the Leukemia/Lymphoma Molecular Profiling Project, Departments of Pathology and Microbiology, Genetics, Internal Medicine, and Preventive and Societal Medicine, University of Nebraska Medical Center, Omaha, NE
    Metabolism Branch and Laboratory of Pathology, Center for Cancer Research, and Biometrics Research Branch, Department of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD
    Department of Pathology, University of Arizona, Tucson, AZ
    Departments of Pathology and Medical Oncology, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
    Department of Pathology, University of Würzburg, Würzburg, Germany
    Department of Pathology, Hospital Clinic, University of Barcelona, Barcelona, Spain
    Department of Pathology, Norwegian Radium Hospital, Oslo, Norway

    ABSTRACT

    BACKGROUND: The role of BCL2 as a predictor of survival in diffuse large B-cell lymphoma (DLBCL) is controversial. DLBCL is heterogeneous, and the expression of BCL2 is variable within the two major subgroups of DLBCL, germinal center B-cell–like (GCB) and activated B-cell–like (ABC) DLBCL, as well as primary mediastinal DLBCL.

    PATIENTS AND METHODS: In this study, we investigated the correlation of BCL2 expression with survival in the two major subgroups of DLBCL, as well as the mechanisms of BCL2 expression.

    RESULTS: There was no significant correlation between BCL2 protein expression and overall survival within the GCB subgroup, but BCL2 expression had a significant adverse effect on overall survival within the ABC subgroup (P = .008). This correlation was also observed at the mRNA level (P < .04). The difference remained significant when the analyses were performed at different cutoff values. The t(14;18) was frequently observed in the GCB subgroup and was highly associated with BCL2 expression. Patients with ABC DLBCL did not exhibit t(14;18) but had a markedly higher frequency of chromosome 18q21 amplification, on which BCL2 resides. Thus, alternative mechanisms such as 18q21 amplification or activation of the nuclear factor-kappa B pathway, as reported previously, seem to be mainly responsible for the upregulation of BCL2 expression in the ABC subgroup.

    CONCLUSION: Treating all DLBCL as a single entity ignores the mechanistic differences in BCL2 upregulation and obscures the prognostic significance of BCL2 expression. Hence, the significance of BCL2 and other biomarkers should be assessed in the context of DLBCL subgroups in future studies.

    INTRODUCTION

    Diffuse large B-cell lymphoma (DLBCL), a lymphoma characterized by an aggressive clinical course, is the most common type of non-Hodgkin’s lymphoma.1 However, DLBCL displays considerable heterogeneity with respect to clinical presentation, morphology, and molecular and cytogenetic characteristics. Recent gene expression profiling (GEP) studies have identified the following three distinct subgroups of DLBCL: germinal center B-cell–like (GCB), activated B-cell–like (ABC), and primary mediastinal (PM) DLBCL.2-5 The GCB subgroup is characterized by expression of a germinal center B-cell molecular signature, frequent association with the t(14;18)(q32;q21),6,7 and a better prognosis than the ABC subgroup.3 The molecular signature of the ABC subgroup is distinctly different and is characterized by overexpression of a group of genes that are upregulated in peripheral-blood B cells activated by mitogenic stimulation in vitro. The PM subgroup can be distinguished from the other subgroups by its unique GEP that is characterized by downregulation of a number of B-cell receptor components or its downstream signaling molecules and upregulation of groups of genes involved in the interleukin-13 cytokine pathway, a number of the tumor necrosis factor family members, and tumor necrosis factor–induced proteins. However, patient survival is similar to that of GCB DLBCL.4,5

    BCL2 is an antiapoptosis factor that is important in normal B-cell development and differentiation.8-12 The t(14;18)(q32;q21) brings the BCL2 gene under the control of immunoglobulin heavy-chain gene (IgH) enhancers and leads to overexpression of BCL2 protein. However, BCL2 expression can also be upregulated by alternate mechanisms, as is often observed in the ABC subgroup of DLBCL, which lacks this translocation. BCL2 overexpression provides a survival advantage for malignant B cells and is thought to play a critical role in resistance to chemotherapy. As a result of these biologic functions, BCL2 overexpression should be of prognostic importance in DLBCL. However, the correlation of BCL2 expression with survival in patients with DLBCL is controversial, with studies showing no difference in overall survival13-15 or decreased overall survival.16-19 A few studies with large series of patients have shown inverse association of BCL2 expression with disease-free survival but not with overall survival.20-22 In our previous studies, we observed that BCL2 expression is highly associated with the t(14;18)(q32;q21) in the GCB subgroup, but the translocation had no impact on survival.6 However, the influence of BCL2 expression on survival within the ABC subgroup of DLBCL is not clear. The existing controversy regarding BCL2 expression and survival may be related to studying DLBCL as a single entity. In contrast, analyzing BCL2 expression in the context of DLBCL subgroups may clarify its clinical relevance. In this study, we reanalyzed the correlation of BCL2 protein and mRNA expression with survival in the two major biologic subgroups of DBLCL and investigated the possible mechanisms for BCL2 overexpression in the ABC DLBCL subgroup.

