Rituximab with chemotherapy improves survival of non-germinal center type untreated diffuse large B-cell lymphoma

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Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disorder that can be subdivided into several subtypes based on DNA microarray-based gene expression profiling: germinal center B-cell-like (GC) DLBCL and activated B-cell-like (ABC) DLBCL. Patients with GC DLBCL have been shown to exhibit a more favorable clinical outcome than patients with non-GC DLBCL.1 As with cDNA microarray, immunohistochemistry can also be used to distinguish the GC/non-GC subtype of DLBCL and to predict survival,2 but there have been few reports comparing clinical outcomes in cases of GC DLBCL and non-GC DLBCL treated with rituximab.3, 4 We retrospectively analyzed the treatment effect of rituximab with conventional chemotherapy on these two DLBCL subtypes by means of immunohistochemistry.

We analyzed patients with untreated DLBCL who were diagnosed according to the WHO classification.5 Patients who were diagnosed in our hospital between 2001 and 2005 were treated with rituximab plus TCOP (R-TCOP) (cyclophosphamide, therarubicin, vincristine and prednisolone), and those who were diagnosed in our hospital between 1997 and 2002 were treated with TCOP alone. R-TCOP consisted of a combination of rituximab (375 mg/m2 administered on day 1 of each chemotherapy cycle) and TCOP (cyclophosphamide 750, therarubicin 50 and vincristine 1.4 mg/m2 administered on day 2 and prednisolone 100 mg/m2 administered on days 2–6). TCOP consisted of TCOP alone. We reduced the chemotherapeutic dosage to two out of three patients who were more than 65 years of age. Tumor response was assessed at the end of treatment according to the International Workshop criteria.6

Immunohistochemical staining was performed using heat-induced antigen retrieval, as described previously.7, 8 Details of the immunohistochemical staining are provided in Table 1. The scoring was based on the algorithm described by Hans et al.2 Bcl-6, CD10 and melanoma associated antigen (mutated) 1 (MUM1) were considered positive if 30% or more of tumor cells were stained with the respective antibody, and bcl-2 was considered positive if 30% or more tumor cells were stained. Given that bcl-6 and CD10 are markers of GC B cells, a case was considered to be a case of GC DLBCL if CD10 alone was positive or if both bcl-6 and CD10 were positive. If both bcl-6 and CD10 were negative, the case was considered to be a case of non-GC DLBCL. If bcl-6 was positive and CD10 was negative, the expression of MUM1 was used to determine the group, that is if MUM1 was negative, the case was considered to be a case of GC DLBCL, and if MUM1 was positive, the case was considered to be a case of non-GC DLBCL.

Table 1 Antibodies used to stain formalin-fixed, paraffin-embedded sections

The results of immunohistochemical staining and response rates were compared between groups using χ2 test and Fisher's exact test. Event-free survival (EFS) was calculated from the time of diagnosis to the date of disease progression, death or last contact. Overall survival (OS) was calculated as the time from diagnosis to the date of death or last contact. Survival curves were constructed using the Kaplan–Meier method and compared by log-rank test. Differences were considered significant at P<0.05. A general-purpose statistical software package, StatFlex Version 5.0 (Artech Co., Osaka, Japan), which provides results compatible with SPSS, was used for the data analyses.

The R-TCOP group comprises 38 patients. The median follow-up time for surviving patients in this group was 13 months. The TCOP group comprises 31 patients. The median follow-up time for surviving patients in this group was 25 months. Of the 69 cases in total, 26 (38%) and 43 (62%) were considered cases of GC DLBCL and non-GC DLBCL, respectively, on the basis of immunohistochemical analysis. In the R-TCOP group, 18 (48%) and 20 (52%) cases were classified as cases of GC DLBCL and non-GC DLBCL, respectively. In the TCOP group, eight (26%) and 23 (74%) cases were classified as cases of GC DLBCL and non-GC DLBCL, respectively. Bcl-2 was expressed in 63% of cases in the R-TCOP group, that is in 61% of GC DLBCL and 65% of non-GC DLBCL. Bcl-2 was expressed in 53% of cases treated with TCOP alone, that is in 50% of GC DLBCLs and in 70% of non-GC-DLBCLs. The patient characteristics (age, sex, performance status, Ann Arbor clinical stage, B symptoms, bone marrow involvement, extranodal disease, International Prognostic Index (IPI) and lactate dehydrogenase (LDH)) did not differ significantly between groups (that is, between patients grouped by treatment or by DLBCL subtype) (Table 2).

