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Clonal Evolution in B-Lineage Acute Lymphoblastic Leukemia by
Contemporaneous VH-VHGene Replacements and VH-DJH
Gene Rearrangements
By Youngnim Choi, Steven J. Greenberg, Tian-Long Du, Pamela M.Ward, Phyllis M. Overturf,
Martin L. Brecher, and Mark Ballow
B-cell acute lymphoblastic leukemia (B-ALL), more frequently than any other B-lineage neoplasm, exhibits oligoclonal Ig heavy chain (IgH) gene rearrangement
in 15% t o
43% of all cases studied. To study the molecular processes
that promote multiple IgH rearrangements, a comprehensive sequence analysis of a B-AU case was performed in
which seven clonal IgH gene rearrangements were identiThe genetic profiles suggested that a single leukemic
progenitor done evolved into several subclones through
dual processes of variable (Vn) t o preexisting diversity-joining (DJH) gene segment rearrangement and Vn t o VH gene
replacement. Predominant IgH-V usageand the uniquely rearranged donotypespecific VHDJHregion gene sequences
were identified using a novel DNA-based geneamplification
strategy. Polymerase chain reaction ( E R )was directed by
an 1gH-J generic p
m
r
i e
r and a complement of family-specific
IgH-V primers that defined the major B 4 1 IgH-V gene usage. C l o n a l i of rearranged VHDJH bands was
substantiated by high resolution denaturant gel electrophoretic analysis. Sequence patterns of the amplified VnDJH
fragments segregated into two groups defined by common
DJn sequences.Partial N region homology at the V& junction as well as shared D J H sequences firmly established V, t o
VnDJH genereplacement as a mechanismgenerating clonal
evolution in one group. In the second subset, oligoclonalii
was propagatedby independent VH generearrangementst o
a common DJn precursor. Thecontributions of all dona1 IgVHDJn repertoires for each group was approximately 50%
and reflected a symmetric distribution of leukemic subclones generated by either process. Thus, oligoclonal rearrangements evolved by two independent, yet seemingly
contemporaneous molecular genetic mechanisms.
All seven
clones displayed nonfunctional Ig-VHDJH recombinations.
These observations may have relevance
t o the recombinatorial opportunities available during normal &cell maturation.
0 1996 by The American S m M y of Hematology.
S
Several mechanisms have been proposed to explain the
presence of multiple, clonally rearranged IgH-VDJ genes in
B-ALL. The IgH locus, located on chromosome 14, may be
genetically duplicated as a consequence of the cytogenetic
abnormalities identified in B-ALL? However, polysomy of
chromosome 14 occurs with relatively low frequency and
cannot account for the majority of cases of B-ALL in which
multiple rearrangements are encountered.” Biclonality has
been described in Epstein-Barr virus-associated B-cell
lymphoproliferative disorders and some B-cell lymphomas,l’.12
Alternatively, multiple rearrangements may reflect
repetitive rearrangements of the IgH loci among leukemic
progenitor
There is evidence to suggest
that
oligoclonal rearrangement may result from multiple independent rearrangements of a gennline precursor cell?” rearrangement of VH to a preexisting DJH segment or VH gene
repla~ement.’’~’~
We have reported a novel molecular diagnostic strategy to detect and characterize clonal expansions
from a variety of B-cell and T-cell malignancie~~.~
and to
profile V, gene family repertoires in physiologic and virallyinfected disease states.”In B-cell malignancies, an initial
primary polymerase chain reaction (PCR), directed by an IgJH generic primer and a complement of family-specific IgVHprimers, defines the major B-cell Ig-vH gene family usage. The clonal nature of each rearranged VHDJH band is
verified by high resolution denaturant gel electrophoresis.
Clonotype-specific primers are designed from sequence data
from theVHDJH juxtaposed regions, ie, the third complementarity determining region (CDR In),and used in a secondary
clonotype primer-directed PCR (CPD-PCR) to detect, with
extreme specificity and sensitivity, the unique B-cell clone.7
Using this approach, the predominantB-cell &-v, usage
and the uniquely rearranged, clonotype-specificVHDJHgene
sequences may be identified in patients with B-ALL.
