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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 952-958
Clonal Diversity of Ig and T-Cell-Receptor Gene Rearrangements
Identifies a Subset of Childhood B-Precursor Acute Lymphoblastic
Leukemia With Increased Risk of Relapse
By
Elaine Green,
Carmel M. McConville,
Judith E. Powell,
Jillian R. Mann,
Philip J. Darbyshire,
A. Malcolm R. Taylor, and
Tatjana Stankovic
From the Department of Haematology/Oncology, Birmingham Children's
Hospital NHS Trust, Ladywood Middleway, Birmingham; and the Division of
Medical Genetics, Department of Public Health and Epidemiology, and CRC
Institute for Cancer Studies, University of Birmingham, UK.
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ABSTRACT |
Current prognostic indicators such as age, sex, and white blood cell
count (WBC) fail to identify all children with more aggressive forms of
B-precursor acute lymphoblastic leukemia (ALL), and a proportion of
patients without poor prognostic indicators still relapse. Results
obtained from an analysis of 65 pediatic B-precursor ALL patients
indicated that subclone formation leading to clonal diversity, as
detected by Ig and T-cell receptor (TCR) gene rearrangements, may
represent a very useful prognostic indicator, independent of age, sex,
and WBC. Disease-free survival was significantly shorter in those
patients showing clonal diversity at presentation. Furthermore, clonal
diversity was detected not only in the majority of high-risk patients
who relapsed but was also associated with a high probability of relapse
in standard-risk patients. Sixty-five percent (13/20) of standard-risk
patients who also showed clonal diversity subsequently relapsed,
whereas the percentage of relapses among standard-risk patients without
clonal diversity was much lower at 19% (7/36). Continued clonal
evolution during disease progression is an important feature of
aggressive B-precursor ALL. All 5 patients with clonal diversity who
were followed up in our study showed a change in the pattern of
clonality between presentation and relapse. This implies an important
role for clonal diversity as a mechanism of disease progression through
the process of clonal variation and clonal selection.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
SIGNIFICANT IMPROVEMENTS have been made
over the past 2 decades in the treatment of childhood acute
lymphoblastic leukemia (ALL).1 In particular the most
common subtype, B-precursor ALL, generally has a good prognosis, with a
survival rate of approximately 70%.1-3 An important
contribution to this improvement has been the recognition of a subgroup
of patients, at high risk of relapse, who require more aggressive
treatment. These high-risk patients may be identified on the basis of
classical clinical parameters such as age, sex, white blood count,
immunophenotype, cytogenetics, and DNA index.3,4
For the remaining 30% of patients, improvement in survival rates will
require progress in two areas. First, further stratification of
standard-risk patients, a proportion of whom show unpredictable relapse
on current treatment protocols despite the absence of conventional poor
prognostic indicators (approximately 32% in our series), and second,
the development of more effective treatment strategies based on a
better understanding of the biological/genetic factors influencing
disease progression.
Lymphoid malignancies offer a unique opportunity for the investigation
of disease evolution and progression at the genetic level as a
consequence of the occurrence of recombination within the immune system
gene loci. During normal B- and T-cell development, recombination in Ig
and T-cell receptor (TCR) genes gives rise to unique combinations of
variable (V), diversity (D), and joining (J) gene segments. The
junctional regions of each of the gene segments are different in each
lymphocyte or lymphocyte clone, and hence may be regarded as
tumor-specific markers.5-8
It is becoming increasingly clear, however, that leukemogenesis is a
dynamic process, with continued evolution of Ig and TCR rearrangements.9,10 This is also likely to reflect more
general genetic change giving rise to the outgrowth of more aggressive, treatment-resistant tumor subclones. Several studies have shown, either
by Southern blotting or polymerase chain reaction (PCR) amplification,
multiple rearrangements of Ig and/or TCR genes at presentation
in up to 40% of patients with B-precursor ALL.11-17 There
is some evidence to suggest that the resulting clonal diversity may be
associated with a poor prognosis in children with B-precursor ALL.11,14,16 In this study, we investigate whether clonal diversity can be used to identify patients with poor prognosis, particularly among those who are classified as standard risk using current prognostic indicators.
