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Journal of Clinical Oncology, Vol 24, No 25 (September 1), 2006: pp. 4135-4142 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.05.5897 Prognostic Impact of Germinal Center–Associated Proteins and Chromosomal Breakpoints in Poor-Risk Diffuse Large B-Cell Lymphoma
From the Departments of Hematology and Pathology, University Medical Center Groningen, University of Groningen, Groningen; Department of Trials and Statistics–HOVON Data Center, Erasmus MC–Daniel den Hoed Cancer Center, Rotterdam; and the Department of Hematology, University Medical Center Utrecht, Utrecht, the Netherlands Address reprint requests to Gustaaf W. van Imhoff, MD, Department of Hematology, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, the Netherlands; e-mail: g.w.van.imhoff{at}int.umcg.nl
Purpose Outcome of diffuse large B-cell lymphoma (DLBCL) with a germinal center B-cell (GCB) expression profile is superior to that of non-GCB DLBCL. This conclusion is mainly derived from patients with mixed international prognostic index (IPI) risk profiles treated with CHOP-like therapy (cyclophosphamide, doxorubicin, vincristine, and prednisone). We wondered whether the prognostic impact of the expression profile would hold out in a homogeneous cohort of poor-risk DLBCL patients treated with high-dose sequential therapy (HDT) and autologous stem-cell transplantation (ASCT) as first-line therapy. Patients and Methods DLBCL from 66 newly diagnosed poor-risk patients, treated in two sequential prospective Dutch Hemato-Oncology Association (HOVON) trials, were studied retrospectively for expression of CD10, bcl6, MUM1/IRF4, bcl2, Ki67, and CD21+ follicular dendritic cells (FDC) by immunohistochemistry, and for the breakpoints of BCL2, BCL6, and MYC by fluorescent in situ hybridization (FISH). Lymphomas with any follicular component were excluded. Results A GCB immunophenotype profile was found in 58% and non-GCB immunophenotype profile in 42% of the tumors. Clinical characteristics of both groups were similar. Complete response (CR) rate was higher in patients with CD10+ tumors (58% v 30%; P = .03). A GCB immunophenotype profile, its constituting markers CD10 more than 30% and MUM1 less than 70%, and bcl2 less than 10% were each associated with a better overall survival (OS). FDC networks, equally present in GCB and non-GCB tumors, had superior CR (73% v 31%; P = .01), but disease-free survival rates were lower and there was no difference in OS rates. None of the breakpoints had a prognostic impact on outcome. Conclusion Also in patients with poor-risk DLBCL treated with HDT and ASCT, the GCB immunophenotype and bcl2 expression retained a major impact on survival.
Diffuse large B-cell lymphoma (DLBCL), the most common type of aggressive non-Hodgkin's Lymphoma (NHL), exhibits distinct biologic and clinical heterogeneity.1 Currently, the international prognostic index (IPI) is still the most important tool to predict response to treatment for aggressive NHL and to classify patients into subgroups with distinctly different prognoses.2 However, even within these IPI risk groups a substantial variability in outcome has been observed. Thus, finding new tools to better classify DLBCL patients in different prognostic subgroups is important. Many studies have focused on the significance of the intrinsic characteristics of the tumors, eg, protein or gene expression and chromosomal breakpoints, however, the clinical relevance of many of these markers is inconsistent. Moreover, few markers retain sufficient prognostic significance individually after adjustment for the overriding prognostic impact of the IPI risk score.3,4 Gene expression profiling offers prognostic value in DLBCL independently of IPI. Tumors with profiles closely resembling that of germinal center B-cells (GCB) have a better outcome than those with profiles resembling activated peripheral B-cells (ABC).5-7 However, not all studies could confirm a prognostic value.8,9 Furthermore, this analysis is not easily incorporated in routine practice as it depends on the availability of frozen tissue and sophisticated laboratory and statistical methods. Based on gene expression studies, Hans et al10 developed an algorithm to discriminate GCB from non-GCB type DLBCL, based on CD10, bcl6, and MUM1/IRF4 expression measured by immunohistochemistry (IHC). Outcome of DLBCL with a GCB protein expression profile was superior to non-GCB tumors and concurred with gene expression profiles as determined by microarray analyses.10 Controversial findings have been reported using the same or slightly different IHC algorithms,11-15 which might be caused by differences in clinical characteristics, variations in treatment, IHC techniques, or cutoff levels for scoring. Moreover, most data published so far have been derived from patients treated with standard (ie, CHOP-like; cyclophosphamide, doxorubicin, vincristine, and prednisone) first-line treatment. Whether up-front high-dose sequential therapy (HDT) and autologous stem-cell transplantation (ASCT) as first-line treatment might overcome the poor prognostic features conferred by cell of origin is still unknown. We recently reported on two sequential prospective trials of the Dutch Belgian Hemato-Oncology Cooperative Group (HOVON), HO27 and HO40, investigating HDT and ASCT as first-line treatment in patients with poor-risk advanced-stage aggressive NHL with age-adjusted IPI scores of 2 to 3.16 In this article, we report the results of a retrospective analysis addressing the prognostic significance of individual protein expression, GCB versus non-GCB immunophenotype, and chromosomal breakpoints in the DLBCL patients of both studies.
