Annals of Oncology Advance Access originally published online on July 22, 2008
Annals of Oncology 2008 19(11):1941-1946; doi:10.1093/annonc/mdn413
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hematologic malignancies |
A clinicopathological study of nm23-H1 expression in classical Hodgkin’s lymphoma
1 Department of Hematology, Comprehensive Cancer Center, International Medical Center, Saitama Medical University, Saitama
2 Adult Lymphoma Treatment Study Group, Nagoya, Japan
* Correspondence to: Dr N. Niitsu, Department of Hematology, Comprehensive Cancer Center, International Medical Center, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298, Japan. Tel: +81-42-984-4526; Fax: +81-42-984-4526; E-mail: nniitsu{at}saitama-med.ac.jp
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Abstract |
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Background: We carried out immunohistochemistry to examine the expression of nm23-H1 in Hodgkin and Reed–Sternberg cells in patients with classical Hodgkin’s lymphoma (CHL).
Patients and methods: We evaluated 128 patients with CHL [87 patients with nodular sclerosis (NS) and 41 patients with mixed cellularity (MC)] for CD15, CD20, Ki-67, EBER, TIA-1, and nm23-H1 by immunohistochemistry.
Results: CD15 was expressed in 79%, CD20 in 11%, Ki-67 in 93%, EBER in 34%, TIA-1 in 11%, and nm23-H1 in 60% of the CHL patients. NS patients showed a significantly higher rate of nm23-H1 expression than MC patients (P < 0.001). The serum nm23-H1 level was significantly higher in patients with positive nm23 expression. Univariate analysis showed that stage IV, poor performance status, low hemoglobin level, low serum albumin level, age of 45 years or older, TIA-1-positive status, and nm23-H1-positive status were associated with significantly shorter progression-free survival. Multivariate analysis with these factors showed TIA-1 and cytoplasmic nm23-H1 expression to be significant and independent prognostic factors.
Conclusions: Our results indicate that nm23-H1 expression is a prognostic factor for CHL and that it is as important as serum nm23-H1, both of which are useful for planning the treatment strategy.
Key words: classical Hodgkin's lymphoma, EBER, nm23-H1, TIA-1
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Approximately 7%–8% of all lymphoma patients in Japan have classical Hodgkin's lymphoma (CHL) [1]. On the other hand, in European and North American populations, CHL comprises 25% of all lymphoma patients. The treatment strategy of CHL is currently determined by the disease stage. The ABVD (doxorubicin, bleomycin, vindestine, and dacarbazine) regimen [2] is administered in the advanced stage of the disease. In recent years, the international prognostic score (IPS) was reported by Hasenclever and Diehl [3], and it would be possible to make a stratified treatment plan for high risk of CHL patient. However, there are few reports on biological prognostic factors of CHL, probably because CHL has a comparatively good prognosis compared with non-Hodgkin's lymphoma.
We previously established an enzyme-linked immunosorbent assay (ELISA) technique for determination of the serum level of nm23-H1 protein [4, 5] and reported that the serum level of nm23-H1 in patients with aggressive lymphoma was significantly higher than that in healthy controls and that a high nm23-H1 level was associated with poor prognosis in aggressive lymphoma [5]. In our previous study on cytoplasmic nm23-H1 expression in diffuse large B-cell lymphoma (DLBCL) by immunohistochemical staining, we found that patients with positive cytoplasmic staining had significantly poorer prognosis than patients with negative staining [6]. However, there are still many unanswered questions, including the question of whether serum nm23-H1 originates from lymphoma cells directly and whether there is a correlation between serum nm23-H1 and intracellular nm23-H1 expression by the tumor cells.
In the present study, we report that cytoplasmic nm23-H1 expression was high in patients with CHL and that the expression level was significantly correlated with the clinical outcome. These findings suggested that cytoplasmic nm23-H1 may be a valuable prognostic factor in patients with CHL.
