Annals of Oncology Advance Access originally published online on March 29, 2007
Annals of Oncology 2007 18(5):931-939; doi:10.1093/annonc/mdm012
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© 2007 European Society for Medical Oncology
hematologic malignancies |
Immunophenotype as prognostic factor for diffuse large B-cell lymphoma in patients undergoing clinical risk-adapted therapy
1 Departments of Internal Medicine I (Hematology/Oncology)
2 Department of Biometry and Statistics
3 Department of Pathology, Freiburg University Medical Center, Freiburg, Germany
* Correspondence to: Dr H. Veelken, Department of Hematology/Oncology, University Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Germany. Tel: +49-761-270-7175; Fax: +49-761-270-7177; E-mail: hendrik.veelken{at}uniklinik-freiburg.de
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Abstract |
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Background: For patients with diffuse large B-cell lymphoma (DLBCL), the International Prognostic Index (IPI) predicts the likelihood for cure with chemotherapy. Biological parameters, including expression of Bcl-6, Bcl-2, CD10, major histocompatibility complex class II, and categorization as germinal center (GC) type have been described as IPI-independent prognostic factors.
Patients and methods: Biological parameters were evaluated retrospectively by immunohistochemistry in 60 consecutive DLBCL patients of the prerituximab era. Forty-one of 60 patients underwent a risk-adapted treatment strategy including autologous stem-cell transplantation for high-risk patients (age-adjusted IPI = 2–3; slow response to chemotherapy).
Results: Bcl-6 expression was associated with superior overall survival (OS) independently of the IPI. Inferior progression-free survival (PFS) was independently correlated with high expression of Bcl-2 and low positivity for HLA-DR and CD10. Distinction into GC and non-GC DLBCL on the basis of Bcl-6, CD10, and IRF-4 expression had no independent prognostic value. Within the risk-adapted treatment strategy, only HLA-DR retained a prognostic impact on OS (P = 0.0058) and PFS (P = 0.0002).
Conclusions: In 60 patients with DLBCL treated with risk-adapted therapy, immunohistochemical subcategorization of DLBCL into GC and non-GC type has little clinical value. The IPI-associated risk appears to be mitigated by intensified upfront therapy. Low HLA-DR expression is associated with poor outcome after intensified upfront therapy. Therefore, additional treatment modalities appear to be required.
Key words: Bcl-6 protein, diffuse large-cell lymphoma, hematopoietic stem-cell transplantation, HLA-DR antigens, immunohistochemistry
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introduction |
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For the last 25 years, anthracyclin-based polychemotherapy has represented the standard of care for diffuse large B-cell lymphoma (DLBCL) and cures 40%–50% of DLBCL cases [1]. For patients undergoing such therapy, the international prognostic index (IPI) defines prognostic subgroups with long-term disease-free survival ranging from 15% to 65% [2]. A simplified risk assessment is the age-adapted International Prognostic Index (aaIPI), which is on the basis of Ann Arbor stage, performance status, and lactate dehydrogenase serum activity only.
Intensification of chemotherapy with autologous stem-cell transplantation (autoSCT) has been shown to improve the prognosis for intermediate/high and high-risk patients with chemosensitive disease in randomized clinical trials [3, 4]. These approaches, however, are associated with increased toxicity. While higher treatment-associated risks may be acceptable for patients with poor prognosis according to the IPI, a more accurate risk assessment would be desirable for the large intermediate-risk group to identify patients who might benefit from dose intensification, and to avoid overtreatment of the remaining group.
