T-ALL with biallelic TCRD deletion (n = 13).
Complete VH-(DH-)JH and incomplete
DH-JH rearrangements.
Detailed PCR analysis of the IGH locus in the 26 patients with
Southern blot documented IGH gene rearrangements was based on
13 primer combinations covering the vast majority of complete VH-(DH-)JH joinings and potentially all
incomplete DH-JH rearrangements. Heteroduplex PCR
analysis showed a total of 39 clonal homoduplexes, reflecting 7 (18%)
complete VH-(DH)-JH joinings and 32 (82%)
incomplete DH-JH rearrangements. Complete
VH-(DH)-JH rearrangements were found in 4 CD3 T-ALL and 2 TCR+ T-ALL
patients, with 1 TCR+ T-ALL showing biallelic
VH-(DH-)JH joinings. Heteroduplex PCR analysis for incomplete DH7-27-JH rearrangements is
shown in Fig 2.
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| Fig 2.
Heteroduplex PCR analysis of DH7-27-JH
cross-lineage rearrangements in T-ALL. Subsequent to agarose gel
electrophoresis, samples containing PCR products were subjected to
heteroduplex PCR analysis, separated in a 6% polyacrylamide gel, and
visualized by ethidium bromide staining. Based on the size of clonal
PCR products, DH7-27-JH rearrangements (~250 bp)
were identified in T-ALL patients T009, T038, T042, T062, T150, and
T184 as well as in a T-cell prolymphocytic leukemia (T-PLL). In
addition to homoduplexes resulting from DH7-27-JH
rearrangement, the germline DH7-27-JH1 and
DH7-27-JH2 homoduplexes were consistently present,
except for cases with biallelic IGH rearrangements and a very
high tumor load (ie, patient T150). To obtain a clonal sequence of
DH7-27-JH rearrangements, homoduplexes of the
correct size were excised from the polyacrylamide gel, eluted, and
sequenced. ss, single-strand DNA.
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Sequence analysis confirmed monoclonality in 7 complete
VH-(DH-)JH rearrangements, which involved 7 different gene segments from 4 families: VH1-3,
VH1-69, VH3-13, VH3-23, VH3-33,
VH4-4, and VH6-1. None of the rearrangements was
potentially functional. Six sequences were out-of-frame joinings,
whereas the single rearrangement with an in-frame
VH-JH contained a stop codon in the junctional region.
DH6-19 and DH7-27 are preferentially used in IGH
gene rearrangements in T-ALL.
Sequence analysis of the junctional regions of complete
VH-(DH-)JH recombinations allowed
identification of a D segment in 5 of 7 joinings. Moreover, 1 incomplete rearrangement was of the DH-DH-JH
type. The frequencies of different DH family members found
among the 38 identified DH sequences in the complete and incomplete cross-lineage IGH gene rearrangements are summarized in Table 2. Usage of the DH6
family was most prominent (47%), with the DH6-19 gene segment
being preferentially used (45% of all identified DH
sequences; Table 3). The second most
frequently used DH segment was DH7-27 (21%). Seven
rearrangements (18%) contained DH segments of the
DH1 family, whereas 5 other rearrangements used various
segments of the remaining 4 DH families (Table 2). Taken
together, only 5 of the 38 rearranged DH segments belonged to
the most upstream part of the DH region, whereas all other 33 DH gene segments (87%) belonged to the most downstream part of the DH region (Table 2). No relationship between age and
DH gene segment usage was observed.
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Table 3.
Junctional Region Sequences of Oligoclonal IGH
Gene Rearrangements in a T-ALL Patient (T061) Illustrating the
Ongoing Recombination Process
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The sizes of the DH-JH junctional regions ranged from
0 to maximally 32 nucleotides, with an average of 7.6 nucleotides.
Three of 37 DH-JH junctions (8%) did not have any
randomly inserted N-region nucleotides. P-nucleotides, indicating the
absence of deletion, were present in 7 DH-JH joinings
(19%).
