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Analysis of Clonal Rearrangements of the Ig Heavy Chain Locus
in Acute Leukemia
By S.E. Height, G.J. Swansbury, E. Matutes, J.G. Treleaven, D. Catovsky, and M.J.S. Dyer
Clonal rearrangements of the Igheavy chain (/GM locus OCcur in nearly all cases of B-cell precursor acute leukemia
(BCP-ALL). Some of these rearrangements may bedetected
by polymerase chain reaction (PCR) using VH gene framework 111 (FRIII) and J H consensus primers. However, about
20% of BCP-ALLs fail t o amplify with thistechnique. To determine thecauses of these PCR failures and t o investigate
any possible association with specific subgroups ofdisease,
we analyzed 72 acute leukemias of defined immunophenotypeand
cytogenetics, comparing FRlll with VH-family
leader-specific PCR methods and Southern blotting. Of 37
BCP-ALL cases, 6 (16.296) failed totally t o amplify with FRlll
and J H primers. Noneof these cases amplified with VHleader
primers. Additionally, all cases retained germline VH6 genes
and 5 of 11 rearranged alleles amplified with a consensus
DHprimer, indicating that
these rearrangements represented
biallelic DHJH recombinations. Among the 6 FRlll and VH
leader PCR-negative BCP-ALLcases, there was no common
immunophenotype or consistent cytogenetic abnormality,
although all showed structuralchromosomal abnormalities
and 3 of 5 successfully karyotyped had abnormalities of chromosome 12p. 13 cases with t(9;22)(q34;qll) (Philadelphia
chromosome-positive [Ph'l) and IGH rearrangements (9
BCP-ALL and 4 biphenotypic cases) were also analyzed. Of
23 rearranged IGH alleles, 19 (82%) were positive by FRlll
PCR, and all4 remaining alleles were amplified byVH leader
primers. Use of the leader primers in these Ph+ cases also
detected 3 additional clonal rearrangements that were not
anticipated from Southern blotting; such unexpected bands
were not observed in 21 other Ph- cases. The additional
bands represented "new" and unrelatedVHrearrangements
rather than VH-VHreplacement events. We conclude that biallelic DHJHrearrangements occur in a subgroup ofBCP-ALL;
in these cases, the activation of the full VHDHJH recombination mechanism had notoccurred. Therefore, these cases of
BCP-ALL were arrested at an early stage of B-cell differentiation. In contrast, all Ph+ BCP-ALLs and biphenotypic acute
leukemias, whichmay represent thetransformation of
multipotent hemopoietic stem cells, had undergone VHDHJH
recombination. Of 9 Ph+BCP-ALL cases,3 also showed ongoing VHDHJHrearrangement, reflecting the persistent expression of the VHDHJH recombinase. Finally, sequence analysis
of 33 rearranged VHDHJH genes showed thatonly 3 including
2 Ph'BCP-ALL maintained an intact open-reading frame.
Loss of the open-reading frame occurred not only because
of out-of-frame VHDH and DHJH joining, but also because of
VH gene mutation and deletion. These data show that most
BCP-ALLs may represent the neoplastic transformation of
BCPs destined t o die in thebone marrow.
0 1996 b y The American Society of Hematology.
T
sity arising from the apparently random assortment of VH,
DH, and JH segments, along with the insertion of both N and
P nucleotides at the time of recombination, results in each
VHDHJH junction being unique.'" Therefore, the VHDHJH sequence provides a good clonal marker. Several polymerase
chain reaction (PCR) methods have been designed to amplify
VHDHJH junctions and have been widely used to detect minimal residual disease, particularly in the context of BCPacute lymphoblastic leukemia (BCP-ALL).""' The most
commonly usedmethod involves primers to a conserved
region of the framework 111 (FRIII) region of the VH gene
segments and a consensus JH primer that amplifies the third
complementarity-determining region (CDRIII)." Although
readily performed, limitations of the methodare (1) that only
a proportion of rearranged IGH alleles are amplifiable and
(2) that up to 30% of BCP-ALL cases may be completely
negative." Lack of CDRIII amplification may reflect loss of
the FRIII primer sequence because of VHpseudogene usage,
VH gene deletion arising from "nibbling" at the time of
recombination, DH-JH rearrangement, or rarely in BCP-ALL,
chromosomal translocation to the IGH locus.'' Alternatively,
loss of the JHprimer sequence may arise because of JH deletion that may occasionally be biallelic.IR
Because VHDHJH recombination is normally a developmentally regulated process, theidentification of FRIIVJH
PCR-negative cases of BCP-ALL resulting from arrested
DHJHrecombination might be of clinical significance in that
these cases may represent BCPs arrested at a veryearly
stage of differentiation, before the activation of the VH-DH
recombinase. Therefore, we sought to determine the causes
of CDRIII PCR failure in a series of 72 cases of acute
leukemia of different lineages and different stages of differentiation by comparing the results obtained by Southern blot-
HE HUMANIG HEAVY-CHAIN locus (IGH)lies adjacent to the telomere of the long arm of chromosome
14 at 14q32.3. The locus consists of dispersed gene segments
(51 functional VH genes, at least 30 pseudogenes, some 30
to 50 DH segments, and 6 JH segments) that must undergo
somatic recombination to produce functional Ig molecules."'
Somatic recombination occurs very early in B-cell ontogeny
and has been shown to be developmentally reg~lated.4.~
In
particular, DH-JH recombination precedes VH-DH recombinatiom6 Furthermore, although not necessary for the early
stages of B-cell differentiation, functional Ig molecules resulting from productively rearranged IGH genes are necessary for the continued survival of B-cell precursors (BCPs)
that otherwise undergo apoptosis within the bone marrow,
which is the fate of most BCPS.'.~
Clonal IGH rearrangements may be detected in virtually
all B-cell-lineage malignancies.' The combinatorial diver-
From the Academic Department of Haematology and Cytogenetics, Haddow Laboratories,
Institute of Cancer Research-Royal Marsden Hospital, Sutton, Surrey, UK
Submitted August 9, 1995; accepted February 12, 1996.
Supported by Royal Marsden Hospital Trust Funds and the Kay
Kendall Leukaemia Trust.