    PATIENTS AND METHODS

    Patient Information

    We have included 240 patients with DLBCL from our cDNA microarray database, which is a part of our previous GEP project.23 Of these 240 patients, BCL2 protein expression data by immunohistochemistry was available in 138 patients, including 57 patients with GCB DLBCL, 44 patients with ABC DLBCL, 10 patients with PM DLBCL, and 27 patients with unclassifiable DLBCL. In the same series with GEP data, 129 patients with fluorescence in situ hybridization (FISH) data on BCL2 translocation were evaluated for gain or amplification of the chromosome 18q21 region. Because the number of PM DLBCL patients was too few for analysis and the unclassifiable patients were not well defined, we limited our analysis to the GCB and ABC subgroups. This study was approved by the Institutional Review Board of the University of Nebraska Medical Center (UNMC).

    Immunohistochemical Staining of Tissue Sections

    All immunohistochemical staining was performed at UNMC, and a prior comparative study of the staining in three different laboratories was performed to validate the reproducibility of staining and evaluation (National Cancer Institute, University of Barcelona, and UNMC).24 Tissue microarrays were prepared from patients with adequate archival paraffin-embedded tissue. Five-micrometer sections were cut from each tissue microarray and stained with antibodies to BCL2, as described previously.25 CD20 stains were performed to evaluate each core for involvement by tumor, and the percentage of tumor cells stained with each antibody was recorded in 10% increments. Disagreements were resolved by joint review on a multihead microscope. For each patient, the core with the highest percentage of tumor cells stained was used for analysis. Immunostains were considered positive if 30% or more of the tumor cells were stained by the antibodies.25

    Detection of Chromosome 18q21 Translocation and Copy Number Changes

    Among the 240 patients studied by GEP, 129 patients were studied by interphase FISH for the t(14;18)(q32;q21). We examined these patients for the gain or amplification of chromosome 18q21. FISH analysis was performed as previously described.6 Briefly, the dual-color LSI IgH Spectrum Green/LSI BCL2 Spectrum Orange Dual-Fusion Translocation Probe (Abbott-Vysis, Downers Grove, IL) was used to detect the t(14;18), and the CEP 18 Spectrum Aqua Probe (Abbott-Vysis) was used simultaneously to evaluate the chromosome 18 copy number. Interphase nuclei of normal cells showed two red signals for BCL2 and two green signals for IgH. The presence of the t(14;18) produced two yellow fusion signals (red + green) on 14q32 and 18q21. The presence of three red signals of 18q21 along with two aqua signals for centromere 18 was classified as a gain, and the presence of four or more red signals of 18q21 with two aqua signals was classified as amplification.

    Analysis of BCL2 and 18q21 Region Gene Expression by cDNA Microarray

    We evaluated BCL2 mRNA levels measured by cDNA microarray in all of the DLBCL patients. Expression levels were log2 transformed, and the mean was centered across samples. Of the three different BCL2 clones (232714, 342181, and 1336385) on the Lymphochip microarray,3 only clone 232714 with a more 5' sequence could measure message with a truncated 3' end as a result of translocation at the major breakpoint region.7 However, clone 232714 is located far from the 3' end of the transcript and may fail to accurately measure the normal BCL2 transcripts. We used the mean value of the array elements for the two BCL2 clones that are located close to the 3' end (342181 and 1336385) of the transcript to represent the gene expression level for patients in whom this mean value was greater than the measurement by clone 232714. The measurement of 232714 was used when it was higher than the other two clones. In addition to BCL2 mRNA, we also examined the expression of other genes in the 18q21 region represented on the Lymphochip and used supervised analysis to compare the mRNA expression within each subgroup of DLBCL.

    Survival Analysis

    The Kaplan-Meier method was used to estimate the overall survival of patients, and the log-rank test was used to compare survival differences between the different BCL2 protein expression group.6 Similar analysis was performed using BCL2 mRNA expression data from the Lymphochip. Patients were divided into halves or quartiles according to their BCL2 mRNA expression levels for the latter analyses. A Fisher’s exact test was used to evaluate the unequal distribution of ABC and GCB samples among the quartiles.

