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Blood First Edition
Paper, prepublished
online
March
9, 2004;
DOI 10.1182/blood-2003-11-3857
Utilization of Ig Heavy-Chain Variable, Diversity and Joining Gene
Segments in Children with B-lineage Acute Lymphoblastic Leukemia:
Implications for the Mechanisms of VDJ Recombination and for
Pathogenesis
Aihong Li*, Montse Rue^, Jianbiao Zhou*, Hongjun Wang*, Meredith A Goldwasser^,
Donna Neuberg^, Virginia Dalton+, David Zuckerman*, Cheryl Lyons+, Lewis B
Silverman+, Stephen E Sallan+ and John G Gribben*
From the Division of Medical Oncology*, Biostatistical Science^, and Pediatric
Oncology+, Dana-Farber Cancer Institute, Department of Medicine, Brigham and
Women’s Hospital* and Division Hematology/Oncology, Department of Medicine, The
Children's Hospital+, Harvard Medical School, Boston, MA, for the Dana-Farber ALL
Consortium
Short title:
VHDHJH gene usage in childhood ALL
Key word:
IgH, VHDHJH gene segments, B-cell, childhood acute lymphoblastic
leukemia.
Scientific heading: Immunobiology
Supported by NIH grant CA68484
Copyright (c) 2004 American Society of Hematology
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Corresponding author: John G Gribben, MD, DSc, Department of Medical Oncology,
Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Phone: 617 632
3033; Fax: 617 632 3222; e-mail: [email protected]
Word count:
Abstract: 182
Total text: 3780
2
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ABSTRACT
Sequence analysis of the immunoglobulin heavy chain genes (IgH) has demonstrated
preferential usage of specific variable (V), diversity (D) and joining (J) genes at different
stages of B cell development and in B cell malignancies, and this has provided insight
into B cell maturation and selection. Knowledge of the association between
rearrangement patterns based on updated databases and clinical characteristics of
pediatric acute lymphoblastic leukemia (ALL) is limited. We analyzed 381 IgH
sequences identified at presentation in 317 children with B-lineage ALL and assessed the
VHDHJH gene utilization profiles. The DHJH-proximal VH segments and the DH2 gene
family were significantly over represented. Only 21% of VH-JH joinings were potentially
productive, a finding associated with a trend towards an increased risk of relapse. These
results suggest that physical location at the VH locus is involved in preferential usage of
DHJH-proximal VH segments whereas DH and JH segments usage is governed by positionindependent molecular mechanisms. Molecular pathophysiology appears relevant to
clinical outcome in patients who have only productive rearrangements and specific
rearrangement patterns are associated with differences in the tumor biology of childhood
ALL.
Corresponding author’s e-mail: [email protected]
3
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INTRODUCTION
Immunoglobulin (Ig) genes are assembled from germline variable (V), diversity (D) and
joining (J) gene segments during early B-cell differentiation by a site-directed DNA
rearrangement mechanism known as VDJ recombination.1 Further recombination at the
heavy chain (H) locus is prevented by a productive VHDHJH rearrangement which also
triggers rearrangements at the light (L) chain loci, most often
followed by .2,3 During
these joining steps, antibody diversity is generated by the following mechanisms: (1)
somatic recombination of multiple VH, DH, JH segments; (2) nucleotide deletions of 3’end VH segment; (3) non-germline-encoded (N) nucleotide insertions by terminal
deoxynucleotidyl transferase (TdT); (4) germline-encoded (P, palindromic) nucleotide
additions; (5) transcription of D regions in any of three potential open reading frames; (6)
fusion and inversion of D regions; and (7) somatic mutation.4-8
A complete map of the human Ig VHDHJH loci has now been constructed on chromosome
14q32.2 in a telomeric-to-centromeric direction.9,10 The VH region contains 123 VH
segments, of which 79 are pseudogenes and 44 have an open reading frame.10 The VH
genes are grouped into seven VH families based on their high sequence homology and not
by their location on chromosome 14q32. VH3 is the largest family followed by VH4 and
VH1.9,10 VH6-1 is the most proximal to the DHJH loci.
9,10
The DH region contains 27 DH
segments, of which 25 have been shown to be involved in creation of human antibody.11
Seven DH families are classified based on sequence homology. DH7-27 is closest to the JH
locus.