Address reprint requests to M.J.S. Dyer, MD, DPhil, Academic
Department of Haematology and Cytogenetics,HaddowLaboratories, Institute of Cancer Research-RoyalMarsden Hospital, Sutton,
Surrey, SM2 5NG, UK.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8712-0037$3.00/0
5242
Blood, Vol 87, No 12 (June 15), 1996: pp 5242-5250
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5243
IGH LOCUS IN ACUTELEUKEMIA
ting using a J H probe with those of PCR using FRIIVJH and
V,., leader-specific/JH primer combinations.
MATERIALS AND METHODS
Clinical and Diagnostic Data
A total of 72 cases of acute leukemia were classified following
morphological, immunophenotypic, and cytogenetic analysis. The
immunophenotyping was performed with a panel of monoclonal
antibodies, and a scoring system was adopted to identify biphenotypic case^.'^.^^ The cases were initially classified as follows: 41
BCP-ALL; 12 T-cell precursor-ALL (TCP-ALL); and 19 acute myUsing this system, 4 cases of BCP-ALL
eloid leukemia (M).
were considered to be biphenotypic by coexpression of multiple
myeloid markers. Similarly, 2 cases of AML were considered to be
biphenotypic by coexpression of multiple lymphoid markers. Details
of initial clinical and diagnostic data of the cases of BCP-ALL are
shown in Table 1.
Amongst the BCP-ALL cases (no. I through 41), there were 17
adults and 24 children (aged <15; age range, 2 to 70 years; mean
age, 16.6 years). Cytogenetic analysis identified 19 translocations;
12 cases had t(9;22) or variant translocations and 1 had
t(8; 14)(q24;q32). Five hyperdiploid cases were identified; however,
additional copies of chromosome 14 were only identified in 1 case.
Three of the four biphenotypic cases had t(9;22), the fourth was
bcr-abl negative as determined by reverse transcription-PCR (F. van
Rhee, personal communication, May 1994).
TCP-ALL cases consisted of 9 adults and 3 children (age range,
4 to 27 years; mean age, 18.6 years). Cytogenetic analysis identified
two translocations; one t(1 l; 14)(p13;qll) and one t(9;22)(q34;qll).
Clones were detected in 4 other cases. No cases were considered to
be biphenotypic.
AML cases consisted of 17 adults and 2 children (age range, 3
to 60 years; mean age, 33 years). Cytogenetic analysis identified
nine translocations including 2 with t(9;22)(q34;qll), and clones
were identified in an additional 5 cases. Two biphenotypic cases
were identified, both with t(9; 22)(q34; ql1).
In several cases, material obtained at diagnosis was also passaged in SCID mice:’ and DNA was extracted from serial passages for comparative analysis by Southern blot and PCR as described below.
DNA Blotting
High molecular weight DNA was extracted from a mononuclear
fraction of either bone mmow or peripheral blood by conventional
methods. High molecular weight DNA (15 pg) was digested for 3
to 4 hours at 37°C with restriction enzymes BglII, EcoRI, HindIII,
and BarnHI. Southern blotting was performed as described.’*Hybridization to a JH probe (clone C76R51A)” and, in selected cases, to
a TCRD probe JdS1623 and a CDKN2 probe” was performed. A
VH6gene probe kindly provided by Dr P.W. Tucker (Southwestern
University, Dallas TX)” was used to probe cases with apparent
biallelic DHJHrearrangement.
PCR
The following are the sequences of the primers used in this study.
FRIII PCR. FRIII PCR sequences are as follows: FRIII 5’ACACGGC(CT)(GC)TGTA”TACTGT-3’ ’*; and JH consensus 5’ACCTGAGGAGACGGTGACCAGGGT-3‘.’4
V, leader and FRI PCR. The VH leader PCR sequences were
as follows: vH1 leader, 5’-CCATGGACTGGACCTGGA-3’; VH2
leader, 5’-ATGGACATACTITGTTCCAC-3‘; vH3 leader, 5‘CCATGGAGTTTGGGCTGAGC-3’; V& leader, 5’-ATGAAA-
The FRI PCR sequences were as follows: VH1F R I , 5”CCTCAGTGAAGGTCTCCTGCAAGG-3’; and V H FRI,
~ 5”GGTCCCTGAGACTCTGCTGTGCA-3’13in combination with the JH consensus
primer.
DH PCR. The choice of DH primer was based on the fact that
the DHL,X families and the DHpszsegments are the most commonly
usedin BCP-ALL and TCP-ALL, respectivelyz5.”; therefore, the
primer was designed to the spacerheptamersequence 5’ of the DHL,
X, Q families. However, if other DH segments were used, these
would not be detected. If the PCR had amplified germJine DNA,
the smallest product obtained would have been 152 bp (the distance
between DHQS2
and JH1). All PCR products were smaller than this,
which precluded contamination with residual normal cells. The sequences used were as follows: DH primer, 5’-(GR)(GR)G(G/
A)GG(T/C)(T/C) TGT GTC ACT GTG-3’;J,1456 primer, 5’-ACCTGAGGAGACGGTGACC-3’; JH2 primer, 5’-GCTGGACAGAGAAGACTGGGA-3‘; and JH3 primer, 5’-AGCTCCAGGACAGAGGACG-3’.
PCR to check DNA integrity. PrimersfromtheacetylcoA
acetyltransferasegene were used tochecktheintegrity
of the
DNA; the sequences were as follows: 5’-GACGGGCTAACTGATGTCTAC-3‘ and 5’-ATTAGCCAAGCAGTTGCCAGC-3’.
All DNA samples used in this study were amplifiable with this
primer combination.