In the course of evaluating the patterns of VHDJH rearrangements in B-ALL cases exhibiting multiple IgH rearrangements, one case was identified in which at least seven
rearranged VHDJH sequences were present. Sequence data
fied.
OMATIC RECOMBINATION of Ig heavy chain (IgH)
variable (VH),diversity (D) and joining (JH) gene segments during B-lymphocyte ontogeny produces a vast array
of rearranged VHDJH sequences.’” The total genetic pool of
circulating B cells is highly polyclonal in nature and each
mature B-cell lineage that has undergone differentiation is
distinguished by a unique clonotypic VHDJH sequence. Conventional dogma asserts that heme-oncologic processes, eg,
leukemia, represent clonal expansions of single transformed
cells. Karyotype analysis3and glucose-6-phosphatedehydrogenase isotyping4 support a single parental cell origin in
acute lymphoblastic leukemia (ALL).However, although
greater than 98% of all B-lineage acute lymphoblastic leukemias (B-ALL) undergo rearrangement of IgH genes?-7 15%
to 45% of all B-ALL cases studied by Southern blot hybridization analysis exhibit oligoclonality in IgH gen~type.~.’
From theDepartments of Neurology, Microbiology and Immunology, Molecular Diagnostics, and Pediatrics, Roswell Park Cancer
Institute. Buffalo, the Department of Pediatrics and Division of Allergy and Immunology, Children’s Hospital of Buffdo, Buffalo;and
the Department of Neurology, State University of New York at Buffalo, Buffalo, NY.
Submitted July 14, 1995; accepted November 7, 1995.
Supported in part bygrants from the Endowmentfor the Neurosciences (North Bellmore, NY), the Margaret D u f i and Robert Caneron Troup Memorial Fund and the Buffalo General Hospital (Buf
falo, NY), Grant No. 229850, and the Association for Research of
Childhood Cancer, lnc (Buffdo,M).
Address reprint requests to Steven J. Greenberg, MD, Laboratory
of Neuroimmunology and Neurovirology, Department of Neurology,
Roswell Park Cancer Institute, Elm and Carlton St, Buffalo, NY
14263.
The publication costs of this article weredefrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisemnt” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
8 19% by The American Society of Hematology.
0006-4971~706-O.00/0
2506
Blood, Vol87, No 6 (March 151, 1996: pp 2506-2512
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CLONAL EVOLUTION IN B-ALL
2507
controls, consistingof the reaction mixture without DNA template,
were tested for each series of PCR amplifications.
Confirmation of leukemic clones by high resolution &naturant
C
%yielding predominant Iggel electrophoresis. Amplicons of P
VHDJH rearranged bands were further analyzed by denaturant gel
electrophoresis to further resolve multiple rearrangements sharing a
common Ig-V" gene family and to confirm the monoclonal nature
Of the Ig-vHDJH amplified band.%A totd Of 5 pL aliqUOts Of amplicons were electrophoresed 55",
at constant powerat 65 watts for 2.5
hours through 8% polyacrylamide denaturant sequencing gels (Ultra
MATERIALS AND METHODS
Pure Gel-mix 8, Gibco-BRL, Grand Island, NY). Gels were dried
onto sequencing gel filter paper (Bio-Rad, Richmond, CA) and autoPatient material. A3-yearold girl withB-lineage ALL was
radiographed for 3 days on XAR-2 film.
evaluated by the Division of Pediatric OncologyService, Children's
Cloning and sequencing the Ig-V&JH sequences. Once identiHospital of Buffalo, Buffalo, N Y , and received a bone marrow biopsy. Bone marrow replacement by leukemic cells was greater than fied,thepredominantIg-VHDJHamplifiedproductwasseparated
80%. Flowcytometric analysis showed the following immunopheno-from minor contaminating bands and excess PCR primers by excising the major band from polyacrylamide gel and then subjecting the
type: CD10+, DR+, CD24+, CD19+, CD9-, CD22+, CD45+,sIg-,
amplicon-contained gel slice to filtration centrifugation using ultraand CD7-. Analysis for cytoplasmic p protein was not performed.
