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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1391-1398
BCL-2 Expression Correlates With Lower Proliferative
Activity in the Intermediate- and High-Grade Non-Hodgkin's
Lymphomas: An Eastern Cooperative Oncology Group and Southwest
Oncology Group Cooperative Laboratory Study
By
Jane N. Winter,
Janet Andersen,
John C. Reed,
Stanislaw Krajewski,
Daina Variakojis,
Kenneth D. Bauer,
Richard I. Fisher,
Leo I. Gordon,
Martin M. Oken,
Shuwei Jiang,
David Jeffries, and
Peter Domer
From the Robert H. Lurie Cancer Center, Northwestern University,
Chicago, IL; The Burnham Institute, La Jolla, CA; Cardinal Bernardin
Cancer Center, Loyola University Medical Center, Maywood, IL; The
Virginia Piper Cancer Institute, Abbott-Northwestern Hospital,
Minneapolis, MN; and the Eastern Cooperative Oncology Group Statistical
Center, the Dana-Farber Cancer Institute, Boston, MA.
 |
ABSTRACT |
An inverse relationship between BCL-2 expression and cell
cycle transition has been suggested by recent studies in murine models.
To investigate the clinical relevance of these laboratory studies, a
group of 116 paraffin-embedded non-Hodgkin's lymphoma (NHL) biopsy
specimens (Working Formulation Groups D-H, and J) from a cooperative
group study of cellular DNA content were analyzed for the 14;18
translocation using polymerase chain reaction (PCR)-based methods and,
if sufficient tissue remained, for BCL-2 and BAX expression by immunohistochemistry. The results of these studies were then compared with the results of the previously performed flow
cytometric analysis of ploidy and proliferative activity (S-phase-fraction). BCL-2 expression was inversely associated with proliferative activity (P = .001; n = 41), but there
was no association between staining for Bax and %S-phase. Ploidy was not associated with either BCL-2 or BAX expression. The
t(14;18) was detected in 21 of the 54 cases in which PCR-amplifiable
DNA was recovered; 20 of these occurred at the major breakpoint region and 1 at the minor breakpoint region. High levels of BCL-2 or BAX expression occurred independently of t(14;18). There was no association between t(14;18) and either ploidy or proliferative activity. The inverse relationship between BCL-2 expression and proliferative activity in the intermediate- and high-grade NHLs is
consistent with recent studies suggesting that Bcl-2 both retards entry
into the cell cycle and inhibits apoptosis.
| |
INTRODUCTION |
THE PROTEIN ENCODED by the BCL-2
(B-cell lymphoma/leukemia-2) gene is a major regulator of programmed
cell death, the process by which many chemotherapeutic agents
ultimately effect tumor cell kill.1,2 BCL-2 was
first identified because of its association with the t(14;18)
chromosomal translocation in which the BCL-2 gene at 18q21 is
juxtaposed with the Ig heavy chain locus at 14q32, resulting in
deregulation of transcription and overexpression of the Bcl-2
protein.3-5 This balanced translocation is characteristic of the majority of follicular non-Hodgkin's lymphomas (NHLs) and a
subset of intermediate- and high-grade lymphomas.6,7 Bcl-2 protein levels may also be elevated in NHLs that do not harbor the
t(14;18), suggesting that other mechanisms may result in
overexpression.8 Recently, high levels of the Bcl-2 protein
detected by immunostaining have been associated with early relapse in
the intermediate- and high-grade NHLs. 9-12
Proliferative activity measured by flow cytometric analysis of cellular
DNA content or Ki-67 expression correlates inversely with clinical
outcome in the NHLs in some series.13-15 To confirm early
reports that proliferative activity is a significant independent determinant of clinical behavior in the intermediate- and high-grade NHLs, a large prospective study of cellular DNA content in archival paraffin-embedded biopsy specimens from uniformly staged and treated patients was initiated through the Eastern Cooperative Oncology Group
(E6486). Although flow cytometric analysis of ploidy and proliferative
activity showed a probable association between increasing proliferative
activity (%S-phase) and shortened survival, clinical parameters were
more powerful prognostic indicators than cellular DNA content
analysis.16 No association with disease-free survival or
time to treatment failure was observed. Using residual
paraffin-embedded material from 116 of the patients enrolled on E6486,
polymerase chain reaction (PCR)-based detection of the 14;18
translocation and immunohistochemical analysis of the Bcl-2 and Bax
proteins were performed. Comparison was then made with the results of
the previous flow cytometric analysis of ploidy and proliferative activity, showing an association between BCL-2 expression and low proliferative activity. These findings are consistent with recent
data suggesting that Bcl-2 retards entry into the cell cycle in
addition to inhibiting apoptosis.17-19
| |
MATERIALS AND METHODS |
Patient materials.
Cases enrolled on two phase-III intergroup clinical trials
(E648320 and E348721) for previously untreated
patients with intermediate- and high-grade NHLs were eligible for entry
onto an ancillary study of cellular DNA content provided that paraffin
blocks from the initial diagnostic specimen were available for
submission and that the embedded tissue had a minimal transverse
diameter of 0.4 cm and sufficient depth to provide a minimum of three
50-micron sections for cellular DNA content analysis and two routine
hematoxylin and eosin sections (5 microns). For technical reasons,
material fixed in B5-formalin or Zenker's fixative was not acceptable.
Patients with antecedent low-grade NHL were ineligible. Cases
in which material remained in the block after sections were cut for DNA
content analysis were processed first for molecular analysis
for the t(14;18) by the PCR and second for immunohistochemical staining
with reagents identifying the Bcl-2 and Bax proteins. Five-micron
sections for routine staining with hematoxylin and eosin were obtained
from each biopsy specimen to show the presence of lymphoma within the material to be studied.
Flow cytometric analysis.
