Leukemia (1997) 11, 1793–1798
 1997 Stockton Press All rights reserved 0887-6924/97 $12.00
BTS TECHNICAL REPORT
BTS
Leukemia
A novel nested-PCR strategy for the detection of rearranged immunoglobulin heavychain genes in B cell tumors
C Voena1, M Ladetto1, M Astolfi1, D Provan2, JG Gribben2, M Boccadoro1, A Pileri1 and P Corradini1
1
Dipartimento di Medicina ed Oncologia Sperimentale, Divisone Universitaria di Ematologia, Azienda Ospedaliera San Giovanni Battista, Torino,
Italy; and 2Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA
Several methods have been developed for the detection of
minimal residual disease (MRD) in B cell tumors. Chromosomal
translocations or the rearrangement of the immunoglobulin
heavy chain (IgH) and T cell receptor genes are generally
employed. We report a novel PCR method to detect MRD using
IgH genes. IgH rearranged variable region (VDJ) were amplified
from tumor specimens using consensus primers for variable
and joining region genes. Complementarity-determining
regions (CDR) were identified and used to generate tumor-specific primers. Two-round amplifications using primers derived
from CDRs and joining or constant regions were performed for
MRD detection. IgH nested-PCR approach was tested on a
panel of 75 B cell tumors including acute lymphoblastic and
chronic lymphocytic leukemias, non-Hodgkin’s lymphomas
and multiple myelomas. A VDJ sequence was obtained in 62
out of 75 cases (83%). Sensitivity using DNA or cDNA templates
was 10−5 and −6, respectively. This method is specific and sensitive and provides a simple, non-radioactive approach for the
evaluation of MRD in B cell tumors.
Keywords: B cell malignancies; IgH nested-PCR; minimal
residual disease
Introduction
B cell malignancies are a heterogeneous group of disorders
characterized by the proliferation of clonal cells at various
stages of differentiation. In recent years, intensified chemoradiotherapy programs followed by autologous or allogeneic
bone marrow transplantation have been successfully
employed for the treatment of these tumors. Although complete remissions (CR) are often achieved, disease relapse
remains the leading cause of death in these patients.1–3
The analysis of minimal residual disease (MRD) has
assumed a growing role in the evaluation of tumor cell contamination of autografts and during molecular follow-up of
patients otherwise in CR.4–6 It has recently been shown that
patients with chronic myelogenous leukemia, acute
promyelocytic leukemia, acute lymphoblastic leukemia or follicular non-Hodgkin’s lymphoma may remain PCR-positive
after chemotherapy or transplantation procedures. The persistence of such a positivity has been correlated with a higher
frequency of relapse.5–8
Correspondence: C Voena, Cattedra di Ematologia, Via Genova 3,
10126 Torino, Italy
Received 17 February 1997; accepted 16 June 1997
In B cell malignancies, several methods have been
developed for the detection of MRD.9 Tumor-specific breakpoints of chromosomal translocations and clonal rearrangements of immunoglobulin heavy chain (IgH) genes are used
as molecular markers.4,5,10,11 The chromosomal translocations
most frequency employed are the t(14;18) present in about
50% of B cell non-Hodgkin’s lymphomas (NHL), the t(9;22)
present in 5% of childhood and 30% of adult acute lymphoblastic leukemias (ALL), the t(1;19) in 25% pre-B ALL, the
t(4;11) in 2–5% ALL and the recently described t(12;21) in
25% childhood precursor B-ALL.5,12–16 Moreover, chromosomal translocations can be employed only in a subset of
patients with B cell malignancies whereas the rearrangment
of IgH genes may provide a tumor marker in about 90% of
patients since it is a lineage-specific marker.
