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Journal of Clinical Oncology, Vol 23, No 31 (November 1), 2005: pp. 8012-8017 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.02.1626 Prognostic Significance of BACH2 Expression in Diffuse Large B-Cell Lymphoma: A Study of the Osaka Lymphoma Study GroupFrom the Departments of Pathology, Hematology and Oncology, and Functional Diagnostic Science, Osaka University Graduate School of Medicine; 1st Department of Internal Medicine, Kansai Medical University; Department of Clinical Hematology and Clinical Diagnostics, Osaka City University; Department of Hematology, Kinki University School of Medicine; Department of Clinical Laboratory, Osaka Minami Medical Center; Department of Hematology, Sakai Municipal Hospital, Osaka; and Department of Biomedical Chemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan Address reprint requests to Katsuyuki Aozasa, MD, Department of Pathology (C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; e-mail: aozasa{at}molpath.med.osaka-u.ac.jp
PURPOSE: BACH2, a B-cell–specific transcription repressor, is abundantly expressed in lymphocytes of B-cell lineage as well as B-cell lymphoma cell lines. BACH2 possesses an inhibitory effect on proliferation of Raji cell lines derived from Burkitt's lymphoma. In this study, the prognostic significance of BACH2 expression was examined in diffuse large B-cell lymphoma (DLBCL). PATIENTS AND METHODS: BACH2 expression was immunohistochemically examined on the paraffin-embedded sections obtained by biopsy from 108 patients (62 males and 46 females; age range, 23 to 85 years) with DLBCL. Staining intensity in the cytoplasm of the tumor cells was categorized as equal to or stronger (level 1) or weaker (level 2) than that in the endothelial cells in the same specimens. RESULTS: Level 1 and 2 expression of BACH2 was found in 32.4% and 67.6% of patients, respectively. Patients with level 1 expression showed significantly better disease-free and overall survival rate than those with level 2 expression (both P < .05). Multivariate analysis revealed BACH2 expression level together with performance status, elevated serum level of lactate dehydrogenase, and treatment response to be independent factors for prognosis of the patients. CONCLUSION: BACH2 expression level is a useful marker to predict disease-free and overall survival of patients with DLBCL.
The incidence rate of malignant lymphomas in Japan has increased from 1975 to 1995 (ie, 1.5 times for males and 1.66 for females).[1] The annual incidence rate of malignant lymphomas in Japan was 8.7 for males and 5.2 for females per 100,000 population in 1998, and the mortality rate was 6.17% in 2001.[2] In the new WHO classification for lymphoid neoplasms proposed in 1995,[3] diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide, including Japan. Because DLBCL comprises heterogenous groups of diseases and shows a wide range of response patterns to treatment, additional patient characterization is essential to establish effective modalities of treatment. DLBCL is categorized as one of the aggressive non-Hodgkin's lymphomas (NHLs). At present, the International Prognostic Index (IPI) is most widely used for prediction of outcome in patients with aggressive NHL.[4] It incorporates patient age, performance status, serum lactate dehydrogenase (LDH), clinical stage, and number of extranodal lesions, but does not include immunophenotype and pattern of gene expression. Recently, analyses of expression profiles of a large number of genes have been used for understanding in detail the mechanism of lymphomagenesis and lymphoma progression. cDNA microarrays have been used to investigate gene expression profiles in human cancers. On the basis of the gene expression profiles, DLBCL could be categorized into germinal center and activated B-cell signatures[5]: the latter show poorer prognosis than the former. Expression levels of proto-oncogenes Bcl6 and Bcl2 were reported to be correlated with survival of patients with DLBCL.[6,7] BACH2 encodes a human B-cell–specific transcription factor and is mapped to chromosome 6q15.[8] BACH2 expression is transcriptionally regulated by the Bcr/Abl oncogene, and this regulator is impaired in chronic myelogenous leukemia.[9] BACH2 induces apoptosis in response to oxidative stress, thus BACH2 could be regarded as a candidate tumor suppressor gene.[10] Sasaki et al[8] previously reported the loss of BACH2 expression in the Burkitt's lymphoma–derived cell line, Raji, and showed that enforced BACH2 expression resulted in marked reduction of clonogenic activity. From these findings, they suggested that the loss of BACH2 expression might contribute to B-cell lymphomagenesis. Recently, we showed the integration of Epstein-Barr virus genome into the intron 1 of BACH2 gene located on chromosome 6q15.[11] This might result in loss of BACH2 expression in the Raji cells. In this study, prognostic significance of BACH2 expression was examined immunohistochemically in 108 patients with DLBCL, and its correlation with prognosis of patients was evaluated.