All reactions were performed in final volumes of 50 mL reaction buffer A (10 mmol/L Tris-HC1 [pH, 10.01, 50 mmol/L KCl,
2.0 mmol/L MgCI,, 0.1% Triton [FRIII/JH consensus PCR and
F R I V H ~ / JPCR]),
H
B (IO mmol/L Tris-HC1 [pH, 8.31, 50 mmol/
L KCl, 1.5 mmol/L MgC12,0.01% gelatin [V, leader/J, consensus
PCR andacetylcoAacetyltransferasePCR]),
C (10 mmollL
Tris-HC1 [pH, 9.01, 50 mmol/L KC], 2.0 mmol/L MgCIz, 0.1%
Triton [FRI VHl/JH consensus PCR]), or D (10 mmol/L Tris-HCI
[pH, 10.01, 50 mmol/L KC1, 1.0 mmoln MgCIz, 0.1% Triton
[DH/JH1456, DH/JH2,or DH/JH3PCR]). Eachreactionincluded
0.2 mmol/L of each deoxynucleotide triphosphate, 100 pmol of
each primer, 2 U of Taq Polymerase (Promega, Madison WI),
and 0.5 to 1 pg of sample DNA. Reactions were overlaid with
mineraloil,andcycling
was performed in a BiometraTRIOthermoblock(Gottingen,Germany).Denaturation
for allreactions was at 93°C initially for 3 minutes and during30 subsequent
cycles for 20 seconds. Annealing temperatures were as follows:
for FRIIUJH consensus PCR, FRI vH1, and 3/JHPCR, VH3,4, and
6 leader/J, consensusPCR,andacetyl
coA acetyltransferase
PCR, 55°C; for vH1 leader/JHconsensusPCR, 56°C; for VH2
leader/JHconsensus PCR, 58°C; for vH5 leader/J, consensus
6 50°C; and for DH/JH2PCR and
PCR, 59°C; for D ~ / J ~ 1 4 5PCR,
D H / J HPCR,
~
53°C.
A11 annealing and elongation reactions were performed for 20
seconds and 30 seconds, respectively, for 30 cycles, and the final
elongation step was performed at 72°C for 5 minutes. Appropriate
positive and negative controls were included, and each PCRwas
performed on at least two separate occasions. Aliquots of the reactions were analyzed by electrophoresis in 3%agarose and 8%nondenaturing polyacrylamide (FRIIVJHconsensus) or 10% nondenaturing
polyacrylamide (DH/JHPCR) or 1.2% agarose gel (V, leader/JHconsensus PCR) or 1.5% agarose gel (acetyl coA acetyltransferase PCR);
the aliquots were then stained with ethidium bromide and visualized
under UV light.
Cloning and Sequencing of PCR Products
Products of the FRIIVJH PCRandVHleader/J,PCR
reactions
were ligated into pGEM-T vector (Promega), XL1-Blue cells were
transformed, and 3 to 7 white colonies were picked. Double-stranded
sequencing was performed in both directions using the dideoxyCACCTGTGGTTCIT-3’; vH5 leader, 5’-ATGGGGTCAACCGCchain termination method with the ‘l7 and SP6 primers using Taq
CATCCT-3’; VH6 leader, 5’-ATGTCTGTCI‘CCTTCCTCAT-3’.’5DNA polymerase (Taquence 2.0; US Biochemicals, Cleveland, OH).
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5244
HEIGHT ET AL
Table 1. Clinical, Immunophenotypic, and Abbreviated Cytogenetic Data for 41 Patients With BCP-ALL Showing Results of DNA
Blot Hybridization WithJH and the No. of Products Obtained With FRlll and VH Leader PCR
Case
No.
Age and Sex
WBC
109/L
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
2f
21-17
2f
2m
3m
3f
3m
4f
4m
4m
4m
4f
4f
5f
5m
5f
6f
7m
llm
12m
14m
10.6
12.8
85
26.5
4.8
10.6
14.8
12.6
1.7
5.9
12.5
4.9
5.3
1.3
200
2.1
9.6
9.5
6.8
44.4
6.2
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
14m
14m
l4f
16m
18m
19f
19f
21f
21m
22f
26m
29m
36m
3 1m
40f
41f
50f
54f
60f
70f
2.2
325
132
8.4
8.0
4.4
37
19.6
10.2
3.4
4.9
97
47.2
13.8
10.4
2.7
1.o
17
12.8
112
lmmunophenotype
2
2
3
2MY'
2
2
2MY+
2
2
2
2
Karyotype
No diagnostic study
High hyperdiploid clone
46,XY,t(9;15)(pZ;ql)
No diagnostic study
High hyperdiploidclone
46,XX,de1(12)(pl)
Fail
47,XX,t(2;3)(q35;q25),+21 [c)
No diagnostic study
High hyperdiploid clone with de1(6)(q2)
45,XY,-9,add(ll)p(13-15)de1(15)(ql5)
2
2
2
381
3
5
2
2
4
3MY+
2MY'
5MY'
281
2
2
2MY'
3
46,XX,de1(6)(q2)
No diagnostic study
45,XX,add(12)(pl),-18
46,XY,t(9;22)(q34;qll)
46,XX,de1(6)(qlq2)
46,XX,de1(6)(q2?q5)
High hyperdiploid clone with trisomy 14
High hyperdiploid clone
ss,XY,del(2)(pZ),t(8;14)(q24;q32)
ND
+
ND
+
+
++
-
++
+
++
++
++
++
++
-
++
+
+++
ND
-
-
++
ND
-
-
+
+
+
++
+++
+
++
++
++
+
ND
+
+
46,XX,add(2)(q37),add(4)(q33),t(9;22l(q34;qll)
46,XY,t(2;9;22)(pll;q34;qll)
281
2
2
1
2
3MY'
46,XY,t~9;22)(q34;ql1~/47,idem,+der(22)
48,XY,+5,t(9;22)(q34;qll),+mar
46,XX,t(9;22)(q34;ql1)/46,idem,i(7)(plO)
46,XX,t(4;ll)(q2l;q23)
No clone
46,XX,t~9;22)(q34;ql1~,i(17)(p10~
44,XX,dic~9;12~~p13;p172~,der~16~t~16;17~~q22;q21~,-17
46,XX,t(9;22)(q34;qll)
+
++
++
de1(22)(q21)
Near-triploid clone
46,XY,t(17;22)(q25;qll)
2
5MY
+
V, Leader/
JH PCRt
-
2MY'
2MY'
281
2
FRIIII
JH
PCRS
~.XY.del~6~~q2lq25~.add~12~~q24~,add~14~~q32~,add~l7~~pl~,
46,XY.t(9;22)(q34;qll)
No clone
46,XX,t(5;12)(q31;p13)
46,XX.t(9;22)(q34;ql1)/46,idem,add(9)(p2)
Fail
48,XY,+X,-7,t(9;22)(q34;ql1),+15,+18
46,XX,t(9;12)(ql;pl)
Fail, bcr-abl-negative
2
DNA
Blott
+
+
++
+
++
++
+
+
+++
++
+
++
++
+
++
++t
c++
-
-
++
+
+
ND
++
++
++
+
+
++
++
++
++
++
ND
+++
-
-
+
++
The cases in which there is a discrepancy between theDNA blot and FRlll PCR data are shown in bold.