    Overall survival was defined as the time from diagnosis to death from any cause or, for patients remaining alive, the time from diagnosis to last contact. SAS software (SAS Institute Inc, Cary, NC) was used for data analysis.

    To adjust for the testing of multiple cut points within the ABC subgroup, we performed a permutation test to determine whether the significance of the best cutoff was greater than what would be expected by chance. This was performed by permuting the relationship between protein values and survival, so that each protein value is associated with a random survival value. Using this permuted data, we then found the cut point that gave the largest log-rank statistics. This was repeated 5,000 times for different random permutations. The proportion of these random permutations that produced a larger optimal log-rank statistics than the optimal statistics found in the unpermuted data was reported as the permutation P value.

    2 tests were used to test the independence of categoric variables. The Spearman rank correlation test was used to test the agreement of BCL2 mRNA and BCL2 protein measures.

    RESULTS

    Patient Characteristics

    We examined BCL2 and 18q21 region mRNA expression levels in 240 patients with DLBCL that was profiled with the Lymphochip.3 The DLBCL patients were divided into GCB DLBCL (n = 93; 38.7%), ABC DLBCL (n = 82; 34.2%), PM DLBCL (n = 19; 7.9%), and unclassifiable (n = 46; 19.2%) subgroups.23 Of the 240 patients, BCL2 immunohistochemical data was available in 138 patients (GCB, n = 57; ABC, n = 44; PM, n = 10; and unclassifiable, n = 27). In the same series of patients, FISH data for the t(14,18) and gain or amplification of 18q21 region were available in 129 patients (GCB, n = 53; ABC, n = 41; PM, n = 11; and unclassifiable, n = 24).6 Among these 240 patients, 108 had data from all three databases. No significant differences were observed for any of the clinical parameters in the group with immunohistochemical data or FISH data when compared with the entire group of DLBCL patients.

    The major clinical characteristics of patients according to DLBCL subgroup classification and BCL2 protein or mRNA expression are listed in Tables 1 and 2. There was no significant difference in clinical characteristics among the different subsets of patients except for the higher lactate dehydrogenase levels and stage in the BCL2-protein–positive patients (30% cutoff) in the ABC subgroup.

    Correlation of BCL2 mRNA and Protein Expression in DLBCL

    In an earlier study, we observed that BCL2 protein expression showed a good correlation with mRNA levels in normal and malignant B cells.26 Although mRNA measurement in the microarray assay was a continuous variable and protein expression by immunoperoxidase staining was measured in discrete increments, we observed a significant association between these two variables in both the ABC and GCB subgroups (P < .0001). In general, increased mRNA expression was associated with increased protein expression; however, exceptions were also observed (Supplemental Table). These discrepancies may be a result of the methodologic limitations or experimental variance or a result of the mutation in the BCL2 gene that inhibited protein and antibody association.27 It is also possible that, in some cases, undefined mechanisms may be responsible for post-transcriptional regulation of BCL2 expression.

    Prognostic Significance of BCL2 Expression

    In our previous study, we observed that the t(14;18) is highly associated with increased BCL2 mRNA and protein expression in GCB DLBCL but does not have any prognostic impact.6 In this study, we evaluated the correlation between BCL2 expression and survival in DLBCL as a single entity or as GEP-defined subgroups. Using BCL2 protein expression by immunostaining and a 30% cutoff for positive patients, there were no significant differences between the BCL2-positive and BCL2-negative patients with regard to overall (P = .09) or event-free survival (P = .08) when DLBCL was taken as a single entity (Fig 1A). Similar results were also observed for the GCB subgroup (Fig 1B). However, a clear association between overall survival and BCL2 protein expression was observed in the ABC subgroup (Fig 1C). We also examined additional cutoff values for BCL2 protein expression to see whether this relationship held true when other cut points were used. We found that other cutoff values remained significant predictors of survival (P = .002 at 10% cutoff v P = .008 at 30% and P = .03 at 50% cutoff; Fig 1D). Statistical analysis of BCL2 protein expression as a continuous variable or by adjusting for multiple comparisons via a permutation test also showed a significant association with overall survival in the ABC subgroup (P = .019 and P = .008, respectively).

    We also analyzed the prognostic significance of BCL2 expression at the mRNA level from Lymphochip data. Because BCL2 mRNA expression is a continuous variable, we divided the patients arbitrarily into halves or quartiles according to BCL2 transcript levels. When DLBCL was analyzed as a single entity, patients with high BCL2 levels had a significantly worse overall survival (P = .05; Figs 2A and 2B). Patients with ABC DLBCL tend to have high BCL2 expression and are over-represented in the groups with the upper half of the transcript levels (P < .001; Table 3). Therefore, the survival curves in Figures 2A and 2B may reflect survival differences between the GCB and ABC subgroups (Table 3). When the patients were separated into GCB and ABC subgroups and reanalyzed, BCL2 expression was not associated with survival in the GCB subgroup (P = .98) but was significantly associated with overall survival in the ABC subgroup (P = .04; Figs 2C and 2D), which was similar to the findings with immunohistochemical staining (Figs 1A and 1D).