11
Six JH segments are functional.12,13 The rearranged heavy chain consists of the
4
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three high variable regions, complementarity determining regions (CDR1, CDR2, and
CDR3), flanked by less variable framework regions (FR1, 2, 3 and 4).4 The CDRs,
especially CDR3, are considered as the core portion responsible for antigen recognition.4
The CDR3 sequence is unique to each rearrangement and therefore identifies individual
B cells or clonal B-cell expansion.4,14,15
The VHDHJH gene repertoires are restricted and developmentally regulated at early stages
of differentiation.3,16 In the most immature B-cell precursors (pro-B cells), the IgH genes
remain germline or there is only DH-JH joining.2,17 At the next stage, in early pre-B cells
VH genes join to the joined DH-JH to complete the IgH rearrangement.2,18 Numerous
studies in both murine and human, have shown stage-specific trends in usage of V, D and
J genes, the degree of N nucleotide addition, and the rate of somatic mutation.8,16-20 In the
murine system, a biased use of JH-proximal VH segments and a high frequency of absence
of N sequences at DJH joining were demonstrated at fetal stages of development.19-21 In
humans, a similar trend was found by a marked overrepresentation of some VH (VH3,
VH5 and VH6), DH (DH7-27) and JH (JH3 and JH4) segments and by a short CDR3 length
in fetal liver B cells and in immature B cells. 16-18
Previous studies have demonstrated a preferential usage of specific VH genes in B-cell
malignancies.15,22,23 In mantle cell lymphoma, VH3-21, VH3-23, VH4-34, VH4-59 and
VH5-51 segments have shown to be most widely used. 24,25 In B-cell chronic lymphocytic
leukemia (CLL), patients with unmutated compared to somatically mutated VH genes
have a worse prognosis.26,27 A biased utilization of VH1-69 combined with selected DH
5
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gene segments and JH6 has been found in unmutated cases,28 and patients with mutated Ig
VH3-21 genes had significantly shorter survival than other mutated patients.29 In Blineage acute lymphoblastic leukemia (B-ALL), a privileged usage of VH6 gene has been
shown in both adult and childhood patients.30-32 Sequence analysis of the CDR region
demonstrated that DH6 (DN1), JH4, and JH6 appeared to be over-represented compared
with the expected frequency of use according to the size of each DH or JH gene
family.14,33 Several studies have suggested that childhood B-ALL is derived from early
fetal life or immature B cells.31-33 In one study, in frame and out of frame CDR3 joinings
were observed in one-third and two thirds of the rearrangements in pre-B ALL similar to
the frequency of occurrence in non-malignant early pre-B cells,33 whereas another study
found that in frame CDR3 rearrangement occurred in 78% in children and 64% in adults,
similar to that observed in healthy B-cells (75%).34
Most previous studies of V gene usage in B-ALL have been performed using databases
with limitations for the newly identified germline genes and the completed gene map and
in particular have focused on gene family usage rather than location on the chromosome.
In particular, awareness of connections among the IgH rearrangement patterns and
clinical characteristics has been relatively limited in ALL compared to the more recent
studies in CLL. In order to derive clone specific oligonucleotides from IgH
rearrangements for minimal residual disease (MRD) detection, we prospectively
sequenced VHDHJH regions at presentation in 317 children with B cell lineage ALL
treated sequentially in a single protocol. The aims of this study were to describe VHDHJH
rearrangement profiles in children with B-lineage ALL; to evaluate biological and
6
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structural features in clonal expanded B cells by comparison with previous findings of the
human Ig VH repertoire9,10 and to investigate the association between rearrangement
patterns and clinical characteristics.
MATERIAL AND METHODS
Patients and samples
Bone marrow (BM) and/or peripheral blood (PB) samples were obtained at presentation
and IgH rearrangements sequenced in 317 children with B-lineage ALL enrolled
consecutively in DFCI/ALL Consortium Protocol 95-01. Institutional Review Board
approval and informed consent were obtained for treatment and for procurement of the
samples in all cases.