During B cell ontogeny, the genes that encode for IgH
undergo rearrangement of variable (V), diversity (D) and joining (J) segments, to generate antigen-specific receptors. The
rearranged variable region (VDJ) is composed of four relatively
conserved regions, called framework (FR), interrupted by three
complementarity-determining regions (CDR). CDRs codify for
the antigen-binding site and are unique for each B cell
clone.17 In B cell malignancies, CDRs are utilized to design
patient-specific primers and probes for the amplification and
hybridization procedures for MRD detection.11,18 Although
such procedures are sensitive and specific, the hybridization
step is expensive, time-consuming and requires the use of a
radioactive labeled probe. Nested-PCR has been recently used
for MRD detection in order to bypass the hybridization step.
Nested-PCR is a simple nonradioactive procedure which is
currently used when tumor-specific translocations are present,
providing the same sensitivity and specificity of amplification
hybridization procedures.19
In the present study, we show a nested-PCR approach for
the detection of MRD using IgH genes. Double amplification
using specific primers derived from tumor CDRs (CDRII and
CDRIII) has been employed. Our approach has been tested
on a panel of 75 B cell tumors including seven ALL, 22 NHL,
nine chronic lymphocytic leukemia (CLL) and 37 multiple
myeloma (MM).
BTS Technical Report
C Voena et al
1794
Table 1
Amplification of the tumor VDJ primers derived from the leader region
VH.LBa
VH1
VH2
VH3
VH4
VH5
VH6
5′-CCATGGACTGGACCTGGAGG-3′
5′-ATGGACATACTTTGTTCCAG-3′
5′-CCATGGAGTTTGGGCTGAGC-3′
5′-ATGAAACACCTGTGGTTCTT-3′
5′-ATGGGGTCAACCGCCATCCT-3′
5′-ATGTCTGTCTCCTTCCTCAT-3′
VH.LSb
VH1
VH2
VH3
VH4
VH5
VH6
5′-CTCACCATGGACTGGACCTGGAG-3′
5′-ATGGACATACTTTGTTCCACGCTC-3′
5′-CCATGGAGTTTGGGCTGAGCTGG-3′
5′-ACATGAAACA(C/T)CTGTGGTTCTTCC-3′
5′-ATGGGGTCAACCGCCATCCTCCG-3′
5′-ATGTCTGTCTCCTTCCTCATCTTC-3′
a
See Ref. 22.
See Ref. 23.
b
Materials and methods
Patients and nucleic acid extraction
Seventy-five patients with B cell tumors were selected for this
study. PB, BM and lymph node samples were obtained during
standard diagnostic procedures. They had different degrees of
tumor infiltration, ranging from 2 to 90%. BM and/or PB cells
were separated on a Ficoll–Hypaque density gradient. DNA
was obtained by cell lysis, phenol extraction and ethanol precipitation.20 RNA was isolated using RNAzol B method
(Biotex Laboratories, Houston, TX, USA). Total cDNA synthesis was carried out as follows: total RNA (5 mg) was
reverse-transcribed with 20 pmol of reverse transcription
primer (Cm1: 5′-AAGGAAGTCCTGTGCGAGG-3′, Cg1: 5′GGAAGTAGTCCTTGACCAG-3′, Ca1: 5′-AGAAGCCCTGGACCAGGCA-3′). A 50-ml reaction was performed in
10 mmol/l dithiothreitol, 1 mmol/l deoxynucleoside triphosphates (dNTPs; Pharmacia LKB Biotechnology, Uppsala,
Sweden), 1 3 reverse transcriptase buffer (50 mmol/l Tris-HCl,
6 mmol/l MgCl2, 40 mmol/l KCl) final concentration, adding
20 U of ribonuclease inhibitor (RNasin; Promega, Madison,
WI, USA) and 200 U Moloney murine leukemia virus reverse
transcriptase (Superscript; GIBCO BRL, Gaithersburg, MD,
USA). The reaction mixture was incubated for 1 h at 37°C.
After cDNA synthesis, reverse transcriptase was heat-inactivated for 5 min at 95°C.