free-MCfilter units, 30,000 molecular weight cut off (Millipore,
Cytogenetic analysis ofanunstimulatedbonemarrow
culture by
Bedford, MA). The purified, amplified Ig-V,DJ, sequence was diconventional methods showed a normal female karyotype( 4 6 , X X ) .
rectly ligated into pCR I1 plasmid (Invitrogen, San Diego, CA). In
DNA and oligonucleotide preparation. Marrow cells were fracinstances in which the Ig-VHDJH clonal band was relatively weak,
tionated by densitygradientcentrifugation,uandhighmolecular
the purified amplicon was subjected to an additional amplification
weight DNA was isolatedby the sodium dodecyl sulfakdproteinase
directed by the corresponding Ig-vH family-specific primer and the
K method, purified by phenoYchloroform extraction, precipitated in
Ig-J, generic primer, repurified as above and ligated into plasmid.
absolute ethanol, and resuspended in10:1 TE (10 mmom Tris-HC1
Subsequent to ligation and bacterial transformation into INV a F'
and 1 mmolR. EDTA) bufferas previously described?'Oligonucleocells (Invitrogen,San Diego, CA), the transformants were selectively
tide primers were synthesized
by the phosphoramidite methodon an
expanded in Kanamycin containing agar and productive clones seautomated 391 DNA synthesizer (Applied Biosystems, Foster City,
lected by extinction of the &galactosidase marker. Ig-V,DJH insertCA) and subsequently released from CPG columns by NH.,OH decontaining plasmids were extracted from bacterial transformants
by
pmtection. 5' trityl-retained oligonucleotideswere purified by chrothe plasmid miniprep technique,= and the Ig-VHDJH segments were
matography using Nensorb Prep cartridges (Dupont, Boston,
MA)
directly sequenced from the purified plasmid by the dideoxy chain
and then concentratedby lyophilization under vacuum.
termination method.% Sequencing primers span either the T7 proProfile of the &-VH gene family repertoire by gene amplification.
moter or theSP6 promoter plasmid regions. The other reagents used
The derivationsof the negative strand Ig-JH generic primer and the
forthe DNAsequencingreactionsweresuppliedwiththeUSB
been reported in detail
seven Ig-V,family-specificprimershave
Sequencing Kit (United States Biochemical Corp, Cleveland, OH).
elsewhere.' Briefly, a set of seven PCR amplifications defined by
At least three separate clones were fully sequenced to obtain
a conan IgJ, generic primer and each of seven different family-specific
sensus genetic sequence for each Ig-V,DJ, segment.
primers was performed to profile Ig-V, repertoire usage. To bias
Southern blot analysis. A total of 10 pg of gDNA was digested
amplification of the leukemic clone, the Ig-JH generic primer was
separately with EcoRI, BamHI, and HindIII restriction endonucleS e n d labeled with yp2deoxyadenosine triphosphate (dATP) by
treatment with polynucleotide kinase and relatively low input target ases (Boehringer Mannheim, Indianapolis,IN), fractionated by electrophoresis through an 0.8%agarose gel, and blot transferred to
DNA was used. In this manner the contributionof polyclonal B cell
Zetabind (CUNO, Meridium, CO). Blots were probed with a
ranDNA was minimized while preserving maximal sensitivity? All Igdomly primed 32P-labeledBamHYHindIII JH cDNA probe."
V,DJ, PCRs were performed against 100 ng DNA template in 100
Sequence analyses. The derived Ig-VHDJH sequences were anapL adjusted to final concentrationsof 10 mmovL Tris-HCI, pH 8.3,
lyzed for their corresponding germline V,,D,and
J, component
50 mmoK KCI, 1.5 mmoVL MgCl2, 0.001% gelatin, 200 pmolR.
deoxynucleotide triphosphates, and nmoK
100 each primer to which using GenBank (EMBL) FASTAsearch and translatedby Mac Vector software (International Biotechnologies Inc, New Haven, CT).
1 U Taq polymerase (Perkin-Elmer Cetus,
Norwalk, C T ) was added.
Temperature cyclingwas performed ina DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, CT)as follows: an initial denaturation
RESULTS
for 5 minutes at 94T, 1 minute at %"C, 1 minute at 65°C. and 1
The multiple Ig-VHDJHrearrangements from a patient with
minute at 72°C for 31 cycles.