DNA preparation
Mononuclear cells were isolated by Ficoll gradient centrifugation (Pharmacia, Uppsala,
Sweden), lysed and DNA extracted and purified according to the manufacturer’s
instructions using the NucleoSpin kit (BD Biosciences, Palo Alto, CA)
7
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PCR analysis of IgH gene rearrangements
To identify patient leukemia-related IgH gene rearrangements, diagnostic BM and/or PB
samples were PCR-amplified using a series of seven VH family FR1 consensus primers
and a JH consensus primer in a modification of a method previously described.35 Primers
were purchased from a commercial supplier (Invitrogen, Carlsbad, CA). The PCR
conditions and methods used for detection of PCR products have been previously
described. 36
Direct sequencing of PCR product
Clonal PCR products were excised and purified using QIAquick gel extraction kits
(QIAGEN, Valencia, CA). Purified PCR fragments were sequenced directly by the DanaFarber/Harvard Cancer Center Core Sequencing Facility (Boston, MA). Sequence
reactions were analyzed on an Applied Biosystem 3700 capillary sequencer using Big
Dye Terminator Chemistry version 2 (Applied Biosystems, Foster City, CA). The
relevant consensus forward and reverse primers were used as sequence primers to obtain
the sequence of both strands. Nucleotide sequences were aligned using the DNAstar
software (DNASTAR, Inc., Madison, WI).
Interpretation of sequence data
8
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VH, DH, and JH segments were identified with a closest matching known human germline
genes using the Immunogenetics Database (http://imgt.cines.fr, IMGT, European
Bioinformatics
Institute,
Montepellier,
France),
the
IGBlast
search
(http://www.ncbi.nlm.nih.gov/igblast/, National Center for Biotechnology Information,
Bethesda,
MD)
or
V
BASE
directory
using
DNAPLOT
(http://www.mrc-
cpe.cam.ac.uk/DNAPLOT, Center for Protein Engineering, Cambridge, UK). The
following criteria were used for DH gene determination: a minimal homology of six
matches in a row or seven matches interrupted by one mismatch. The CDR3 length was
calculated according to previously described criteria.6
Statistical analysis
Descriptive statistics (percentages, medians and ranges) were used to describe VDJ gene
utilization profiles. The Chi-square test was used to compare two categorical variables
and for two by two tables we used the Fisher’s exact test.37 The Wilcoxon rank-sum and
the Kruskal-Wallis tests were used to compare a continuous variable with a categorical
variable with two or more categories, respectively.38 The exact binomial distribution was
used to assess differences between a specific observed percentage and an expected
percentage. The Kaplan-Meier method and the log-rank test were used to estimate and
compare time to relapse according to productivity of the rearrangements.39,40 All tests
were two-sided except the exact binomial, which was one-sided.
9
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RESULTS
381 IgH sequences were identified at presentation from 317 children with B-lineage ALL
enrolled in DFCI/ALL Consortium protocol 95-01. A high identity (>98%) to the human
germline gene segments was found in 375 (98.4%) of the 381 sequences. We identified
only six sequences (1.5%) with identity in a range of 86-96%. Of note four of these cases
used V3-11, raising the possibility of polymorphisms at this locus.
VH gene usage.
The frequency of the usage of the specific VH segments is shown by their position on
chromosome 14 in a telomeric-to-centromeric direction in Figure 1.
10
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Figure 1. VH gene segment usage profile by physical location on chromosome 14 in 381
rearrangements from 317 children with B-lineage ALL showing a privileged use of the
DHJH-proximal VH segments. (p) pseudogenes
When this region was divided into four clusters each of approximately 200kb, we
observed a privileged use of the DHJH-proximal VH segments in cluster D, with 47%
versus expected 25% use (p<0.001 using the Binomial test). Fifty-two germline VH
segments were used by the 381 IgH sequences. VH6-1 (35, 9.19%), VH3-13 (32, 8.4%)
and VH4-34 (22, 5.77%) were the three most overused VH segments in pediatric B-ALL.
Of the 52 VH segments used, 9 were pseudogenes used by 24 sequences. In the DHJHproximal VH segments, VH6-1 segment was used in 35 of the 177 sequences, followed by
VH3-13 (n=32), VH3-11 (n=19), VH1-2 (n=18), VH2-5 (n=16), VH1-3 (n=12), VH3-9
(n=12) and VH3-15 (n=10) segment. The rest of these VH segments (VH1-14, VH5-a, VH210, VH1-8, VH2-5 VH3-7 and VH4-4) in this region were used by less than 10 sequences.
Of the 381 IgH sequences, VH3 family gene segments were identified in 192 clones
(50%), followed by VH1 (62, 16%), VH4 (60, 16%), VH6 (35, 9%), VH2 (22, 6%) and
VH5 (10, 3%). VH3 is the largest family in children with B-lineage ALL, with its usage
occurring at frequency commensurate with its germline family size.10 We observed a
privileged usage of VH6 (9%) in children with B-lineage ALL compared to PBL (0.8%,
p=0.006 using the Chi-square test). Although murine data suggest that this gene segment
is overused also in normal B cells in early life,30 there are no published human studies to
11
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assess whetherVH6 use in ALL is different from what would be seen in fetal and
childhood B cells.