Patient Selection Patients were included in either one of two consecutive HOVON trials, HO27 and HO40, for previously untreated poor-risk aggressive NHL.16 In summary, patients had aggressive NHL, were age 18 to 65 years, had WHO performance status 0 to 2, had Ann Arbor stage III or IV disease, and had serum lactate dehydrogenase (LDH) of at least 1.5 times the upper limit of normal (ULN). Treatment consisted of high-dose sequential chemotherapy followed by HDT and ASCT (trial HO27) or the same treatment preceded by three cycles of intensified CHOP (trial HO40). One hundred forty-seven patients were included, 117 of whom had DLBCL. Both studies were carried out in accordance with the modified Declaration of Helsinki. The institutional boards of each participating institution approved both study protocols and all patients gave written informed consent.
Histology
Immunohistochemistry and Chromosomal Breakpoints Slides were evaluated semi-quantitatively by two or three independent observers (E.-J.G.B., P.M.K., and G.W.v.I.) and were grouped as follows: for bcl2, tumors with 0% to 10%, 11% to 50%, and 51% to 100% positive tumor cells; for MUM1/IRF4, 0% to 30%, 31% to 50%, 51% to 70%, and 71% to 100% positive tumor cells; for Ki67, 0% to 50%, 51% to 90%, and 91% to 100%. Bcl6 and CD10 were considered positive when more than 30% of the tumor cells were positive. CD21 was used for the staining of follicular dendritic cell (FDC) networks within the tumor fields using a visual score of 0, +, ++, or +++; staining of tumor cells was ignored. Classification of GCB versus non-GCB was based on the algorithm of Hans et al, 10 using CD10, bcl6, and MUM1/IRF4 expression with 30% cutoff values for the number of positive tumor cells (Fig 1).
For the detection of chromosomal breakpoints in MYC/8q24, BCL2/18q21, and BCL6/3q27, all cases were studied by segregation fluorescent in situ hybridization (FISH) on paraffin tissue sections in accordance with recently published methods of our group.17
Study End Points and Statistical Considerations
Clinical Characteristics Between 1994 and 2001, 147 patients were included in trials HO27 and HO40. Of 117 patients with DLBCL, 73 tissue samples were retrieved, ultimately retrieving adequate tissue from 66 patients (56%), of which 58% were from DLBCL patients from trial HO27 and 55% from HO40. Clinical characteristics of the patients are listed in Table 1. All patients had advanced disease with highly elevated LDH levels and age-adjusted IPI scores  2. Median follow-up of the 24 patients still alive (seven patients in HO27 and 17 in HO40) was 59 months (range, 23 to 132).
Distribution of Markers IHC and FISH results were assessable in almost all cases (Table 2). For the IHC markers, the percentages of positive tumors were similar in both trials. Slightly more MYC and BCL2 breakpoints were observed in the HO27 study and more BCL6 breakpoints were observed in the HO40 study. However, absolute numbers were small.
GCB Versus Non-GCB Phenotype Algorithm Applying the IHC algorithm described by Hans et al,10 with cutoff values at 30% of positive tumor cells, 38 patients had GCB and 28 patients had non-GCB DLBCL (Fig 1). The majority of GCB cases (84%) were classified as such based on CD10 positivity. Only six CD10-negative cases were classified as GCB-type, based on a combination of bcl6 positivity and MUM1 negativity. In 11 (34%) and four (13%) of the 32 CD10+ cases, MUM1 was positive at 30% and 70% cutoff levels, respectively. Eight of the 34 CD10-negative cases (24%) were bcl6 negative, whereas 20 tumors (59%) were both bcl6 and MUM1 more than 30% positive; both were classified as non-GCB. Although slightly more patients with GCB tumors had bulky disease, no significant differences were observed in clinical characteristics or treatment according to trial between GCB and non-GCB tumors (Table 3).
Expression of Other Proteins and Chromosomal Breakpoints The distribution of the other IHC markers and chromosomal breakpoints according to GCB versus non-GCB phenotype are listed in Table 4. Bcl2 protein overexpression may be the result of BCL2 rearrangement or other mechanisms,10,18-20 and as such confer a different clinical behavior.21 All 10 BCL2 breakpoint–positive tumors (17%) expressed bcl2 protein. BCL2 breakpoints clustered in GCB, whereas breakpoint-negative but bcl2 protein–positive tumors were more frequent in the non-GCB group.