The protein expression levels of several genes in DLBCL cells as assessed by immunohistochemistry, including the antiapoptotic proto-oncogene Bcl-2 [5–9], the germinal center (GC)-associated gene Bcl-6 [9, 10], and major histocompatibility complex (MHC) class II [11, 12] have been identified as IPI-independent prognostic factors. Furthermore, microarray transcriptome analyses have revealed biological subtypes of DLBCL, most notably one category with a gene expression signature resembling GC B cells and another of activated B cells (ABC). Independently of the IPI, GC-type DLBCL has a better prognosis than the ABC-like type tumors [13–15]. A distinction between GC- and non-GC-type DLBCL may also be possible by immunohistochemical detection of subtype-associated gene expression at the protein level, potentially permitting subclassification from archival formalin-fixed biopsy material [16]. This composite phenotype requires staining for expression of the GC genes Bcl-6 and CD10 and the interferon regulatory factor IRF-4. Alternatively, an RT-PCR-based quantitative assessment of the transcripts of six genes has been developed which permits an IPI-independent prognostic subcategorization but does not formally categorize DLBCL into defined subgroups [17].
Of the immunohistochemical markers with prognostic importance, both Bcl-2 and Bcl-6 are associated with DLBCL subclassification. In contrast, MHC class II expression, which has also been identified as an important prognostic parameter in one of the microarray-based subgroup classifications [14], is not associated with either GC or non-GC features. Therefore, the composite immunohistochemical approach does not take expression of MHC class II into account, nor does the six-gene RT-PCR predictor [16, 17].
In order to test the usefulness of biological subclassification of DLBCL by immunohistochemistry in clinical practice, we analyzed outcome of DLBCL patients at our institution in relationship to the IPI and the tumor immunophenotype. Since the value of identified prognostic factors may change with different treatment strategies for a particular disease, the meaning of immunohistologic risk factors was also evaluated with respect to an aaIPI risk-adapted treatment strategy which has been the standard of care at Freiburg University Medical Center throughout the study period.
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patients and methods |
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patients and clinical parameters
All patients with DLBCL according to the World Health Organization classification with an available paraffin-embedded tumor biopsy who were treated in the Department of Hematology/Oncology from 1991 to 2002 were identified from the database of Freiburg University Medical Center. Patients with DLBCL originating from transformation of an indolent lymphoma with primary involvement of the central nervous system and with human immunodeficiency virus-associated lymphoma were excluded. For patients whose last visit was >12 months ago, additional follow-up data were obtained by contacting their primary care physician.
Overall survival (OS) was defined as the time interval from diagnosis to death or the last contact. Progression-free survival (PFS) was defined as the time interval from diagnosis to death from any cause or relapse/disease progression according to standardized criteria [18], whichever came earlier. Patients were censored for OS at the time of the last contact if currently alive and for PFS if no relapse/disease progression had occurred at the time of their last visit. Survival data between defined subgroups were compared with the log-rank test using GraphPad Prism software 4.01 (GraphPad Software, San Diego, CA). Multivariate analyses were carried out by a Cox regression model with backward elimination using SAS 8.2 software (SAS Institute, Cary, NC). The study was in accordance with the ethical standards set by the institutional ethics committee and was conducted according to the declaration of Helsinki. All patients gave informed consent.
treatment strategy
Patients with newly diagnosed DLBCL were treated according to an aaIPI risk-adapted treatment algorithm if no medical contraindications for the appropriate chemotherapy were present. All patients received anthracyclin-based induction chemotherapy with 6 weeks of the VACOP-B protocol or three cycles of the combination chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen. Patients with an initial aaIPI score of zero to one without bulky disease (defined as lesion of 10 cm or more in any direction) who achieved a partial or complete remission to induction received another 6 weeks of VACOP-B or three to five cycles of CHOP. For patients of this low-risk subgroup who had clinical stage I–II at presentation, involved-field radiation therapy with 36–40 Gy was offered alternatively. All other patients (aaIPI 2–3, aaIPI 0–1 and bulky disease, aaIPI 0–1 not achieving at least a partial response after induction) received intensification with two cycles of VIP-E or VCP-E [19] including autologous stem-cell harvest, followed by high-dose BEAM chemotherapy and autoSCT [20]. Sites of bulky disease received consolidating radiation therapy of 20–36 Gy.