Abbreviations: f, female; m, male; ND, not done.
* For immunophenotype: CDlO-, CD22-, cytoplasmic p- ALL = 1; CDlO+ ALL = 2; cytoplasmic p+ ALL = 3; smlg+ ALL = 4; ALL not assigned
t o a subgroup = 5; one or two myeloidmarkers+, MY'; biphenotypic, 81.
t For DNA blot: G,germline; R, monoallelic rearrangement; R/R, biallelic rearrangement; R/R/R, oligoallelic rearrangement. A lower case "g"
or "r" represents weak band.
t For PCR: -, no PCR product obtained; +, single band identified; ++, two bands identified; +++, three bands identified.
To confirm that the observed deviations from germline VHsequences
Hall, Cambridge, UK) to identify V, and JH segments. DH segments
werenotanartifactdue
to Taqpolymeraseincorporationerrors,wereidentifiedaccording
to thecriteriadescribed."
DNA sequence was obtainedfrombetween 3 to 7 clones derived
from two separate
reactions;
PCR
no differences in DNA sequence
RESULTS
were observed. Sequencingreactionswere
runin 6% denaturing
polyacrylamide
sequencing
gels (8 m o m urea). Sequence analysis
A total Of 72
Of
leukemia were
iniwas
performed
using the Wisconsin Sequence Analysis Package
tially by Southem blot and m I 1 PCR. ne
for the
(Genetics ComputerGroup,Madison,WI)viacomputing
facilities
41 BCP-ALL cases are summarized inTable 1.
providedbytheHumanGenomeMappingResourceCenter(Hinxton
Nine cases (6 BCP-ALL and 3 TCP-ALL) with 15 re-
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EUKEMIA
IGH LOCUS IN ACUTE
Table 2. Results of DH/JH1456,J&? and JH3 PCR for Cases of BCP-ALL and TCP-ALL With IGHRearrangements by Southern Blot But
Negative PCR, With Both FRlll and VH Leader/& Consensus PCR Also Showing Resulis of TCRS DNA Blot
Case No.
2MY'
2
3MY'
Kawotyw
Immunophenotype*
BCP-ALL
7
12
14
21
IGH
DNA Blott
Fail
FRlll and
VH Leadar/JH
DdJH
PCRS
1456PCRS
-
-
-
-
ND
ND
+
-
D~JIN~D~
DN2D3
-
+
-
GIG
GIG
-
+
-
DNIJI
+
-
-
ND
ND
g/R/R
G/R/R
46,XY,de1(6)(q21q25),add(12)(q24).add(14)(q32),
glR1R
-
+
g/R/R
g/R
-
++
-
-
DdJH3 TCR6 DNA
PCRS
Blott
-
-
46,XX,de1(6)(q2)
-
DdJJ
PCRS
-
add(17)(pl).de1(22)(qZl)
2MY'
31
40
TCP-ALL
43
7
49
51
2
46,XX,t(9;12)(ql;pl)
44,XX,dic(9;12)(p13;p1?2),der(l6~t(16;17~
(q22;q21),-17
7MY+, LY'
G/R/R
46,XX,del(6)(ql5q33~,t~9;22~~q34;qll~,del~ll~~q14~,
6
de1(12)(p13)/46,idem,del(l7)(pl)
Fail
ND
RIR
SIR
-
-
-
-
-
* For immunophenotype: CD10'BCP-ALL = 2; cytoplasmic b+ BCP-ALL = 3; CD2- TCP-ALL = 6; CD2+TCP-ALL = 7; one or two myeloid
markers', MY+; one or two lymphoid markers', LY'.
t For DNA blot: D, deleted; G,germline; R, monoallelic rearrangement; RIR, biallelic rearrangement; WRIR, oligoallelic rearrangement; lower
case "g" or "r" represents weak band.
For PCR: -, no PCR product obtained; +, single band identified; ++, two bands identified.
*
arranged IGH alleles on DNA blot completely failed to amplify with FRIII PCR. Furthermore, a total of 25 of 78 (32%)
rearranged alleles observed on Southern blot failed to amplify by this method. To investigate the apparent failure of
FRIII PCR, further experiments were performed, initially,
using the VH leader family primers with the JH consensus
primer and, subsequently, using a DH primer with JH-specific
primers in cases that remained persistently VHPCR-negative.
The results have been analyzed according to different subgroups; (1) the 9 FRIII-negative cases, (2) the 14 Philadelphia chromosome-positive (Ph+) cases, and (3) cases with
discrepancies between the DNA blot and FRIII PCR results.
FRIZZ-Negative Cases
A total of 9 cases (6 BCP-ALL and 3 TCP-ALL) with
IGH rearrangements by DNA blot failed to amplify with the
FRIII PCR. All 9 cases also failed to amplify with all the
V H leader primers. In the absence of cytogenetically demonstrable translocations involving the IGH locus at 14q32.3 in
6 of the 9 cases, these data strongly suggested biallelic DHJ H rearrangement. Such DH-J H rearrangements were confirmed first by the use of a VH6-specificprobe on Southern
blot; all cases showed retention of this gene, confirming the
lack of more distal VH rearrangement that would result in
v H 6deletion. Second, PCR with a consensus DH primer that
should amplify the most commonly used DH segments was
performed. The results of analysis with DH PCR are shown
in Table 2. DH PCR products were obtained in 6 of 9 cases
representing 7 of 15 rearranged IGH alleles. DH-JH rearrangements have been previously reported as a recurrent rearrangement in TCP-ALL with IGH rearra~~gernent.~'
Therefore, further analysis focused on the 6 cases of BCP-ALL.
