Diffuse large B-cell lymphoma (DLBCL) can be molecularly subtyped as either germinal center B-cell (GCB) or non-GCB. The role of rituximab(R) in these two groups remains unclear. We studied 204 patients with de novo DLBCL (107 treated with first-line CHOP; 97 treated with first-line R-CHOP), patients being stratified into GCB and non-GCB on the basis of BCL-6, CD10, and MUM1 protein expression. The relationships between clinical characteristics, survival data, and immunophenotype (IHC) were studied. The 5-year overall survival (OS) in the CHOP and R-CHOP groups was 50.4% and 66.6% (
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphomas, representing 30% of all newly diagnosed cases and more than 80% of aggressive lymphomas [
Nearly all the studies of prognostic indicators in DLBCL, including the IPI, have been based on the clinical outcome following treatment with CHOP [
In the present study, we explore the outcome of addition of rituximab into CHOP regimen among GCB and non-GCB in our institutes and address whether IHC-defined GC versus non-GC distinction of DLBCL could be used to predict a patient’s outcome in response to a combination of rituximab and chemotherapy.
We retrospectively studied 204 patients with de novo DLBCL diagnosed at Sun Yat-sen University Cancer Center between 1998 and 2005. All of these patients received either the CHOP (
Formalin-fixed, paraffin-embedded tumor sections were available in all cases and were reviewed by two pathologists, who confirmed that all cases were de novo DLBCL according to the 2008 WHO lymphoma classification system.
The extent of disease had been determined at first presentation by physical examination, serum lactate dehydrogenase concentration, full blood count, and computed tomography of the chest and abdomen. For each patient, the following characteristics were noted from the medical records: age at diagnosis, sex, Ann Arbor stage at presentation, therapy, achievement of complete remission, occurrence of relapse, and time to death or loss to followup.
Formalin-fixed, paraffin-embedded tumor sections were examined for the expression of CD10, BCL-6, and MUM1. Briefly, slides were deparaffinized by xylene and rehydrated. Tissue sections were antigen-retrieved in Tris EDTA buffer (10 mM/L, pH 8.0) and incubated by heat induction for 20 minutes (CD10 and MUM1) or 40 minutes (BCL-6). The mouse anti-IRF4 antibody (diluted 1 : 400; clone M17, DAKO, Glostrup, Denmark), mouse anti-BCL-6 antibody (diluted 1 : 40; clone M17, DAKO, Glostrup, Denmark), and mouse anti-CD10 antibody (diluted 1 : 50; Zymed) were used. Sections of reactive tonsil and PAS were used as positive and negative controls, respectively.
The proportion of positively stained tumor cells was estimated by two pathologists who had no knowledge of the corresponding clinical data. Disagreements were resolved by reanalysis of the staining. Tumor cells positive for the markers were evaluated semiquantitatively with a cutoff of 30%. All cases were subdivided into GCB or non-GCB subtypes as described by Hans; briefly, the phenotype of GCB was defined as CD10 positive (regardless of the other two markers) or CD10−BCL6+MUM1−. The non-GCB phenotype was defined as CD10−BCL6−MUM1+ or lack of expression of all three markers.
The response criterion of non-Hodgkin’s lymphomas was applied to determine the occurrence of complete remission (CR), partial remission (PR), stable disease (SD), or progressive disease (PD). Overall survival (OS) was measured from the date of diagnosis until the last followup or death from any cause. Progression-free survival (PFS) was determined as an interval between the date of diagnosis and relapse, or death.
The relationships between the three markers, the subdivision and OS were assessed by Kaplan-Meier graphs. Fisher’s exact test was used to identify significant correlations between variables. The
Of the 204 patients, 107 were treated initially with the standard CHOP regimen and 97 were treated with R-CHOP. The clinical data, including IPI, was retrospectively evaluated in all patients. A total of 202 of the 204 patients had all the necessary data available to calculate the IPI; two of 204 patients had no record of serum lactate dehydrogenate concentration. For the CHOP group, 107 DLBCL patients aged 16–84 years (median age, 54 years) were included. The follow-up period ranged from 6 to 167 months (median, 53 months). For the R-CHOP group, 97 DLBCL patients aged 20–83 years (median, 59.0 years) were studied. The follow-up period for the R-CHOP group ranged from 3 to 106 months (median, 56 months). Patient and disease characteristics for both treatment cohorts, including the five clinical parameters that comprise the IPI, are listed in Table
Characteristics of DLBCL patients treated with CHOP or R-CHOP.
