The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.

DIFFUSE large cell lymphomas (DLCL) represent a diverse spectrum of lymphoid neoplasms with variable clinical, histologic, immunophenotypic, cytogenetic, and molecular genetic features.1,2 Therapy for these non-Hodgkin's lymphomas (NHLs) has greatly improved over the last two decades, with over half the patients experiencing long-term cure.3-6 Unfortunately, 40% to 50% of patients are not cured by multi-agent chemotherapy regimens, thus highlighting the need to develop models that identify potential patients better served by risk-adjusted therapies. Survival can be predicted on the basis of clinical characteristics, as recently established by the International Non-Hodgkin's Lymphoma Prognostic Factors Project.7 This model is useful for identifying at-risk patients who may benefit from more intensive therapy, but does not address the underlying biology of these heterogeneous diseases. Thus, an assessment of clinical factors is unlikely to be helpful in the design of specific therapies aimed at the molecular defects that characterize DLCLs.

The bcl-2 gene was originally discovered by virtue of its involvement in the (14; 18) (q32;q21) translocation.8-11 This cytogenetic abnormality results in deregulated expression of Bcl-2 protein and is found in the majority of follicular lymphomas (FL) and a variable number (10% to 40%) of DLCLs.12-33 Expression of Bcl-2 protein is independent of the translocation, as evidenced by its expression in a number of normal tissues, as well as a spectrum of lymphoproliferative disorders without a t(14; 18).34 A high level of Bcl-2 protein confers a survival advantage on B cells by inhibiting apoptosis and more generally may block a common cell death pathway induced by chemotherapy, conferring clinical drug resistance on cells over-expressing Bcl-2 protein.35,36 Bcl-2 protein expression has been shown to predict for poor outcome in acute myeloid leukemia, but conflicting results have been reported for acute lymphoblastic leukemia.37-39 

Previous studies of bcl-2 gene rearrangement in DLCLs have been hampered by patient selection, nonuniform treatment strategies, variable molecular techniques for assessing bcl-2, and the inclusion of patients with antecedent low-grade follicular lymphoma.17-19,21-32 Recent studies have used more stringent inclusion criteria, but were analyzed using only mbr breakpoints.33 Much less is known about Bcl-2 protein expression in DLCL, but most studies have shown little impact on survival.30,31,40 Recently, several studies have suggested that Bcl-2 protein expression is an important predictor of disease-free survival (DFS), but a significant effect on overall survival was not seen.33,41 42 The purpose of this study was to determine the clinical utility of bcl-2 gene rearrangement (both mbr and mcr) and Bcl-2 protein expression for predicting overall survival (OS), DFS, and relapse-free survival (RFS) in a cohort of advanced-stage DLCL patients treated with uniform chemotherapy at a single institution.

Patients.This study includes 145 consecutively encountered patients with diffuse aggressive lymphomas diagnosed between 1981 and June 1989 at the British Columbia Cancer Agency. This institution is the primary referral center for NHLs in the province of British Columbia, seeing the majority of patients with DLCL. Eligibility criteria were the following: age 16 to 70 years; diffuse lymphoma of large cell type (diffuse mixed, diffuse large cell, and immunoblastic lymphoma, Working Formulation categories F, G, or H); advanced disease with stage III, IV, or II with B symptoms or a mass greater than 10 cm; no prior treatment for lymphoma; and no congestive heart failure.43 Lymphomas related to acquired immune deficiency syndrome or organ transplantation were excluded. Patients with antecedent low-grade lymphoma or discordant lymphoma at diagnosis were also excluded. Eligible patients were treated with MACOP-B (n = 121)3 or VACOP-B (n = 18)4 as previously reported. This consisted of a 12-week outpatient regimen of oral and intravenous medications including prednisone, doxorubicin, and cyclophosphamide, alternating with vincristine plus either bleomycin or moderate-dose methotrexate with leucovorin rescue.3 Patients given VACOP-B received etoposide instead of methotrexate, but were otherwise treated the same. Ninety percent of the patients received more than 80% of the planned dose of chemotherapy. Details of the patients' characteristics and treatment delivery and outcome have been previously published.3 4 Of the total of 145 patients, 6 were excluded as no blocks were available for analysis (n = 139). Clinical features and histology were prospectively collected and entered into a computerized database at diagnosis and at follow-up. Bcl-2 gene rearrangement, Bcl-2 protein expression, and immunophenotype were retrospectively determined and recorded in a separate database constructed without knowledge of clinical outcome. These databases were later merged to allow this analysis of outcome.