BCL6 and MYC breakpoints were detected in 26% and 15%, respectively. Four of the nine MYC-positive cases harbored additional breakpoints: three BCL2, and one BCL6 (Table 4). Apart from incidental copy number gain for BCL2 (n = 2), MYC (n = 4), and BCL6 (n = 4), two cases with gene amplification of BCL6 were observed. FDC networks were present in 17 tumors (26%) and were equally frequent in GCB and non-GCB tumors (Table 4). Five FDC+ cases had bone marrow involvement of lymphoma. None of the three cases that could be analyzed showed discordant morphology. FDC+ cases had breakpoints of MYC, BCL2, or BCL6 in zero, four, and four cases, respectively. Slightly more GCB type DLBCL had a Ki-67/MIB-1 proliferation rate of more than 90% (46% v 32%).
Prognostic Impact of Biologic Markers on Treatment Outcome
GCB conferred better OS than non-GCB phenotype (P = .04; Fig 2). CD10 positivity (Fig 3A) and MUM1 less than 70% (Fig 3B; but not MUM < 30%), and bcl2 protein less than 10% (Fig 3C), were associated with superior OS. By applying MUM1 more than 70% instead of more than 30% as the cutoff level for non-GCB classification in the Hans algorithm, nine non-GCB patients changed to GCB type. The predictive value of survival of non-GCB cases based on this MUM1 more than 70% algorithm (HR, 2.11; 95% CI,1.12 to 3.98) might be slightly stronger than the value based on the original algorithm, with a threshold of more than 30% (HR, 1.8; 95% CI, 1.02 to 3.45).
Outcome of bcl2 protein+ GCB cases with (n = 10) or without (n = 8) a BCL2 breakpoint was not significantly different (median survival, 13 v 21 months, respectively; P = .37). Although CR rate was higher in tumors with CD21+ FDC networks, DFS was inferior (P = .03) and OS was similar (Fig 4). None of the chromosomal breakpoints had a prognostic impact on outcome.
We investigated the prognostic relevance of immunophenotype and chromosomal breakpoints in a well-defined group of DLBCL patients with a homogeneous poor-risk IPI score, whose disease was treated up-front with HDT and ASCT. GCB immunophenotype as determined by the Hans algorithm, which includes CD10, bcl6, and MUM1/IRF4 as individual markers,10 conferred a significantly better survival than non-GCB immunophenotype. Additionally, we found a strong positive association between CD10 expression and response to treatment and survival. Expression of MUM1/IRF4 and bcl2 at cutoff levels of 70% and 10%, respectively, were inversely correlated with survival. Breakpoint analysis of BCL6, BCL2, and MYC had no detectable prognostic impact. Studies on the clinical relevance of biologic markers in DLBCL are frequently hampered by confounding factors, such as type of treatment and case-mix of patients with different IPI risk scores. This might partially explain why different groups studying the same marker(s) or combinations thereof reported controversial results. In our study, clinical presentation and age-adjusted IPI scores of both cohorts of poor-risk DLBCL patients were almost identical. Treatment, however, was not equal and might have influenced the results; in trial HO40 more chemotherapy (three intensified CHOP courses) had been administered before ASCT. Despite similar CR rates, a significantly better DFS, EFS, and OS in trial HO40 was observed.16 Nevertheless, the studied tumor markers were almost equally distributed in both trials and all immunophenotypic markers, including GCB- versus non-GCB phenotype, had similar prognostic relevance in both cohorts. Moreover, repeated analyses of prognostic factors with adjustment for trial did not alter the results. Grouping of DLBCL by gene expression profiling into GCB versus non-GCB derived tumors has originally been based on algorithms containing a myriad of genes.5 Subsequently, a simple IHC algorithm, based on the expression of only three different proteins (CD10, bcl6, and MUM1/IRF4) could classify tumors in the same groups with similar prognostic results.10 Our study confirms these findings for survival in a group of poor-risk patients treated with up-front HDT and ASCT. The prognostic impact of the algorithm not only depends on the relative prognostic value of the individual markers, but also on their hierarchical position in the algorithm and, especially in the case of MUM1/IRF4, on the applied cutoff value. CD10, a marker of follicular center B-cell differentiation,22 is placed at the first hierarchical level of the algorithm. Reports concerning the prognostic value of CD10 in DLBCL are controversial,10-12,15,23-26 possibly due to differences in IPI risk factors. In our study of homogeneous age-adjusted IPI poor-risk DLBCL patients, CD10+ immunophenotype correlated most strongly with improved remission rate and outcome, supporting its hierarchical position in the algorithm. Expression of bcl6, a protein essential for germinal center formation,27 was observed in 85% of our cases at a cutoff level of 10%, which is in agreement with the literature. All cases harboring BCL6 translocation expressed bcl6 protein, which might be caused by promoter substitution. Additional genetic alterations including point mutations in regulatory sequences of BCL6 might