Characteristics | Total | CHOP | RCHOP | ||||||
---|---|---|---|---|---|---|---|---|---|
CHOP | RCHOP |
|
GCB | Non-GCB |
|
GCB | Non-GCB |
|
|
Sex | |||||||||
Male | 65 | 60 | 0.871 | 26 | 39 | 0.769 | 19 | 41 | 0.633 |
Female | 42 | 37 | 18 | 24 | 14 | 23 | |||
Age | |||||||||
Young | 77 | 58 | 0.066 | 32 | 45 | 0.833 | 21 | 37 | 0.579 |
Elder | 30 | 39 | 12 | 18 | 12 | 27 | |||
LDH | |||||||||
Normal | 66 | 56 | 0.565 | 28 | 38 | 0.728 | 23 | 33 | 0.086 |
High | 41 | 41 | 16 | 25 | 10 | 31 | |||
Stage | |||||||||
I | 46 | 18 | 20 | 26 | 9 | 9 | |||
II | 36 | 39 | 16 | 20 | 14 | 25 | |||
III | 18 | 26 | 6 | 12 | 4 | 22 | |||
IV | 7 | 14 | 2 | 5 | 6 | 8 | |||
Site | |||||||||
Nodal | 82 | 79 | 0.400 | 34 | 48 | 0.896 | 26 | 53 | 0.629 |
Extra nodal | 25 | 18 | 10 | 15 | 7 | 11 | |||
IPI | |||||||||
Normal | 82 | 55 | 0.002 | 39 | 43 | 0.014 | 23 | 32 | 0.063 |
High | 25 | 42 | 5 | 20 | 10 | 32 |
The expression patterns of CD10, BCL-6, and MUM1 among the patients treated with CHOP and R-CHOP are summarized in Tables
Results of the different immunohistochemistry staining in relation to overall survival in CHOP and R-CHOP subgroups.
Characteristics | Expression | CHOP ( |
R-CHOP ( |
||||
---|---|---|---|---|---|---|---|
|
OS |
|
|
OS |
|
||
CD10 | Negative | 71 | 47.9 | 0.297 | 67 | 61.5 | 0.139 |
Positive | 36 | 54.8 | 30 | 77.6 | |||
| |||||||
BCL-6 | Negative | 72 | 52.5 | 0.350 | 64 | 67.8 | 0.858 |
Positive | 35 | 49.2 | 33 | 64.9 | |||
| |||||||
MUM1 | Negative | 59 | 60.0 | 0.252 | 56 | 67.0 | 0.780 |
Positive | 48 | 40.2 | 41 | 65.8 | |||
| |||||||
Cell origin | GCB | 44 | 65.0 | 0.011 | 33 | 76.5 | 0.141 |
Non-GCB | 63 | 40.9 | 64 | 61.3 |
Cox proportional hazard regression analysis for CHOP and R-CHOP groups.
CHOP | R-CHOP | |||||||
---|---|---|---|---|---|---|---|---|
RR | 95% CI |
|
RR | 95% CI |
|
|||
Lower | Upper | Lower | Upper | |||||
OS | ||||||||
GCB versus non-GCB | 2.125 | 1.196 | 3.776 | 0.010 | 1.712 | 0.757 | 3.872 | 0.197 |
Sex (male versus female) | 2.057 | 1.192 | 3.550 | 0.010 | 0.550 | 0.240 | 1.260 | 0.158 |
Age (young versus elder) | 3.350 | 1.911 | 5.871 | 0.000 | 1.972 | 0.853 | 4.562 | 0.112 |
Stage (early versus advance) | 1.932 | 1.066 | 3.502 | 0.030 | 0.559 | 0.173 | 2.068 | 0.418 |
PFS | ||||||||
IPI (0-1 versus 2–5) | 1.156 | 0.438 | 3.051 | 0.770 | 2.408 | 1.187 | 4.883 | 0.015 |
GCB versus non-GCB | 2.463 | 1.385 | 4.382 | 0.003 | 1.664 | 0.739 | 3.747 | 0.219 |
Sex (male versus female) | 1.869 | 1.102 | 3.170 | 0.020 | 0.562 | 0.247 | 1.276 | 0.168 |
Age (young versus elder) | 3.464 | 1.994 | 6.017 | 0.000 | 2.473 | 1.223 | 5.002 | 0.012 |
RR indicates relative risk; CI: confidence interval; OS: overall survival; PFS: disease-free survival.