Usage of JH gene segments in cross-lineage IGH gene
rearrangements.
The frequencies of different JH gene segments in IGH
gene rearrangements in T-ALL are summarized in
Table 4. The JH4 gene segment was
found most frequently in approximately one third of joinings, followed
by JH6 in 20% of cases. The remaining 4 JH segments
were almost equally used, each comprising 10% to 15% of the
rearrangements.
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Table 4.
Usage of Different JH Gene Segments in
Incomplete and Complete Cross-Lineage IGH Gene Rearrangements
in T-ALL as Compared With Precursor-B-ALL, Human BM Precursor-B
Cells, and PB B Lymphocytes
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Oligoclonality in cross-lineage IGH gene rearrangements in T-ALL.
SB analysis and heteroduplex PCR analysis showed fully concordant
results in 20 of 26 cases with cross-lineage IGH gene
rearrangements in T-ALL. In 1 case with a single rearranged band on SB,
we were not able to amplify the clonal rearrangement with the applied primer sets. In the remaining 5 cases, the number of clonal
PCR-detected homoduplexes was higher than the number of rearranged
bands in SB, which may suggest the presence of minor subclones
undetectable by SB. In 1 of these seemingly oligoclonal cases, the
identified incomplete and complete rearrangements (1 and 2 in Table 3)
shared the same DH-JH fragment, suggesting ongoing
VH to DH-JH joining. In 4 other patients,
the detected rearrangements were not related in their used gene
segments. This may reflect secondary DH-JH joining,
with concomitant deletion of a pre-existing DH-JH rearrangement.
Based on the combined SB/PCR results, we found evidence for IGH
oligoclonality at diagnosis in 27% (7/26) of T-ALL patients with this
type of cross-lineage recombination. This includes the above-mentioned
5 cases with the higher number of PCR-detected homoduplexes than the
number of rearranged bands in SB, and 2 additional PCR-positive cases
with weak bands on SB analysis, apparently derived from
subclones.20
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DISCUSSION |
We investigated a large group of 118 T-ALL patients for the presence of
cross-lineage IGH gene rearrangements. Based on SB analysis, we
identified such rearrangements in 22% of T-ALL, which is slightly
higher than previously reported.11,12 The vast majority
(82%) of IGH gene rearrangements in T-ALL concerned incomplete DH-JH joinings. However, complete
VH-(DH-)JH recombinations were also
documented in our group of T-ALL patients. The usage of VH gene segments was seemingly random and not limited to the ones most
proximal to the JH cluster. Nevertheless, 6 of 7 involved VH gene segments were derived from the proximal 3'
portion of the IGH locus, a pattern that is seen in first
trimester fetal VH-(DH)-JH
rearrangements.31 None of the seven
VH-(DH-)JH rearrangements was potentially
functional. To our knowledge, this is the first extensive evidence for
the occurrence of clonal complete IGH gene rearrangements in
T-ALL. They were previously reported in a single case of
T-lymphoblastic non-Hodgkin's lymphoma, which is the lymphomatous counterpart of T-ALL.32
In both the VH-(DH-)JH and
DH-JH rearrangements, we found a strikingly
preferential usage of the more downstream DH gene segments (87% of identified DH sequences), especially DH6-19
(45%) and DH7-27 (21%). The DH6-19 gene segment is
one of the gene segments that were recently discovered thanks to
complete sequencing of the DH region.23
Retrospective analysis of nearly 900 IGH junctional regions of
B-lineage cells showed DH6-19 involvement in approximately 5%
of the rearrangements, which is significantly higher than would be
expected on a random basis.23 The same holds true for 8 other DH segments, but 6 of them were not found in our T-ALL
patients. The preferential usage of DH6-19 could not be
explained by a more optimal RSS at the 3' end in comparison to
other DH segments, either. Further studies are needed to
define whether this gene segment is also preferentially rearranged in
precursor-B cells or whether this finding only relates to the
cross-lineage phenomenon in T-ALL. In contrast to DH6-19,
earlier reports indicated that the DH7-27 gene segment is
involved in a significant proportion of IGH gene rearrangements
in T-ALL.13,14 Interestingly, we also found this gene
segment in a cross-lineage IGH gene rearrangement of a
TCR+ T-prolymphocytic leukemia (Fig 2). The
DH7-27 gene segment is also preferentially used by fetal B
cells but is rarely observed in adult BM and PB.33-36 In
the analysis of nearly 900 IGH junctional regions of B-lineage
cells, DH7-27 was found in only 0.5% of the rearrangements.23 Because DH7-27 consists of only
11 nucleotides, it is more difficult to identify this gene segment in a
junctional region after moderate trimming during V(D)J recombination,
if stringent assignment criteria are used.