have accounted for the expression in the many other DLBCL cases in our and other series.28 The reported prognostic impact of bcl6 protein expression is controversial.11,12,29 We observed no prognostic impact of bcl6. Furthermore, the contribution of bcl6 in the IHC algorithm on the outcome of classification GCB versus non-GCB immunophenotype in our study was rather small, mainly as a result of the very small percentage of bcl6 negative cases. Expression of MUM1/IRF4, a marker of terminal differentiation of B cells,30 is inversely correlated with GCB-type DLBCL in gene expression arrays. MUM1 protein expression is correlated with adverse prognosis in DLBCL according to most,10,12,31 but not all, reports.11,15 Cutoff values differ from 20% to 30%10-12,15 to 80%.31 In our study, a cutoff value of 30% had no prognostic value. Using a cutoff value of 70% we observed a small group of patients with a very short median survival of 10 months. This effect was independent of CD10 expression. Higher cutoff levels for MUM1 might improve its performance as a single prognostic marker, as well as in prognostic algorithms as suggested by our results. Recently, Moskowitz et al32 observed no correlation between GCB versus non-GCB phenotype and outcome in 88 patients with relapsed/primary refractory DLBCL eligible for second-line treatment followed by ASCT. The authors hypothesized that the prognostic value of the IHC algorithm was overcome by HDT and ASCT. In our study of patients treated with HDT and ASCT in first-line treatment, this apparently was not the case. Whether the prognostic value of GCB or other biologic markers in DLBCL would disappear with new treatment modalities remains to be investigated. Notably, neither our patients, nor those of Moskowitz et al, had been treated with anti-CD20 monoclonal antibodies (rituximab). The results of other markers investigated in our study are of particular interest. We confirmed the strong negative correlation of bcl2 expression with survival, which was reported previously.3,11,14,31,33-36 We also analyzed the prognostic relevance of bcl2 expression in conjunction with BCL2/18q21 breaks, because expression of bcl2 protein might also be the result of distinct other mechanisms and as such confer a different prognosis.18-21 The negative effect of bcl2 protein expression on survival was observed both in the presence and absence of BCL2 translocations. Moreover, within the 18 bcl2-expressing GCB-type DLBCL, no difference in outcome was observed between cases with and without the BCL2 breakpoint. Thus, we could not confirm the negative correlation with survival of BCL2 breakpoints within the GCB group as reported by Barrans et al.21 This suggests a dominant effect of overexpression of bcl2 protein, irrespective of the underlying mechanism. Adding rituximab to chemotherapy strongly improves outcome in DLBCL.37-39 As improvement was most pronounced in patients with tumors expressing bcl2 protein, the negative effect of bcl2 protein expression might diminish in future patients treated with rituximab.40 The percentages of DLBCL with breakpoints for MYC, BCL2, and BCL6 were comparable with those found in a larger series from a population-based Dutch registry.3 MYC rearrangements in DLBCL may be secondary genetic events and may be associated with aggressive clinical behavior. Half of the MYC-positive cases in our study had additional breakpoints of BCL2 or BCL6. Median survival rates for the nine patients with MYC rearrangement (including the four cases with double hits) tended to be inferior to rates for patients without the MYC rearrangement (nonsignificant). Due to small numbers, our study had insufficient power to detect possibly moderate but clinically relevant differences between the subgroups. The prognostic impact of CD21+ FDCs was unexpected. Although not associated with GCB immunophenotype, the clinical data of these cases mimicked the high response rates but increased relapse rates of follicular lymphomas naturally containing such mesh works. Utmost care was taken to exclude lymphomas having any follicular growth pattern or component. In addition, we could exclude discordant lymphoma in three of the five FDC+ cases with bone marrow involvement. Katzenberger et al41 reported the presence of FDC mesh works in DLBCL with BCL6/3q27 translocation. We did not find such a correlation. Nevertheless, the abundance of FDC mesh works in some of our DLBCL cases, even at extranodal sites without naturally occurring FDCs, might indicate an intricate active interaction between the tumor cells and FDCs.41 In conclusion, in this clinically homogeneous group of poor-risk DLBCL patients, we observed a strong prognostic influence of intrinsic biologic markers such as bcl2 expression and GCB immunophenotype, which was not overcome by up-front HDT and ASCT therapy.
The authors indicated no potential conflicts of interest.
Presented in part at the 9th International Conference on Malignant Lymphoma, Lugano, Switzerland, June 9-11, 2005. G.W.v.I. and E.G.B. contributed equally to this study. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
=.05 was considered significant. 