Histology and immunohistochemistry.Tissue biopsy samples were fixed in buffered formalin or B5 fixative, routinely processed, sectioned at 3 μm, and stained with hematoxylin and eosin. All of the analyses were performed on biopsy specimens obtained before therapy. Cell lineage was assigned in each case using paraffin section immunostaining with a routine streptavidin-biotin peroxidase detection system and diaminobenzidine as chromogen. Monoclonal antibodies (MoAbs) included CD20 (L26), MB-2, CD45RO (A6, UCHL-1), CD45 (LCA), CD30 (Ber-H2), EMA, and polyclonal anti-CD3 (Dako, Carpinteria, CA). Cases were assigned a B-cell immunophenotype if positive staining was seen with CD20 and/or MB-2 but no T-cell antibodies. A T-cell immunophenotype was recorded if positive staining was seen with either CD45RO antibodies or polyclonal CD3 without B-cell staining. NHLs that failed to stain with any lineage marker were assigned a Null immunophenotype. Diagnosis of anaplastic large cell lymphoma (ALCL) was made using accepted histologic criteria and confirmed with CD30 staining.44 

Bcl-2 immunostaining was performed on diagnostic biopsies using polyclonal anti–Bcl-2 antibodies (rabbit polyclonal anti–Bcl-2; Pharmingen, San Diego, CA) after microwave antigen retrieval as previously described.45 Previous studies had shown equivalent staining with MoAb clone 124 (Dako) in B5 fixed tissues, but superior staining in buffered formalin-fixed material.46 Sections were scored as negative if no large neoplastic cells stained; +1 (1% to 10% positive cells); +2 (11% to 30%); +3 (31% to 70%); and +4 (>70% positive large cells). Of the 139 total cases, 23 were excluded because Bcl-2 staining failed to stain the normal small lymphocytes that serve as positive internal controls, leaving 116 cases for analysis of Bcl-2 protein expression.

bcl-2 gene rearrangements.DNA was extracted from formalin-fixed paraffin blocks as follows: three 20-μm sections were cut from each block containing representative tissue, with the microtome blade changed between cases to avoid contamination. One section from each case was deparaffinized and DNA extracted by routine methods.12 

Polymerase chain reaction (PCR) was performed with an automated thermal cycler (Perkin-Elmer Applied Biosystems Division, Foster City, CA) using a modification of published conditions as previously reported.12,47 For each PCR analysis, controls included a blank (no DNA template), a known positive control, and a 510-bp fragment of the β-globin gene used to assess for the presence of amplifiable DNA.12 Half of the amplified DNA was electrophoresed in 2% agarose and photographed under UV light after ethidium-bromide staining. A positive result was indicated by the presence of a single band of appropriate size in a lane of the gel.

Based on the intensity of the amplified β-globin fragment, the volume of template DNA in each PCR was adjusted appropriately so as to obtain optimum amplification with the primers for the mbr and mcr regions of the bcl-2 gene as follows: mbr: 5′- CCAAGTCATGTGCATTTCCACGTC- 3′; mcr: 5′-ACAGCGTGGTTAGGGTTAGGTCGTA-3′; consensus JH: 3′-ACCTGAGGAGACGGTGACC-5′.

Cases that failed to amplify the β-globin fragment when tested at three different dilutions were considered failures and were not amplified with the bcl-2–specific primers. Cases with amplifiable DNA that were negative after two attempts with the bcl-2 primers were considered bcl-2–negative. Amplifiable cases that showed a unique band with the bcl-2–specific primers were considered bcl-2–positive. The specificity of these amplified products was confirmed by transfer of the gel to a nylon membrane and probing with a radiolabeled oligonucleotide internal to the bcl-2 primers.

Statistical analysis.OS was calculated from the date of diagnosis until the patient's death or last follow-up. DFS was calculated as the interval between diagnosis and relapse, progression if the patient had less than a complete response, or death due to toxicity of treatment. RFS was calculated only for patients achieving a complete remission (CR) and was the interval between diagnosis and relapse of the disease. Survival curves were calculated by the method of Kaplan and Meier.48 Statistical comparison between curves was made by the log-rank test.49 Determination of significant differences in the distribution of clinical prognostic factors between groups was determined by the Pearson chi-squared test. Multivariate survival analysis was performed with the use of a stepwise proportional hazards model.50 

A total of 139 patients were identified for whom there was adequate histologic material available for analysis. Their clinical characteristics are shown in Table 1. The median age was 52 years at diagnosis (range, 20 to 69 years), and 121 patients (87%) achieved a CR. After a median follow-up of 81 months (range, 1 to 183 months), the 8-year OS, DFS, and RFS were 55%, 58%, and 51%, respectively.

Table 1.

Clinical Characteristics of 139 Patients With DLCL

CharacteristicNo. of Patients (%)
Age: 
≤60 yr 99 (71) 
>60 yr 40 (29) 
Sex: 
Male 87 (63) 
Female 52 (37) 
Site: 
Nodal 96 (69) 
Extranodal 43 (31) 
Stage: 
IIB 50 (36) 
III 35 (25) 
IV 54 (39) 
B symptoms 66 (47) 
Serum LDH: 
Normal 37 (27) 
Elevated 102 (73) 
Performance status (ECOG): 
0,1 113 (81) 
2-4 26 (19) 
Extranodal sites: 
0,1 99 (71) 
>1 40 (29) 
BM involvement 14 (10) 
CR 121 (87) 
CharacteristicNo. of Patients (%)
Age: 
≤60 yr 99 (71) 
>60 yr 40 (29) 
Sex: 
Male 87 (63) 
Female 52 (37) 
Site: 
Nodal 96 (69) 
Extranodal 43 (31) 
Stage: 
IIB 50 (36) 
III 35 (25) 
IV 54 (39) 
B symptoms 66 (47) 
Serum LDH: 
Normal 37 (27) 
Elevated 102 (73) 
Performance status (ECOG): 
0,1 113 (81) 
2-4 26 (19) 
Extranodal sites: 
0,1 99 (71) 
>1 40 (29) 
BM involvement 14 (10) 
CR 121 (87) 

Abbreviations: LDH, lactate dehydrogenase; ECOG, Eastern Cooperative Oncology Group; BM, bone marrow.