The analysis of JH segment usage showed a more frequent use of
JH4 and JH6 gene segments, which is also the case in
normal and leukemic B-lineage cells (Table 4).35-37
However, in our T-ALL group, a significant proportion of joinings
involved the most upstream JH1 and JH2 gene segments
(28%), which are rarely used by B lymphocytes (~1%) and B-cell
precursors (3% to 4%).35-37 In conclusion, the high
frequency of incomplete DH-JH rearrangements together with the frequent usage of the more downstream DH
gene segments and the most upstream JH1 and JH2 gene
segments suggest a predominance of the most immature IGH
rearrangements in T-ALL. This particular DH-JH
rearrangement pattern appears to be nonrandom and is not comparable
with any known stage of B-cell ontogeny or B-cell
differentiation.31
Because the types of preferential IGH gene rearrangements have
now been identified, it would be relatively easy to screen T-ALL
patients for the presence of cross-lineage IGH gene
rearrangements and apply them as PCR targets for monitoring of minimal
residual disease (MRD) in T-ALL. Three primer combinations
(DH1, DH6, and DH7 in combination with a
JH consensus primer) can identify 85% of incomplete
DH-JH rearrangements in T-ALL. However, we observed oligoclonality in the IGH locus in 27% of T-ALL patients with rearranged IGH genes. In 1 case, we found evidence for
continuing VH to DH-JH recombination,
whereas VH replacements have been described previously during
disease progression of a T-lymphoblastic lymphoma.32 Secondary rearrangements via continuing VH to
DH-JH joining, VH gene replacements, and de
novo IGH gene rearrangements have been reported for
precursor-B-ALL.38-40 These processes may lead to the
emergence of clones with secondary IGH rearrangements. In a
previous study, we compared the IGH gene rearrangement patterns between diagnosis and relapse in 40 ALL patients and found that at
least one major IGH rearrangement was stable in most
cases.41 Therefore, cross-lineage IGH gene
rearrangements might be useful as supplementary MRD target in addition
to leukemia-specific TCRG and TCRD gene rearrangements
and TAL1 gene deletions.24,42
Cross-lineage IGH gene rearrangements occurred most frequently
in TCR+ T-ALL (50% of patients) and in 20% of
CD3 T-ALL, but we found them in a single case of
TCR+ T-ALL (4%). Moreover, there was a striking
association between the presence of rearranged TCRD genes and
the occurrence of IGH recombination. A similar association was
previously found for cross-lineage gene rearrangements in acute myeloid
leukemia, suggesting that TCRD and IGH
genes are concomitantly accessible for V(D)J recombinase in early
hematopoietic precursors.43 Furthermore, in the
genotypically most mature T-ALL subgroup with biallelic TCRD
gene deletions, an IGH gene rearrangement was observed in only
1 of 26 cases. Because IGH gene rearrangements are also rare in
mature T-cell malignancies (~5%),11 this suggests that
the IGH locus may be accessible to V(D)J recombinase activity
only in cells at earlier stages of T-cell differentiation. In this context, TCR+ T-ALL should be regarded as cells that
branched off T-cell development at an early stage of completion of
TCRD and TCRG gene rearrangement processes, when the
recombinase activity is still retained.18
Incomplete DH-JH rearrangements are one of the
earliest events during normal B-cell development and are already found
in CD34+/CD19/CD10+