B-lineage ALL are depicted in Fig 1. PCR-amplified BA total of 40 pL aliquots of all seven PCR amplifications were
ALL bone marrow-derived DNA generated intense signals
electrophoresed through a nondenaturant 5% acrylamide gel. Gels
were dried and exposed overnightto XAR-2 film (Eastman Kodak,
corresponding to usage by four IgH-V gene families (Vl,
Rochester, W ) .AutoradiographswerethenscanneddensitometV2, V3, and V,) upon autoradiography after electrophoresis
rically using visual light transillumination, converted into a computer
through a nondenaturant polyacrylamide gel. Subsequently,
Scanner Tiff file (Microtek
BLW),and quantitatedby Scan Analysis
seven distinct bands (A-G) were detected on further resolusoftwm. Relative Ig-vH gene family usage was calculated
by dividtion of the V,, V2, V3, and V, -related amplicons by electroby thesumof the signal
ing the signal intensity of each family
phoresis through a denaturant sequencing gel:two clonal
intensities of the seven families. In this manner, Ig-V, gene family
bands rearranged a member ofthe IgH-VI family (A and B),
usage was profiled.
one rearranged an IgH-V2 family member (C), three used an
To assure quality ofDNA and integrity ofthePCRs, internal
JgH-V3family member (D, E,and F), and one used an IgHcontrol amplifications were run concurrently. Each genomic DNA
V4 family member (G) (Fig 1). The relative combined IgHsample was amplified using an intra-exon primer set that frames a
325-bp target sequence within the actin gene. In addition, reagent
V family band intensities were V, > Vj > V2 > V4. By
from seven rearrangements provided a comprehensive analysis of the evolutionary processes leading to oligoclonality
in IgH genotype and suggested the coexistence ofVH to
preexisting DJHsegment rearrangement and VHgene replacement. These observations suggest that VH-VH gene replacement and VH-DJHgene rearrangement are not mutually exclusive events and that both mechanisms may be operative
contemporaneously.
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CH01 ET AL
2508
VI
v2
v3
v4
V5
V6
V7
v1
v2
v3
v4
G
Fig 1. 19% gene family usage andoligoclonal Ig-VHDJH gene rearrangements
in a B-ALLcase.(Left Panel) Profileofthe Ig-VHDJHrepertoires:
Aliquots of amplicons generated by each family-specific lg-VH primer and *-labeled generic
l g J Hprimer were electrophoresed through a
nondenaturant polyacrylamide gel and then autoradiographed (lanesV1-W). PCR amplification of B-ALLbone marrow-derived DNA displayed
intense signals in lanes V1, V2, V3, and V4. Two less intense high molecular weight bands in V1 are artifacts in a nondenaturant gel, which
disappeared in a denaturant gel. (Right Panel) High resolution denaturant gel electrophoresis: Separationof amplicons through a denaturant
sequencing gel showed seven distinct clonal Ig-VHDJH rearrangements:two rearranged using a member of the V1 family (A and B), three
rearranged V3 family members (D, E, and F), and V2 and V4 were used once(C and G, respectively).
contrast, Southern blot analysis showed four unique restriction fragment length bands on Hind111 digestion and three
unique bands on digestion withEcoRIandwithBurnHI
(Fig 2).
The amplified, clonally rearranged VHDJH segments were
subcloned andthen sequenced. The weaker C and G rearranged bands were cloned subsequent to reamplification of
the corresponding products derived from the primary PCR.
Consensus sequences were established by sequencing at least
three clones for bands A, B, C, and G . The VHDJH rearranged
segments that used the IgH-V3 family gene did not migrate
as separate bands by polyacrylamide gel electrophoresis and
were cloned as a mixture. A 314-bp consensus sequence
corresponding to the F band and two different 327-bp consensus sequences, El and E2, corresponding to the E band
were obtained by sequencing a total of I 1 randomly selected
subclones. A VHDJH sequence corresponding to band D, the
weakest of IgH-V3 rearranged bands, was not retrieved from
among the multiple IgH-VS subclones. An additional PCR
amplification would not be expected to enhance the likelihoodof selecting for a band D subclone as the ratios of
bands F El: E2: D would not change. ,411 clonal Ig-VHDJH
gene sequences segregated into only two DJH recombinatorial elements: three VH genes were juxtaposed to the DXPIJ4 segment and four VH genes recombined to the DXP4-J4
segment (Fig 3).