Patients From November 1, 1999 to August 31, 2003, 1,383 patients with lymphoproliferative diseases were registered with the Osaka Lymphoma Study Group, which was established by main hospitals and medical institutes in Osaka, Japan, in 1999. Among these patients, 1,175 patients were diagnosed with malignant lymphomas, of whom 1,066 (90.7%) were NHL and 109 (9.3%) were Hodgkin's lymphoma. Four hundred ninety patients had DLBCL. One hundred eight patients with DLBCL, for whom adequate clinical information was available, were selected for the present study. There were 62 males and 46 females, with age ranging from 23 to 85 years (median age, 63 years). The primary site of tumors was lymph node in 68 patients and extranodal organs (six stomach, four pharynx, four testis, three paranasal sinuses, three breast, three spleen, three ileum, two thyroid gland, two liver, two bone marrow, two tonsil, two skin, one kidney, one CNS, one parotid gland, and one tongue) in 40 patients. Histologic specimens obtained by biopsy were fixed in 10% formalin, and routinely processed for paraffin embedding. Histologic sections cut at 4 µm were stained with hematoxylin and eosin, and immunoperoxidase procedures were used. All of the histologic sections were reviewed by one of the authors (K.A.), and classified according to the WHO classification. Adequate clinical findings were available for all patients. On the basis of the records of physical examinations, surgical notes, and pathologic examinations of the specimens, the Ann Arbor staging system was applied in all 108 patients: stage I in 28 patients, stage II in 34 patients, stage III in 16 patients, and stage IV in 30 patients. The IPI score was calculated with four adverse factors (age > 60 years, Ann Arbor stage III and IV, Eastern Cooperative Oncology Group performance score 2 to 4, and serum LDH elevation) present at the time of diagnosis.[4] All patients had received anthracycline-based chemotherapy, mostly a regimen including doxorubicin, cyclophosphamide, vincristine, and prednisone or similar regimens. Clinical outcome was evaluated according to the guidelines of the International Workshop to standardize response criteria for NHL.[12] The patients were observed until December 31, 2004; the follow-up periods for survivors ranged from 0 to 108 months (median, 13 months). Seventy-four patients were alive at the end of the observation period.
Immunohistochemical Analysis
Quantitative Reverse Transcription Polymerase Chain Reaction for BACH2 Briefly, 10 µg of Dnase I-treated total RNA was used for reverse transcription with Superscript III (Invitrogen). An aliquot representing 100 ng of input RNA was amplified by quantitative real-time polymerase chain reaction (PCR) using the TaqMan PCR Reagent Kit and Assay-on-Demand Gene Expression Products (Applied Biosystems, Foster City, CA).[15,16] RNA extracted from chronic tonsillitis was used as a standard. After reverse transcription, standard cDNA was diluted serially to obtain five standard solutions for use in PCR to generate the reference curve. The relative amount of cDNA in each sample was measured by interpolation in the standard curve,[16] then the relative ratio of BACH2/ß-actin expression was calculated for each DLBCL sample.
Statistics
The presenting characteristics of the 108 patients with DLBCL are summarized in [Table 1]. Complete response for the chemotherapy was obtained in 62% of the patients. Five-year overall survival rate in the 108 patients was 51.7%.
BACH2 Expression Thirty-five (32.4%) and 73 (67.6%) of 108 patients were regarded as having level 1 and level 2 of BACH2 expression, respectively ([Fig 1]). Quantitative RT-PCR was performed in 14 patients with level 1 and six patients with level 2 expression. The relative ratio of BACH2/ß-actin expression in patients with level 1 and level 2 expression was 18.9 ± 7.9 and 1.6 ± 5.6 (mean ± standard deviation), respectively. The mRNA expression level was higher in level 1 patients than in level 2 patients, but the difference was not statistically significant (P = .1284). Raji cells did not express BACH2, which was consistent with the previous report.[8] The MIB-1 labeling index was 53 ± 30 and 52 ± 31 (mean ± standard deviation) in level 1 and level 2 patients, respectively. There was no significant difference between two groups of patients.
Uni- and Multivariate Analyses for Prognostic Factors Correlation between BACH2 expression and clinicopathologic factors is summarized in [Table 1]. There was no significant correlation between BACH2 expression level and other clinicopathologic factors. Univariate analysis revealed that the patients with level 1 expression showed a better 5-year survival rate than those with level 2 expression (70.2% v 45.3%; P < .05). Ann Arbor stage, extranodal involvement, size of largest tumor, performance status, serum LDH level, IPI, and treatment response were also significant for overall survival ([Table 2]; [Fig 2]). Multivariate analysis was performed with the factors proven to be significant in the univariate analysis: BACH2 expression, performance status, serum LDH level, and treatment response were independent prognostic factors for overall survival ([Table 3]).