Three 50-micron sections were obtained from formalin-fixed blocks,
deparaffinized, and dissociated using a previously reported modification of the method described by Hedley et al.22,23 After dissociation, nuclei were permeabilized with Triton X-100 (0.1%), incubated with RNAase A, and stained with propidium iodide (50 µg/mL) as described in previous reports.23 The presence of lymphoma within the material was shown by examination of adjacent hematoxylin and eosin stained sections.
Cellular DNA content and proliferative activity (S-phase fraction) were
quantitated using a Coulter Epics 752 flow cytometer (Coulter, Miami,
FL), and analyzed using the Multicycle Software (Phoenix
Flow Systems, San Diego, CA) as previously reported.16 Cell
cycle analysis for determination of the S-phase fraction was based on
the average S-phase; consequently, for DNA-aneuploid cases this
measurement was based on the weighted average of S-phase fraction of
the DNA diploid and DNA aneuploid cellular subpopulations.
PCR for detection of t(14;18).
DNA was prepared from paraffin-embedded tissue sections by incubating
15-micron sections in 1 mL of xylene followed by three incubations with
100% ethanol. The sample was air dried and suspended in 100 µL of 50 mmol/L Tris-HCL pH 8.5, 1 mmol/L EDTA, 0.5% Triton X-100, 1 mg/mL
proteinase K.
After overnight incubation at 37°C, the proteinase K was
inactivated by boiling for 10 minutes and samples were stored at 4°C (Phenol/CHCl3 extraction followed by ethanol
precipitation of this material offered no improvement in template
quality). The suitability of the template DNA was determined by
amplification of a 267-bp human  -globin product using 10 µL of
diluted and 100-fold diluted paraffin block rescued DNA
template.24 If neither template concentration amplified the
-globin product, the case was considered unsuitable for t(14;18)
PCR. The same two templated concentrations were used before a
-globin-positive case was considered negative for t(14;18).
Templates that were amplifiable were then assessed for the presence of
t(14;18) chromosomes involving the BCL-2 major breakpoint region (mbr) using the protocol of Crescenzi et al25 and
the primers MBR5: 5 -TTAGAGAGTTGCTTACGTG and SKJH:
5-ACCTGAGGAGACGGTGCCAGGGT. The products were then subjected to
electrophoresis in 2% agarose, transferred to nylon membranes, and
probed with an end-labeled BCL-2 mbr oligonucleotide
(5-GCCTGTTCAACACAGAC).25 DNA from the t(14;18)/mbr
cell lines SU-DHL-6 or RL-7 was used as a positive control.
Those specimens that were amplifiable but negative for the t(14;18)
using the BCL-2 mbr primers were then evaluated for the presence of a t(14;18) involving the minor breakpoint cluster region
(mcr) using a primer MC12 from the mcr
5-GATGGCTTGCTGAGAGGTAT-3 and an Ig JH primer
(SKJH) by the method of Ngan et al.26 The products were
analyzed by Southern blotting with a 32P-end labeled
BCL-2 mcr oligonucleotide probe
(5-GACTCCTTTACGTGCTGGTACC).26 DNA from the mcr
t(14;18) containing cell line, SU-DHL-16, served as a positive control.
In all PCR experiments, DNA from the myeloid leukemia cell line HL60
served as a negative control. All experiments included a no-template
DNA control.
Immunohistochemical staining.
Five-micron tissue sections mounted on poly-L-lysine-coated glass
slides were deparaffinized, dehydrated, and stained as previously described using polyclonal antisera raised in rabbits against synthetic
peptides corresponding to amino acids 41 to 54 of the human Bcl-2
protein and to amino acids 43 to 61 of the human Bax protein.27,28 The slides were scored according to the
number of neoplastic cells that stained positively with the antisera. To be consistent with previous reports, cases with high levels of
BCL-2 or BAX expression were distinguished from those
considered to have minimal expression. A breakpoint of 20% positive
neoplastic cells proved to be a reproducible cutoff.
Statistical methods.
Univariate associations between dichotomous variables were evaluated
with Fisher's exact test. Associations involving ordered categorical
variables were evaluated with Wilcoxon's rank sum test.29
| |
RESULTS |
Clinical characteristics.
Paraffin-embedded material was available for 116 of 257 cases enrolled
on the parent study of ploidy and proliferative activity in the
intermediate- and high-grade NHLs (E6486) for which blocks were
received. These specimens represent the diagnostic material from
previously untreated patients with NHLs representing histological groups D through H and J by the Working Formulation30
(Table 1). All specimens underwent
secondary pathology review to confirm the histological diagnosis.
Molecular studies.
PCR-based assays were used to detect the t(14;18) in paraffin-embedded
material.25,26 The sensitivity of these assays was in the
range of one t(14;18)-positive cell per 100,000 cells making it highly
unlikely that a t(14;18) in non-neoplastic cells would be detected
(Fig 1A).31 DNA was prepared
from 115 cases but amplification was successfully achieved in fewer
than half of these (Table 2). The t(14;18)
was detected in 21 of the 54 cases in which amplification occurred (Fig
1B). Some positive cases showed two bands most likely resulting from
priming from more than one JH gene. Twenty of the
translocations occurred at the major breakpoint region, and only one at
the minor breakpoint region. No two patients had identical t(14;18) PCR
products, and t(14;18) PCR products ranged in size from 80 to 500 base
pairs.
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| Fig 1.
(A) Southern blot of t(14;18) major breakpoint region PCR
products to assess assay sensitivity. Dilutions of positive control (SU-DHL-6) DNA in negative control (HL60) DNA. 1, 1:103; 2, 1:104; 3, 1:105; 4, 1:106; 5, HL60 DNA; arrow, t(14;18) PCR product. (B)
Example of Southern blot of t(14;18) major breakpoint region PCR
products from patient biopsy specimens: 1, RL-7 positive control; 2, negative control; 3 to 8, patient PCR products. Size standards at left
in base pairs.