Amplification and sequencing of the tumor VDJ
VDJs were amplified starting from genomic DNA or total
cDNA depending on sample availability. Amplifications were
performed as previously described.18 Briefly, 1 mg of genomic
DNA or 1 ml of total cDNA, was amplified using two sets of
consensus sense primers derived from the IgH leader region
and FR1 respectively21–24 (Tables 1 and 2), and an antisense
Table 2
primer derived from the JH 3′ end (JH3 5′-ACCTGAGGAGACGGTGACCAGGGT-3′). The reaction was carried out for
33 cycles (denaturation 94°C for 30 s, annealing 65°C for 30 s
and extension 72°C for 30 s) with a final extension of 7 min.
The prevention of PCR contamination was performed as previously described.25 PCR products were analyzed by electrophoresis on 2% agarose gel. Direct sequencing of amplified
DNAs was performed using the Promega fmol sequencing system as previously described.26 Reactions were carried out in
a thermocycler at 68°C annealing temperature for 15 cycles.
When the sequence quality did not allow a complete reading
of CDR regions, DNA was reamplified with primers containing EcoRI and HindIII restriction sites, and cloned in a
Bluescript SK vector (Stratagene, San Diego, CA, USA).20
Restriction enzyme analysis was carried out on plasmid DNAs
prepared by the alkaline lysis method, and miniprep plasmid
DNAs were then sequenced. The analysis of sequencing was
performed using the PC-GENE software (IntelliGenetics,
Mountain View, CA, USA).
Detection of residual tumor cells
Nested-PCR for IgH rearrangement was carried out as follows:
for DNA samples, 1 mg of genomic DNA was amplified using
a consensus primer derived from the tumor VH family and an
antisense derived from the 3′ end of the JH region (JH3). The
first reaction was carried out for 28 cycles (denaturation 94°C
for 30 s, annealing 64°C for 30 s, extension 72°C for 30 s) with
a final extension of 7 min. One ml of amplified DNA was then
reamplified using CDRII sense and CDRIII antisense primers
using 2.5 U of AmpliTaq Gold (PE Applied Biosystems, Roche
Molecular Systems, Branchburg, NJ, USA). The second reaction was carried out for 35 cycles (denaturation 94°C for 30 s,
annealing depending on specific primers melting temperature
(Tm) for 30 s, extension 72°C for 30 s) with a final extension
of 7 min. For cDNA samples, 1 ml of cDNA (prepared from
Amplification of the tumor VDJ primers derived from the first framework region
VH.FDa
VH1
VH2
VH3
VH4a
VH4b
VH5
VH6
a
See Ref. 21.
See Ref. 20.
b
5′CCTCAGTGAAGGTCTCCTGCAAGG-3′
5′-TCCTGCGCTGGTGAAAGCCACACA-3′
5′-GGTCCCTGAGACTCTCCTGTGCA-3′
5′-TCGGAGACCCTGTCCCTCACCTGCA-3′
5′-CGCTGTCTCTGGTTACTCCATCAG-3′
5′-GAAAAAGCCCGGGGAGTCTCTGAA-3′
5′-CCTGTGCCATCTCCGGGGACAGTG-3′
VH.FSb
VH1
VH2
VH3
VH4a
VH4b
VH5
VH6
5′-CAGGTGCAGCTGGTGCA(C/G)(A/T)CTG-3′
5′-CAG(A/G)TCACCTTGAAGGAGTCTG-3′
5′-CAGGTGCAGCTGGTG(C/G)AGTC(C/T)G-3′
5′-GAG(C/G)TGCAGCTGCAGGAGTC(C/G)G-3′
5′-CAGGTGCAGCTACA(A/G)CAGTGGG-3′
5′-GAGGTGCAGCTG(G/T)TGCAGTCTG-3′
5′-CAGGTACAGCTGCAGCAGTCAG-3′
BTS Technical Report
C Voena et al
5 mg of total RNA as described above) was amplified using
CDRII primer with an antisense primer derived from the first
exon of the IgH tumor constant region (CH1, the same primer
used in RT reaction). Amplification was carried out for 28
cycles (denaturation 94°C for 30 s, annealing depending on
specific primer Tm, extension 72°C for 30 s). Second PCR reaction was performed using a primer derived from CDRIII and
an internal antisense primer derived from the IgH constant
region (CH2) (Cm2: 5′-CAGGAGACGAGGGGGAAA-3′, Cg2:
5′-ACCACG5′-CAGAGGTGCTCCTGGAGCA-3′,
Ca2:
TTCCCATCTGGCTG-3′). The first and the second reactions
were performed using AmpliTaq Gold (PE Applied Biosystems)
Specific primers for the second reaction were selected, when
possible, with a slightly higher melting temperature to minimize inappropriate annealing of primers used during the first
amplification. PCR products were visualized on a 3% Metaphor agarose gel (FMC Bioproducts, Rockland, ME, USA)
stained with ethidium bromide (0.5 mg/ml). Two polyclonal
DNAs were always used as negative controls.