Alignment of the CDR 111 consensus sequences showed
a detailed pattern of base pair homologies that permitted the
prediction of the series of events leading to oligoclonality
in IgH genotype (Fig 3). In the group that shared the DXPIJ4 configuration, oligoclonality arose through three independent VH to DJH (VH-DJH) rearrangements based on homology that diverged within the germline DXplsegment: clonal
sequence G incorporated the entire DXpl
germline sequence,
while clonal sequences A and E2 deleted 6 bp and I 1 bp,
respectively, from the 5' portion of the DXplsegment. In
addition, clonal sequence A was the product of an unusual
D(N)DJ juxtaposition followed by rearrangement of an IgHV , gene into the DDJH segment.
GERMLINE
ALL
nn
. .. .
-Qr
e
Fig 2. Identification of multiple IgH generearrangements by
Southern blot. Genomic DNA from the index AU-patient and from
the promyelocytic cell line HL60 (germline control) were restriction
enzyme digested; H, Hindlll; E, EcoRI; B, BamHI. Restriction digests
wereelectrophoresed,Southern
blot transferred,andhybridized
with a UP-labelledJHprobe. ALL DNA generatedfour unique restriction fragment length bands upon Hindlll digestion and three unique
bands upon digestion with EcoRl and with BamHI.
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CLONAL EVOLUTION IN B-ALL
2509
N
V
-1
D
I I
DXPl
TATMXATATmOACTGGTTAlTA
PA1
'I[
-TAT
1
TAT-]
A
TAT-lQ;
GTKiAAc
V1
v3
TAT-ICMG
1
V1
B
TATlWlegaga
['
v2
c TATITACX;TOICK:
EI
v3
TATL-~~
1
w
-
4
I
hPl
4
h
p
OATATTTTDACTOOTTAlTA
TDICTICmGOGCUUYUI
GATKGCCCCGTAAGCOQXFFAa%CG
TAT-]
rAn-ATA%nAnA
DLRS
A
W
o
A
c
G
A
G
A
m
v3
F(PA2)
4
myJTICX%OOCCIIGOU
v4
G
E2
J
1 1
" Z ' a n A n A
qB4
m
Q4242
"SAGS
GTATTACGATITITGGAG-AT
m
m
- 4
-4
W 4
4
T A ~ ~ G m o o T T A TIOICTAC70000CCMOOU
T
4
qB4
TAW-GTGGGHAT
TmilcTkCTOOQOCCMQUA
T A a - % A T
T
m
c
T
li
4
€2.
Fig 3. Evolution of VHDJHoligoclonalii in B-ALL. (Top Panel) Multiple Ig-vHDJH rearrangements (designated A, B, C, El,
F, and G) from
a patient with B-lineage ALL are depicted schematically. Two different sequences of equal size were subsequently resolved from the original
band E and are designated as E l and E2. The seven clonal Ig-VHDJHrearrangements are distinguished by different VH-family members, but
segregated into two groups according t o their DJHrecombinatorial elements. Dxpl-J4 and Dxp4-J4segments were shared by three (A, E2, G )
and four (B, C, El, F) rearrangements, respectively. Relative usage, shown to the right ofeach lg-VHDJHrearrangement, was deduced from
band intensity on high resolution denaturant gel and
represented
is
as a percentage of the sum of all clonal
bands. (Bottom Panel) Sequences
corresponding t o t h eCDR3 are aligned in order of presumed clonal evolution.
DJHnucleotides arein bold. In the Dxpl-J4 group, homology is
progressively disrupted within the D gene segment with the additional presence of a unique D t o D rearrangement in the A sequence,
suggesting independent VH gene rearrangements t o a common DJHprecursor as the mechanism propagating oligoclonalheterogeneity. In
the DXP4-J,group, partial N region homology at the VHD junction (underlined) and conservation of the spliced 3' V,-DPlO pentanucleotide
derived from B (lower case) suggest VH t o VHDJHgene replacements as the mechanism generating clonal evolution. The internal heptamer
sequences at the3'-end of theVH segments are boxed.