There were no consistent cytogenetic or immunophenotypic features in this group. However, all 5 cases that were
successfully karyotyped showed structural abnormalities,
and3 had abnormalities of chromosome 12p. CD34 was
positive in 3 and negative in 2 cases analyzed. Analysis of
the TCRD rearrangements was performed in 4 cases and
showed germline configuration in 2 and incomplete V2D3
rearrangements (the configuration associated withBCPALL)28in 2, 1 of which was associated with deletion of the
other allele and the other which occurred in combination
withan incomplete TCRD D2J1 rearrangement. All cases
retained both alleles of CDKNZ in germline config~ration.'~
Ph+ Cases
Of 72 cases, 14 were Ph+; these included 9 BCP-ALL, 1
TCP-ALL, and 5 biphenotypic cases. Excluding the case of
Ph+ TCP-ALL that has been discussed above (Table 2, case
no. 43), this group showed a total of 23 rearranged alleles
by Southern blot; of these, 19 (76%) were amplified by FRIII
PCR. Of the remaining 4 alleles, all 4 amplified with the VH
leader primers. An unexpected finding was the detection of
2 and 1 additional clonal rearrangements in 2 cases of BCPALL (cases no. 23 and 39, respectively). In addition, PCR
on SCID mouse passage material from a further BCP-ALL
(case no. 33) also led to the identification of 2 newrearrangements (33 c and d) in addition to those present in the diagnostic material (33 a and b). Cloning and sequencing did not
show any evidence of a clonal relationship, eg, VH-VH replacement or open-and-shut m e ~ h a n i s m ,between
~ ~ * ~ ~the different clones in these 3 cases that appeared to be totally
unrelated (Fig 1). In contrast, analysis of 21 Ph- BCP-ALL
cases did not lead to the detection of any additional unexpected rearrangements.
Causes for Discrepancies Between DNA Blot and FRIII
PCR in BCP-ALL
Apart from the 6 FRIII PCR-negative cases discussed
above, comparison of the DNA blot and FRIII PCR data
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HEIGHT ET AL
5246
CASE
FRIll PRIMER SITE
D
N
N
J N CONSENSUS PRIMER
Is3
0 % ~ In
Vn
ACACGGCCTGTA'ITACTCT
23.
&p&!+CCTOTOTATIrcTG'K
C T A c m o T C C ? T C ~ ~ f f ~ f f i T ~ C C ~ TH S~i V lCl b ~ T ~ DKf
C ~ ~ ~ 2~
TCAGCCATAGT-CCCCA
23c A C ~ M C C C O T O I A I I ~ C T O I O C O P I O P I O * C I CACAGACTACGATITTEGAGIOGTTATTWAGTTG
13.
CCGTGCCCr25AGTATA~AGTaorrarocnnccccowrcc
1lc ACACWCTQIUTATIAC'ZOTCCA
39
.
MOATCCAC"?TWITATTAl"GCAAG
a a m d
A
C
T
A
A
C
-
T
I
1
-
O
~
A
~
C
C
T
A
=
~
~
~
~
~
I
C
~
C
~
C
~
3-53
I ~
I
~
C3 - ~
23 I
WCCAUYjlUCC~TCICCUTCTCCICIQO1
ADGOCGTA
3 9c &qCACO(ICTOICTATIACTUTG
A
T
~CTlll'GAT0Tcx;ooocclu~CTCTGUICACCUTCTC r4WI
TCAMA
19b LCACWCTOTUTATTACTQTGC
6b
~ A ~ ~ ~ C ~ ~ C X ~ ~ C ~ ~ ~ U C C C I 3-7
~ ~ DCI RCx ~
U V TD LC R I~ ~C4V8 C T ~ ~ T
=
T
AGACTCCOGCGGCXOSTG-
Ild A C I C O O C T 0 T U T A I I A C I O ~
DW.
1-18
mCTACWTATffiAffiTr;Tcmo04ccm
TAAGAGAn%l\WLTACCACCAGTWCTCT
ATACUOCIVTQIATTACTGIG
31b ACACQOCCUIOIAT
4b
U X X i C C A G W 2 M C C C m O3T4U4C C O Z C I M U O O r
TGCCACUC
llb A C A C I Q T C C T C ~ G C O A G A G
SAACIYXj~CGACCCC~-CCCmOTUCEOC
^kW1
1-18
~DWt.DWlmv
~ I
~
6b
DA
C ~ ~ O 6b
O ~
DWO
3a
1-2
4b
5b
3.7
O G ~ C O A C C C ~ C C C ~ T ~ C W T C I C 1~- 2~3 O O DT W l
TAACACACTCGTCA-ACGC
5b
Fig 1. Continuing VH-DHJH rearrangementsin Ph+ BCP-ALL (sequence
of cloned CDRlll regionst. FRlll and
JHprimer sequences are underlined
in bold facing. Homology to VH segments 3' of FRlll primer site are shown attached to FRlll primer site. DH and JWsegment homology is
underlined; mismatches are shownnot underlined. N regions are indicated attnched to DH segments h o t underlinadl. Gene segment usage
is shown to the right of each CDRlll sequence. Where several products were obtained, a11 CDRlll sequences are shown.
identifiedatotal of 14 patientswith BCP-ALL inwhom
there was a discrepancy between the number of rearranged
alleles detected by the two methods. In all cases, fewer rearranged alleles were detected by FRIII PCR than were predicted by Southem blotting. Intotal, 16 of29 rearranged
alleles were detected by FRIII PCR in these cases. Table 3
shows the correlation of the two series of results.
To determine the possible causes of failure of FFUI PCR,
VH leaderPCR was performed and the products cloned and
sequenced. A total of 13 BCP-ALL cases with concordant
DNA blot and FRIII PCR results were also studied in parallel. PCR using the VHleader primers led to the detection of
a higher proportion of rearranged alleles in these cases (26
of 29 [90%]). In 9 of 14 cases, VHleader PCR identified the
corresponding number of products predicted by Southern
blot, and, by cloning and sequencing the products, it was
possible to identify the reasons for the failure of FRIJI PCR
in these cases. However, in 4 of 14 cases, fewer rearranged
alleles were amplified than expected; these included cases
no. 9, 16,20, and 22 in which only one product was obtained
although tworearrangements were known to be presentfrom
the DNA blot data. However, one allele in case no. 20 would
not be expected to amplify because of its involvement in
the t(8;14)(q24;q32). In 1 of 14 cases (case no. 39), more
rearrangements were detected by VH leader PCR than were
predicted from DNA blot.
Sequencing of the products of the VH leaderPCR in cases
with discrepancies showed deletion of between 3 and 18
bases from the 3' end of the FRIII primer site. In 9 other
cases, mutations within the primer site were also identified,
but these did not affect the FRIII PCR. Sequencing of the
cases with concordance between DNA blot and FRIII PCR
showed intact FRIII primer sites.