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Amplification occurred in only 2 of 12 follicular large-cell (Group D)
and 2 of 5 diffuse small-cleaved cell (Group E) lymphoma specimens.
Both Group D and one of the two Group E biopsy specimens were positive
for the t(14;18). Fifty-seven percent of the amplifiable cases from
Group F (4 of 7), 31% from Group G (8 of 26), and 38% from Group H (6 of 16) were also positive. The t(14;18) was not detected in the one
amplifiable specimen from Group J.
Immunohistochemical studies.
Immunohistochemistry was performed on 56 cases in which material
remained after processing for molecular studies. Expression was scored
as high (>20% cells positive), intermediate (1% to 20% cells
positive), or negative (summarized in Table 2). High levels of
BCL-2 expression were found in 35 of 56 (63%) cases including
all three follicular large-cell cases (Group D) and both diffuse,
small-cleaved cell cases (Group E) that were immunostained. Intermediate expression was seen in 10 of 56 cases (18%). Seven of 56 (13%) cases were immunonegative. Four cases were inevaluable for
technical reasons. The majority of cases analyzed contained high levels
of BAX expression (19 of 25; 76%). Twelve of the 19 cases with
high levels of BAX expression also had intense Bcl-2 immunostaining. Four cases with >20% of neoplastic cells positive for Bax were negative for Bcl-2, whereas two cases with low or negative
staining for Bax were positive (>20%) for Bcl-2. No association was
found between Bcl-2 and Bax immunostaining. In all Bcl-2 immunopositive cases, discrete cell associated cytoplasmic staining was present. The
anti-Bax staining was typically uniform from cell to cell; however,
considerable cell-to-cell variability was the norm for the Bcl-2
immunostaining. All Bcl-2 and Bax immunonegative cases contained
positive staining in occasional benign small lymphocytes, thus
validating the immunostaining results.
Figure 2 provides examples of the Bax and
Bcl-2 immunostaining.
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| Fig 2.
Representative immunoperoxidase with anti-Bcl-2 and
anti-Bax antisera. (A) anti-Bcl-2 study of lymphoma negative for Bcl-2 (arrowhead indicates positive staining in background small lymphocyte); (B) same case as (A) showing positive staining in greater than 20% of
lymphoma cells with anti-Bax antisera; (C) anti-Bcl-2 study of
lymphoma showing Bcl-2 positivity in fewer than 20% of the neoplastic
cells (arrowheads indicate positive large atypical cells); (D) same as
(C) showing positive staining in greater than 20% of lymphoma cells
with anti-Bax antisera; (E) anti-Bcl-2 study of lymphoma showing Bcl-2
positivity in greater than 20% of the neoplastic cells; (F) same case
as (E) showing lymphoma with greater than 20% of cells positive for
Bax.
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Associations between variables.
BCL-2 expression was inversely associated with proliferative
activity (P = .001; Table 3). Cases
with >20% neoplastic cells positive for Bcl-2 had the lowest median
S-phase, whereas those that were entirely negative for Bcl-2 had the
highest %S-phase. This analysis was based on the 41 cases for which
both flow cytometric quantitation of %S-phase and immunohistochemical
analysis for Bcl-2 were available. This association was maintained if
the five cases representing Working Formulation categories D and E were excluded from the analysis. There was no evidence of an association between anti-Bax immunopositivity and proliferative activity (P = .79 by Wilcoxon's test). Consistent with the results of the larger
parent study of ploidy and proliferative activity, 57% of cases
included in this study were aneuploid by flow cytometry and the median
%S-phase was 10.2 (interquartile range: 6.4-17.6). Ploidy was not
associated with positive immunostaining for either Bcl-2 or Bax.
The presence of the t(14;18) rearrangement at the major breakpoint
region was also not associated with either DNA ploidy or proliferative
activity. The percent of neoplastic cells positive for Bcl-2 or Bax by
immunohistochemistry was independent of the t(14;18); a high percentage
of cells stained with anti-Bcl-2 in cases that were negative for the
translocation.
| |
DISCUSSION |
In this study, immunohistochemically detected BCL-2 expression
was associated with low proliferative activity (%S-phase) in biopsy
specimens from untreated patients with intermediate- and high-grade NHLs. This result is consistent with recent in vitro data showing that the Bcl-2 protein retards entry into cell
cycle.17-19 These findings also confirm trends reported in
previous immunohistochemical studies of follicular
lymphomas32,33 in which BCL-2 expression was
associated with proliferative activity. Bcl-2 has been shown to inhibit
programmed cell death under a wide range of circumstances, but a common
molecular mechanism has not yet been established. Recent studies
linking activators of cell cycle progression to the induction of
programmed cell death have led investigators to hypothesize that Bcl-2
may exert at least part of its antiapoptotic effects through regulation
of either cell cycle entry (G0 to G1 ) or cell
cycle progression at the G1 to S-phase transition. The association of BCL-2 expression with low proliferative activity in the intermediate- and high-grade NHLs seen in this study is consistent with this proposal and provides indirect support for a model
in which Bcl-2 suppresses cell proliferation in vivo. The
implications of this association for both the process of
lymphomagenesis and clinical behavior remain to be elucidated.
Overexpression of wild-type BCL-2 in the B cells of transgenic
mice has been shown to inhibit cell death leading to an excess accumulation of lymphocytes without an increase in
proliferation.34 Similarly, BCL-2 overexpression
has been shown to protect interleukin-3 (IL-3)-dependent lymphoid
cells from apoptosis induced by growth factor deprivation without
promoting IL-3-independent cell growth. When cells that overexpress
BCL-2 are first deprived of IL-3 and then reexposed
to the cytokine, they exhibit a prolonged lag phase before
S-phase.35 An elegant genetic murine T-cell model providing a gradient of BCL-2 expression has been used to show that the level of BCL-2 expression determines the duration of
G0 to S-phase transition, with cells not expressing
BCL-2 showing a shortened G0 to S-phase transition
and cells that overexpress wild-type BCL-2 showing a delay in
progress into cell cycle.17 Studies using other lymphoid
systems including one B-cell model have also shown a Bcl-2-induced
prolongation of the G0/G1 transition or G1-phase.18,19,36 In one of these studies,
Bcl-2-induced accumulation of cells at the
G0/G1 stage was reversed by the Bax protein,
which heterodimerizes with Bcl-2 promoting apoptosis.19
Therefore, the association of BCL-2 expression with a lower
proliferative index would appear to be reasonable in light of current
molecular theory.