Histologic subclassification showed 23 (16%) with diffuse mixed (DM), 87 (63%) diffuse large cell (DL), 19 (14%) immunoblastic, and 10 (7%) anaplastic large cell lymphomas (ALCL). Paraffin section immunophenotyping was successful in assigning lineage in 125 of 139 (90%) cases, including 115 (83%) B-cell, 10 (7%) T-cell, and 14 (10%) with a null immunophenotype.

The analysis for bcl-2 gene rearrangement was performed on all 139 cases, with analyzable DNA present in 118 of 139 (85%) cases, including 14 positive samples. All cases with either an ALCL histologic subtype or T-cell immunophenotype having interpretable DNA results were negative for a bcl-2 gene rearrangement. If the PCR analysis is restricted to those cases with a B-cell immunophenotype and intact DNA, then 14 of 102 (14%) demonstrated a bcl-2 gene rearrangement, including 11 with an mbr and 3 with an mcr breakpoint (Fig 1). The relationship between clinical factors and the presence of a bcl-2 gene rearrangement is shown in Table 2. The international prognostic index factors (age > 60 years, elevated serum lactate dehydrogenase, stage III or IV, poor performance status, or >1 extranodal site) were evenly distributed between the bcl-2 positive versus negative groups, with the exception that the bcl-2–positive cases tended to be younger (P = .03). Interestingly, the bcl-2–negative cases were more frequently associated with concordant large cell lymphoma in the bone marrow as compared with the cases with a bcl-2 translocation, but this difference did not achieve statistical significance. The complete remission rate in both groups is virtually identical (see Table 2). Moreover, OS (57% v 57%, P = .85), DFS (42% v 47%, P = .73), and RFS (43% v 51%, P = .61) at 8 years were similar for bcl-2–positive and bcl-2–negative cases, respectively. The OS and DFS curves are shown in Figs 2 and 3, respectively, and include a separate curve of the 21 (15%) cases without analyzable DNA.

Fig. 1.

Agarose gel (2%) showing the 14 patients with a bcl-2 rearrangement by PCR. The top gel demonstrates all the bcl-2–positive cases. Bands are seen using mbr primers in all lanes except 3, 10, and 13. These three cases are shown in the lower gel and are positive for mcr rearrangements.

Fig. 1.

Agarose gel (2%) showing the 14 patients with a bcl-2 rearrangement by PCR. The top gel demonstrates all the bcl-2–positive cases. Bands are seen using mbr primers in all lanes except 3, 10, and 13. These three cases are shown in the lower gel and are positive for mcr rearrangements.

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Table 2.

Clinical Characteristics of 118 Patients With DLCL Who Had DNA That Was Analyzable for t(14; 18) by PCR (bcl-2 gene rearrangement)

CharacteristicNo. t(14; 18) Positive (%)No. t(14; 18) Negative (%)P
 
Total 14 (100) 104 (100)  —  
Age >60 yr 34 (33) .03 
Stage III or IV 7 (50) 66 (63) NS 
Elevated LDH 10 (71) 76 (73) NS 
Performance status (ECOG) 2-4 3 (21) 17 (16) NS 
Extranodal sites >1 3 (21) 31 (30) NS 
BM involvement 10 (10) NS 
CR rate 12 (86) 88 (85) NS 
CharacteristicNo. t(14; 18) Positive (%)No. t(14; 18) Negative (%)P
 
Total 14 (100) 104 (100)  —  
Age >60 yr 34 (33) .03 
Stage III or IV 7 (50) 66 (63) NS 
Elevated LDH 10 (71) 76 (73) NS 
Performance status (ECOG) 2-4 3 (21) 17 (16) NS 
Extranodal sites >1 3 (21) 31 (30) NS 
BM involvement 10 (10) NS 
CR rate 12 (86) 88 (85) NS 

Abbreviation: NS, not significant.

Fig. 2.

OS of the 139 patients analyzed for the presence of a bcl-2 rearrangement by PCR.

Fig. 2.

OS of the 139 patients analyzed for the presence of a bcl-2 rearrangement by PCR.

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Fig. 3.

DFS of the 139 patients analyzed for the presence of a bcl-2 rearrangement by PCR.

Fig. 3.

DFS of the 139 patients analyzed for the presence of a bcl-2 rearrangement by PCR.