DH gene usage.
Of the 381 IgH sequences, DH segments could be identified in 304 sequences. For 77
(20%) of the 381 sequences insufficient D segment sequence was available for definitive
identification of the DH gene segment used, either because of aberrant VDJ
recombinations or from exonuclease activity. A single DH gene was identified in 301
sequences and the unusual DH-DH joining sequences were found in only three sequences.
For these three sequences using more than one DH genes, the JH-proximal DH segment
was considered for gene usage analysis to assess DJ recombination. The DH2 gene family
was used most frequently at 35% (105), followed by DH3 (32%, 97), DH6 (12%, 37), DH1
(8%, 23), DH5 (5%, 15), DH7 (4%, 12) and DH4 (4%, 12), respectively. The DH2 and DH7
genes were over-represented whereas DH4 and DH5 gene segments were underrepresented in B-lineage ALL CDR3 regions compared to usage in PBL12 (p<0.001 using
Chi-square test) as shown in Figure 2.
12
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Figure 2. DH gene family usage in 381 rearrangements in ALL compared to published
results from peripheral B lymphocytes.12
Similar to the VH gene families, assignment to DH gene families is based upon sequence
homology and not their chromosomal loci. The telomeric-to-centromeric position of these
genes is shown in Figure 3 with the percentage of utilization of each gene. Four clusters
were assigned, each of approximately 15kb. A privileged usage of JH-distal DH segments
was observed in cluster A (37%) and in cluster B (37%) versus expected 25% (p<0.001
using the exact binomial test). Of 27 human germline DH gene segments, 26 were used by
the B-ALL IgH sequences including two pseudogenes (DH1-14 and DH6-25). None of
the sequences used DH4-4 segment. Seven DH segments (DH2-2, DH3-3, DH2-8, DH3-9,
13
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DH3-10, DH2-15 and DH3-22) were over-represented in comparison to individual family
size as shown in Figure 3 (p<0.001 using the exact binomial distribution).
Figure 3. DH gene segment usage profile by physical location on chromosome 14 in 304
rearrangements from children with B-lineage ALL showing a privilege utilization of the
JH-distal DH segments. The dotted line indicates statistical significance of D gene
segment utilization compared to expected use by the exact binomial distribution
(p<0.001). (p) pseudogenes.
Joining gene usage
Usage of the JH4 gene family was 39% (148), followed by JH6 (34%, 131), JH5 (20%,
78), JH2 (3%, 11), JH3 (2%, 7) and JH1 (2%, 6), respectively. The JH gene usage in
14
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children with B-lineage ALL is significantly different from the JH usage found in normal
PBL12 where JH4 is the most prominent JH gene used in 52.5% versus JH6 in 22.2%
(p<0.001 using the Chi-square test, Figure 4).
Figure 4
60
50
percent
40
This study
PBL
30
20
10
0
JH1
JH2
JH3
JH4
JH5
JH6
Figure 4. JH gene family usage in 381 rearrangements in this study compared to published
results from peripheral B lymphocytes.12
CDR3 length
The CDR3 length was calculated in size of base pairs for 381 sequences showing a
median of 30.0bp. Excessively long CDR3 length (>100bp) was found in 4 sequences
and by Blast search aberrant VDJ recombination was identified, including incorporation
of DH intron sequences in one case. For patients with multiple IgH sequences the average
of the CDR3 lengths was considered. We observed a significant association between
CDR3 length and the VH or DH family utilization but not by their JH genes (Table 1). A
15
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short CDR3 was correlated with utilization of VH6 and DH7-27 segments (p<0.01 using
the Kruskal-Wallis test).
Table 1. Correlation of CDR3 length and VHDHJH utilization in childhood B-lineage ALL
Gene family
n
Mean
SD
62
22
192
60
10
35
29.7
31.9
36.8
32.1
33.4
22.3
15.9
12.0
37.7
16.3
15.2
11.9
CDR3 length
(bp)
Median
p
VH
VH1
VH2
VH3
VH4
VH5
VH6
26.5
31.5
30.0
31.5
33.5
21.0
0.006
DH
DH1
DH2
DH3
DH4
DH5
DH6
DH7
23
108
97
12
15
37
12
25.3
41.4
37.1
31.3
27.9
35.0
20.9
10.1
29.4
24.5
12.8
9.8
36.8
8.7
24.0
36.5
34.0
28.5
27.0
26.0
20.5
<0.0001
JH
JH1
JH2
JH3
JH4
JH5
JH6
6
11
7
148
78
131
48.0
33.5
31.1
29.9
32.0
37.1
21.4
19.4
16.6
15.3
28.4
40.3
44.0
34.0
33.0
27.0
30.0
31.0
0.16
CDR, complementarity-determining region.
bp, base pairs.