An association between low proliferative activity and apoptotic indices
has been previously shown in one small series of gastrointestinal lymphomas of mucosa-associated lymphoid tissue (MALT)37 and in two studies of follicular NHLs,32,33 but not in previous studies of the intermediate- and high-grade
NHLs.38-40 In a compilation of 40 MALT lymphomas (19 low-grade, 21 high-grade), proliferative indices quantitated by
immunostaining for the Ki-67 antigen and apoptotic indices calculated
by in situ end-labeling were loosely correlated (r = 0.586;
P < .001).37 The low proliferative activity characteristic of the low-grade lymphomas and the high frequency of the
t(14;18) with its associated BCL-2 expression are likely to
favor an association between proliferative activity and apoptotic indices whenever low- and intermediate-grade NHLs are combined. Nevertheless, Barrans et al32 could not show a
statistically significant correlation between the number of cells
expressing BCL-2 and either p53 or MIB1(anti-Ki-67) in
follicular center cell lymphomas, although cases with high levels of
expression of BCL-2 did tend to have lower rates of
proliferation.32 Among diffuse large-cell lymphomas,
proliferative activity was significantly lower in cases in which the
t(14;18) was detected and was comparable with that observed in
follicular lymphomas.41 In a large series of NHLs with
diffuse architectural patterns, an apparent inverse correlation between
growth fraction and percentage of Bcl-2 immunopositive cells was noted
but this association lost statistical significance when the low- and
high-grade lymphomas were examined separately.38 Mitotic
and apoptotic indices quantitated by counting mitoses and the number of
pyknotic cells per 30 high-powered fields were loosely correlated in a
nonlinear fashion in a series of diffuse centrocytic and/or
centroblastic NHLs.39 In the intermediate-grade subset of
biopsy specimens from patients with relapsed or refractory NHL
undergoing salvage therapy, there was a trend towards an inverse correlation between anti-Ki-67 staining and BCL-2
expression.40
In contrast to the previously reported series, the NHLs examined here
were restricted to the intermediate and high-grade NHLs (groups D-H and
J). The parent study from which these specimens were drawn included all
intermediate- and high-grade categories, excluding lymphoblastic
lymphoma, based on the eligibility for the clinical trials in which
these patients participated.16,20,21 Proliferative
activity, measured by flow cytometry correlates with the
morphologically based low-, intermediate-, and high-grade categories of
the Working Formulation.42 Categories D and E have
proliferative activity that is intermediate between that of the diffuse
mixed (Category F) and diffuse large-cell lymphomas (Category G) and
the follicular low-grade NHLs.42 Whereas the frequency of
the t(14;18) is greater among Groups D and E than the other
intermediate-grade NHLs, a greater proportion of Group D and E NHLs are
likely to stain for Bcl-2 using immunohistochemical techniques.43-45 A preponderance of cases of low S-phase
with a high likelihood of Bcl-2 positivity might favor an association between these two characteristics. Hence, the analysis was performed with and without these two categories, and the inverse correlation was
maintained even when cases from categories D and/or E were excluded. Of note, the one case from category J included in the Bcl-2
versus proliferative activity analysis was Bcl-2 positive, with an
exceptionally low %S-phase (8.8) for this histological subtype. None
of the cases included in this series were known to have evolved from
low-grade NHLs, although a proportion of large-cell lymphomas may
derive from previously undetected follicular lymphomas that have
retained the t(14;18).46
The Bcl-2 homolog and heterodimerization partner, Bax, is a cell death
promoter.47,48 The ratio of Bax to Bcl-2
determines the relative sensitivity or resistance of cells to apoptotic
stimuli. In previous in vitro studies, Bax has been shown to override
the inhibitory effects of Bcl-2 on the cell cycle.19 The
majority of cases in which material was available for immunostaining
with anti-Bax antibodies was strongly positive, but too few cases were analyzed overall to evaluate the Bcl-2/Bax ratio and its association with proliferative activity. In recent studies, BAX expression has been associated with increased proliferative
activity.49 In the NHLs, the ratio of Bcl-2 to Bax seems
likely to play an important role in determining both proliferation and
susceptibility to chemotherapy.2,47
PCR amplifiable DNA was isolated from the paraffin-embedded specimens
in only 47% of the cases in this series. These specimens were obtained
from both academic and community centers belonging to the Eastern
Cooperative Oncology Group and the Southwestern Oncology Group in the
United States and South Africa; differences in the fixation methodology
between institutions may underlie the low success rate. Although all
blocks contained formalin-fixed material, the duration of fixation was
not specified and may have varied widely from institution to
institution. The generation of a PCR product is dependent in part on
the duration of formalin fixation.50 In addition, prolonged
fixation may interfere with the amplification of larger products such
as those generated by the minor breakpoint region
primers.51 This may explain the lower than expected
percentage of cases positive for the t(14;18) minor breakpoint cluster
type translocations.7
False-negative PCR reactions may also occur when the translocation
breakpoint is not encompassed by the PCR primer used. Comparative studies of PCR and Southern blot and/or cytogenetic detection of the t(14;18) indicate that, depending on the protocol, PCR can
detect 75% to 83% of translocations.52,53 In addition, there will be false negatives resulting from DNA degradation during fixation which precludes the amplification of larger products. A study
comparing fresh versus formalin-fixed material from the same cases that
used very similar DNA extraction and PCR protocols to those used in
this study indicated up to 20% false negatives related to fixation and
embedding techniques.54 This may be especially problematic
in the detection of translocations producing long PCR products that are
beyond the range of confirmed amplification of the DNA isolated in our
series. These factors may have also contributed to the detection of
only one minor breakpoint cluster type t(14;18). However, one large