Gene Segment Usage in BCP-ALL
Analysis of 33sequencedrearranged VHDHJH products
(Table 4) showed a bias in the VHsegment usage. The vH3
family accounted for 15 of the sequences, 3 of which had
homology with VH3-23, 2 with
VH3-22(and shared thesame
base-pair mismatches atthe3'
end) and2withVH
3-7.
Within the 8 vH1 family products, VH 1-18 was represented
three times, and, of the 8 V& sequences, 3 shared homology
with VH 4-34 and 2 with VH 4-31.
Of note was the position of some of the VH segments in
relation to J H in the germline; it has been suggested that
developmentalmechanisms operate inthegenerationof
Table 3. Correlation Between Numberof Rearranged Alleles Detectod by Southern Blotting andFRWJHPCRin BCP-ALL
Southern Blot Results
FRllI/JH PCR Result
PCR-negative
1 PCR product
2 PCR products
3 PCR products
Total no. of cases
Germline
Monoellelic
Rearrangement
Biallelic
Rearrangement
Oligoallelic
Rearrangement
Total
(GIG)
(GIRI
-
1
6
-
(m)
5
12
14
(GIRIWR)
No. of
Cases
-
6
18
2
16
1
41
-
-
-
1
0
7
31
3
The cases in which there is concordance between the Southern blot and FRlll PCR results are shown in bold.
Abbreviations: G, germline; R, rearrangement.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
52 47
IGH LOCUS IN ACUTELEUKEMIA
Table 4.
V. Family
1
1
l
1.6
0.6
1.5
1.4
1
1
1
1
1
2
3
3
3
3
3
3
3
3
3
3
3.6
3
3
3
3
3
4
4
4
4
4
4
4
4
7
vHGene Sequent Usage, Mismatches From Germline Sequences, and ORFs from33 Fully SqUenCd VH PCB Products
VH Segment
Hslg V~152
HsV14lb
1-18
HumlgHDLN
1-18
1-18
1-2
1-46
HumlgVH2F
3-7
3-13
3-43
3-30
3-53
3-23
3-64
3-22
3-16
3-22
3-53
3-23
3-7
3-23
HslgDP34
Hsu03896
4-61
4-34
4-34
4-39
4-31
4-31
4-34
7-56
96 Mismatch
ORF
2.8
4.7
1.8
0.3
0
0
1.1
0.2
1.5
-
1.5
1.2
2.9
3.3
V,'Deletions/lnsertions*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
9.7
2.9
4.3
3950.6
l.3
3.5
0
0
1.o
2.3
2.2
?
DEL 87bp (48-135)
-
Distance From JH Ikb)
?
160
310
?
310
310
125
640
DEL 13bp (188-201)
190
260
600
470
725
395
790
385
290
-
-
+
-
-
DEL 1bp(248.281) 385
INS 1bp (327)
-
725
+
-
-
-
190
395
-
-
-
-
-
-
-
-
-
-
(165-168)
-
DEL 4bp
800
?
760
505
505
550
470
470
505
740
-
-
-
-
-
-
-
-
-
~
Abbreviations: ?, unknown; +, ORF maintained; -, loss of ORF; DEL, internal V" deletion; INS, VH insertion.
* Numbers indicate the position of the deletion or insertion within the germline sequences, as given in Cook and T ~ m l i n s o nOnly
. ~ 3 of 33
alleles analyzed retained an ORF. These were derived from case no. 20 [slg' B-ALL with t(8;14)(q24;q32)] that showed a VH 3-23 genewith 3.3%
mismatch from the germline sequence; case no. 24 [clg'/slg- CD10'
biphenotypic leukemia with t(9;22)(q34;qll)l that showed a VH~-16
gene
with 9.7% mismatch from the
germline, and case no. 39(a pre-6-cell, [CD19+/CD10~16CP-ALLwhichcoexpressed myeloid antigens) that showed
a VH3-7 gene with 1.3% mismatch from the germline. All other sequences showed mutations, insertions, or deletions within the V" genes
resulting in loss of the ORF. There was no obvious difference in frequency of VH mutation according to VH genefamily.
VHDHJH rearrangements in BCP-ALL, leading to preferential
utilization of the gene segments closest to J H . ~However,
~ * ~ ~
in this study, only 4 of 33 rearrangements used VH gene
segments less than 200 kb from J H ; 6 rearrangements used
gene segments from the distal, telomeric region of the VH
locus, at least 600 kb from J H (Table 4).
Of the 33 VFb-JH rearrangements sequenced, 3 retained
an open-reading frame(ORF). In the other 30 rearrangements,
the ORF was lost because of stop or missense codons distributed throughout the length of theVHgene andor internal deletions within the VHgene. For example, case no. 28 was found
to have a VH2-DLR4-JH6brearrangement, with a deletion of
87 bp. This deletion commenced at the VHintron-exon boundary and included the whole intron with the exception of the
last base; the remaindexof the VHgene was intact but showed
deviation from the most similar germlinevH2 gene. Deviation
from known germlineVHgenes was detected in
29 of 33 (88%)
VH-DH-JH rearrangements sequenced, the percentage deviation
ranged from 0.3% to 9.7%(mean, 2.3%). In all cases, whether
O W + or OW-, the point mutations were not clustered within
the CDR regions but were distributed along the length of the
VH gene^.'^,^^
Analysis of DH segment usage showed that the DXP and
DLR families represented 29% and 24% of rearrangements,
respectively. DH-DH fusions were present in 8 of 41 (19%)
sequences, and no DH segment was identifiable in 5 of 41
(12%). Analysisof J H segment usage showed that 53%, 25%,
and 15% of rearrangements used JH4,JH6and JH5,respectively, with underrepresentation ofJ H 1 , 2 , and 3. There were
no mutations in the J H segments. Of 41 BCP-ALL sequences,
5 (12%) had no N regions or a P nucleotide at the DHJH
junction, but all cases showed N regions at the VHDH junction. Terminal deoxynucleotidyl transferase was expressed
in all cases.
DISCUSSION
The impetus to this studywasthat in BCP-ALL some
rearranged IGH alleles and, indeed, up to 20% of cases fail
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
5248
totally to amplify with FRIWJH PCR primers. Unlike the
non-Hodgkin lymphomas, in which translocations to the IGH
locus are common, such translocations are rare in BCP-ALL
and cannot be implicated in the high failure rate. Therefore,
we sought to determine the causes of FRIII PCR failure in
this disease by comparing the results obtained by DNA blot
(which allowed the number of rearranged IGH alleles to be
estimated) and those obtained byPCR methods using VH
leader, FRIII and D,-specific primers. Second, we sought to
determine whether total absence of signal with FRIII primers
correlated with a specific subgroup of BCP-ALL.