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MATERIALS AND METHODS |
Patient material.
Bone marrow samples were collected from 65 patients who presented with
B-lineage ALL at Birmingham Children's Hospital between 1989 and 1996. Patients were selected only on the basis of availability of material
for study. A total of 207 patients were treated in this period
according to UK Medical Research Council UKALL Trial protocols (UKALL
X, XI) or on the MRC Infant Leukaemia Trial. The 5-year disease-free
survival (DFS) of 142 cases that were not included in the study, due to
unavailibility of material, was 65% (95% confidence
interval = 55.2  74.8).
For the purposes of the analyses performed in this study, patients were
classified as being at high risk of treatment failure on the basis of
the presence of one or more of the following parameters: age less than
1 year, Ph chromosome, translocation t(4;11), or a hazard score of
greater than or equal to 0.8 (calculated retrospectively). The Oxford
hazard score,4 as implemented in the MRC UKALL 1997 Trial,
relates risk of treatment failure for children over 1 year to age,
gender, and leucocyte count as follows: Hazard Score = 0.22 × loge (WBC + 1.0) + 0.0043 × Age2 0.39 × Sex (male = 1; female = 2). The
boundary value of 0.8 defines a group of patients with 5-year DFS of
less than 40%.
Bone marrow mononuclear cells were isolated by
centrifugation on Ficoll density gradients, and DNA extracted by
standard methods.18 Chromosome analyses of bone marrow
samples were performed using standard G-banding techniques.
Southern blot analysis.
The following probes were used for Southern blot hybridization: a
2.7-kb BglII/Pst I fragment specific for the
JH region (M13CTGR51A), a 0.8-kb
HindIII/EcoRI fragment for the C region (pRH10), an 8-kb EcoRI fragment for the C region, a 1.5-kb Sac I
fragment for the J region (JdS16), a 5-kb EcoRI fragment for
J region (JRR), a 0.7-kb HindIII/EcoRI fragment
for the J region (M13H60), and a 0.7kb HindIII/EcoRI
fragment for the C region, all kindly provided by Dr T.H. Rabbitts
(MRC Laboratory of Molecular Biology, Cambridge, UK).
IgH gene amplification.
PCR amplification of the third complementarity determining region
(CDR-III) was performed using either primers P1 and P2 for direct
amplification or primers P1/P3 followed by P1/P419 for
seminested PCR as shown in Fig 1. The whole
of the VNDNJ region was amplified using seminested PCR and primers
P5/P3 for the first round followed by the primers P5/P2 for the second
round of amplification (Fig 1).
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| Fig 1.
Position and sequence of primers for single round (P1/P2)
and seminested (P1/P3, P1/P4, or P5/P3, P5/P2) PCR amplification of the
third complementarity region (CDR-III) of the IgH gene.
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TCR and amplification.
The V2-D3 rearrangement was amplified using primers
5 GAG TCA TGT CAG CCA TG AG (forward), and 5AGG GAA ATG
GCA CTT TTG CC (reverse). The primers used for TCR amplification
were 5CTG TGA CAA CAA GTG TTG TTC CAC and 5GTG CTT CTA
GCT TTC CTG TCT C,20 with internal primers for
nested PCR: 5GAG TAC GCT GCC TAC AGA GAG G and 5CCA CTG
CCA AAG AGT TTC TT. PCR amplifications were performed using 100 ng of
genomic DNA. The amplified products were resolved on 8% acrylamide or
3.5% Metaphor (FMC) agarose gels, and bands of interest were excised
and eluted in water for sequencing.
CDR-III sequencing.