To evaluate the prognostic efficacy of the three factors, we performed survival analyses based on the individual markers alone and in combination. First, we evaluated the role of additional rituximab in DLBCL patients. We compared patient outcomes between the CHOP group and the R-CHOP group. A significant difference in outcome was observed between the two groups. According to the Kaplan-Meier estimates, the 5-year OS rates were 54.0% in the CHOP group and 66.6% in the R-CHOP group (
We demonstrated that subgrouping determined by the cell of origin on the basis of IHC successfully predicted the prognosis of DLBCL patients treated with the standard CHOP regimen. Clinical outcomes defined immunohistologically as GCB versus non-GCB subtypes are shown in Figures
Kaplan-Meier survival curves for all DLBCL patients treated with CHOP or R-CHOP. (a) OS of all DLBCL patients treat with CHOP (
To study the impact of rituximab on the predictive value of subclassification on the basis of the cell of origin, we examined the survival outcomes according to treatment in the GCB or non-GCB subgroups, as defined by IHC stains. Among the GCB subgroup, no significant difference was found in the 5-year OS of patients treated with CHOP and R-CHOP which were 66.5% and 76.5% (
Kaplan-Meier survival curves for GCB or non-GCB subgroup DLBCL patients treated with CHOP or R-CHOP. (a) OS of GCB subgroup treat with CHOP (
We also explored the prognostic significance of the IPI. We used IPI scoring system, instead of individual included factors in IPI, and subgrouped the patient into low risk (IPI score 0 and 1) and high risk (IPI score more then 2). In the CHOP group 34, 47, 21, 5, and 0 patients had an IPI score 0 to 4, respectively. The 5-year OS was 66.5%, 45.8%, and 30.0% for IPI scores 0, 1, and 2 (
By the Cox proportional hazards regression model, in the CHOP group, the IHC-defined GCB phenotypes and clinical characters such as males, young patients, and early stages were associated with a significantly favorable survival rate, independently of other IPI parameters. Whereas these factors were not independently significant prognostic factors in the R-CHOP group (Table
DLBCL is a heterogeneous disease, as the microarray analysis showed that patients with DLBCL expressing a gene expression profile (GEP) of germinal center B cells (GCB) have a longer survival than patients of activated B cells (ABC) [
The aim of our study was to identify whether cell-of-origin distinction has prognostic impact on DLBCL patients treated with combination of rituximab and chemotherapy. We confirmed that patients with DLBCL treated with CHOP alone can be stratified into low-risk and high-risk subgroups by cell of origin. In line with previous Western clinical studies [
Several studies have suggested that prognostic factors for patients with non-Hodgkin’s lymphoma have changed for those treated with rituximab, and patient characteristics may differ between those treated with chemotherapy or rituximab. Czuczman et al. analyzed the characteristics of 166 patients with non-Hodgkin’s lymphoma (including 130 patients with follicular lymphoma) in a phase III trial of rituximab; results showed that the Follicular Lymphoma International Prognostic Index did not correlate consistently with the response to rituximab or the response duration [
The mechanism is unknown but a chemosensitizing effect of the antibody was suggested in previous study [
With the gain insight of these molecular characteristics, many studies are ongoing to explore new treatments among the poor prognosis of IHC-defined non-GCB subtype DLBCL patients (NCT00931918, NCT00736450). These studies will assess the effectiveness of R-CHOP in combination with bortezomib and antisense BCL-2 antibody in previously untreated non-GCB DLBCL patients.
Although IHC algorithms and possibly other methods are promising tools for predicting cell of origin of DLBCL as part of routine pathologic diagnosis, improvements of these techniques are needed. In line with other studies, the main problem in our study was the poor methodological standardization due to nonuniform technology. Prospective studies would obtain more reliable information than retrospective studies. Our results demonstrate that prognostic factors based on the cell of origin correlate with significantly different OS rates in patients treated with CHOP; however, no difference is observed in the survival rates of patients treated with R-CHOP, which indicates that rituximab may improve the poor prognosis of patients with high-risk DLBCL. To properly reevaluate the existing prognostic factors, prospective studies using uniform technology and standardized methodology will be required.
Y. Huang, S. Ye, and Y. Cao contributed equally to this study.
The authors indicated that no actual or potential conflict of interests exists.
This work was supported by the Medical Science Foundation of Guangdong Province (B2009087) and the Guangdong Natural Science Foundation (S2011040004296).