    Gain and Amplification of the Chromosome 18q21 Region in ABC DLBCL As a Mechanism for BCL2 Expression

    We investigated chromosome 18q21 gain or amplification in 129 DLBCL patients with t(14;18) data. We found that 18q21 was much more frequently amplified in the ABC subgroup (nine of 41 patients; 21%) than in the GCB subgroup (one of 53 patients; 1.8%; P = .002; Fig 3). The only patient in the GCB subgroup with this genetic abnormality was also positive for the t(14;18). Chromosome 18q21 amplification was associated with increased BCL2 mRNA levels and could well account for elevated BCL2 protein expression in this subset of ABC DLBCL patients. Gain of an extra copy of the BCL2 gene was observed in 27 patients with DLBCL, and the majority of these patients were in the ABC subgroup (19 of 41 patients in ABC subgroup and eight of 53 patients in GCB subgroup; P = .005). In the GCB subgroup, four patients with an extra copy of 18q21 also had the t(14;18). The other four GCB patients lacked this translocation and had significantly lower expression of the BCL2 protein compared with patients having the t(14;18). We also analyzed mRNA expression of other genes located in the 18q21 region from the Lymphochip data in the entire DLBCL series (240 patients) and observed that increased mRNA expression of this genomic region was highly associated with the ABC subgroup and 18q21 amplification (Fig 4).

    DISCUSSION

    BCL2 is an antiapoptotic protein and belongs to a large family of proteins involved in the regulation of programmed cell death.28 BCL2 plays a major role in the response of malignant cells to a variety of stresses that may lead to apoptosis, including chemotherapy.29 Our previous studies showed that BCL2 rearrangement with the t(14; 18)(q32;q21) is found in approximately 20% of DLBCL patients6,7 and occurs predominantly in the GCB DLBCL subgroup (approximately 35%) but not in the ABC subgroup. This translocation in the GCB DLBCL subgroup correlates well with BCL2 mRNA and protein expression,6 indicating that this is the major mechanism in the upregulation of BCL2 expression in this subgroup. However, ABC DLBCL patients also have high levels of BCL2 expression in the absence of t(14;18)(q32;q21), indicating alternative mechanisms of BCL2 upregulation.

    The various reports in the literature on BCL2 expression as a prognostic indicator in DLBCL have been contradictory.15-22,30,31 The majority of the above reports have also disputed the prognostic significance of the t(14;18) in DLBCL, with some showing no effect18,19,21 and others showing significant clinical correlation.15,30,32,33 Our previous studies have shown a good correlation between the t(14;18) and BCL2 protein expression in GCB DLBCL but with no difference in survival between t(14;18)-positive and -negative patients in this subgroup. Reanalyzing the data using BCL2 protein expression as a discriminator also showed no difference in survival in this subgroup. We used a similar analytic approach in this study with regard to the ABC DLBCL subgroup. BCL2 was frequently highly expressed in this subgroup, and we demonstrated an inverse relationship between protein expression and survival that was not observed when the entire DLBCL group was examined. Furthermore, this relationship can be observed at different cutoff values for BCL2 expression. These observations may explain, at least in part, the reason for the controversy in the literature regarding the prognostic value of BCL2 protein expression in DLBCL.

    We have observed a relationship between BCL2 protein expression and survival in the ABC subgroup of DLBCL even when we use multiple cutoff values. However, immunohistochemical assays are not easy to quantitate accurately. In this study, we have recorded only the percentage of positive cells but did not attempt to evaluate the intensity of staining because of the methodologic limitations and the difficulty in accessing the intensity of staining in tissues from multiple institutions. Because our previous study demonstrated a good correlation (r = 0.8) between BCL2 protein and mRNA expression, it would be of interest to examine the relationship between BCL2 mRNA expression, which is more readily quantifiable, and survival in DLBCL. DLBCL patients in the upper half or in the highest quartile according to the expression values for BCL2 mRNA showed worse survival. When the subgroups were analyzed separately, BCL2 expression level was associated with worse survival only in the ABC subgroup, in agreement with the protein expression data. Thus, this study clearly indicates that future evaluations of biomarkers in DLBCL should take subgroup classification into consideration. There are recent reports that the addition of rituximab to chemotherapy for DLBCL may be of benefit, mainly in the subgroup with BCL2 expression.34 It is of interest to further investigate this finding, especially in the framework of subgroup distinction. BCL2 protein–positive patients in the ABC subgroups also showed an association with higher lactate dehydrogenase levels and stages of disease, both of which are known adverse prognostic factors.35 Thus, BCL2 expression could be a biomarker of more aggressive disease in the ABC subgroup but not the GCB subgroup.