SD, standard deviation.
P values were obtained using the Kruskal-Wallis tests, two sided.
16
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Biclonality and oligoclonality
A single IgH sequence was identified in 256 cases from diagnostic tumor samples,
whereas two sequences were found in 58 cases and three sequences were found in three
children. However, it should be noted that the technical approach we use identifies only
major clonal rearrangements and does not detect small subclones. In this study we
observed no correlation between bi-or oligo-clonality and clinical features except
chromosome 9 deletions and in particular noted no association with relapse (Table 2).
17
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Table 2. Clonality and clinical characteristics of Children with B - lineage ALL
Overall
Patients with one
sequence
Patients with
more than one
sequence
317
256 (81%)
61 (19%)
4.3 (0.01-17.9)
4.3 (0.01-17.9)
4.1 (0.9-16.5)
167 (53%)
150 (47%)
134 (52%)
122 (48%)
33 (54%)
28 (46%)
n
Age (years)
Median (range)
p-value
0.56*
Sex
0.89
Male
Female
WBC (×109/L)
0-<20
20-<50
50<100
100
211
54
28
23
Relapse
No
Yes
275 (87%)
42 (13%)
225 (88%)
31 (12%)
50 (82%)
11 (18%)
Cytogenetic analysis
Hyperdiploid>50
<50
Diploid
Pseudo
Hypodiploid
Structure abnormal
11q23
t(1;19)
6q9p12p
Insufficient
ND
n
53
16
83
35
13
80
8
2
9
7
10
94
17
n
40
11
72
27
11
60
8
2
7
3
7
74
15
n
13
5
11
8
2
20
0
0
2
4
3
20
2
0.38
(67%)
(17%)
(9%)
(7%)
174
40
21
20
(68%)
(16%)
(8%)
(8%)
37
14
7
3
(60%)
(23%)
(11%)
(5%)
0.21
(%)
(17)
(5)
(26)
(11)
(4)
(25)
(3)
(1)
(3)
(2)
(3)
(30)
(5)
(%)
(16)
(4)
(28)
(11)
(4)
(23)
(3)
(1)
(3)
(1)
(3)
(29)
(6)
(%)
(21)
(8)
(18)
(13)
(3)
(33)
(0)
(0)
(3)
(7)
(5)
(33)
(3)
0.34
0.20
0.14
0.65
1.00
0.14
0.36
1.00
0.69
0.03
0.41
0.54
0.54
WBC, white blood cell count. ND, not done.
*p value was obtained using the Wilcoxon rank-sum tests for age. The Fisher’s exact
tests and the Chi-square test were used for categorical variables.
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In 52 (85%) of the 61 patients with more than one sequence at presentation, the IgH
sequences were unrelated. Only nine cases of the samples identified at presentation
involved ongoing IgH rearrangement mechanisms, phenomena described previously to
account for clonal evolution.41-44 Sequence analyses demonstrated two cases showing VHVH replacement (case 99 and 177), 6 cases showing VH to DHJH joining (case 190, 246,
266, 269, 392 and 400) and one case showing an “open-and-shut” mechanism (Table 3).
Although any of these sequences identified at presentation could be used for MRD
detection, no relapses have occurred in any of these nine children.