Sensitivity experiments were performed diluting both cell
lines and tumor cells from patients (with at least 90% of circulating tumor cells) with normal marrow cells (up to one tumor
cell in 106 normal marrow cells). Cell mixtures were pelleted
and resuspended in 10 ml of H2O and then lysed by heating
at 95°C for 5 min with 10 U of ribonuclease inhibitor (RNasin;
Promega). The whole lysate from each serial dilution was
amplified with specific nested primers for DNA strategy,
otherwise reverse transcribed into cDNA in a total volume of
20 ml. The whole cDNA was then amplified following the previously described conditions in a 100 ml final volume.
Oligonucleotide synthesis
Oligonucleotides were chemically synthesized with a 391
PCR-MATE EP, DNA synthesizer (Applied Biosystems, Foster
City, CA, USA) on a 0.2 mmol scale, according to the users’
manual.
Results
Identification of VDJ sequence
VDJ regions were amplified using two sets of 5′ consensus
primers derived from the leader or FR1 region of IgH genes
and a 3′ primer from the JH region. First, specimens were
amplified with 5′ consensus primer from FR1 and tumor VDJ
was obtained by direct sequencing in 31 patients; then
remaining amplified VDJs were cloned in a plasmid vector.
For each patient 10 to 15 clones were sequenced and a predominant clone was found in 15 patients. Alternatively, those
patients whose VDJ sequence was impossible to obtain with
FR1 amplification, were amplified with 5′ consensus primer
from the leader region and then sequenced directly or cloned.
Tumor VDJ was obtained by direct sequence only in five
patients, while cloning and sequencing was successful in 11
patients. Overall, complete sequence of the tumor VDJ was
obtained in 62 patients out of 75 (83%) (77% of NHL, 86%
of ALL, 66% of CLL and 89% of MM patients) (Figure 1). Probably, either the presence of somatic mutations or the use of
unknown variable regions were the cause of the lack of amplification in 13 patients. There was no correlation between a
successful amplification of VDJ region and the number of
tumor cells in the diagnostic sample. Sequence analysis, per-
formed as described in the Materials and methods section,
showed that the most common VH family used was VH3
(61%) for all B cell malignancies studied, in particular 57%
of ALL, 50% of NHL, 49% of MM and 55% of CLL. The most
represented JH was JH4, especially in MM (49%) and NHL
(36%), followed by JH6 (29% in ALL, 19% in MM, 33% in
CLL and 9% in NHL). Tumor-specific primers for nested-PCR
to detect MRD were derived from CDRs sequences. For each
patient CDRII sequence was used to synthesize a sense
primer, while the CDRIII sequence was used to synthesize
either a sense or an antisense primer as described in the
Materials and methods section.