Gel-purified PCR products were either sequenced directly using the
automated chain termination sequencing system (Applied Biosystems Inc,
Warrington, UK) or following subcloning into pUC18. Sequenced
rearrangements were assigned to their component variable (V), diversity
(D), and joining (J) region sequences by comparison with published
sequences.20-27
Criteria for detection of clonal diversity.
The criteria for the identification of clonal diversity of leukemic
blast cells by either Southern blotting or PCR amplification of immune
system gene rearrangements were: (1) the presence of an excess of bands
on Southerns or number of PCR products in relation to the number of
alleles, ie, more than 2 bands on Southerns or more than 2 PCR products
when only 2 copies of the relevant chromosome were present, and (2) the
existence of 2 bands of unequal density on Southern blots.
Statistical methods.
DFS was defined as the time between diagnosis and the first relapse, or
if no relapse occurred, between diagnosis and the censor date of August
31, 1997. For patients who did not enter remission, the DFS was defined
as zero; patients who died without relapse were censored at the date of
death. Differences in the prognostic cofactors (white cell count, age,
sex, high-/standard-risk category) between the oligoclonal and normal
group were analyzed using the Chi-squared and Fisher's exact test and
Mann-Whitney U-test. Differences in DFS between oligoclonal and
nonoligoclonal patients were evaluated using Kaplan-Meier survival
curves and the log-rank test. The effects of prognostic cofactors were
investigated using stepwise Cox's proportional hazard regression.
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RESULTS |
Detection of clonal diversity.
To ensure optimal detection of clonal diversity we analyzed immune
system gene rearrangements by both Southern blotting, using seven
immune system gene probes (see Materials and Methods) and JH, TCR J, and TCR J PCR assays whenever the amount
of material allowed such extensive study. Complete analysis was
performed in 33 patients with B-precursor ALL, whereas in a further 32 patients, for whom only small samples were available, the PCR method
was used. The results obtained indicated that while clonal diversity was detected in the majority of cases by both methods, a number of
cases was detected only by Southern blotting or only by PCR. Six of 33 fully analyzed patients (18%), for example, were detected only by
Southern blotting; the negative PCR result in these cases was probably
a consequence of loss of primer binding site(s) or incomplete
DJH rearrangement. PCR methods did, however, show greater sensitivity in the detection of minor subclones, which may account for
the observation of clonal diversity in 3 patients using IgH PCR
amplification but not with Southern blotting. The most informative assays, in terms of subclone detection, were Southern blotting with
IgJH, IgC, and TCR J probes and PCR amplification
across the CDR-III region of the JH locus. Using a
combination of these assays we identified oligoclonality in 40%
(26/65) of patients. However, as the Southern blotting showed a higher
frequency of subclone detection than PCR analysis, we cannot rule out
the possibility that we may have underestimated the frequency of clonal
diversity in the 32 patients analyzed by PCR only.
Clonal diversity and DFS in pediatric B-precursor ALL.
In all patients the presence of clonal diversity was investigated at
diagnosis and, in some cases, at intervals during the course of their
treatment. The results obtained from a total of 65 patients
(Table 1), (29 of whom have been reported
previously16) indicated that approximately 40% of patients
(26/65) showed clonal diversity at the presentation of their disease.
Eighteen out of 26 (69%) oligoclonal patients relapsed or died without
entering remission compared with only 10 of the remaining 39 patients
(26%) without clonal diversity. Patients were followed up for between
18 and 90 months from diagnosis, (median follow-up was 59 months;
80.4% of surviving patients had  30 months follow-up). The
association between clonal diversity at presentation and shorter DFS
was shown to be significant by a log-rank test (P = .0025; Fig 2A).
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| Fig 2.
(A) DFS in 65 children with ALL. The broken line
represents survival in patients with clonal diversity (n = 26), and
the solid line represents survival in patients without clonal diversity (n = 39). (B) DFS in 56 patients with standard-risk ALL. The broken line represents survival in standard-risk patients with clonal diversity (n = 20), and the solid line represents survival in standard-risk patients without clonal diversity (n = 36).