In contradistinction, the second group of clonal segments originated by a series of VH-VH replacements. This
was evident from partial N-region homology (Fig 3) that
was present in addition to DXp4J4 sequence homology
shared among the clonal B, C, and El members. Clonal
sequence F contained the entire Dxp4germline sequence.
Clonal sequences B, C, and El each deleted a S-bp GATAT sequence from the S' portion of the DXp4 segment.
The pentanucleotide sequence CGAGA in clonal sequence B (Fig 3) is derived from the extreme 3'-end of
the human IgH-VI-DPIO.'* While both clones C and El
retain the entire N insertion sequence GAGAC found in
sequence B, clone C also retains the pentanucleotide sequence CGAGA from the 3' end of V I-DPIO, whereas
clone El retains only the very last A nucleotide (Fig 3).
Interestingly, all sequences containedtheinternalhep-
tamer signal sequence TACTGTG which may have mediated additional VH to VHDJH rearrangements.'s.'h
The quantitative nature of this molecular genetic strategy
to study V, gene family usage among polyclonal B-cell
populations has been reported previously." The relationship
between the VII-DJH rearranged group and the VH-VHreplacement group can be expressed as the sum of all clonal
!g-VHDJHrepertoires found in each group. The contribution
of VH-DJIirearranged clones (A + E2 + G ) was 47% and
the contribution of VH-VH replacement clones (B + C + El
+ F) was SO% (Fig 3). Thus, the approximate 1:1 stoichiometric relationship may suggest a state of dynamic equilibrium between the process of VH-DJlirearrangement and that
of VH-VI, replacement, which represent recombinatorial
events on at least two separate alleles.
All seven clonal Ig-VHDJII sequences were translated. Six
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CH01 ET AL
2510
sequences, A, B, C, E2, F and G, were found to be out of
frame. Only the clonal E l sequence was joined in-frame,
but the E l Ig-VHDJHsequence used a pseudo-V gene.Therefore, all B-ALL clonal Ig-vHDJHsequences detected were
nonfunctional.
DISCUSSION
The extreme oligoclonal nature in IgH genotype of the BALL case presented here was suggested by Southern blot
analysis and aflirmed and defined further by a DNA-based
PCR methodology.' The predominant B-cell Ig-VHDJHgene
rearrangements were further resolved by high resolution denaturant gel electrophoresis and seven clonal IgH gene rearrangements were identified. Comparative sequence analysis
suggested that a single leukemic progenitor clone with genetic configuration of DXPIJ4 (PAl)and V3DXP4J4 (PA2) in
each allele evolved into several subclones represented by
two groups of related Ig-VHDJHsequences and thatleukemic
clonal evolution was propagated by two coincident mechanisms, that of VHto preexisting DJH segment rearrangement
and VH to VH gene replacement. The coexistence and contemporaneous operation of both mechanisms giving rise to
multiple IgH rearrangements has not been documented previously.
The following series of events are proposed to account
for the oligoclonal VHDJH sequences observed in this case.
A VHto DJH recombinatorial event results in the deletion of
all intervening D segments. Thus, it follows that the juxtaposition of Dm5-DmIin sequence A was generated independently from sequences Gand E2 by arearrangement of
DmSto the common progenitor DxplJ4segment, followed by
rearrangement of VI-DPl5 to DDJH. Separately, sequence
E2 could not have evolved from sequence G as a result of
VHto VHgene replacement and exonuclease digestion. From
the germline Ig-vH locus map, DP-77, the VH gene in sequence E2, retains a 3' location relative to DP-63, the VH
gene in sequence G, thereby excluding replacement of the
VHgene in G by E2?9 Collectively, these results imply that
each VH segment rearranged independently to the common
DXPIJ4
recombinatorial element, PAl, from among a pool
of leukemic progenitor cells, thus, giving rise separately to
subclones G, A, and E2.