Table 2 Patient characteristics according to GC or nGC and treatment (total, n=69)

The overall response to treatment was 92% for patients treated with R-TCOP and 58% for those treated with TCOP alone. In the R-TCOP group, there was no significant difference in the response to treatment between patients with GC DLBCL and those with non-GC DLBCL (P=0.5). However, in the TCOP group, there was a significant difference in the response to treatment between patients with GC DLBCL and those with non-GC DLBCL (P<0.01). Among patients with non-GC DLBCL, the response to treatment was significantly greater for those treated with R-TCOP than for those treated with TCOP alone (P<0.01); however, among patients with GC DLBCL, no significant difference was observed in response to the two treatments (P=0.6). Two-year OS and EFS were significantly longer for patients treated with R-TCOP than for those treated with TCOP alone. With TCOP, the 2-year OS of patients with GC DLBCL was superior to that of patients with non-GC DLBCL. With R-TCOP, no significant differences in 2-year OS and EFS were observed between patients with GC and those with non-GC DLBCL. In GC DLBCL cases, 2-year OS and EFS were not significantly different between treatments, but in non-GC DLBCL cases, the 2-year OS and EFS were significantly longer for patients treated with R-TCOP than for those treated with TCOP alone (Table 3, Figure 1). Recently, similar data regarding the loss of prognostic value for the distinction of the origin of immunohistochemically defined cells has been reported in patients treated with rituximab plus CHOP.3

Table 3 Patient characteristics and univarate analysis of clinical outcomes per group (total, n=69)
Figure 1
figure1

OS curves according to subtype and treatment. OS, overall survival.

Furthermore, in the present study, in the low IPI (low and low-intermediate risk) and non-GC cases, 2-year OS and EFS did not differ significantly between treatments. However, in high IPI (high and high-intermediate risk) and non-GC cases, 2-year OS and EFS were significantly longer for patients treated with R-TCOP than for those treated with TCOP alone (OS: P<0.01; EFS: P<0.01). Hans et al.2 reported that non-GC DLBCL patients with a high IPI had a particularly poor outcome, with a median OS of only 1 year, when treated with non-rituximab regimens. In our study, non-GC DLBCL patients with a high IPI who were treated with R-TCOP exhibited significantly better outcomes compared to those patients treated with TCOP alone. Our results suggest that the improved overall outcome obtained by R-TCOP treatment was due primarily to the beneficial effect of rituximab combined with chemotherapy in cases of non-GC DLBCL, especially for patients with a high IPI.

Bcl-2 was expressed in 63% of cases in the R-TCOP group; that is, in 61% of GC DLBCL patients and 65% of non-GC DLBCL patients. Bcl-2 was expressed in 53% of cases treated by TCOP alone, that is, in 50% of GC DLBCLs and in 70% of non-GC DLBCLs. There were no differences in 2-year OS between bcl-2-positive and bcl-2-negative cases (TCOP, P=0.5; R-TCOP, P=0.4). In a recent study, expression of bcl-2 in cases of GC DLBCL did not predict OS or EFS, but cases of non-GC DLBCL that expressed bcl-2 exhibited significantly worse OS and EFS.2 In the present study, bcl-2 expression itself had no prognostic effect on OS and did not predict OS in relation to GC or non-GC DLBCL or in relation to R-TCOP or TCOP treatment.

Non-GC DLBCL includes ABC DLBCL. ABC DLBCL is characterized by activation of nuclear factor-κB (NF-κB) target genes, including those that encode interferon regulatory factor 4 (IRF4/MUM1), cell adhesion molecule CD44, anti-apoptotic genes, such as bcl-2 and bcl-xl, and the cell cycle-associated gene, cyclin D2. Genes in the NF-κB pathway are overexpressed in ABC-like DLBCL cell lines.1, 9 Jazirehi et al.10 reported that rituximab inhibits the constitutive NF-κB signaling pathway in selected non-Hodgkin's lymphoma B-cell lines, leading to increased sensitivity to chemotherapy. Moreover, Liu et al.4 reported that PRDM1β gene expression correlated with a short survival time in non-GC patients treated with CHOP, but not in those treated with rituximab plus CHOP, and that rituximab suppressed PRDM1β expression, concomitant with NF-κB inactivation. Rituximab inhibits the constitutive NF-κB signaling pathway in non-Hodgkin's lymphoma cell lines; therefore, we believe our results point to the clinical importance of NF-κB as a therapeutic target for DLBCL.

The present study confirms that improved overall outcome with R-TCOP may be due primarily to the beneficial effect of the addition of rituximab to chemotherapy in cases of non-GC DLBCL. Our findings support the notion that apoptosis and inhibition of cell proliferation, including NF-κB, play important roles in the therapeutic effect of rituximab.

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Saito, B., Shiozawa, E., Usui, T. et al. Rituximab with chemotherapy improves survival of non-germinal center type untreated diffuse large B-cell lymphoma. Leukemia 21, 2563–2566 (2007) doi:10.1038/sj.leu.2404844

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