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Prognostic significance of clonal diversity in patients with
standard-risk B-precursor ALL.
In view of these results it was important to determine whether
prognostic information obtained from the investigation of clonal diversity could be used to improve the stratification of patients as
compared with currently used indicators.
Initially we compared the relationship between DFS and five prognostic
indicators: clonal diversity, age, sex, white blood cell count, and
high-risk status (as defined in Materials and Methods) in a stepwise
Cox's regression analysis. Both white blood cell count and clonal
diversity were independently predictive of outcome (clonal diversity,
P = .013; white blood cell count, P = .005),
whereas the effects of age, sex, and high-risk status were not
significant. The relative risk of treatment failure in patients showing
clonal diversity, independent of white blood cell count, was 2.73 (95%
confidence interval = 1.24 6.00).
We next looked in more detail at patients classified as standard risk
(ie, age >1 year, absence of t(4;11) and t(9;22), hazard score < 0.8). Again, DFS was shown to be significantly shorter in patients with
clonal diversity (P = .002; 5-year DFS = 32% and
72% for patients with and without clonal diversity, respectively; Fig
2B). In our series of 56 patients with standard-risk ALL, 65% (13/20)
of oligoclonal patients subsequently relapsed without achieving
remission. This was in contrast to a relapse rate of only 19% (7/36)
observed among the patients with a standard-risk and monoclonal
disease.
A Cox's regression analysis of patients with standard risk indicated
that clonal diversity was more predictive of outcome than any other
parameter (clonal diversity P = .002;white blood cell count
P = .028; sex P = .560; age P = .921). In the
presence of clonal diversity no other factor was significantly
prognostic. The relative risk of treatment failure in clonal diversity
versus monoclonal patients was 3.66.
In summary, an important feature of these results was the fact that the
improved prognostic information contributed by the assessment of clonal
diversity appeared to relate largely to a group of patients currently
classified as standard risk. Consequently, the use of this indicator
may make a significant contribution to the identification of patients
within this group who have a greater risk of treatment failure.
Clonal diversity and disease progression.
Having shown that clonal diversity was closely associated with
aggressive disease, we next looked in more detail at the pattern of
clonal evolution between presentation and relapse. We sought to
investigate whether clonal diversity is always generated from (1) the
major clone, (2) could be generated from a tumor cell at an earlier
stage of development, and (3) the extent to which clonal diversity
contributed to clonal selection and emergence of the final highly
resistant clone.
The pattern of clonality was followed by IgH CDR-III PCR through
various stages of disease progression in six oligoclonal patients, five
with oligoclonality at presentation and one with oligoclonality at the
end of treatment. In all analyzed patients the number and/or
size of IgH CDR-III fragments differed between presentation and
relapse, although in four out of six cases at least one fragment of
identical size was observed (Table
2).
To determine the relationship between individual subclones,
JH rearrangements from various stages of disease were
sequenced in three patients and the sequences aligned to V, D, and J
regions. Sequencing of the major clones at different stages of the
disease in patient 48 revealed that all detectable subclones appeared to be related, since they all shared common D (DLR4) and J (J6) sequences (Table 3). However, the greater
preservation of the D sequences in one of the relapse clones suggested
that this was not derived directly from a presentation clone but rather
was present at an earlier stage of leukemogenesis. Interestingly, this
patient was initially classified as a standard-risk ALL, with no other
recognizable features of aggressive disease. The pattern of clonality
started to change 2 weeks after presentation, during induction
treatment (Fig 3), and the patient
eventually relapsed 54 months later, showing an entirely different
pattern of bands to that observed previously (Fig 3).
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| Fig 3.