Prognostic Significance of BACH2 Expression in the IPI Among IPI intermediate- and high-risk groups with number of risk factors 2 to 3 and 4 to 5, respectively, patients with level 1 expression showed a more favorable prognosis than those with level 2: P < .05 for intermediate- and P < .01 for high-risk group ([Fig 3]).
This series consisted of a group with a higher number of patients older than 60 years of age and early-stage disease (I and II) than those in the literature[4,19] (ie, 62% v 41% to 55% and 57% v 34% to 45%). Regarding histologic type, centroblastic type predominated immunoblastic type in the present and the previous report.[19] The 5-year survival rate in the present and previous series was similar (ie, 51.7% and 52%, respectively). Given that it is evident that first-, second-, and third-generation chemotherapeutic regimens for unselected patients with aggressive NHL show comparable outcomes,[20-22] the variety of regimens containing doxorubicin as used in this study is unlikely to have influenced the analysis. DLBCL comprises heterogenous groups of diseases and shows a wide range of response patterns to chemotherapy. Since new anticancer drugs and combination therapies for lymphomas have been developed, it has become more important to identify high-risk patients who are resistant to the conventional therapy and need such new treatment modalities. IPI was initially developed as a prognostic model incorporating widely recognized clinical features, but proved to be not as effective in stratifying patients with DLBCL for therapeutic trials.[23] Previous immunohistochemical study using polyclonal antihuman BACH2 antibody (F69-2[13]) raised in rabbit showed the successful detection of BACH2 expression in BACH2-overexpressing Raji cell clones at the protein level. Thus, this antibody was used on the paraffin-embedded sections from DLBCL, and gave the clear staining results as shown in [Figure 1]. BACH2 possesses one nuclear localization signal and one cytoplasmic localization signal (CLS). BACH2 is localized mainly in the cytoplasm through its C-terminal evolutionarily conserved CLS.[13,24] Oxidative stress induces the nuclear accumulation of BACH 2 by inhibiting the CLS activity. The CLS might be active in lymphoma cells, and thus show cytoplasmic staining for BACH2. Positive correlation of BACH2 expression between mRNA and protein level was found, but statistical significance was not found. There might be at least three possible reasons for this result: the presence of substantial amounts of non-neoplastic cells concomitant with tumor cells; the efficacy of mRNA translation into protein might be different from patient to patient; and stability of BACH2 might be different in lymphoid cells because of ubiquitination of BACH2.[25] The aim of this study was to evaluate whether BACH2 expression level, in combination with the IPI, could be used for risk stratification of DLBCL. Tumor cells in approximately 30% of patients showed a relatively strong staining (level 1), and these patients showed a more favorable prognosis than those with level 2 expression. Both the BACH2 expression of tumor cells and IPI were the independent predictors for survival (P < .05). Furthermore, patients with intermediate and high risk according to the IPI could be further stratified into favorable and unfavorable prognosis groups based on BACH2 expression. BACH2 expression is restricted to B cells among hematopoietic cells.[8,10] BACH2-expressing cells show reduced proliferation and undergo spontaneous cell death.[10,13] Kamio et al[13] reported that BACH2 modifies the in vitro cytotoxicity of anticancer drugs (including doxorubicin, which is commonly used in chemotherapy for DLBCL) by promoting the oxidative stress-induced cell death. Raji cell clones overexpressing BACH2 were more sensitive to etoposide, doxorubicin, and cytarabine than control Raji cells. These findings provide a rationale why patients with DLBCL expressing the high level of BACH2 show the more favorable prognosis than those with lower expression level. The chemotherapeutic agents used in the present patients included doxorubicin. Frequency of complete remission at initial chemotherapy was 74.2% in patients with BACH2 level 1 expression and 56.2% in patients with level 2 expression (P = .0649). In conclusion, BACH2 expression could be determined on routinely processed, paraffin-embedded samples, thus could be widely used for prognostic stratification of DLBCL cases.
The authors indicated no potential conflicts of interest.
We thank Yoichi Tani for technical advice.
Supported by Grants No. 15406013 and 16390105 from the Ministry of Education, Science, Culture and Sports, Japan. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test and Fisher's exact probability test. Overall survival rates were calculated by the Kaplan-Meier method,[