In contrast, a series of VH-V, replacements appears to
havebeen responsible for the generation of the group of
related sequences B, C, El, and F. Members of this group
are distinguished by the DXP4J4 rearranged
element. Sequence
F retains the greatest length of sequence homology with the
germline DXP4 sequence.Thus, it is postulated that sequence
F may represent a second allele of the progenitor leukemic
clone, PA2. Sequence B, resulted from replacement of V3DP42, the VHgene in F, with VI-DP10, the VHgene in B.
Physical constraints imposed by the g e d i n e Ig:-vH locus
map (see below) restricts a possible independent rearrangement of VI-DP10 to the preexisting DXP4J4segment and
associated exonuclease nibbling and random N-insertionsof
GAGAC. Clone C arose from a cleavage between the 3' end
of the internal heptamer and the terminal pentanucleotide
CGAGA of VI-DP10, plus N-insertions consisting of bases
TTCCCC upstream to the breakpoint, followed by a V2DP27 for V1-DP10 replacement (Fig 3). Further, it is postu-
lated that sequence E l arose from a CGAG 1 A cleavage
within the 3' VI-DPlO pentanucleotide derivedfrom sequence B, plus N-insertions consisting of bases CATGGAGGT upstream to the breakpoint, followed by a V3-DP34
for V1-DP10 replacement (Fig 3).
The physical locations of the VH genes on the VH locus
map substantiate the proposed ordered genesis of the clonal
sequences F B (C and E l ) by VH-VH replacements. The
VHgenes rearranged in sequences F and B are located 750
kb (DP42) and 900 kb (DPlO) upstream of VH6,
respectively.
This relationship is compatible with the proposition that sequence Bwasgenerated by replacing V3-DP42withthe
upstreamV1-DP10. V2-DE7 andV3-DP34,usedinsequences C andE l , respectively, belongto an 80-kb insertion/
deletion polymorphismnotincluded in the publishedIgVH physical map.29 Nonetheless, the location of the 80-kb
insertion polymorphism was thought to coincide withthe
position of V1-DP10.29 We propose, as a corollary to the
corresponding hierarchy of VH-VH replacements, ie,
DP42 DPlO (DP27 + DP34), that the 80-kb insertion
polymorphism is located distal to DPlO inthe V, locus,
which includes the polymorphic region.
Recently, Umiel et aI3' reported the expression of recombination activating gene-l (RAG-l) in 20 of 21 ALL cases,
independent of developmental phenotype or genotype. They
also detected Terminal deoxynucleotidyl transferase (TdT)
activity in 18 of 21 ALL cases. Thus, most ALL cases seem
to exhibit the requisite enzymatic machinery to undergo repetitive Ig heavy chain gene recombinations.
The circumstances that promote continued immunoglobulin gene rearrangements remain unclear. Teleologically, all
viable B cells must undergo functional rearrangement of the
VHDJH genes, the expression of which, in association with
a surrogate light chain, mediates allelic exclusion during
normal B-cell ~ n t o g e n y . ~In"the
~ ~present case, all seven
clones displayed nonfunctional VHDJH recombinations. This
propensity to form nonfunctional Ig heavy chain recombinants was documented in a group of 16 ALL patients with
multiple clonal Ig-VHDJHrearrangements compiled by other
investigator^.^.^"'^ Although expression of cytoplasmic p,
the protein product associated with functionally rearranged
genes, was not assessed in the presentcase, most ALL cases
with multiple IgH gene rearrangements do not express cytoplasmic p and its expression is observed in only about 20%
of B-lineage ALL case^.^.'"^ One possible explanation
drawn from these observations is that in a majority of cases
in ALL, recombination of Ig variable, diversity, and joining
genes may continue until either a functional rearrangement
is achieved or the available pool of IgH-V, D, and J genes is
deleted. That pursuit of a functional Ig-VHDJHrearrangement
may lead to near depletion of the IgH loci is supported by
the absence of clonal evolution amongmany established
transformed lymphocyte lines. For example, the T-ALLderived cell lines Jurkat and HPB-ALL express T-cell antigen receptor (TCR). These cell lines have retained their respective TCR-P chain genotypes over decades of cell culture
and countless cell divisions, in the presence of high levels
of TdT, RAG- 1, and RAG-2 mRNA (authors' unpublished
data). Extensive analyses of the TCR-P locihaveshown
extensive deletions in both alleles of Jurkat and HPB-AL
+
+
+
+
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2511
CLONAL EVOLUTION IN B-ALL