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patients and methods |
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patients
We collected consecutive and untreated patients with Hodgkin’s lymphoma (HL) (n = 133) who were managed by the Adult Lymphoma Treatment Study Group (ALTSG) in Japan from 1997 to 2006. The lymphomas were classified according to the World Health Organization classification as defined elsewhere. Of the 133 patients with HL, 128 patients had CHL [87 had nodular sclerosis (NS) and 41 had mixed cellularity (MC)] and five had nodular lymphocyte-predominant (NLP) HL. Clinical staging was carried out according to the Ann Arbor classification system. The patients with HL were classified according to the IPS. The patients with CHL were treated with ABVD [2] or the BEACOPP (doxorubicin, cyclophosphamide, etoposide, bleomycin, vincristine, procarbazine, and prednisolone) regimen [7]. No patients receive radiation in order to avoid the toxicity of radiation and who could only receive chemotherapy. Pathologic evaluation of the materials from each patient was carried out at several central review meetings by six hematopathologists in the ALTSG Pathology Review Board. The median follow-up time was 74 months (range 16–124 months). All patients gave informed consent for both treatment and sample collection in accordance with institutional policy.
immunohistochemical staining
Formalin-fixed, paraffin-embedded sections were subjected to immunoperoxidase studies using the avidin–biotin peroxidase complex method. mAbs against CD3, CD15, CD20, CD30, Ki-67, TIA-1, and nm23-H1 (Seikagaku Corporation, Tokyo, Japan) were used. We counted 30 Hodgkin's and Reed–Sternberg (H–RS) cells and if 20 (67%) or more of the 30 cells were positive for nm23-H1, the specimen was considered to be positive for cytoplasmic nm23-H1. The presence of Epstein–Barr virus (EBV) small RNAs (EBER) was determined by in situ hybridization using EBV-encoded small nuclear early-region oligonucleotides on formalin-fixed, paraffin-embedded sections.
ELISA for determination of the serum nm23-H1 level
We previously established an ELISA procedure to determine the nm23-H1 protein level in the serum [5]. Briefly, 96-well plates (Corning Co., Corning, NY) were coated with a monoclonal anti-nm23-H1 antibody (Seikagakukougyo Co., Tokyo, Japan), washed four times with phosphate-buffered saline (PBS), and incubated with 25% Block Ace solution (Dainihon Seiyaku, Osaka, Japan). Serum samples were diluted two-fold with PBS and 50 µl aliquots were added to the wells. After incubation at room temperature for 1 h, the wells were washed four times with PBS containing 0.05% Tween 20 (T-PBS). Samples were then incubated at room temperature for 1 h with a polyclonal rabbit anti-nm23-H1 antibody (Santa Cruz Biotechnologies Inc., Santa Cruz, CA), washed four times with T-PBS, and incubated with alkaline phosphatase-conjugated anti-rabbit IgG (BioRad Lab, Richmond, CA). After four washes with T-PBS, alkaline phosphatase activity was detected with diethanolamine as a substrate and an alkaline phosphatase detection kit (BioRad Lab). The reaction was stopped with 50 µl of 0.4 N NaOH. Absorbance was measured at 405–415 nm with a correction wavelength of 620–630 nm using a microplate reader.
statistical analyses
Differences in characteristics between the two groups were examined by the 
2 test, Fisher's exact test, and Mann–Whitney U test, and P < 0.05 was taken to indicate significance. Complete response (CR) was defined as the absence of detectable disease on the basis of clinical, radiological, and histological criteria. Judgment criteria used for the analysis were progression-free survival (PFS) and overall survival (OS). Progression was defined as progression of the lymphoma in nonresponding patients and in partial response patients, a relapse in CR patients, or death from any cause without progression. PFS was calculated as the duration from randomization to the date of the first progression. Survival analysis was carried out according to the Kaplan–Meier method. The statistical significance of the differences in survival was determined by the log-rank test. Multivariate analysis of the prognosis was carried out using Cox proportional hazards regression model. All statistical analyses were carried out with SAS software (version 6.10, SAS Institute, Cary, NC).
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clinical characteristics according to histology
The characteristics of the patients with CHL (n = 128) are summarized in Table 1. There were 84 male and 44 female patients with a median age of 49 years (range 18–84 years). Histopathologically, they included 87 patients with NS (median age 42 years; male-to-female ratio 1.49) and 41 patients with MC (median age 57 years, male-to-female ratio 3.56). The patients with NS showed a significantly younger age at onset (P < 0.001) and higher ratio of females (P = 0.04). NS was significantly associated with high white blood cell count (P = 0.03) and presence of mediastinal mass (P = 0.02).