Disease relapses after full anthracyclin-containing primary therapy were treated with two cycles of DHAP chemotherapy. Objective tumor responses in patients without prior autoSCT were treated with high-dose chemotherapy and autoSCT if no contraindications were present. Eligible patients with relapse after previous autoSCT were offered allogeneic SCT with reduced conditioning according to the FBM protocol [21].
Patients who had contraindications to this algorithm or who did not consent to SCT were managed individually according to the recommendations of the institutional lymphoma panel.
histopathological analysis
For all patients, fresh serial 3-µm sections were cut from the original diagnostic paraffin-embedded tissue blocks which had been fixed in 10% buffered formalin. The adequacy of the material and the confirmation of the diagnosis of DLBCL after Giemsa staining were carried out by a hematopathologist (AS-G). For immunohistochemistry, sections were deparaffinized and rehydrated through graded alcohols. Heat-mediated antigen retrieval was carried out (except for CD3 and CD20) by boiling the sections in a steamer in target retrieval solutions (Dako, Glostrup, Denmark) for detection of CD10, CD79a and Ki-67 (pH 6.0) or for all other antibodies (pH 9.0). CD3 immunostaining was carried out after digestion with 0.05% proteinase K (Sigma-Aldrich Chemie, München, Germany) in phosphate-buffered saline (pH 7.4).
The primary unconjugated antibodies and their working dilutions are given in Table 1. After incubation for 20 min with 10% blocking sera from the respective animal species (Biotrend, Cologne, Germany), sections were stained semiautomatically (Autostainer instrument, Dako, Glostrup, Denmark) with the primary antibodies and biotinylated anti-mouse and anti-rabbit detection antibodies. Bound detection antibodies were detected on the basis of the labeled streptavidin–biotin method [22] (ChemMate K5005 Alkaline Phosphatase/Red detection kit, Dako, Glostrup, Denmark). Nuclei were counterstained with Mayer's hemalaun solution. Semiquantitative assessment of the fraction of antigen-positive tumor cells in increments of 10% (decentiles) was carried out by two investigators (AS-G, SVD) without knowledge of the clinical outcome.
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outcome according to aaIPI
The 60 patients included in this analysis represented the entire spectrum of clinical risk factors and were evenly distributed with respect to the aaIPI score that formed the basis for the treatment algorithm (Table 2). With regard to histological subtype of DLBCL, 42 cases had centroblastic, five immunoblastic, seven anaplastic variant, five primary mediastinal, and one case T-cell rich morphology. All patients received anthracyclin-based polychemotherapy. In accordance with the risk-adapted treatment strategy, 19 patients received upfront autoSCT (Figure 1). With a median follow-up of 57 months (range 2–162 months), the 5-year OS was 73% with a 95% confidence interval (CI) of 61.2% to 84.8% and the 5-year PFS was 53.8% (95% CI 40.9% to 66.7%) in this consecutive patient group (Figure 2). The aaIPI discriminated between the subgroups for OS and PFS (Figure 2).
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outcome according to immunophenotype
A bimodal distribution was noted for the expression of the proteins studied with the exception of Ki-67 (Figure 3). The cut-off between high and low antigen expression for statistical comparisons was therefore defined according to the hiatal pattern. For Ki-67, cases comprising 80% or more positive cells were categorized as high expression.
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In univariate analyses, high expression of Bcl-6 or HLA-DR was associated with superior OS (Table 3). In addition, positivity for HLA-DR and CD10 and low expression of Bcl-2 and IRF-4 were correlated with a superior PFS. Expression patterns of Ki-67 and hTERT had no detectable prognostic value. The subclassification into GC and non-GC DLBCL according to the composite immunohistochemistry index (Figure 4) was also associated with outcome in univariate analyses (Figure 5).
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Multivariate analyses identified an aaIPI score of two to three [hazard ratio (HR) = 3.233; P = 0.0205] and high expression of Bcl-6 (HR = 0.281; P = 0.0214) as independent prognostic factors of similar importance with respect to OS. Expression of CD10 (P = 0.0711) and HLA-DR (P = 0.1476) had a lesser impact, whereas Bcl-2 expression (P = 0.85) and the composite classifier of GC and non-GC DLBCL (P = 0.71) did not emerge as independent prognostic indicators for OS in multivariate analyses.