cell lineages. It would appear that successful TCR-VpDJp
rearrangements were achieved only after several recombinatorial events and that these clones were constrained from
further recombinations, aside from achieving functional
TCR-VpDJBrearrangement, due to lack of additional available TCRB-V, D, and J genes. Hematological malignancies
represent clonal expansions of cells often arrested at various
stages of maturational development and are characterized by
those operative molecular processes which correspond to
various stages of normal differentiation. For example, isotype switching has been noted in chronic lymphoblastic leukemia, the malignant counterpart of a mature B - ~ e l lAlso,
.~~
ongoing somatic mutation has beenreported in follicular
non-Hodgkin's lymphoma and reflects the developmental
stage of an antigen-selected B-cell.= If the generation of
multiple IgH rearrangements in ALL corresponds to a normal developmental stage-related differentiation process, one
would predict that the continuing IgH-VDJ recombinatorial
events would be mediated by rearrangements of V, to preexisting DJH segments or by V, gene replacements, except in
those few cases in which transformation occurred at the proB-cell stage.Indeed, this conclusion is borne out by our own
unpublished observations and by those of
The same process driving continuous gene rearrangements
in B-ALL may also participate in the course of normal bone
marrow lymphocytopoiesis in which approximately 80% of
cells die, presumably by apopt~sis.~'*~*
Cell loss during Bcell lymphocytopoiesis as a consequence of aberrant Ig gene
rearrangement is also suggested by the observation that severe combined immunodeficiency (SCID) mice or RAG-l
knock-out mice lack apre-B I1 cell ~ o m p a r t m e n t .Factors
~~.~
that contribute to the production of nonfunctional rearrangements include the fact that, by chance alone, the majority of
developing B cells will have out-of-frame joints in D-JH,
VH-DJH,or VL-JL and the fact that 33% of the germline V,
locus consists of pseudo-V, genes.29Indeed, it has been our
experience that 27% (n = 6) used pseudo-VH genes among
clonal IgH-VDJ rearrangements analyzed (n = 22) (authors'
unpublished data). However, B-cell repertoires recombining
apseudo-V, gene would undergo apoptotic death, unless
subsequent recombinatorial events provideda functional
IgVHDJHgene prod~ct.4'"~
It is therefore plausible to consider that maturing B cells, having the capacity, often undergo multiple V,-DJH gene rearrangements and V,-V,
gene replacements, mimicking the events in ALL, in an attempt to escape the dire consequences of a failed, nonfunctional Ig-VHDJHrearrangement.
The progressive series of V, gene replacements in the
case presented here involved V, gene segments located close
to the telomere, ie, D m 7 and DP34 used in sequences C
and El, respectively, and suggests the importance of this
process during normal B-cell development. Because the
product of VH gene replacement is difficult to distinguish
from the original VHDJH sequence in normal conditions, the
physiological significance of VH to VHDJHrearrangement
remains speculative. However, the potential importance of
this mechanism is underscored by the highly conserved internal V, heptamer sequence found in most mouse and human
VH genes.l5.l6Also, study of the Ig rearrangements in IgM'
normal mouse bone marrow cells has shown that most of
the IgM+ cells retained their unexpressed IgH allele in a
DJH-rearranged configuration." Assuming multiple recombinations occur naturally during B-cell bone marrow maturation, this would suggest that V,-VH replacement may be
preferred to V, to DJ, rearrangement, despite the relatively
less efficient signal sequence rendered by the internal heptamer. The striking degree of oligoclonal rearrangements
often observed in ALL may represent the malignant counterpart of multiple somatic immunoglobulin gene recombinatorial events in the normal course of B-cell maturation.
ACKNOWLEDGMENT
The authors express their deep appreciation
for the excellent secreof the
tarial services provided by Grace Lombard0 in all aspects
preparation of this manuscript.
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1996 87: 2506-2512
Clonal evolution in B-lineage acute lymphoblastic leukemia by
contemporaneous VH-VH gene replacements and VH-DJH gene
rearrangements
Y Choi, SJ Greenberg, TL Du, PM Ward, PM Overturf, ML Brecher and M Ballow
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