North American mapping study found only 7 of 91 (8%) of
cytogenetically proven t(14;18) lymphomas to have the minor breakpoint
cluster type translocations.53 Although these technical
considerations suggest that there were false negatives in the molecular
analysis, the overall percentage of cases positive for the t(14;18) in
this report (39%) is consistent with earlier cytogenetic, Southern,
and PCR based studies of diffuse B-cell lymphoma.55-57 The
fact that there were cases with high level BCL-2 expression
without evidence of the t(14;18) is consistent with the results of
other studies and supports the hypothesized existence of mechanisms
other than chromosomal translocation for dysregulated BCL-2
expression.8,57
Three large clinical trials have now established the prognostic
significance of BCL-2 expression in the previously untreated diffuse large-cell lymphomas.9-11 Disease-free survival but
not overall survival has been inversely associated with BCL-2
expression in these studies. Although drug resistance related to Bcl-2
has been proposed as the underlying basis for this clinical behavior, at least one study of relapsed and refractory patients has shown no
relationship between the level of Bcl-2 protein and response to a novel
chemotherapeutic regimen.40 In some series, the prognostic significance of p53 mutations were also investigated and showed an
association with drug resistance and/or a poor overall
survival.40,58 The tumor suppressor gene p53
induces cell cycle arrest and apoptosis and its loss has been
associated with drug resistance.59 In the present series,
insufficient tissue prevented the investigation of p53 mutations and
expression of p21/WAF1, its functional effector. WAF1 has been
implicated in growth arrest through interference with transition from
G1 into S-phase, and may have prognostic significance either alone or
in combination with the identification of p53 mutations.60
There were too few cases in this series to investigate BCL-2
expression and proliferative activity as predictors of clinical outcome using a multivariate analysis. Molecular analysis and later
immunohistochemical staining for Bcl-2 and Bax were appended to the
flow cytometric studies of ploidy and proliferative activity, which
were the initial and primary intent of the parent
protocol.16 Hence, only a small number of cases had
residual material in the submitted block, and in many cases the limited
tissue was exhausted during preparation of DNA. Whereas these tissues
were submitted by a large number of institutions, there was
considerable variation in the quality of fixation resulting in poor
preparations for both PCR and flow cytometric analysis in some cases.
This further limited the number of cases with both S-phase quantitation
and immunohistochemistry.
To be consistent with previous publications,9-12 cases with
minimal staining with anti-Bax and anti-Bcl-2 were distinguished from
those with greater numbers of positive cells. A cutoff of 20% was
chosen, reflecting the fact that a significant number of cases had 10%
to 15% positive cells. With this cutoff, 67% of evaluable cases
showed staining with anti-Bcl-2 in greater than 20% of the neoplastic
cells and were judged to be strong positives, which is in the same
range as that reported in the larger series by Hill et al10
and by Hermine et al.9
Most studies, including the parent study of DNA content from which
these specimens were drawn, have established proliferative activity as
a prognostic indicator in the NHLs.13-16 In our previous study,16 a trend associating low proliferative activity
with good early survival and very high S-phase with shortened survival was shown leading us to conclude that proliferative activity is probably predictive of survival although clinical parameters
represented by the International Index proved to be more powerful
prognostic indicators than %S-phase. No variable was predictive of
disease-free survival or time to treatment failure. In a much smaller
study by Miller15 in which the monoclonal antibody Ki-67
was used to assess proliferative activity, growth fraction was a
powerful predictor of survival. In contrast, Bcl-2 has been associated with disease-free survival but not overall survival in most of the
larger clinical trials.10-12 The associations between Bcl-2 immunopositvity and low proliferative activity and between
proliferative activity and overall survival are not inconsistent with
this finding. Bcl-2 expression may identify a subset with low
proliferative activity with a predisposition to early relapse, but not
necessarily a shortened survival. This subset would have a clinical
behavior not unlike the indolent lymphomas. The apparent discrepancies between the reported series will need to be resolved in the context of
large studies in which Bcl-2 immunostaining, Southern analysis for the
t(14;18), and proliferative activity are measured on fresh tissue.
Although the numbers of specimens studied both for Bcl-2
immunopositivity and S-phase were too few to draw clinical inferences regarding prognosis, the association between BCL-2 expression and low S-phase in clinical specimens complements the results of
laboratory investigations into the relationship between apoptosis and
cell cycle pathways. The effect of Bcl-2 on cell cycle transition may
underlie its apparent effect on chemosensitivity and clinical outcome. In the future, a combination of molecular and
immunohistochemical variables may be shown to more accurately predict
patient response to therapy and survival than clinical parameters.
Classification of NHLs by biological characteristics such as BCL-2
expression may be of greater clinical significance than morphological
categories.
| |
FOOTNOTES |
Submitted April 30, 1997;
accepted October 8, 1997.
Coordinated by the Eastern Cooperative Oncology Group (Robert L. Comis,
MD, Chair) and supported in part by the American Cancer Society,
Illinois Division, and Public Health Service Grants No. CA17145,
CA23318, CA49883, CA 20365, CA2111519, CA60421, CA 32102, and CA66636
from the National Cancer Institute, National Institutes of Health, and
the Department of Health and Human Services. Its contents are solely
the responsibility of the authors and do not necessarily represent the
official views of the National Cancer Institute.
Address reprint requests to Jane N. Winter, MD, Rm 1456A, Wesley
Pavilion, Northwestern Memorial Hospital, 250 E Superior St, Chicago,
IL 60611.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
REFERENCES |
1.