With respect to PFS, high aaIPI scores (HR = 2.328; P = 0.0371) and expression levels of Bcl-2 (HR = 2.502; P = 0.0302), CD10 (HR = 0.302; P = 0.0331), and HLA-DR (HR = 0.373; P = 0.0279) were independently associated with PFS. Expression of Bcl-6 (P = 0.74) and the composite GC phenotype (P = 0.70) exerted no detectable influence on PFS.
impact of risk-adapted treatment strategy
The risk-adapted strategy was implemented as an attempt to offer patients with a high clinical risk a better chance of long-term disease-free survival. In the prerituximab era, the highest impact on outcome would be expected to be achieved through high-dose chemotherapy with autoSCT. The oldest patient who received autoSCT was 62 years old in this series. Therefore, we reanalyzed the data for the 30 patients who were treated according to the risk-adapted algorithm and were assumed to be eligible for autoSCT, i.e. <63 years of age. By definition, this group was on average younger than the remaining patients, but no significant differences were found with respect to other clinically important parameters (Table 2).
The 5-year OS and PFS within this group were 78.3% (95% CI 62.7% to 93.9%) and 66.7% (95% CI 49.8% to 83.6%), respectively, without a detectable influence of the aaIPI (Figure 6). Among the immunohistochemical parameters only expression of HLA-DR was associated with improved OS and PFS; all other parameters were not associated with outcome (Table 4). Bcl-6 expression could not be evaluated since all tumors in this subgroup stained positive for this marker.
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Given its balanced distribution with respect to risk groups defined by the clinical parameters of the IPI, our single-center patient population does not appear to differ in major aspects from other retrospective studies on prognostic factors for DLBCL [14, 15, 23, 24]. These studies reported OS ranging from 42% after 2 years to 54% after 5 years for patients treated with anthracyclin-based polychemotherapy without monoclonal antibodies. Even with all necessary caution concerning comparability of such series, the 5-year OS of our patients appears to be comparatively high with 73%. This relatively good outcome is possibly attributable to the risk-adapted treatment strategy at our institution with upfront high-dose chemotherapy and autoSCT for eligible patients with unfavorable IPI. Indeed, within the subgroup of patients who could be treated according to this approach, the 5-year OS of higher risk patients was with 83.3% (95% CI 62.2% to 100%) remarkably similar to prognostically favorable patients (OS 79.3%; 95% CI 61.2% to 97.4%). Taken together, the observation that the outcome of this patient subgroup was independent of the IPI strengthens the assumption that intensified chemotherapy may improve the prognosis of such patients beyond the selective setting of a prospective trial.
In addition to the IPI, the recognition of biologically defined prognostic DLBCL subgroups would offer further opportunities to define risk-adapted treatment strategies. Regarding available diagnostic techniques for this purpose, immunohistochemistry would be most convenient followed by a panel of RT-PCR reactions; whereas array-based technologies may not easily be applied routinely to the majority of DLBCL cases. We therefore evaluated well-known immunohistochemical markers separately and combined to a composite index for their association with clinical outcome. In multivariate analyses, Bcl-6 expression and possibly CD10 and HLA-DR expression emerged as IPI-independent favorable markers. In contrast to the original report and a confirmatory analysis [16, 24], the composite index (CD10, Bcl-6, IRF-4) for distinction between GC- and non-GC-type of DLBCL had no independent prognostic value in our series. To exclude the possibility that this discrepancy was due to the choice of cut-off levels for negative versus positive cases, which were chosen according to the bimodal distribution of marker expression in our series, we reanalyzed our data with the fixed cut-off of 30% for CD10, Bcl-6, and IRF-4 as in published series [16, 24]. However, this change resulted in only minor differences and rather reduced the significance level for most parameters (data not shown).