Double strategy to detect MRD with IgH nested-PCR
Nested-PCR using CDRII and CDRIII primers was successfully
performed in all patients in which VDJ sequence was available. For each patient, the specificity of the method was
evaluated by amplifying two polyclonal DNAs under the same
conditions of tumor DNA and no amplifications were
observed. In order to determine whether the amplification for
MRD detection is more efficient when performed on RNA
with an RT-nested PCR based approach or on genomic DNA
with a simple nested-PCR, two experimental strategies were
adopted and compared. Both methods were used depending
on sample availability. On DNA samples the strategy, called
DNA strategy, was based on a first amplification with a sense
primer derived from patient clonal VH family and an antisense
primer derived from the 3′ end of the JH region. Samples were
then reamplified with patient-specific CDR primers: CDRII
sense and CDRIII antisense primers (Figure 2). On RNA
samples the approach, called cDNA strategy, involved a
reverse transcription reaction to obtain tumor-specific IgH
cDNA followed by a two-round amplification. Both amplifications were performed with 5′ sense primers derived from
patient-tumor CDRs (CDRII and CDRIII) and 3′ antisense primers derived from the first exon of the tumor IgH constant
region (Figure 3). All the patients examined gave a band of
the expected size. No false positive results were observed in
any strategy. Overall, we could perform both strategies on 40
patients (31 MM, seven NHL, two CLL). DNA strategy was
used for all the ALL patients as most of them do not express
the m chain in the cytoplasm. DNA strategy was also used
when only DNA samples were available (two MM, 10 NHL,
four CLL). In order to compare the sensitivity of both strategies,
two amplifications were performed on serial dilutions on
either DNA or RNA of a Burkitt lymphoma cell line (BJAB).
The sensitivity was higher with the cDNA strategy as it
allowed the detection of one tumor cell in 106 normal marrow
cells, while the DNA strategy reached the sensitivity of one
tumor cell in 105 normal marrow cells. Sensitivity experiments
were also performed on 10 patients, diluting tumor cells with
normal marrow cells. The results were similar to those
obtained with the cell line: a 10 times lower sensitivity was
usually obtained with DNA strategy. We also compared these
data with the results previously obtained in 55 patients using
the hybridization procedure: the tumor clone was amplified
with CDRII and JH or CH primers and then hybridized to a
CDRIII probe. IgH nested-PCR results were identical showing
the same sensitivity and specificity of the hybridization
procedure.
An alternative approach suitable for DNA and RNA templates was also performed, based on a nested-PCR with sense
primers derived from the tumor CDRs (CDRII and CDRIII) and
1795
BTS Technical Report
C Voena et al
1796
Figure 1
Strategy used to amplify and sequence CDRII and CDRIII regions from 75 patients with B cell malignancies.
Figure 2
DNA strategy. Nested PCR for immunoglobulin genes
using genomic DNA. (a) Schematic representation of nested-PCR for
the detection of residual tumor cells. Genomic DNA was first amplified using 5′ tumor VH family primer and 3′ JH primer (VH, JH3), and
then an aliquot of the first reaction was reamplified using inner primers derived from tumor CDRII and CDRIII. Amplified DNA was then
run on 3% Metaphor gel. (b) PCR dilution experiment demonstrating
assay sensitivity and specificity in the BJAB cell line. MW, molecular
weight marker; P, BJAB DNA; PL, unrelated polyclonal DNA; N, no
DNA control lane.
Figure 3
cDNA strategy. RT-nested PCR for immunoglobulin
genes using RNA-cDNA samples. (a) Schematic representation of
nested-PCR for the detection of residual tumor cells. Total cDNA was
first amplified using outer primers (CDRII, CH1) and then an aliquot
of the first reaction was reamplified using inner primers (CDRIII, CH2).
Amplified DNA was then run on 3% Metaphor gel. (b) PCR dilution
experiment demonstrating assay sensitivity and specificity in the BJAB
cell line. MW, molecular weight marker; P, BJAB DNA; PL, unrelated
polyclonal cDNA; N, no DNA control lane.
BTS Technical Report
C Voena et al
two different antisense primers from the JH 3′ end. This strategy failed to yield satisfactory results: nested-PCR products
were usually very short (50–60 bp) due to deletion of the 5′
end of the JH region and due to few N insertions between
D segments and JH regions during IgH genes rearrangement.
Resulting bands were often so faint they were hardly
visualized on 3% Metaphor gel (data not shown).
Discussion
In the present study, we describe a novel nested PCR-method
for MRD detection in B cell malignancies using the rearrangement of IgH genes. This method employs tumor-specific primers derived from the tumor CDRs.