Yang E,
Korsmeyer SJ:
Molecular thanatopsis: A discourse on the BCL2 family and cell death.
Blood
88:386,
1996[Free Full Text]
2.
Reed JC:
BCL-2: Prevention of apoptosis as a mechanism of drug resistance.
Hematol Oncol Clin North Am
9:451,
1995[Medline]
[Order article via Infotrieve]
3.
Tsujimoto T,
Finger LR,
Yunis J,
Nowell PC,
Croce CM:
Cloning the chromosome breakpoint of neoplastic B-cells with the t(14;18) chromosome translocation.
Science
226:1097,
1984[Abstract/Free Full Text]
4.
Bakhshi A,
Jensen JP,
Goldman P,
Wright JJ,
McBride OW,
Epstein AL,
Korsmeyer SJ:
Cloning the chromosomal breakpoint of t(14;18) human lymphomas: Clustering around JH on chromosome 14 and near a transcriptional unit on 18.
Cell
41:899,
1985[Medline]
[Order article via Infotrieve]
5.
Cleary ML,
Sklar J:
Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18.
Proc Natl Acad Sci USA
82:7439,
1985[Abstract/Free Full Text]
6.
Yunis JJ,
Frizzera G,
Oken MM,
McKenna J,
Theologides A,
Arnesen M:
Multiple recurrent genomic defects in follicular lymphoma.
N Engl J Med
316:79,
1987[Abstract]
7.
Weiss LM,
Warnke RA,
Sklar J,
Cleary ML:
Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas.
N Engl J Med
317:1185,
1987[Abstract]
8.
Zutter M,
Hockenbery D,
Silverman G,
Korsmeyer SJ:
Immunolocalization of the Bcl-2 protein within hematopoietic neoplasms.
Blood
78:1062,
1991[Abstract/Free Full Text]
9.
Hermine O,
Haioun C,
Lepage E,
d'Agay M-F,
Briere J,
Lavignac C,
Fillet G,
Salles G,
Marolleau JP,
Diebold J,
Reyes F,
Gaulard P:
Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin's lymphoma.
Blood
87:265,
1996[Abstract/Free Full Text]
10.
Hill ME,
MacLennan KA,
Cunningham DC,
Vaughan Hudson B,
Burke M,
Clarke P,
Di Stefano F,
Anderson L,
Vaughan Hudson G,
Mason D,
Selby P,
Linch DC:
Prognostic significance of BCL-2 expression and bcl-2 major breakpoint region rearrangement in diffuse large cell non-Hodgkin's lymphoma: A British National Lymphoma Investigation study.
Blood
88:1046,
1996[Abstract/Free Full Text]
11.
Kramer MHH,
Hermans J,
Parker J,
Krol ADG,
Kluin-Nelemans JC,
Haak HL,
van Groningen K,
van Krieken JHJM,
de Jong D,
Kluin PM:
Clinical significance of bcl-2 and p53 protein expression in diffuse large B-cell lymphoma: A population based study.
J Clin Oncol
14:2131,
1996[Abstract/Free Full Text]
12.
Gascoyne RD,
Adomat SA,
Krajewski S,
Krajewska M,
Horsman DE,
Tolcher AW,
O'Reilly SE,
Hoskins P,
Coldman AJ,
Reed JC,
Connors JM:
Prognostic significance of Bcl-2 protein expression and Bcl-2 gene rearrangement in diffuse aggressive non-Hodgkin's lymphoma.
Blood
90:244,
1997[Abstract/Free Full Text]
13. Braylan RC: Lymphomas, in Bauer KD, Duque RE, Shankey TV (eds):
Flow Cytometry: Principles and Applications. Baltimore, MD, Williams & Wilkins, 1993, p 203
14.
Duque RE,
Andreef M,
Braylan RC,
Diamond LW,
Peiper SC:
Consensus review of the clinical utility of DNA flow cytometry in neoplastic hematopathology.
Cytometry
14:492,
1993[Medline]
[Order article via Infotrieve]
15.
Miller TP,
Grogan TM,
Dahlberg S,
Spier CM,
Braziel RM,
Banks PM,
Foucar K,
Kjeldsberg CR,
Levy N,
Nathwani BN,
Schnitzer B,
Tubbs RR,
Gaynor ER,
Fisher RI:
Prognostic significance of the Ki-67 associated proliferative antigen in aggressive non-Hodgkin's lymphomas: A prospective Southwest Oncology Group trial.
Blood
83:1460,
1994[Abstract/Free Full Text]
16.
Winter JN,
Andersen J,
Variakojis D,
Gordon LI,
Fisher RI,
Oken MM,
Neiman RS,
Jiang S,
Bauer KD:
Prognostic implications of ploidy and proliferative activity in the diffuse, aggressive non-Hodgkin's lymphomas.
Blood
88:3919,
1996[Abstract/Free Full Text]
17.
Linette GP,
Li Y,
Roth K,
Korsmeyer SJ:
Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.
Proc Natl Acad Sci USA
93:9545,
1996[Abstract/Free Full Text]
18.
Mazel S,
Burtrum D,
Petrie HT:
Regulation of cell division cycle progression by bcl-2 expression: A potential mechanism for inhibition of programmed cell death.
J Exp Med
183:2219,
1996[Abstract/Free Full Text]
19.
Borner C:
Diminished cell proliferation associated with the death protective activity of Bcl-2.
J Biol Chem
271:12695,
1996[Abstract/Free Full Text]
20.
Gordon LI,
Harrington D,
Andersen J,
Colgan J,
Glick J,
Neiman R,
Mann R,
Resnick GD,
Barcos M,
Gottlieb A,
O'Connell M:
Comparison of a second-generation combination chemotherapeutic regimen (m-BACOD) with a standard regimen (CHOP) for advanced diffuse non-Hodgkin's lymphoma.
N Engl J Med
327:1342,
1992[Abstract]
21.