Clonal evolution identified by FRA3/JH PCR at
various stages of disease in patient 48. Lane 1, DNA size marker; lane
2, four clones identified at presentation; lane 3, two major clones
together with 2 minor subclones identified at week 2; lane 4, two
clones identified at relapse; lane 5, a smear of PCR products in
control sample from normal bone marrow.
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In the second patient, patient 65, the clone from presentation was
accompanied at the time of relapse, 61 months later, by a new,
independent subclone with the involvement of different D and J regions
(Table 3). The presence of two independent subclones indicated the
derivation of clonal diversity from an immature, nonrearranged tumor
progenitor. Bearing in mind the time of the relapse in both patients,
54 and 61 months, respectively, the possibility of a secondary leukemia
was considered. However, secondary leukemia was unlikely in either of
these patients because in patient 48 all identified subclones from
presentation to relapse were related, and in patient 65 the
presentation clone was still detectable at the time of relapse.
In a further two patients, 44 and 50, an increase in the number of
amplified JH rearrangements was observed at the end of treatment. Interestingly, patient 44 presented with a single leukemic clone, and clonal diversity was identified only later at the end of
treatment. Subsequently, both children relapsed within 1 year. Sequencing of the terminal clones in patient 44 revealed coexistence of
the initial clone with a new independent subclone (Table 3).
| |
DISCUSSION |
Current approaches to the treatment of pediatric ALL involve the
stratification of patients into different risk groups so that treatment
regimens may be tailored to match the specific needs of each group.
Those patients at highest risk of relapse, for example, will require
more aggressive treatment to improve their chances of long-term
survival. Equally important, however, is the need to avoid
over-treating those children in the better prognostic groups in order
to minimize long-term side-effects.28-30 Discrimination
between the different risk groups is not straightforward, and several
different systems have been proposed.4,31,32 None of these
appear to be entirely satisfactory, however, since a proportion of
children currently classified as low or standard risk still relapse.
In a follow-up to previous reports11,14,16 that suggested
that the detection of clonal diversity in pediatric B-precursor ALL
patients might provide a new indicator of poor prognosis, we have
sought to investigate this association further and to develop a
straightforward strategy that would allow the routine assessment of
clonal diversity in large numbers of patient samples. Results obtained
indicate that combination of a PCR-based approach, involving the
amplification of the CDR-III region of IgH (using consensus FR1, FR3,
and JH primers), and Southern blotting using IgH, Ig, and TCR
allowed a high level of detection of clonal diversity comparable to
previously published reports.11,13
A study of 65 pediatric B-precursor ALL patients confirmed that clonal
diversity was significantly associated with a poor outcome. A reduction
in long-term survival (>5 years) of almost 50% was observed as
compared with patients who did not show clonal diversity (Fig 2A).
Statistical analyses confirmed that clonal diversity was independant
of, and more predictive of poor outcome, than any other prognostic
indicator tested.
It was apparent from this study however, that the prognostic
information obtained differed in some ways from that provided by other
prognostic indicators. Of particular interest was the observation that
a proportion of patients regarded as standard risk showed clonal
diversity and also had significantly shorter DFS; 5-year survival for
these patients was only 32% as compared with 72% in standard-risk
patients without clonal diversity.