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Of the 139 total cases, 116 ( 83%) had positive staining of small lymphocytes in paraffin sections and were included in the analysis of Bcl-2 protein expression. This included 63 (53%) without large cells staining (negative or 0), 26 (23%) with ≤10% large cells immunopositive (+1), 8 (7%) with between 11% and 30% cells staining (+2), 14 (12%) with 31% to 70% cells staining (+3), and 6 (5%) with >70% of the large neoplastic cells immunopositive (+4). Overall, 53 (47%) of the cases showed some expression of Bcl-2 protein. If a cut-off of >10% Bcl-2 immunopositive tumor cells is used for analysis, then 88 cases (76%) were scored as negative and 28 (24%) were positive for expression of Bcl-2 protein. The relationship between clinical characteristics, histologic subtype and Bcl-2 protein expression is shown in Table 3. The differences in 8-year OS (60% v 34%, P < .01), DFS (66% v 32%, P < .001), and RFS (59% v 25%, P < .001) are statistically significant, demonstrating a worse outcome for Bcl-2 immunopositive cases. These data are shown in Figs 4-6. The 8-year OS and DFS for the 23 cases that failed internal control staining of small lymphocytes for Bcl-2 protein was 57% and 55%, respectively. The relationship between the presence of a bcl-2 gene rearrangement and Bcl-2 protein expression is shown in Table 4. Of note, 6 cases with a bcl-2 rearrangement by PCR failed to express Bcl-2 protein, although 3 of these cases had between 1% and 10% immunopositive large cells (scored as +1).

Table 3.

Clinical Characteristics and Histologic Subclassification of 116 Patients With DLCL With Results Interpretable for Bcl-2 Protein Expression

CharacteristicNo. Bcl-2 Positive (%)No. Bcl-2 Negative (%)P
Total 28 (100) 88 (100)  —  
Age >60 yr 7 (25) 26 (30) NS 
Stage III or IV 13 (46) 59 (67) NS 
Elevated LDH 25 (89) 64 (73) .02 
Performance status (ECOG) 2-4 6 (21) 15 (17) NS 
Extranodal sites >1 7 (25) 26 (30) NS 
BM involvement 2 (7) 9 (10) NS 
CR rate 25 (89) 75 (85) NS 
Diffuse mixed 7 (25) 10 (11) NS 
Diffuse large cell 20 (71) 54 (62) NS 
IBL 16 (18) NS 
ALCL 1 (4) 8 (9) NS 
CharacteristicNo. Bcl-2 Positive (%)No. Bcl-2 Negative (%)P
Total 28 (100) 88 (100)  —  
Age >60 yr 7 (25) 26 (30) NS 
Stage III or IV 13 (46) 59 (67) NS 
Elevated LDH 25 (89) 64 (73) .02 
Performance status (ECOG) 2-4 6 (21) 15 (17) NS 
Extranodal sites >1 7 (25) 26 (30) NS 
BM involvement 2 (7) 9 (10) NS 
CR rate 25 (89) 75 (85) NS 
Diffuse mixed 7 (25) 10 (11) NS 
Diffuse large cell 20 (71) 54 (62) NS 
IBL 16 (18) NS 
ALCL 1 (4) 8 (9) NS 

Abbreviations: IBL, immunoblastic lymphoma; ALCL, anaplastic large cell lymphoma.

Fig. 4.

OS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

Fig. 4.

OS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

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Table 4.

Relationship Between t(14; 18) (bcl-2 gene rearrangement) and Bcl-2 Protein Expression

t(14; 18) Positivet(14; 18) NegativeUnknown
Bcl-2 positive 7 (5%) 19 (14%) 
Bcl-2 negative 6 (4%)4-150 66 (47%) 14 
NA 19 
t(14; 18) Positivet(14; 18) NegativeUnknown
Bcl-2 positive 7 (5%) 19 (14%) 
Bcl-2 negative 6 (4%)4-150 66 (47%) 14 
NA 19 

Abbreviations: Unknown, DNA failed amplification; NA, not assessable (Bcl-2 staining uninterpretable because of failure to stain control small lymphocytes).

F4-150

Three of the six cases had 1% to 10% immunopositive large cells and were scored as +1.

Multivariate analysis was performed and included the clinical variables recognized as prognostically important in the International Prognostic Factor study7 as well as immunophenotype and Bcl-2 protein expression. We had previously shown that the presence of a T-cell immunophenotype was an independent adverse prognostic factor in DLCLs.51 Bcl-2 protein expression was added to this model and was also found to be independently associated with a worse outcome in DLCL (P < .02).

In this study we sought to address two questions: (1) Does the presence of a bcl-2 gene rearrangment at the time of diagnosis predict for outcome in DLCLs? and (2) Is expression of Bcl-2 protein in DLCL an independent prognostic factor? Despite recently published work in this area, both of these questions remain controversial.30,33,40 41 To address these questions, we studied a cohort of patients who were uniformly staged and treated at a single institution with lengthy follow-up. Although many of the data are in agreement with several recent reports in the literature, this study provides some additional unique observations concerning the prognostic relevance of Bcl-2 protein expression in DLCL.

The reported frequency of t(14; 18) in DLCLs documented by cytogenetics and/or molecular genetics is highly variable in different studies, which probably reflects patient selection, use of various probes (mbr, mcr, or both), different molecular techniques (Southern blot analysis, PCR), and the inclusion of patients with either antecedent follicular lymphoma or discordant lymphoma.14-33 Not surprisingly, those studies that include patients with transformed follicular lymphomas report higher frequencies of bcl-2 gene rearrangement in DLCL.22 The majority of studies that have used PCR to evaluate bcl-2 rearrangements have analyzed only mbr breakpoints.21,22,24,25,30,33 Hill et al33 recently reviewed the literature and reported a cumulative frequency of 204 of 1,030 (19.8%), with a range of 10% to 40% for bcl-2 rearrangement in DLCL. This included their own recently published work from the British National Lymphoma Investigation (BNLI) Study in which they reported a frequency of 17% (27 of 161) using only mbr primers.33 Our data are quite similar, with 14 of 102 (14%) B-cell DLCL cases demonstrating a bcl-2 rearrangement using both mbr and mcr primer pairs. Patients with discordant lymphoma, who typically present with DLCL at a nodal or extranodal site with small cell lymphoma in the bone marrow, were excluded from our study. This latter group may be more frequently associated with a bcl-2 rearrangement, but to the best of our knowledge this has not been reported.