    Alternate mechanisms that promote BCL2 expression, unrelated to the t(14;18), must operate in ABC DLBCL. A recent study has shown that nuclear factor-kappa B (NF-B) is constitutively expressed in ABC DLBCL and has a critical role in its pathogenesis.36 BCL2 is a target gene for NF-B,37 and in many ABC DLBCL patients, BCL2 upregulation may be mediated through the NF-B pathway. Another possible mechanism is the amplification of the chromosomal locus 18q21 on which BCL2 resides. Studies using comparative genomic hybridization have shown that approximately 20% of DLBCL patients have 18q gains.38-40 In this study, we investigated the incidence of 18q21 gain or amplification using the FISH technique. Interestingly, whereas gain of 18q21 was found more frequently in the ABC DLBCL subgroup compared with the GCB DLBCL subgroup, 18q21 amplification was strikingly more frequent in the ABC subgroup. Our microarray data complement this finding by showing upregulation of many genes in the 18q21 region, including BCL2, in patients with amplification or gain of this region. Although we do not have FISH data on all of our patients, patients with this pattern of gene expression likely have gain or amplification of 18q21. Among the other upregulated genes, MALT1 may be of biologic importance. MALT1 is a paracaspase that, together with BCL10, is involved in the activation of NF-B.41 In these patients, the high expression of MALT1 may contribute to the activation of this important pathogenetic pathway and further enhance BCL2 expression.

    In summary, our study has demonstrated that BCL2 is upregulated by different mechanisms in the GCB and ABC subgroups of DLBCL. BCL2 expression is associated with poor survival in the ABC subgroup but not in the GCB subgroup. Although BCL2 may act as an antiapoptotic factor, in the ABC subgroup, it may also serve as a marker for events that are responsible for poor prognosis (eg, NF-B activation or 18q21 amplification). In the GCB subgroup, BCL2 expression is mainly a result of the t(14;18), thus representing a completely different mechanism of expression. BCL2 protein expression may reflect activation of different genetic pathways in the two subgroups of DLBCL and, therefore, has distinct clinical implications. Assessing the prognostic impact of expression of BCL2 or other biomarkers on survival without taking subgroup distinction into consideration may result in erroneous conclusions. Future studies should aim at a more precise definition of the optimal cutoff value for BCL2 positivity. This will require more quantitative measurements of the analyte concentrations in the tumor cells.

    Supplementary Table

    Authors' Disclosures of Potential Conflicts of Interest

    The authors indicated no potential conflicts of interest.

    Author Contributions

    Conception and design: Javeed Iqbal, Dennis D. Weisenburger, Wing C. Chan

    Financial support: Wing C. Chan

    Administrative support: Julie M. Vose

    Provision of study materials or patients: Christine P. Hans, Dennis D. Weisenburger, Timothy C. Greiner, Randy D. Gascoyne, Elias Campo, German Ott, H. Konrad Müller-Hermelink, Jan Delabie, Elaine S. Jaffe, Thomas M. Grogan, Joseph M. Connors, Julie M. Vose, James O. Armitage, Louis M. Staudt, Wing C. Chan

    Collection and assembly of data: Javeed Iqbal, Vishala T. Neppalli, Bhavana J. Dave, Douglas E. Horsman, Andreas Rosenwald

    Data analysis and interpretation: Javeed Iqbal, Vishala T. Neppalli, George Wright, James Lynch, Timothy C. Greiner, Julie M. Vose, Louis M. Staudt, Wing C. Chan

    Manuscript writing: Javeed Iqbal, Vishala T. Neppalli, Christine P. Hans, Dennis D. Weisenburger, Louis M. Staudt, Wing C. Chan

    Final approval of manuscript: Javeed Iqbal, Wing C. Chan

    Acknowledgment

    We thank D. Pickering, W.G. Sanger, Y. Shen, K. Fu, and R.M. Braziel for their contribution and helpful discussion.

    NOTES

    Supported in part by US Public Health Service Grant Nos. CA36727 and CA84967 awarded by the National Cancer Institute, Department of Health and Human Services, Bethesda, MD.

    Both J.I. and V.T.N. contributed equally to this work.

    Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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