It is interesting to speculate whether clonal diversity represented
only a marker of aggressive disease or whether there was a biological
role for subclone formation during tumor progression. The observations
from this study, particularly with respect to the change in the pattern
of clonality between presentation and relapse support the hypothesis
that clonal diversity, indeed, represents a mechanism of disease
progression. It is possible that this mechanism operates through the
process of clonal variation and clonal selection providing a highly
proliferative, resistant malignant clone in the terminal stage of the
disease.33 The circumstances that promote both continued or
de novo immunoglobulin gene rearrangements in clonally unstable cells
are not clear. The process of VHDJH
recombination is regulated at several levels and, therefore, many
factors may be involved. At one level, the appearance in a normal cell
of a productive rearrangement leads to the subsequent inhibition of
recombinase activity. Continued, multiple VHDJH
gene rearrangements, which are nonfunctional, are common in ALL tumor
cells,10 suggesting that inhibition of the recombinase
activity might not take place in leukemic blasts. At another level, it
is possible that factors affecting accessibility of chromosomal regions
containing the signal sequences34 contribute to the
continuously active recombinations during leukemic proliferation. The
same mechanism could cause both subclone formation and a wider genomic
instability, involving genome wide recognition signal-like sequences,
with consequent involvement of other genes important for tumor
proliferation.35 There is a need, therefore, for the identification of factors promoting both clonal diversity and the
aggressive phenotype that could be potentially used in an appropriate
assay in clinical studies. In this respect, the expression of
recombinase (RAG) proteins that are essential for B-cell
differentiation might be one of the factors affected in patients with
clonal diversity.
In two patients, 44 and 65, in whom subclones were sequenced, the
source of clonal diversity might have been an early B nonrearranged progenitor cell. One can speculate that differences in the time before
relapse (39 and 61 months) was related to the requirement for different
additional molecular change and further clonal selection to produce a
more aggressive, treatment-resistant subclone. However, an alternative
explanation for the presence of the further, independent VHDJH rearrangement at the time of relapse in
these patients is that a second, previously nonrearranged or
DJ-rearranged IgH allele underwent a complete
VHDJH rearrangement in the original leukemic clone. Such a possibility cannot be excluded, particularly in the light
of the fact that both the original and the new independent VHDJH rearrangement were present at the time of
relapse in both patients. In contrast to patients 44 and 65, all the
identified subclones in patient 48 were related and showed utilization
of the same DH and JH region. The CDR-III
sequences of the subclones showed substantial variability at the
VHJH junctions suggesting that V-V replacement
was an unlikely mechanism for the generation of clonal diversity in
this patient. The variability of the DJH joints was also
remarkable in patient 48 and, therefore, de novo V rearrangements to a
preexisting DJH rearrangement, as an alternative mechanism,
seems doubtful. A further possibility is that all these subclones were
independently derived from a nonrearranged tumor progenitor cell in an
environment that directed rearrangement to the same VH, D
and J segments as has been previously postulated for another
patient.36
The present observations are restricted to a small number of patients
that were available for the study. To clarify the origin of clonal
diversity and its biological significance it would be informative, in a
further study, to extend sequence analysis of the identified
VHDJH rearrangements to all patients with
clonal diversity irrespective of relapse. It would be important to
determine whether the relationship between subclones and the mechanism
of their generation, in patients who do not relapse, differs from that
observed in patients who do relapse. Such a distinction would provide
further stratification of patients with standard risk and improve the
power of clonal diversity as a predictor of relapse. The conclusions
from this study, therefore, have potential clinical implications,
particularly with respect to the future treatment of standard-risk ALL
patients. Clearly, it is neccessary to perform both larger scale
retrospective and prospective studies to confirm our findings and to
provide a firm basis on which to make any subsequent changes in the
clinical management of pediatric B-precursor ALL.
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FOOTNOTES |
Submitted January 12, 1998;
accepted April 2, 1998.
Supported in part by The British Council and the Cancer Research
Campaign. E.G. was a Price Hall Fellow (Former United Birmingham Hospitals Endowment Fund).
Address reprint requests to Tatjana Stankovic, MD, PhD, CRC
Institute for Cancer Studies, University of Birmingham, Birmingham, UK
B15 2TT.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank the Department of Haematology, Birmingham Children's Hospital
and the Regional Cytogenetics Unit, Birmingham Heartlands Hospital for
immunophenotype and cytogenetics data. Patient data were compiled from
the West Midlands Regional Children's Tumour Research Group Registry.
We also thank Dr C.G. Steward for helpful comments on the manuscript,
and S. Rees and K. Willis for technical assistance.
| |
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Abstract
Introduction
Abstract