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immunophenotypic characteristics
Table 2 summarizes the results of immunohistochemical studies on the 133 specimens. Seventy-seven (60%) of 128 patients with CHL were positive for cytoplasmic nm23-H1 expression in H–RS cells (Figure 1A). The results of immunohistochemical staining for CD30 and TIA-1 in H–RS cells from a patient with NS are shown in Figures 1B and 1C, respectively. Regarding phenotypic features, there were significant differences in the positivity of H–RS cells for EBV and nm23-H1 between the NS and MC patients. EBV was harbored in 68% of the MC patients and in 18% of the NS patients, showing a significant difference (P < 0.001). Furthermore, nm23-H1 expression was observed in a significantly higher percentage of NS patients than MC patients (78% versus 32%, P < 0.001).
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relationship between nm23-H1 expression and clinical characteristics in CHL
The relationship between nm23-H1 expression and clinicopathological factors was investigated in the 128 patients with CHL (Table 3). nm23-H1 expression was significantly associated with high IPS (P = 0.003), presence of bulky mass (P = 0.005), and presence of B symptoms (P = 0.006). There were no significant associations between nm23-H1 expression and age, gender, serum lactate dehydrogenase level, stage, white blood cell count, serum albumin level, or hemoglobin level. The nm23-H1-positive rate among the patients who achieved CR was 57.3%, while the nm23-H1-positive rate among those who failed to do so was 90.1%, and the difference was statistically significant (P < 0.019). Thus, a close relationship between cytoplasmic nm23-H1 expression and therapeutic responsiveness was found.
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The serum nm23-H1 level in the 77 patients with cytoplasmic nm23-H1-positive CHL was 56.24 ± 27.52 ng/ml [mean ± standard deviation (SD)] and that in the 51 patients with cytoplasmic nm23-H1-negative CHL was 22.56 ± 15.45 ng/ml (mean ± SD). The difference was statistically significant (P = 0.0005) (Figure 2). Therefore, this result suggests that a portion of the serum nm23-H1 had been produced directly by lymphoma cells.
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survival in patients with CHL
Among all 128 CHL patients, the 5-year OS and PFS rates were 85.1% and 77.6%, respectively. There was no significant difference in PFS between the NS (n = 87) and MC (n = 41) patients (Figure 3A). The patients were divided into two groups according to the presence or absence of TIA-1 expression. The 5-year PFS rate of the TIA-1-positive and -negative groups was 41.7% (n = 14) and 83.0% (n = 114), respectively (P = 0.001) (Figure 3B). Thus, the TIA1-positive group showed a significantly poorer prognosis than the TIA1-negative group. When the patients were divided into the nm23-H1-positive and -negative groups, the 5-year PFS rate was significantly lower in the nm23-H1-positive group (63.4%) than in the nm23-H1-negative group (93.8%) (P = 0.037, Figure 3C). In the MC patients, the 5-year PFS rate of the nm23-H1-positive and -negative groups was 36.1% (n = 13) and 88.1% (n = 28), respectively (P = 0.006) (Figure 3D). In the NS patients, the 5-year PFS rate of the nm23-H1-positive and -negative groups was 68.4% (n = 64) and 86.1% (n = 23), respectively (P = 0.041) (data not shown). We also compared the PFS of patients who had a high or low level of serum nm23-H1 (cut-off value of 80 ng/ml). The 5-year PFS rates of the high nm23-H1 (
80, n = 31) and low nm23-H1 groups (<80, n = 97) were 49.8% and 94.1%, respectively (P = 0.0001) (data not shown). The 5-year PFS of the EBV-positive group (n = 40) was 73.9% and that of the EBV-negative group (n = 88) was 80.6%, showing no significant difference (Figure 3E). As for IPS, the 5-year PFS of the IPS 4 group (n = 21) was 48.4% and that of the IPS 0–3 group (n = 107) was 87.9%, indicating that the high-risk group showed significantly poorer prognosis (P = 0.002, Figure 3F).