Fisher RI,
Gaynor ER,
Dahlberg S,
Oken MM,
Grogan TM,
Mize EM,
Glick JH,
Coltman CA Jr,
Miller TP:
Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin's lymphoma.
N Engl J Med
328:1002,
1993[Abstract/Free Full Text]
22.
Hedley DW,
Friedlander ML,
Taylor IW,
Rugg CA,
Musgrove EA:
Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry.
J Histochem Cytochem
31:1333,
1983[Abstract]
23.
Bauer KD,
Merkel DE,
Winter JN,
Marder RJ,
Hauck WW,
Wallmark CB,
William TJ,
Variakojis D:
Prognostic implications of ploidy and proliferative activity in diffuse, large cell lymphomas.
Cancer Res
46:3173,
1986[Abstract/Free Full Text]
24.
Bauer HM,
Ting Y,
Greer CE,
Chambers JC,
Tashiro CJ,
Chimera J,
Reingold A,
Manos MM:
Genital human papillomavirus infection in female university students as determined by a PCR-based method.
JAMA
265:472,
1991[Abstract/Free Full Text]
25.
Crescenzi M,
Seto M,
Herzig GP,
Weiss PD,
Griffith RC,
Korsmeyer SJ:
Thermostable DNA polymerase chain amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease.
Proc Natl Acad Sci USA
85:4869,
1988[Abstract/Free Full Text]
26.
Ngan B-Y,
Nourse J,
Cleary M:
Detection of chromosomal translocation of t(14;18) within the minor cluster region of bcl-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas.
Blood
73:1759,
1989[Abstract/Free Full Text]
27.
Krajewski S,
Bodrug S,
Gascoyne R,
Berean K,
Krajewska M,
Reed JC:
Immunohistochemical analysis of Mcl-1 and Bcl-2 proteins in normal and neoplastic lymph nodes.
Am J Pathol
145:515,
1994[Abstract]
28.
Krajewski S,
Blomqvist C,
Franssila K,
Krajewska M,
Wasenius V-M,
Niskanen E,
Nordling S,
Reed JC:
Reduced expression of proapoptotic gene BAX is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma.
Cancer Res
55:4471,
1995[Abstract/Free Full Text]
29.
Mehta CR,
Patel NR,
Tsiatis AA:
Exact significance testing for ordered categorical data.
Biometrics
40:819,
1984[Medline]
[Order article via Infotrieve]
30.
The Non-Hodgkin's Lymphoma Pathologic Classification Project:
National Cancer Institute sponsored study of classifications of non-Hodgkin's lymphomas: Summary and description of a working formulation for clinical usage.
Cancer
49:2112,
1982[Medline]
[Order article via Infotrieve]
31.
Segal G,
Scott M,
Jorgensen T,
Braylan RC:
Standard polymerase chain reaction analysis does not detect t(14;18) in reactive lymphoid hyperplasia.
Arch Path Lab Med
118:791,
1994
32.
Barrans S,
Randerson J,
Evans P,
Blythe D,
Shiach C,
Child JA,
Morgan G,
Jack AS:
Heterogeneity in cell proliferation and expression of p53 and bcl-2 during the indolent phase of germinal center cell lymphoma: An explanation for clinical variability.
Br J Haematol
90:830,
1995[Medline]
[Order article via Infotrieve]
33.
Leoncini L,
Del Vecchio MT,
Megha T,
Barbini P,
Galieni P,
Pileri S,
Savattini E,
Gherlinzoni F,
Tosi P,
Kraft R,
Cottier H:
Correlations between apoptotic and proliferative indices in malignant and non-Hodgkin's lymphomas.
Amer J Pathol
142:755,
1993[Abstract]
34.
McDonnell TJ,
Deane N,
Platt FM,
Nunez G,
Jaeger U,
McKearn J,
Korsmeyer S:
Bcl-2 immunoglobulin transgenic mice demonstrate extended B-cell survival and follicular lymphoproliferation.
Cell
57:79,
1989[Medline]
[Order article via Infotrieve]
35.
Marvel J,
Perkins GR,
Lopez Rivas A,
Collins MKL:
Growth factor starvation of bcl-2 overexpressing murine bone marrow cells induced refractoriness to IL-3 stimulation of proliferation.
Oncogene
9:1117,
1994[Medline]
[Order article via Infotrieve]
36.
O'Reilly LA,
Huang DC,
Strasser A:
The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry.
EMBO J
15:6979,
1996[Medline]
[Order article via Infotrieve]
37.
Du M,
Singh N,
Husseuin A,
Isaacson PG,
Pan L:
Positive correlation between apoptotic and proliferative indices in gastrointestinal lymphomas of mucosa-associated lymphoid tissue (MALT).
J Pathol
178:379,
1996[Medline]
[Order article via Infotrieve]
38.
Leoncini L,
Del Vecchio MT,
Spina D,
Megha T,
Barbini P,
Sabattini E,
Pileri S,
Tosi P,
Kraft R,
Laissue JA,
Cottier H:
Presence of the bcl-2 protein and apoptosis in non-Hodgkin lymphomas with diffuse growth pattern.
Int J Cancer
61:826,
1995[Medline]
[Order article via Infotrieve]
39.
Del Vecchio MT,
Leoncini L,
Buerki K,
Kraft R,
Megha T,
Barbini P,
Tosi P,
Cottier H:
Diffuse centrocytic and/or centroblastic malignant non-Hodgkin's lymphomas: Comparison of mitotic and pyknotic (apoptotic) indices.
Int J Cancer
47:38,
1991[Medline]
[Order article via Infotrieve]
40.
Wilson WH,
Teruya-Feldstein J,
Fest T,
Harris C,
Steinberg SM,
Jaffe ES,
Raffeld M:
Relationship of p53, bcl-2, and tumor proliferation to clinical drug resistance in non-Hodgkin's lymphomas.