precursor cells.44,45 Most of the more mature
CD34+/CD19+/CD10+ B-lineage
precursors contain at least 1 DH-JH rearranged allele and frequently also complete VH-(DH-)JH
rearrangements.45,46 One could therefore speculate that
cross-lineage IGH gene rearrangements in T-ALL might reflect
malignant transformation of a thymocyte derived from a
CD34+/CD19/CD10+ precursor
cell with rearranged IGH genes. This idea may be supported by a
murine model, in which IGH rearrangements were only found at an
intermediate stage of thymocyte development.47
Nevertheless, this phenomenon was not observed in normal human
thymocytes.44 Furthermore, the absence of D-J-Cµ
transcripts in fetal human thymocytes depleted of CD34+-
and/or CD19+-bearing cells suggest that
DH-JH rearrangements in humans may be restricted to
normal B-lineage differentiation.44,48 It has also been
suggested that IGH gene rearrangements in T-cell precursors may
be an aberrant event directing cells into apoptotic pathway, unless
they become immortalized by malignant transformation.3 An
alternative explanation could be that cross-lineage IGH gene rearrangements in T-ALL are postoncogenic events resulting from the
ongoing activity of the common B- and T-cell V(D)J recombinase system
on accessible gene loci.3,49,50 We favor the last hypothesis, because this would explain the virtual absence of IGH gene rearrangements in normal thymocytes and mature T-cell malignancies on the one hand and the presence of IGH
oligoclonality and secondary
VH-(DH-)JH rearrangements in T-ALL
on the other hand.41 The virtual absence of IGH
gene rearrangements in -lineage T-ALL with biallelic TCRD
gene deletions suggests that the recombination system is not active in
this more mature type of T-ALL. Apparently, the recombinational
activity is switched off as soon as the rearrangement and deletion
processes in the TCRA/TCRD locus are completed.
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ACKNOWLEDGMENT |
The authors thank Drs K. Hählen, I.M. Appel, R.M. Egeler,
F.G.A.J. Hakvoort-Cammel, W.J.D. Hofhuis, G.E. van Zanen, B. Löwenberg, P. Sonneveld, G. J. Ossenkoppele, G.J. Schuurhuis, G. Verhoef, Ph. Vandekerckhove, M. Stul, E.J. Petersen, A.W. Dekker, D. Campana, J.C. Kluin-Nelemans, W-D. Ludwig, and C.E. van der Schoot and the Dutch Childhood Leukemia Study Group (DCLSG) for kindly providing T-ALL cell samples. Board members of the DCLSG are I.M. Appel, H. van
den Berg, J.P.M. Bökkerink, M.C.A. Bruin, J.J. Groot-Loonen, S.S.N. de Graaf, K. Hählen, P.M. Hoogerbrugge, W.A. Kamps, F.A.E. Nabben, J.A. Rammeloo, T. Revesz, A.Y.N. Schouten-van Meeteren, A.J.P.
Veerman, M. van Weel-Sipman, and R.S. Weening. We are grateful to Prof
Dr R. Benner, Prof Dr D. So ta-Jakimczyk, and Dr H Hooijkaas for
their continuous support and to Dr E. Moreau and I.L.M. Wolvers-Tettero for technical assistance, T.M. van Os for preparation of the figures, and A.D. Korpershoek for secretarial support.
| |
FOOTNOTES |
Submitted October 29, 1998; accepted March 28, 1999.
Supported by the Dutch Cancer Foundation (Nederlandse
Kankerbestrijding/ Koningin Wilhelmina Fonds), Grant No. EUR 94-852.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Jacques J.M. van Dongen, MD, PhD,
Department of Immunology, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands; e-mail:
vandongen{at}immu.fgg.eur.nl.
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ABSTRACT
INTRODUCTION