Several methods have been developed to evaluate MRD
using the IgH rearrangement. Most of these methods are based
on the amplification of a small portion of the tumor VDJ by
using consensus primers derived from the FR3. Amplification
with a FR3 primer is often unsuccessful because of the presence of many somatic mutations in the primer template that
prevent correct annealing rendering this approach of limited
application, especially in those B cell malignancies in which B
lymphocytes have already been exposed to the hypermutation
mechanism (MM and NHL). In addition, most of the ALL in
which amplification with FR3 primer fail, have a loss of 3′
portion of the primer site, presumably as a consequence of
exonuclease activity at the time of recombination. 27 Moreover, such a method allows the reading of the CDRIII only,
that is usually 20 nucleotides long (sometimes less than 15)
and that can just be employed to derive a tumor-specific
probe for oligonucleotide hybridization.11 The FR3 method
specificity is then based on one tumor-specific marker and this
may increase the risk of false positive results. In order to avoid
these problems we adopted a different strategy based on the
amplification of a longer fragment of the tumor VDJ by using
sense primers derived from the leader or FR1 region. This
approach allows the reading of both CDRII and CDRIII. The
reading of two different tumor-specific sequences for each
patient is essential to develop a sensitive and specific method
for MRD detection characterized by two step specificity: a
tumor-specific amplification using a CDRII-derived primer
and a tumor-specific hybridization using a CDRIII-derived
probe.18,24,28 In order to simplify such an approach, we
devised a nested-PCR method similar to that employed for
chromosomal translocations. This method maintains two steps
of specificity since two primers are derived from hypervariable
regions of the tumor VDJ, but avoids the use of a hybridization
step. Our data show that nested-PCR of the IgH genes is
widely applicable, simple and fast. Furthermore, since it has
provided sensitivity and specificity similar to hybridization
procedures, it could represent a reliable alternative to
hybridization methods. Our comparative study to assess
whether MRD detection on RNA is better than DNA shows
the advantages of a cDNA strategy. cDNA strategy is more
sensitive than a DNA strategy since, in those specimens in
which both methods were applied, amplification of DNA
showed a sensitivity usually 10 times lower (10−5 vs 10−6).
This result is conceivable since within a cell mRNA is more
abundant than DNA. cDNA strategy is, then, a preferable
approach as it allows an increase in the yield of the reaction
and possibly enhances the detection of residual tumor cells.
This strategy is sometimes difficult to achieve because well
preserved samples for RNA extraction are not always available. In both amplification CDRs, derived sense primers and
tumor IgH constant region antisense primers are used, increasing on one hand the sensitivity and on the other decreasing
the risk of false positive results. This procedure always gave
cleaner products reducing the possibility of nonspecific
annealing. Moreover, the use of a IgH constant region primer
avoids the use of JH antisense primer that sometimes fails to
give amplification because of extensive deletion in its
sequence, especially in MM and ALL.29,30
DNA is a good source for MRD detection when only smears
or old (badly preserved) material are available and RNA
extraction could fail. In this case it is advisable to proceed
with RNA and DNA extraction simultaneously. If RNA extraction fails, the DNA strategy provides a possible alternative. It
consists of an initial amplification with the 5′ tumor VH family
primer and the 3′ JH primer, followed by a second amplification using CDRII and CDRIII. The use of a VH consensus
primer in the first PCR may lead to amplification of normal
clones unrelated to the tumor one, thus decreasing the sensitivity of the second specific amplification.
Although the use of the IgH genes as tumor markers still
remains a relatively complex procedure, especially because
of VDJ sequencing, the use of nested-PCR may greatly simplify
the screening for residual tumor cells in remission samples.
This is particularly useful when frequent molecular monitoring
is required.
Acknowledgements
This work has been supported by Associazione Italiana
Ricerca sul Cancro (AIRC, Milano, Italy) and Consiglio
Nazionale delle Ricerche (Progetto Finalizzato ACRO
9500416.PF39) and BIOMED grant ERBCMRXCT940473. MA
is a recipient of a fellowship from AIRC. We are indebted to
Dr SS Sahota and Dr FK Stevenson for sending us the
sequence of framework and leader region families.