Bcl-6 controls GC formation where it transiently represses p53 [25]. This repression prevents p53-mediated B-cell apoptosis in the GC induced by double-strand DNA breaks that occur naturally in the course of somatic hypermutation and class switching [26]. This antiapoptotic effect evidently contributes to lymphomagenesis when Bcl-6 is constitutively expressed by DLBCL cells.
Expression of CD10, a neutral endopeptidase, is another marker of GC B cells and hence participates in the GC-like DLBCL subtype as defined by microarray analysis [13, 14]. CD10 appears to have a lesser predictive importance than Bcl-6 [10]. Whether and how the Bcl-6 and CD10 proteins interact directly or indirectly with anthracylin-based chemotherapy to enhance its efficacy, however, is as yet unknown.
The prognostic role of Bcl-2 overexpression in DLBCL has also been controversial. In accordance to some studies [27, 28], we did not find an influence on OS in our series. However, our sample size may simply have been too small to permit detection of a negative impact of Bcl-2 on OS, which other investigators have described [7, 9, 29]. However, our findings further confirm the recognized association of Bcl-2 expression with inferior PFS [5, 6, 8]. Taken together, the stronger influence on PFS than on OS permits to speculate that the potent antiapoptotic activity of Bcl-2 enables some DLBCL cells to survive chemotherapy and to initiate a clinical relapse later. However, these relapses appear to retain sensitivity to chemotherapy and may still be curable with salvage regimens. A recent analysis has demonstrated that Bcl-2 expression may be associated with inferior OS only in the activated B-cell type of DLBCL as defined by microarray analysis [30]. Since expression profiling could not be carried out and the immunohistochemical index failed to have an independent prognostic value in our series, we were unable to address this issue. In the subgroup of patients treated according to the risk-adapted strategy, Bcl-2 expression was not significantly correlated with PFS, but the sample size may again be too small to detect a minor difference.
HLA-DR expression has been recognized as a prognostically unfavorable DLBCL feature in several retrospective series [11, 12, 14]. In DLBCL arising from immunopriviledged sites, loss of MHC class II expression is frequently attributable to large genomic deletions of the MHC class II gene cluster [31]. In contrast, transcriptional repression of the MHC class II gene cluster, including their master transcription factor, class II transactivator (CIITA), by a common control mechanism appears to occur in DLBCL arising at other sites [32]. Accordingly, microarray analyses demonstrated that survival was associated with expression of each of 35 different genes of the so-called MHC class II signature [14]. Low HLA-DR expression in DLBCL is associated with fewer tumor-infiltrating cytotoxic T cells [12], indicating that the dismal prognosis of HLA-DR-negative DLBCL may be attributable to an impaired immune surveillance. This observation might also indicate that cellular antitumor immune effects operate independently from cytoreductive therapy and would therefore predict that cellular immunotherapy may represent a promising and independent treatment strategy for MHC class II-expressing DLBCL.
Since high-grade lymphomas have been shown to have higher levels of telomerase activity compared with normal B cells and low-grade lymphomas [33] and the hTERT expression has been reported to be a prognostic marker in B-chronic lymphocytic leukemia [34], we added an analysis of hTERT protein expression in our patients but failed to find a prognostic significance.
The combination of rituximab, an anti-CD20 antibody, with anthracyclin-based therapy has greatly improved the treatment results for DLBCL patients [23]. In elderly patients, the benefit of rituximab may be more pronounced for lower IPI scores than for high-risk patients [35]. Recent evidence indicates that Bcl-6-negative and possibly Bcl-2-positive DLBCL cases may benefit most from rituximab [36, 37]. In contrast, the impact of immunochemotherapy on MHC class II-associated risk or GC subtype by microarray technology has not yet been clarified. Our study may indicate that risk-adapted therapy intensification particularly fails to improve the adverse prognostic influence of HLA-DR expression status. While this observation needs independent confirmation, novel therapeutic modalities are nevertheless urgently required for this particularly unfavorable patient subgroup.
Received for publication August 24, 2006. Revision received December 19, 2006. Accepted for publication January 11, 2007.
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