Of the 37 cases of BCP-ALL, 6 (16.2%) were FRIII PCRnegative. These cases showed 11 rearranged IGH alleles by
DNA blot, of which 5 could be amplified with a consensus
DH primer and, therefore, represented DH-JH rearrangements.
The failure to amplify the remaining 6 alleles probably represented a failure of the consensus DH primer; this primer was
designed to amplify the DH segments used in about 60% of
rearranged IGH alleles in BCP-ALL.25However, the failure
of the VH leader primers to amplify successfully any of
the rearranged IGH alleles in the absence of cytogenetically
detectable translocations and the retention of an intact VH6
gene in all cases makes it likely that most, if not all, of these
rearrangements represented DHJH rearrangements.
Because VHDHJH recombination is developmentally regulated, this implies a developmental arrest of the neoplastic
B-cell clones before the activation of the full VHDHJH recombinase. Moreover, because VHDH and DHJH recombinations
occur very early in B-cell differentiation, it might be anticipated that these cases might show a less differentiated immunophenotype and a consistent cytogenetic abnormality. This
wasnot the case, although 3 of the 5 cases successfully
karyotyped showed abnormalities of 12p. Whether the subgroup thus defined wasassociated with a particular therapeutic outcome could not be determined from this small sample.
It was noteworthy that all 6 retained germline p16KDKN2
gene^.*^,'^
Otherwise, of the 14 other cases in which a discrepancy
between DNA blot and FRIII PCR was observed, the use of
the 6 VH leader-specific primers increased the detection of
IGH rearrangements from 55% observed with FRIII PCR to
90% in this group. This may be of importance in monitoring
BCP-ALL for residual disease in which morethan one
marker may be necessary to follow the neoplastic clone with
confidence. Sequencing of the rearranged VH genes that
failed to amplify with the FRIII primers showed that this
was due in all cases to the loss of the 3’ portion of the primer
site, presumably as a consequence of exonuclease activity
at the time of recombination.’ It was not possible to design
a further more 5‘ consensus FRIII primer to overcome this
phenomenon.
Sequencing of 33 of the rearranged VH genes showed a
number of other interesting features. First, mutations (or
strictly deviations from the known germline VH sequences)
and deletions of the rearranged VH genes were detected in
29 (88%) cases. The mutations werenot clustered in the
CDR regions but were found throughout the VHgenes. Only
4 sequences were completely identical to previously sequenced VHgenes. Because the corresponding germline VH
genes were not sequenced, we cannot exclude the possibility
HEIGHT ET AL
that some of the rearranged VH sequences might represent
previously undescribed polymorphisms. However. VH sequencing data from normal individuals have indicated that
such polymorphisms are uncommon.’ Furthermore, it was
unlikely that these rearrangements represented the involvement of VH “orphan genes” on chromosomes 15 and 16,
because this would have required translocation to the JH
segments of the IGH locus at 14q32.3; no such translocations
were observed. Given that the mean deviation from known
germline sequences was 2.3%, this deviation could also not
be explained by Taq DNA polymerase incorporation error
alone. Moreover, several clones from different PCRreactions were sequenced in each case with identical results.
Similar mutations have been previously reported in a series of 9 cases of BCP-ALL studied by FRIPCR.” The
frequency of nucleotide changes within the VHgenes varied
from 0.2%to2.4%, and, as reported here, the mutations
were not clustered to the CDRs but were scattered throughout
the length of the VH genes. In contrast, it should be noted
that 6 rearranged VHgenes (isolated by genomic phage cloning from 2 patients with multiple JH rearrangements) showed
no evidence of somatic mutation.” The reasons for these
differences are not clear. However, the combination of our
data on 33 rearranged VHgenes along with those of Kitchingman” and the observation that somatic mutation can occur
in virally transformed mouse pre-B cells’’ strongly suggests
that somatic mutation canoccur in at least some BCPs before
the expression of surface Ig. Whether the mechanism is identical to that which occurs in more mature B cells after exposure to antigen and results in affinitymaturation of antibody”
isnot known. The lack of clustering of mutations to the
CDRs in BCPs might be anticipated because of the lack of
Ig expression.
A second feature of interest was that only 3 of 33 (9%)
VH-DH-JH genes sequenced in this study retained an ORF,
in part because of the presence of both the mutations and
deletions resulting in stop or missense codons distributed
throughout the VH gene. This percentage of rearrangements
with an O W would be expected of a population of B cells
unselected by antigen. However, a functional Ig molecule is
necessary for the later stages of B-cell maturation7;therefore,
these data indicate that most BCP-ALL represent the neoplastic transformation of BCPs destined to die in the bone
marrow. This is in contrast to the observations of Steenbergen et aP7 who,on the basis of analysis of the CDRIII
product alone, showed maintenance of an ORF in 17 of 68
(25%) cases. Here, however, by the use of VHleader primers,
it was possible to show other causes for the loss of the ORF
in rearranged VHgenes. The transformation of BCPs destined
to undergo apoptosis as a result of failing to complete inframe VH-DH-JH recombination might explain the high frequency of TCR gene rearrangements in these cases, arising
through persistent expression of recombinase.
Finally, as regards VHgene usage, there was no evidence
in this study for preferential usage of JH-proximalVHgenes.
Instead, VHgenes were scattered throughout the IGH locus,
and 36% of rearrangements occurred with VHsegments more
than 500 kb distal of JH. As with other studies on normal Bcell populations VH3-23(which constitutes 26% of the 500
rearranged VH3genes in the rearranged database3) was also
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
IGH LOCUS IN ACUTELEUKEMIA
5249
3. Cook GP, Tomlinson I M : The human immunoglobulin VHrepoverrepresented in this series of BCP-ALL, being observed
ertoire.