References
1 Vose JM, Armitage JO. Role of autologous bone marrow transplantation in non-Hodgkin lymphoma. Hematol/Oncol Clin N Am
1993; 7: 577–590.
2 Gianni AM, Tarella C, Bregni M, Siena S, Lombardi F, Gandola
L, Caracciolo D, Stern A, Bonadonna G, Boccadoro M, Pileri A.
High-dose sequential chemoradiotherapy, a widely applicable
regimen confers survival benefit to patients with high-risk multiple
myeloma. J Clin Oncol 1994; 12: 503–509.
3 Preti A, Kantarjian HG. Management of adult acute lymphocytic
leukemia: present issues and key challenges. J Clin Oncol 1994;
12: 1312–1322.
4 Campana D, Pui CH. Detection of minimal residual disease in
acute leukemia: methodologic advances and clinical significance.
Blood 1995; 85: 1416–1434.
5 Gribben JG, Neuberg D, Freedman AS, Gimmi CD, Pesek KW,
Barber M, Saporito L, Woo SD, Coral F, Spector N, Rabinowe SN,
Grossbard ML, Ritz J, Nadler LM. Detection by polymerase chain
reaction of residual cells with the bcl-2 translocation is associated
with increased risk of relapse after autologous bone marrow transplantation for B-cell lymphoma. Blood 1993; 81: 2449–2457.
6 Brisco MJ,. Condon J, Hughes E, Neoh SH, Sykes PJ, Seshadri R,
Toogod I, Waters K, Tauro G, Ekert H, Morley AA. Outcome prediction in childhood acute lymphoblastic leukaemia by molecular
quantification of residual disease at the end of induction. Lancet
1994; 343: 196–200.
7 Huang W, Sun GL, Li XS, Cao Q, Lu Y, Jang G-S, Zhang F-Q,
Chai J-R, Wang ZY, Waxmann S, Chen Z, Chen S-J. Acute promyelocytic leukemia: clinical relevance of two major PML/RAR-a isoforms and detection of minimal residual disease by retro-
1797
BTS Technical Report
C Voena et al
1798
8
9
10
11
12
13
14
15
16
17
18
transcriptase/polymerase chain reaction to predict relapse. Blood
1993; 82: 1264–1269.
Lo Coco F, Diverio D, Pandolfi PP, Biondi A, Rossi V, Avvisati A,
Rambaldi A, Arcese W, Petti MC, Meloni G, Mandelli F, Grignani
F, Masera G, Barbui T, Pelicci PG. Molecular evaluation of
residual disease as a predictor of relapse in acute promyelocytic
leukemia. Lancet 1992; 340: 1437–1438.
Negrin RS, Blume KG. The use of polymerase chain reaction for
the detection of minimal residual malignant disease. Blood 1991;
78: 255–258.
Cross NCP, Feng L, Chas A, Bungey J, Hughes TP, Goldman JM.
Competitive polymerase chain reaction to estimate the number of
BCR-ABL transcripts in chronic myeloid leukemia patients after
bone marrow transplantation. Blood 1993; 82: 1929–1938.
Yamada M, Wasserman R, Lange B, Richard BA, Womer RB, Rovera G. Minimal residual disease in childhood B-lineage lymphoblastic leukemia. Persistence of leukemia cells during the first 18
months of treatment. New Engl J Med 1990; 323: 448–455.
Pui CH, Crist WM, Look AT. Biology and clinical significance of
cytogenetic abnormalities in childhood acute lymphoblastic leukemia. Blood 1990; 76: 1449–1463.
Bartram CR. Detection of minimal residual leukemia by the polymerase chain reaction: potential implications for therapy. Clin
Chim Acta 1993; 217: 75–78.
Previtera E, Rivolta A, Ronchetti D, Mosna G, Giudici G, Biondi
A. Reverse transcriptase/polymerase chain reaction follow-up and
minimal residual disease detection in t(1;19)-positive acute lymphoblastic leukemia. Br J Haematol 1996; 92: 653–658.