Although some studies have found that bcl-2 gene rearrangement positive cases have a higher rate of relapse, the majority of published series have not found a significant difference in either OS or DFS.14-33 Similarly, the BNLI report found that the presence of a bcl-2 rearrangement had no effect on RFS in DLCL. We also found that the presence of a bcl-2 gene rearrangement had no impact on either OS or DFS, and fails to predict for those patients who will ultimately relapse. We did find some patient characteristics which were associated with a bcl-2 rearrangement. They tended to be younger and had less frequent involvement of their bone marrow, although this latter difference did not reach statistical significance (see Table 2). Of note, the bcl-2–positive cases were evenly distributed between nodal and primarily extranodal presentation at diagnosis (data not shown).52 

Some caution in interpreting the molecular data is required because bcl-2 PCR has a well-recognized false-negative rate. Using cytogenetics as the gold standard, both Southern blot analysis and PCR fail to detect rearrangements in approximately 15% and 25% of cases, respectively.12 The availability of paraffin material limited our analytical strategy to the use of PCR only, but the results are in keeping with those studies using cytogenetics.20 Additionally, the DNA failures were included in the analysis of outcome (see Figs 2 and 3), with similar survival as compared with either the bcl-2 gene rearrangement positive or negative patient groups.

Several studies of Bcl-2 protein expression in DLCL have been reported, but with conflicting results.30,33,40-42 Overall, the reported frequency of Bcl-2 expression has varied between 34% and 69%. Most reported series have used similar techniques and anti–Bcl-2 MoAb reagents. The original report of Ngan et al53 in 1988 used polyclonal antibody reagents and reported a frequency of 34%. In our study we used a rabbit polyclonal antisera with well-documented Bcl-2 specificity and found positive staining in 53 of 116 (47%) of cases. Previous experience with this reagent demonstrated an improved sensitivity in buffered formalin-fixed material as compared with MoAb reagents (Dako; clone 124).46 However, using a cut-off similar to other studies of greater than 10% immunopositive tumor cells, we found only 24% Bcl-2 immunopositivity in DLCLs. Importantly, a significant survival difference was shown for OS, DFS, and RFS at either threshold of Bcl-2 immunopositivity, further supporting the validity of these observations. The original reports of the prognostic significance of Bcl-2 protein expression in DLCL failed to show any impact on survival.30,40 Subsequently, three groups using similar techniques and thresholds of Bcl-2 immunopositivity have reported that Bcl-2–positive cases have a worse disease-free or cause-specific survival, although none were able to demonstrate a significant difference in OS.33,41 42 Methodological differences, uniformity of patient selection, staging and treatment, and longer follow-up may explain why we were able to demonstrate a significant difference in OS at either level of Bcl-2 immunopositivity. Moreover, we were able to show that Bcl-2 protein expression was an independent prognostic factor in DLCL, when incorporated into a model that included the clinically related International Prognostic Factor Index as well as immunophenotype. Although Bcl-2 protein expression was associated with an elevated serum LDH, it remained a statistically significant prognostic factor after multivariate analysis.

Fig. 5.

DFS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

Fig. 5.

DFS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

Close modal
Fig. 6.

RFS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

Fig. 6.

RFS of the 116 patients analyzed for expression of Bcl-2 protein by immunohistochemistry.

Close modal

Table 4 highlights the relationship between bcl-2 gene rearrangements and Bcl-2 protein expression. These data demonstrate that cases with a molecular rearrangement may fail to express the protein. This phenomenon has been previously described in follicular lymphoma, whereby cases with a bcl-2 translocation fail to express Bcl-2 mRNA or protein.54 Mutations in the open reading frame of the translocated bcl-2 gene may be present, leading to absent or diminished production of Bcl-2 protein.55,56 This finding has also been recently described in DLCLs that arise through transformation of follicular lymphoma, and may be one mechanism that leads to absent Bcl-2 protein expression in some cases with the translocation.57 The frequent expression of Bcl-2 protein in cases lacking t(14; 18) is well described, and suggests that mechanisms other than translocation can lead to increased Bcl-2 levels in NHLs.34 Both examples serve to point out that the presence of a bcl-2 gene rearrangement is not synonymous with overexpression of Bcl-2 protein, and vice versa.