Table 3. Sequence analysis showing ongoing rearrangement in 9 children with Blineage ALL
Case
99
177
190
246
266
269
392
400
214
VHgene*
CDR3 (n-DH-n)
5’-JH
Molecular
mechanisms
VH1-69
VH3-52
gatcggcggTTGTACTAATGGTGTATGCT(DH2-8)
gaggct----------------------(DH2-8)
AAC (JH5)
--- (JH5)
VH-VH replacement
VH3-11
VH4-39
agagaccctgaacggTATTTTGACTGGTTATTA(DH3-9)
cggatg------------------------------(DH3-9)
TTT (JH4)
--- (JH4)
VH-VH replacement
VH3-13
VH1-3
gatcgggagaAT(AGC)3TGaGATATTGTAGTGGTGGTAGCTGCTAC(DH2-15)
gatggccc------------------------(DH2-15)
TAC (JH6)
--- (JH6)
VH-DJH joining
VH3-74
VH1-2
gtcgattGGATATTGTAGTAGTACCAGCTGCTAT(DH2-2)
tcacggacaacgcgacag-----------------------(DH2-2)
TAC (JH6)
--- (JH6)
VH-DJH joining
VH1-46
VH5-51
gaaactctaggctaactcggta(n=22bp)
cgaaaaa----------------------(n=29bp)
TGA (JH4)
--- (JH4)
VH-NJH joining
VH3-48
VH4-34
GATATTGTAGTAGTACCAGCTGCTA(DH2-2)
ggcggacc-------------------(DH2-2)
AAC (JH5)
--- (JH5)
VH-DJH joining
VH3-23
VH4-30
ggccgccctacgGTATTACTATGATAGTAGTGGTTATcgg(DH3-22)
gcccct---------------------------(DH3-22)
CTA (JH4)
--- (JH4)
VH-DJH joining
VH4-28
VH3-13
aatttcttcCTATGATAGTAGTGGTTATTAaaaggggg(DH3-22)
tggggggg--------------------------(DH3-22)
TGG (JH5)
--- (JH5)
VH-DJH joining
VH3-30
VH3-30
cTAGTAGTACCAGCTGCTATtttggtcggggtg(DH2-2)
gagggtaggttt--------------------------------(DH2-2)
ACT (JH6)
--- (JH6)
“Open-and-shut”
*, Identity to VH germline genes was 99-100%. CDR, complementarity-determining
region. bp, base pairs.
For case 99 and case 177, DHJH regions were conserved but VH segment was replaced by
the other VH segment, demonstrating a VH-VH replacement mechanism. For case 190, one
sequence used VH3-13, two DH (DH6-13 and DH2-15) and JH6 segment, but in the other
19
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sequence VH1-3 is attached to the identical DH2-15-JH6 region. For case 246, two
sequences shared the identical DH2-2-JH6 region but were attached to two different VH
segments (VH3-34 and VH1-2). In case 266 22 nucleotides in the N region and the JH4
segment were preserved but attached to different VH segments (VH1-46 and VH5-51). For
case 269, 392 and 400, an identical DHJH complex was conserved but attached to two
different VH segments. Sequence analysis in these 6 cases demonstrate a VH-DHJH joining
mechanism. Case 214 presented with two sequences that used the same V, D and J
segment but introduced 12 nucleotides in V-D joining with one C nucleotide deletion
indicating that an “open-and-shut” mechanism was involved in this case.
Functional rearrangements in B cell childhood ALL.
Among the 381 sequences, 302 (79%) were joined in a potentially non-productive
rearrangement including out-of-frame joinings or in-frame joinings containing a stop
codon. Only 21% of the IgH rearrangements could potentially result in production of
heavy chain protein and these cases demonstrated an association with privileged usage of
VH segments with 36% of these sequences utilizing VH4 (Table 4).
Table 4. Association between potential productive rearrangement and VH segment
utilization
Gene family
VH1
VH2
VH3
VH4
VH5
VH6
Total
Potential
productive
4 (5%)
5 (6%)
40 (51%)
22 (28%)
3 (4%)
5 (6%)
79 (21%)
Potential
non-productive
58 (19%)
17 (6%)
152 (50%)
38 (13%)
7 (2%)
30 (10%)
302 (79%)
Percentage of productive
within VH gene family
6%
23 %
21 %
37 %
30 %
14 %
(4/62)
(5/22)
(40/192)
(22/60)
(3/10)
(5/35)
Association between VH family usage and potential productive rearrangements (p=0.002
by Chi-Square test and p=0.001 by Fisher exact test).
20
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In-frame rearrangements only were detected in 59 patients, out-of frame rearrangements
only in 239 and in 19 patients both in-frame and out-of-frame rearrangements were
detected in the same patient (Table 5). We noted an association between the presence of
more than one sequence and whether the rearrangements were in-frame or out of frame
(Table 5).
Table 5. Association between potential productive rearrangement and clonality in
childhood ALL
Clonality
Number of cases
One IgH sequence
More than one IgH
sequence
Potential
productive
59
57 (97%)
2 (3%)
Potential
non-productive
239
199 (83%)
40 (17%)
Mixed with
productive and nonproductive
19
19
(p=0.006 by Fisher’s exact test).