Cimino G, Elia L, Rivolta A, Rapanotti MC, Rossi V, Alimena G,
Annino L, Canaani E, Lo Coco F, Biondi A. Clinical relevance of
residual disease monitoring by polymerase chain reaction in
patients with ALL-1/AF-4 positive-acute lymphoblastic leukemia.
Br J Haematol 1996; 92: 659–664.
Shurtleff SA, Buij S, Behm FG, Rubnitz JE, Raimondi SC, Hancock
ML, Chan GC-F, Pui C-H, Grosveld G, Downing JR. TEL/AML1
fusion resulting from a cryptic t(12;21) is the most common genetic lesion in pediatric ALL and defines a subgroup of patients with
an excellent prognosis. Leukemia 1995; 9: 1985–1989.
Alt FW, Blackwell TK, Yancopoulos GD. Development of the primary antibody repertoire. Science 1987; 238: 1079–1087.
Corradini P, Voena C, Astolfi M, Ladetto M, Tarella C, Boccadoro
M, Pileri A. High dose sequential chemoradiotherapy in multiple
myeloma: residual tumor cells are detectable in bone marrow and
peripheral blood cell harvests and after autografting. Blood 1995;
85: 1596–1602.
19 Gribben JG, Freedman AS, Woo SD, Blake K, Shu RS, Freeman
GJ, Longtine JA, Pinkus GS, Nadler LM. All advanced stage nonHodgkin’s lymphomas with polymerase chain reaction amplifiable
breakpoint of bcl-2 have residual cells containing the bcl-2
rearrangement at evaluation and after treatment. Blood 1991; 78:
3275–3280.
20 Maniatis T, Fritsch EF, Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory: Cold Spring Harbor,
NY, 1989.
21 Hawkins RE, Zhu D, Ovecka M, Winter G, Hamblin TJ, Long A,
Stevenson F. Idiotypic vaccination against human B cell lymphoma. Rescue of variable region sequences from biopsy material
for assembly as single-chain Fv personal vaccines. Blood 1994;
83: 3279–3288.
22 Deane M, Norton JD. Immunoglobulin gene fingerprinting: an
approach to analysis of B lymphoid clonality in lymphoproliferative disorders. Br J Haematol 1991; 77: 274–281.
23 Bahler DW, Campbell MJ, Hart S, Miller RA, Levy S, Levy R. Ig
VH gene expression among human follicular lymphomas. Blood
1991; 78: 1561–1568.
24 Sahota SS, Leo R, Hamblin TH, Stevenson F. Ig VH gene
mutational patterns indicate different tumor cell status in human
myeloma and monoclonal gammopathy of undetermined significance. Blood 1996; 87: 746–755.
25 Kwok S, Higuchi R. Avoiding false positives with PCR. Nature
1989; 339: 237–238.
26 Corradini P, Ladetto M, Voena C, Palumbo A, Inghirami G,
Knowles DM, Boccadoro M, Pileri A. Mutational activation of Nand K-ras oncogenes in plasma cell dyscrasias. Blood 1993; 81:
2708–2713.
27 Height SE, Swansbury GJ, Matutes E, Treleaven JG, Catovsky D,
Dyer MJS. Analysis of clonal rearrangements of the Ig heavy chain
locus in acute leukemia. Blood 1996; 87: 5242–5250.
28 Bakkus MHC, Heirman C, Van Riet I, Van Camp B, Thielemans
K. Evidence that multiple myeloma Ig heavy-chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood
1992; 80: 2326–2335.
29 Kunkel L, Vescio R, Cao J, Hong C, Schiller G, Lichtenstein A,
Berenson J. Unconventional mechanisms used to generate the Ig
heavy chain third complementarity determining region (CDR3) in
B cell malignancy. Blood 1993; 82: 976 (Abstr.).
30 Dyer MJS, Heward JM, Zani VJ, Buccheri V, Catovsky D. Unusual
deletions within the immunoglobulin heavy-chain locus in acute
leukemias. Blood 1993; 82: 865–871.