One selective pressure against Bcl-2 expression could be related to the reported ability of Bcl-2 overexpression to inhibit cell proliferation, probably by causing a G1-phase delay or block.58-61 Thus, while high levels of Bcl-2 provide a survival advantage for malignant cells, they may also result in diminished proliferation and thereby negatively affect cell growth. A reasonable hypothesis therefore is that a t(14; 18) that activates the bcl-2 gene may create a permissive environment for the development of other genetic alterations by blocking programmed cell death, but eventually these tumors may become less dependent on Bcl-2 protein expression for their survival in vivo, and thus find it advantageous to inactivate Bcl-2 so that higher amounts of proliferation are achieved. However, the consequences of Bcl-2 protein downregulation may be deleterious to the tumor, resulting from loss of the anti-apoptotic mechanism and enhanced susceptibility to cell death from an increased tumor growth fraction killed by cell cycle active chemotheraputic agents. The balance between these opposing forces may be important for understanding the relationship between cell death and cell cycle pathways, and may provide insights into novel stategies for overcoming chemoresistance.

In summary, in this study we were unable to show that bcl-2 gene rearrangement status at diagnosis has a significant impact on clinical outcome. However, Bcl-2 protein expression is an important independent predictor of survival in patients with advanced-stage DLCL treated with chemotherapy. Consideration should therefore be given to incorporating this and perhaps other established pathologic variables into the design of treatment strategies aimed at improving the outcome of patients considered at increased risk for treatment failure.

The authors thank Dr P. Klimo for their help in providing clinical data for this study, Dr C. Coppin for assistance with the statistical analysis, and C. Wong for her help with data collection.

Supported by a British Columbia Health Research Foundation grant (R.D.G.) and partial support from a United States National Cancer Institute Grant to J.C.R. (CA-60421).

Address reprint requests to Randy D. Gascoyne, MD, FRCPC, Department of Pathology, B.C. Cancer Agency, 600 W 10th Ave, Vancouver, BC, V5Z 4E6 Canada.