Only 3% of patients with only in-frame rearrangements had more than one sequence
detested, compared to 17% of the cases with only out-of-frame rearrangements (p=0.006
by Fisher exact test). A trend towards a higher probability of risk of relapse was observed
in patients with only productive rearrangements (p=0.08 by the log-rank test), compared
to children in whom at least one non-productive rearrangement was identified at
presentation (Figure 5).
21
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Figure 5. Kaplan-Meier analysis showing a trend towards a higher probability of risk of
relapse in patients with only potential productive rearrangements compared to children in
whom at least one non-productive rearrangement was identified at presentation (p=0.08
by the log-rank test, two sided). Five cases lost to follow up for relapse were excluded
from this analysis.
Figure 5
1.0
Patients with at least one non-productive rearrangement
n=253, 30 relapses
0.8
Patients with potential productive rearrangement
n=59, 12 relapses
Probability
0.6
0.4
0.2
0
10
20
30
40
50
60
70
80
90
Months after diagnosis
22
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DISCUSSION
We assessed the VHDHJH gene usage profiles at presentation from 381 IgH sequences
identified from 317 cases of B-lineage childhood ALL. This is the largest reported series
of IgH sequences in childhood ALL and incorporates knowledge from the now complete
map of the human IgH locus.
When VH gene usage is analyzed by position on the chromosome rather than simply by
VH gene family, we observed a preferential usage of the DHJH-proximal VH segments
including not only VH6-1 segment, the closest segment to the DHJH locus, but also other
VH family segments near the DHJH locus including VH1-2, VH1-3, VH2-5, VH3-7, VH3-9,
VH3-11, VH3-13 and VH3-15 segments. The finding of position-dependent VH gene
utilization supports the hypothesis that chromosomal order might in part regulate the VH
sequences rearrangement.45 VH3 represents the largest gene family and was the most
utilized family in children with B-lineage ALL, in keeping with the size of this VH family
in the germline.9,10 We observed a privileged usage of the VH6 segment, previously
identified as a component of the quite restricted human fetal antibody repertoire.16,20 A
privileged usage of VH6 has also been reported in immature B cells.30 Our observation is
in line with previous reports from precursor B ALL suggesting that ALL transformation
arises at the early stage of B cell development. 30,32,34
The DH2 and DH7 segments were over-represented and DH4 and DH5 segments
underrepresented in childhood B-ALL compared to PBL.11 A privileged usage of DH7
segments has been observed in both murine and human fetal cells (liver, spleen and
23
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marrow B cells).16,19,46-48 However, in the present study we observed a lower frequency
(4%) of utilization of DH7 in cases of ALL, compared to those reported for use in nonmalignant B cells in fetal tissues by Sanz et al (14%)6 and by others (50%).16,48 We also
observed that JH-distal DH segments were over represented. Therefore, it is unlikely that
this can be explained by proximal locus regulation of the recombination machinery at the
DHJH joining stage. In murine immature B cell lines, a secondary DH-JH rearrangement
has been suggested by the finding that an initial DH-JH rearrangement can be deleted and
replaced by a more 5’ DH gene.49 Recent studies have shown that the specific
recombination signal sequences (RSS) and coding ends may play a role in the preferential
joining of specific DH to JH genes,50-52 and the more closely an RSS sequence resembles
the consensus (CACAGTG-spacer-ACAAAAACC), the more often it is recombined in
extrachromosomal substrates.53 Therefore molecular mechanisms rather than location
appear to govern the selection of DH gene segment during early B cell development.