Immunol Today 16:237, 1995
in 3 of 33 cases. There may be some structural reason for
4. Rolink A, Melchers F Generation and regeneration of cells of
the selection of this gene segment. DH and JH gene segment
the B-lymphocyte lineage. C m Opin Immunol 5:207, 1993
usages were consistent with those previously rep~rted.~.~’
5. Ehlich A, Schaal S, Gu H, Kitamura D, Muller W, Rajewsky
The causes for the preferential selection of the 5 ~ and
4 the
K:Immunoglobulin heavy and light chain genes rearrange indepenJ H segments
~
are unknown. Five sequences were identified
dently at early stages of B-cell development. Cell 72:695, 1993
that lacked N regions at the D H - J H junction, which is suppos6. Yancopoulos GD, Alt FW: Regulation of the assembly and
edly a feature of “primitive” rearrangements; and, although
expression of variable-region genes. Annu RevImmunol4:339,1986
7. Kitamura D, Roes J, Kuhn R, Rajewsky K:A B-cell deficient
one sequence was isolated from a 4-year-old, the remainder
mouse by targeted disruption of the membrane exon of the immunowere found in adults with Ph’ leukemia, suggesting that in
globulin p chain. Nature 350423, 1991
these leukemias the rearrangement mechanism retains some
8. Osmond DG: The turnover of B-cell populations. Immunol
featuresofthatwhich
occurs in very early fetal B cells.
Today
14:34, 1993
However, all cases showed N regions at the VHDH junction.
9. van Dongen JJM, Wolvers-Tetter0 ILM: Analysis of immunoThirteen cases of BCP-ALL and biphenotypic acute leukeglobulin and T cell receptor genes. Part 11: Possibilities and limitamias with t(9;22)(q34;qll) showed 23 rearranged IGH altions in the diagnosis and management of lymphoproliferative disleles; all 23 were successfully amplifiedwith either theFRIT1
eases and related disorders. Clin Chim Acta 198:93, 1991
andor the VH leader primers, thus implying full VHDHJH
10. Lafaille JJ, DeCloux A, Bonneville M, Takagaki Y, Tonerecombination. This was confirmed by DNA sequencing of
gawa S : Junctional sequences of T cell receptor y6 genes: implica21 rearranged VH genes. Therefore, these data indicate that
tions for y6 T cell lineages and for a novel intermediate of V-(D)J joining. Cell 59:585, 1989
the acquisition of the t(9;22)(q34;ql l), which is thought to
11. Yamada M, Hudson S, Toumay 0, Bittenbender S , Shane
occur either very early in B-cell differentiation or at the level
S S , Lange B, Tsujimoto Y, Caton AJ, Rovera G: Detection of miniof the multipotent hemopoietic stem cell, nevertheless, did
mal disease in hemopoietic malignancies of the B-cell lineage using
not influence the activity of the VHDHJH recombinase. This
third-complementarity-determiningregion (CDRII1)-specific probes.
is in contrast to theBCP-ALL arrested at the stage of D H J H
Proc Natl Acad Sci USA 86:5123, 1989
rearrangement.
12. Potter MN, Steward CG, Oakhill A: The significanceof detecFurthermore,that VHDHJH recombination may continue
tion of minimal residual disease in childhood acute lymphoblastic
in BCP-ALL with t(9;22)(q34;qll) was suggested
by the
leukaemia. Br J Haematol 83:412, 1993
presence of 3 cases in which additional VH rearrangements
13. Deane M, Norton JD: Immunoglobulin heavy chain variable
leader
unanticipated from Southern blot were detected by VH
region family usage is independent of tumor cell phenotype in human
B lineage leukaemias. Eur J Immunol 20:2209, 1990
PCR. These additional subclones represented “new” VH re14. Deane M, Norton JD: Preferential rearrangement of developarrangements rather than VH-VH replacements in which the
mentally regulated immunoglobulin VH1 genes in human B-lineage
D H - J H segment usually remains intact. In 1 case, the addileukaemias. Leukemia 5:646, 1991
serial passage of the
tional subclones were detected after
15. Campbell MJ, Zelenetz AD, Levy S , Levy R: Use of familyBCP-ALL in SCID mice. Such additional subclones were
specific leader region primers for PCR amplification of the human
not detected in 21 other cases lacking the t(9;22)(q34;qll)
heavy-chain variable region gene repertoire. Mol Immunol 29:193,
and have not been observed in other t(9;22)-negative cases
1992
passaged in SCID mice. These data may indicate thatin
16. Brisco MJ, Condon J, Hughes E, Neoh SH, Nicholson SH,
some cases of BCP-ALL the t(9; 22)(q34;ql l) occurs before
Sykes PJ, Tauro G , Ekert H, Waters K, Toogood I, Seshadri R,
the activationofthe
VHDHJH recombinase; the persistent
MorleyAA, the Australian andNew Zealand Children’s Cancer
expression of the VHDHJH recombinase acting on the germStudy Group: Prognostic significance of detection of monoclonality
in remission marrow in acute lymphoblastic leukemia in childhood.
line IGH genes of the leukemic “stem cell” population may
Leukemia 7:1514, 1993
then account for the generation of new VHDHJH rearrange17. Hayashi Y, Pui C-H, Behm FG, Fuchs AH, Raimondi SC,
ments during the course of the disease.
Kitchingman GR, Mirro .I, Williams DL: 14q32 translocations are
associated with mixed-lineage expression in childhood acute leukaeACKNOWLEDGMENT
mia. Blood 76:150, 1990
18. Dyer MJS, Heward JM, Zani VJ, Buccheri V, Catovsky D:
We gratefully acknowledge the expert assistance of our colleagues
Unusual deletions within the Ig heavy-chain locus in acute leukeM. Dainton and T. Miu in the production of the karyotypes reported
mias. Blood 82:865, 1993
in this study. We thank the following for kindly providing DNA
19. Buccheri V, Matutes E, Dyer MJS, Catovsky D: Lineage
probes: Dr T.H. Rabbitts (Laboratory of Molecular Biology, Camcommitment in acute leukaemia. Leukemia 7:919, 1993
bridge UK) for the Ig JH and TCRD J6S16 probes and Gail Stranks
20. Catovsky D, Matutes E, Buccheri V, Shetty V, Hanslip J,
for the CDKNZ probe. We thank Dr P. Mitchell for providing passage
Yoshida N, Morilla R: A classification of the acute leukaemias for
material from SCID mouse xenografts.
the 1990s. Ann Haematol 62:16, 1991
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1996 87: 5242-5250
Analysis of clonal rearrangements of the Ig heavy chain locus in
acute leukemia
SE Height, GJ Swansbury, E Matutes, JG Treleaven, D Catovsky and MJ Dyer
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