Blood
89:601,
1997[Abstract/Free Full Text]
41.
Grierson HL,
Wooldridge TN,
Hess M,
Ratashak A,
Wooldridge L,
Fordyce-Boyer R,
Bast M,
Armitage JO,
Weisenburger DD,
Sanger WG:
Proliferative fraction and DNA content are lower in B-cell non-Hodgkin's lymphomas with the t(14;18).
Leuk Lymphoma
19:253,
1995[Medline]
[Order article via Infotrieve]
42.
Srigley J,
Barlogie B,
Butler JJ,
Osborne B,
Blick M,
Johnston D,
Kantarjian H,
Reuben J,
Batsakis J,
Freireich E:
Heterogeneity of non-Hodgkin's lymphoma probed by nucleic acid cytometry.
Blood
65:1090,
1985[Abstract/Free Full Text]
43.
Weiss LM,
Warnke RA,
Sklar J,
Cleary ML:
Molecular analysis of the t(14;18) chromosomal translocation in malignant lymphomas.
N Engl J Med
317:1185,
1987
44.
Levine EG,
Arthur DC,
Frizzera G,
Peterson BA,
Hurd DD,
Bloomfield CD:
There are differences in cytogenetic abnormalities among histologic subtypes of the non-Hodgkin's lymphomas.
Blood
66:1414,
1985[Abstract/Free Full Text]
45. (suppl 6)
Gulley ML,
Dent GA,
Ross DW:
Classification and staging of lymphoma by molecular genetics.
Cancer
69:1600,
1992[Medline]
[Order article via Infotrieve]
46.
Matolcsy A,
Casali P,
Warnke RA,
Knowles DM:
Morphologic transformation of follicular lymphoma is associated with somatic mutation of the translocated Bcl-2 gene.
Blood
88:10,
1996
47.
McConkey DJ,
Chandra J,
Wright S,
Funkett W,
McDonnell TJ,
Reed JC,
Keating M:
Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.
J Immunol
156:2624,
1996[Abstract]
48.
Hanada M,
Aime-Sempe C,
Sato T,
Reed JC:
Structure-function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax.
J Biol Chem
270:11962,
1995[Abstract/Free Full Text]
49.
Brady HJM,
Gil-Gomez G,
Kirberg J,
Berns AJM:
Bax-alpha perturbs T cell development and affects cell cycle entry of T cells.
EMBO J
15:6991,
1996[Medline]
[Order article via Infotrieve]
50.
Karlsen F,
Kalantari M,
Chitemerere M,
Johansson B,
Hagmar B:
Modifications of human and viral deoxyribonucleic acid by formaldehyde fixation.
Lab Invest
71:604,
1994[Medline]
[Order article via Infotrieve]
51.
Coad JE,
Olson DJ,
Lander TA,
McGlennen RC:
Molecular assessment of clonality in lymphoproliferative disorders: I. Immunoglobulin gene rearrangements.
Mol Diagn
4:335,
1996
52.
Ladanyi M,
Wang S:
Detection of rearrangement of BCL2 major breakpoint region in follicular lymphomas, correlation of polymerase chain reaction results with Southern blot analysis.
Diagn Mol Pathol
1:31,
1992[Medline]
[Order article via Infotrieve]
53.
Horsman DE,
Gascoyne RD,
Coupland RW,
Goldman AJ,
Adomat SA:
Comparison of cytogenetic analysis, Southern analysis, and polymerase chain reaction for the detection of t(14;18) in follicular lymphoma.
Am J Clin Pathol
103:472,
1995[Medline]
[Order article via Infotrieve]
54.
Lui J,
Johnson RM,
Traweek ST:
Rearrangement of the BCL-2 gene in follicular lymphoma, detection by PCR in both fresh and fixed tissue samples.
Diagn Mol Pathol
2:241,
1993[Medline]
[Order article via Infotrieve]
55.
Yunis JJ,
Mayer MB,
Arnesen MA,
Aeppli DP,
Oken MM,
Frizzera G:
Bcl-2 and other genomic alteration in the prognosis of large-cell lymphoma.
N Engl J Med
320:1047,
1989[Abstract]
56.
Jacobson JO,
Wilkes BM,
Kwiatkowski DJ,
Medeiros LJ,
Aisenberg AC,
Harris NL:
Bcl-2 rearrangement in de novo diffuse large cell lymphomas.
Cancer
72:231,
1993[Medline]
[Order article via Infotrieve]
57.
Tang SC,
Visser L,
Hepperle B,
Hanson J,
Poppema S:
Clinical significance of bcl-2 MBR gene rearrangement and protein expression in diffuse large cell non-Hodgkin's lymphoma: An analysis of 83 cases.
J Clin Oncol
12:149,
1994[Abstract]
58.
Piris MA,
Pezella F,
Martinex-Montero JC,
Orradre JL,
Villuendas R,
Sanchez-Beato M,
Cuena R,
Cruz MA,
Martinez B,
Garrido MC,
Gatter K,
Aiello A,
Delia D,
Giardini R,
Rilke F:
p53 and bcl-2 expression in high-grade B-cell lymphomas: Correlation with survival time.
Br J Cancer
69:337,
1994[Medline]
[Order article via Infotrieve]
59.
Shimamura A,
Fisher DE:
p53 in life and death.
Clin Cancer Res
2:435,
1996[Medline]
[Order article via Infotrieve]
60.
Chilosi M,
Doglioni C,
Magalini A,
Inghirami G,
Krampera M,
Nadali G,
Rahal D,
Pedron S,
Benedetti A,
Scardoni M,
Macri E,
Lestani M,
Menestrina F,
Pizzolo G,
Scarpa A:
p21/WAF1 cyclin-kinase inhibitor expression in non-Hodgkin's lymphomas: A potential marker of p53 tumor-suppressor gene function.
Blood
88:4012,
1996[Abstract/Free Full Text]
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Abstract
Introduction
Abstract