A characteristic JH gene usage pattern (JH4>JH6>JH5>>JH3/2/1) has been reported for
fetal, neonatal, childhood and adult peripheral B lymphocyte CDR3 regions.12,46,54 The
usage of the JH genes in this study is in line with this order. It is likely that molecular
mechanisms also govern the JH gene usage with JH4 RSS more closely reassembling the
consensus sequences followed by the RSS of JH6, JH5, JH3, JH2 and JH1.12,54
In normal B cells, previous studies have demonstrated shorter CDR3 in pre-term infants
than in term infants and adults.55 In immature B cells, a recent study demonstrated that
lack of expression of TdT was associated with the absence or shorter N sequences which
24
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contributed to shorter CDR3 regions, especially at DHJH joining.56 TdT interacts with the
DNA-dependent protein kinase (DNA-PK), particularly with the Ku proteins, suggesting
that TdT expression may modulate gene segment recombination at the ligation step.57
The majority of cases of ALL presenting within the first 3 years of age have been
suggested to arise from in utero transformation events by the finding of a high frequency
(87.5%) of absence of N sequences at DHJH joining, similar to that found in human fetal
life.31,33 This high frequency of absence of N sequences at DHJH joining was not observed
in our study (data not shown). However, we observed that a short CDR3 was correlated
with utilization of VH6 and DH7-27. This observation might be explained by
developmental regulation of the recombination machinery with favored selection of JHproximal VH and DH gene segments and by influence of TdT expression at an earlier
stage of B cell development.17,57
The overall incidence of cases with more than one IgH sequence in the present study was
19%. Although this is lower than in some reported studies (30-60%) using PCR
fingerprinting or cloning strategies that may be better suited to find minor
subclone(s)44,58,59 our findings are in keeping with previous studies using Southern Blot
analysis.60-62 Some studies have shown an association between oligoclonality at
presentation and poor prognosis in ALL patients.63,64 We did not observe an association
between bi- or oligoclonality and relapse, or with any other clinical characteristic except
for the presence of chromosome 9p deletion. The correlation between chromosome 9p
deletion and more than one IgH sequence might be partly explained by the recent finding
that illegitimate VDJ recombinations involved in chromosome 9p21 deletion in lymphoid
25
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leukemia, are targeted at recombination signal sequence (RSS)-like sequences widely
distributed in 9p21.65 Detailed sequence analysis showed that among the cases with more
than one sequence, the majority of sequences were unrelated with ongoing
rearrangements found in only 15% of cases.
Similar to the findings previously reported for immature B cells,66 and in keeping with a
previous study in ALL,33 only 21% of VH-JH rearrangements detected were potentially
productive. Unlike normal B cells, this does not lead to loss of leukemic clone,
demonstrating that ALL does not require signals mediated by Ig signaling for survival. Of
note, a trend towards a higher probability of risk of relapse was found in patients with
only productive rearrangements compared to children in whom at least one nonproductive rearrangement was identified at presentation. The reason why potentially
productive rearrangement would be relevant to clinical outcome is unclear. We did note
an association between detection of more than one sequence and functional IgH
rearrangement. Whether this relates to the stage of differentiation of the B cell at which
malignant transformation occurs, or to lack of allelic exclusion by non-functional VDJ
recombination is currently unknown. In productive rearrangements we observed a
privileged usage of VH4 segments. Of note, VH4-34 was found to be the third overused
VH segments (22/381, 5.8%) and the most frequently over represented V4 family segment
(22/60, 36.7%). The VH4-34 gene segment is located 500kb upstream of the D gene locus
and is markedly over-represented in the normal B cell repertoire.67 A biased usage of the
VH4-34 segment in rearranged VH4 family genes has been reported in immature B cells.17
Increased usage of this gene segment also occurred in pathologic states including
26
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autoimmune
disease
and
B-cell
malignancies
associated
with
autoimmune
phenomena.68,69 The relevance of these findings to ALL is not clear, since ALL is not
thought to be an antigen driven process, although it is possible that this may be important
in the subset of cases with potentially productive Ig gene rearrangements.
In summary, this study describes the VHDHJH gene utilization profile from 317 children
with B-lineage ALL. A biased usage of VH6, and a shift from JH4 to JH6 was observed,
similar to the patterns of immature B cells, in keeping with the widely held views that
leukemic transformation occurs at the early stage of B cell differentiation. The
preferential usage of DHJH-proximal VH gene segments suggests that V to DJ
recombination is somewhat dependent on physical location at the VH locus. However, DH
and JH segments utilization is position-independent and more likely governed by
molecular mechanisms. Moreover, patients with only productive rearrangement
demonstrated a trend towards an increased risk of relapse suggesting that molecular
pathophysiology might be relevant to clinical outcome.
ACKNOWLEDGEMENT
This study was supported by supported by P01 CA68484 from the National Cancer
Institute, Bethesda, MD. We thank Peter Varney for help in the preparation of the
manuscript and all members of the DFCI ALL consortium for providing samples for this
study.
27
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Prepublished online March 9, 2004;
doi:10.1182/blood-2003-11-3857
Utilization of Ig Heavy-Chain Variable, Diversity and Joining Gene
Segments in Children with B-lineage Acute Lymphoblastic Leukemia:
Implications for the Mechanisms of VDJ Recombination and for
Pathogenesis
Aihong Li, Montse Rue, Jianbiao Zhou, Hongjun Wang, Meredith A Goldwasser, Donna Neuberg, Virginia
Dalton, David Zuckerman, Cheryl Lyons, Lewis B Silverman, Stephen E Sallan and John G Gribben
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