1
Harris
NL
Jaffe
ES
Stein
H
Banks
PM
Chan
JKC
Cleary
ML
Delsol
G
De Wolf-Peeters
C
Falini
B
Gatter
KC
Grogan
TM
Isaacson
PG
Knowles
DM
Mason
DY
Muller-Hermelink
H-K
Pileri
SA
Piris
MA
Ralfkiaer
E
Warnke
RA
A revised European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group.
Blood
84
1994
1361
2
Offit
K
Lo
Coco F
Louie
DC
Parsa
NZ
Leung
D
Portlock
C
Ye
BH
Lista
F
Filippa
DA
Rosenbaum
A
Ladanyi
M
Jhanwar
S
Dalla-Favera
R
Chaganti
RSK
Rearrangement of the bcl-6 gene as a prognostic marker in diffuse large-cell lymphoma.
N Engl J Med
331
1994
74
3
Klimo
P
Connors
JM
MACOP-B chemotherapy for the treatment of diffuse large cell lymphoma.
Ann Intern Med
102
1985
596
4
O'Reilly
SE
Hoskins
P
Klimo
P
Connors
JM
MACP-B and VACP-B in diffuse large cell lymphomas and MOPP/ABV in Hodgkin's disease.
Ann Oncol
2
1991
17
5
Klimo P, Connors JM: Updated clinical experience with MACOP-B. Semin Hematol 24:26, 1987 (suppl 1)
6
Fisher
RI
Gaynor
ER
Dahlberg
S
Oken
MM
Grogan
TM
Mize
EM
Glick
JH
Coltman
CA
Miller
TP
Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin's lymphoma.
N Engl J Med
328
1993
1002
7
Shipp
MA
Harrington
DP
Anderson
JR
Armitage
JO
Bonadonna
G
Brittinger
G Cabanillas F
Canellos
GP
Coiffier
B
Connors
JM
Cowan
RA
Crowther
D
Dahlberg
S
Engelhard
M
Fisher
RI
Gisselbrecht
C
Horning
SJ
Lepage
E
Lister
A
Meerwaldt
JH
Montserrat
E
Nissen
NI
Oken
MM
Peterson
BA
Tondini
C
Velasquez
WS
Yeap
BY
A predictive model for aggressive NHL: The International Non-Hodgkin's Lymphoma Prognostic Factors Project.
N Engl J Med
329
1993
987
8
Tsujimoto
Y
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
1984
1097
9
Tsujimoto
Y
Gorham
J
Cossman
J Jaffe ES
Croce
CM
The t(14; 18) chromosome translocations involved in B-cell neoplasms result from mistakes in VDJ joining.
Science
229
1985
1390
10
Bakhshi
A
Jensen
JP
Goldman
P
Wright
JL
McBride
W
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
1985
899
11
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
1985
7439
12
Horsman
DE
Gascoyne
RD
Coupland
RW
Coldman
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
1995
472
13
Pezzella
F
Ralfkiaer
E
Gatter
KC
Mason
DY
Comparison of Southern blotting and the polymerase chain reaction.
Br J Haematol
76
1990
58
14
Bloomfield
CD
Arthur
DC
Frizzera
G
Levine
EG
Peterson
BA
Gaji-Peczalska
KJ
Nonrandom chromosome abnormalities in lymphoma.
Cancer Res
43
1983
2975
15
Levine
EG
Arthur
DC
Frizerra
G
Peterson
BA
Hurd
DD
Bloomfield
CD
There are differences in cytogenetic abnormalities among histologic subtypes of the non-Hodgkin's lymphomas.
Blood
66
1985
1414
16
Koduru
PRK
Filippa
DA
Richardson
ME
Jhanwar
SC
Chaganti
SR
Koziner
B
Clarkson
BD
Lieberman
PH
Chaganti
RSK
Cytogenetic and histologic correlations in malignant lymphoma.
Blood
69
1987
97
17
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
1987
1185
18
Lipford
E
Wright
JJ
Urba
W
Whang-Peng
J
Kirsch
IR
Raffeld
M
Cossman
J
Longo
DL
Bakhshi
A
Korsmeyer
SJ
Refinement of lymphoma cytogenetics by the chromosome 18q21 major breakpoint region.
Blood
70
1987
1816
19
Lee
M-S
Blick
MB
Pathak
S
Trujillo
JM
Butler
JJ
Katz
RL
McLaughlin
P
Hagemeister
FB
Velasquez
WS
Goodacre
A
Cork
A
Gutterman
JU
Cabanillas
F
The gene located at chromosome 18 band q21 is rearranged in uncultured diffuse lymphomas as well as follicular lymphomas.
Blood
70
1987
90
20
Armitage
JO
Sanger
WG
Weisenburger
DD
Harrington
DS
Linder
J
Bierman
PJ
Vose
JM
Purtilo
DT
Correlation of secondary cytogenetic abnormalities with histologic appearance in non-Hodgkin's lymphomas bearing t(14; 18)(q32;q21).
J Natl Cancer Inst
80
1988
576
21
Aisenberg
AC
Wilkes
BM
Jacobson
JO
The bcl-2 gene is rearranged in many diffuse B-cell lymphomas.
Blood
71
1988
969
22
Offit
K
Koduru
PRK
Hollis
R
Filippa
DA
Jhanwar
SC
Clarkson
BC
Chaganti
RSK
18q21 rearrangement in diffuse large cell lymphoma: Incidence and clinical significance.
Br J Haematol
72
1989
178
23
Yunis
JJ
Mayer
MG
Arnesen
MA
Aeppli
DP
Oken
MM
Frizerra
G
bcl-2 and other genomic alterations in the prognosis of large-cell lymphoma.
N Engl J Med
320
1989
1047
24
Kneba
M
Eick
S
Herbst
H
Willigeroth
S
Pott
C
Bolz
I
Bergholz
M
Neumann
C
Stein
H
Krieger
G
Frequency and structure of (14; 18) major breakpoint regions in non-Hodgkin's lymphomas typed according to the Kiel classification: Analysis by direct DNA sequencing.
Cancer Res
51
1991
3243
25
Ohshima
K-I
Kikuchi
M
Kobari
S-I
Eguchi
F
Masuda
Y
Mohtai
H
Kimura
N
Takeshita
M
Bcl-2 gene and prognosis of B-cell lymphoma.
Leuk Lymphoma
5
1991
305
26
Jacobson
JO
Wilkes
BM
Kwiatkowski
DJ
Mederios
LJ
Aisenberg
AC
Harris
NL
bcl-2 rearrangements in de novo diffuse large cell lymphoma.
Cancer
72
1993
231
27
Romaguera
JE
Pugh
W
Luthra
R
Goodacre
A
Cabanillas
F
The clinical relevance of t(14; 18)/bcl-2 rearrangement and del 6q in diffuse large cell lymphoma and immunoblastic lymphoma.
Ann Oncol
4
1993
51
28
Masih A, Weisenburger D, Vose J, Bierman P, Naknmine H, Sanger W, Chan W, Anderson J, Armitage J: Clinicopathologic analysis of the t(14; 18) in uniformly treated de novo diffuse large B-cell lymphoma. Blood 82:133a, 1993 (abstr, suppl 1)
29
Lee
KA
Goepel
JR
Winfield
DA
Hancock
BW
Goyns
MH
Investigation of bcl-2 gene rearrangements in a United Kingdom series of low and high grade non-Hodgkin's lymphomas.
Leuk Lymphoma
11
1993
91
30
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
1994
149
31
Miller TP, Levy N, Bailey NP, Kawasaki E, Baier M, Grogan TM: The bcl-2 gene translocation (T14; 18) identifies a subgroup of patients with diffuse large cell lymphoma having an indolent clinical course with late relapse. Proc Am Soc Clin Oncol 13:370, 1994 (abstr 1249).
32
Johnson
A
Brun
A
Dictor
M
Rambech
E
Akerman
M
Anderson
H
Incidence and prognostic significance of t(14; 18) translocation in follicular center cell lymphoma of low and high grade.
Ann Oncol
6
1995
789
33
Hill
ME
MacLennan
KA
Cunningham
DC
Vaughan
Hudson B
Burke
M
Clarke
P
Di Stefano
F
Anderson
L
Vaughan
Hudson G
Mason
DY
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
1996
1046
34
Pezzella
F
Tse
AGD
Cordell
JL
Pulford
KAF
Gatter
KC
Mason
DY
Expression of the bcl-2 oncogene protein is not specific for the 14-18 chromosomal translocation.
Am J Pathol
137
1990
225
35
Miyashita
T
Reed
JC
Bcl-2 gene transfer increases relative resistance to S49.1 and WEH17.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs.
Cancer Res
52
1992
5407
36
Miyashita
T
Reed
JC
Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line.
Blood
81
1993
151
37
Campos
L
Rouault
J-P
Sabido
O
Oriol
P
Roubi
N
Vasselon
C
Archimbaud
E
Magaud
J-P
Guyotat
D
High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy.
Blood
81
1993
3091
38
Maung
ZT
MacLean
FR
Reid
MM
Pearson
AD
Proctor
SJ
Hamilton
PJ
Hall
AG
The relationship between bcl-2 expression and response to chemotherapy in acute leukemia.
Br J Haematol
88
1994
105
39
Gala
JL
Vermylen
C
Cornu
G
Ferrant
A
Michaux
JL
Philippe
M
Martiat
P
High expression of bcl-2 is the rule in acute lymphoblastic leukemia, except in Burkitt's subtype at presentation, and is not correlated with the prognosis.
Ann Hematol
69
1994
17
40
Piris
MA
Pezella
F
Martinez-Montero
JC
Orradre
JL
Villuendas
R
Sanchez-Beato
M
Cuena
R
Cruz
MA
Martinez
B
Garrido
MC
Gatter
KC
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
1994
337
41
Hermine
O
Haioun
C
Lepage
E
d'Agay
M-F
Briere
J
Lavignac
C
Fillet
G
Salles
G
Marolleau
J-P
Diebold
J
Reyes
F
Gaulard
P
for the GELA
Prognostic significance of bcl-2 protein expression in aggressive non-Hodgkin's lymphoma.
Blood
87
1996
265
42
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
1996
2131
43
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
1982
2112
44
Stein
H
Mason
DY
Gerdes
J
O'Connor
N
Wainscoat
J
Pallesen
G
Gatter
K
Falini
B
Delsol
G
Lemke
H
Schwarting
R
Lennert
K
The expression of the hodgkin's disease associated antigen Ki-1 in reactive and neoplastic tissue. Evidence that Reed-Sternberg cells and histiocytic malignancies are derived from activated lymphoid cells.
Blood
66
1985
848
45
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
1994
515
46
Gascoyne R, Krajewski S, Krajewska M, Berean K, Reed JC: Unique immunostaining discriminates follicular lymphoma from reactive hyperplasia: Utility of anti-bcl-2 antiserum 9716. Mod Pathol 7:108A, 1994 (abstr 626)
47
Greiner
TC
Gascoyne
RD
Anderson
ME
Kingma
DW
Adomat
SA
Said
J
Jaffe
ES
Nodular lymphocyte-predominant Hodgkin's disease associated with large-cell lymphoma: Analysis of Ig gene rearrangements by V-J polymerase chain reaction.
Blood
88
1996
657
48
Kaplan
E
Meier
P
Nonparametric estimation from incomplete observations.
Am J Stat Assoc
53
1958
457
49
Peto
R
Pike
MC
Armitage
P
Breslow
NE
Cox
DR
Howard
SV
Mantel
N
McPherson
K
Peto
J
Smith
PG
Design and analysis of randomized clinical trials requiring prolonged observations of each patient. II. Analysis and examples.
Br J Cancer
35
1977
1
50
Cox DR: Regression models and life tables. J R Stat Soc B34:187, 1972
51
Gascoyne R, Tolcher A, Coupland R, Connors JM: The prognostic significance of immunophenotype in diffuse large cell lymphomas. Mod Pathol 7:109A, 1994 (abstr 629)
52
Raghoebier
S
Kramer
MHH
van Krieken
JHJM
de Jong
D
Limpens
J
Kluin-Nelemans
JC
van Ommen
GJB
Kluin
PM
Essential differences in oncogene involvement between primary nodal and extranodal large cell lymphoma.
Blood
78
1991
2680
53
Ngan
B-Y
Chen-Levy
Z
Weiss
LM
Warnke
RA
Cleary
ML
Expression in non-Hodgkin's lymphoma of the bcl-2 protein associated with the t(14; 18) chromosomal translocation.
N Engl J Med
318
1988
1638
54
Wang
J
Raffeld
M
Medeiros
LJ
Longo
DL
Jaffe
ES
Duffey
P
Stetler-Stevenson
M
Follicular center cell lymphoma with the t(14; 18) translocation in which the rearranged bcl-2 gene is silent.
Leukemia
7
1993
1834
55
Tanaka
S
Louie
D
Kant
J
Reed
JC
Frequent incidence of somatic mutations in translocated bcl-2 oncogenes of non-Hodgkin's lymphomas.
Blood
79
1992
229
56
Reed
JC
Tanaka
S
Somatic point mutations in the translocated bcl-2 genes of non-Hodgkin's lymphomas and lymphocytic leukemias: Implications for mechanisms of tumor progression.
Leuk Lymphoma
10
1993
157
57
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
1996
3937
58
Pientenpol
JA
Papadopoulos
N
Markowitz
S
Willson
JKV
Kinzler
KW
Vogelstein
B
Paradoxical inhibition of solid tumor growth by bcl-2.
Cancer Res
54
1994
3714
59
Borner
C
Diminished cell proliferation associated with the death-protective activity of bcl-2.
J Biol Chem
271
1996
12695
60
Mazel
S
Burtrum
D
Petrie
HT
Regulation of cell cycle progression by bcl-2 expression: A potential mechanism for inhibition of programmed cell death.
J Exp Med
183
1996
2219
61
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
1996
9545
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