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univariate and multivariate analyses of PFS in patients with CHL
Among the patients with CHL, the PFS was significantly worse in patients with the following characteristics: stage IV, poor performance status, low hemoglobin level (<10.5 g/dl), low serum albumin level (<4.0 g/dl), age of 45 years or older, positive TIA-1 expression and positive nm23-H1 expression in H–RS cells (Figure 3). Multivariate analysis with these individual factors showed TIA-1 [hazards ratio 5.09, 95% confidence interval (CI), 1.85–7.25; P = 0.0001] and nm23-H1 expression (hazards ratio 3.31, 95% CI 1.23–5.87; P = 0.0032) to be significant and independent prognostic factors among the 128 CHL patients (Table 4).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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CHL is characterized by heterogeneous clinical, histological, and epidemiological features. NS is the predominant histological subtype. NS is predominant among young adults with CHL, whereas MC is relatively more frequent in children and older adults. In our study, the clinicopathological features and age distribution of the CHL cases in Japan were similar to the patterns described in Western countries.
Recently, therapies for CHL have improved, at least partly because the choices of treatment have broadened to include radiotherapy, the BEACOPP regimen [7], Stanford V regimen [8], and high-dose chemotherapy combined with peripheral blood stem-cell transplantation. However, it is important to be able to predict the prognosis and make a stratified treatment plan that attains a long remission period with the initial treatment. In other words, it is important to choose a therapy that is adequate without causing secondary cancers, infertility, and organ failure such as cardiopulmonary toxicity in long-term survivors. The treatment strategy for CHL at present is generally determined by the stage and IPS. By using the IPS, a fairly accurate prognostication could be made and, hence, it would be possible to make a stratified treatment plan for each patient. However, IPS is a marker based primarily on clinical findings and laboratory data. Recently, prognostic factors for CHL based on the biological characteristics of tumor cells have been identified. Asano et al. [9] identified that expression of cytotoxic antigens in H–RS cells may represent a poor prognostic factor in CHL. TIA-1 is a 15-kD cytotoxic granule-associated protein that is structurally related to the tumor necrosis factor receptor family; TIA-1 induced apoptotic cell death when introduced into permeabilized target cells [10]. Felgar et al. [11] evaluated TIA-1 expression in lymphoid neoplasms by immunohistochemistry of paraffin sections and reported that 64% of T- or NK-cell neoplasms and 13% of CHLs were positive for TIA-1. On the other hand, all B-cell neoplasms and NLP HL were negative for TIA-1 [11]. The results of our previous study are in agreement with this consensus, with TIA-1 expression in 78.8% of T- or NK-cell lymphomas [12]. In the present study, TIA-1 was expressed in H–RS cells in 11% of CHLs, and the TIA-1-positive group showed a significantly poorer prognosis in terms of PFS.
EBV is detected in some HL tumor cells. In North America and Western Europe, 30%–50% of the tumors of HL patients are EBV positive. Asano et al. [9] reported that EBV was detected in H–RS cells in 47% of the patients with CHL, with no association seen with histopathologic group. In the present study, EBV-positive H–RS cells were harbored in 68% of the MC patients and 18% of the NS patients, showing a significant difference. Several studies have suggested that the presence or absence of this virus has no association with OS, while others have suggested an association in very young and in very old patients [9, 13, 14] Our present results are in agreement with these findings. These studies have not defined particular modalities of therapy that are more or less likely to be effective in the treatment of virus-associated tumors.
The first nm23 gene was isolated on the basis of its reduced expression in highly metastatic murine melanoma cell lines, as compared with nonmetastatic ones, and it was subsequently classified as a metastatic suppressor gene [15]. Subsequently, high homology between the nm23 proteins and nucleoside diphosphate kinases has been identified in a number of species [16]. We previously established an ELISA technique to determine the serum level of nm23-H1 protein [4]. The nm23 genes play critical roles in cellular proliferation, differentiation, oncogenesis, and tumor metastasis. The mechanisms responsible for these pleiomorphic effects are not well understood. We previously reported that the serum nm23-H1 level in patients with aggressive lymphoma was significantly higher than that in healthy controls and that a high nm23-H1 level was associated with poor prognosis in patients with aggressive lymphoma [4, 5]. We examined cytoplasmic nm23-H1 expression in DLBCLs [6] and peripheral T-cell lymphomas [12] and found that the serum and cytoplasmic nm23-H1 levels were significant prognostic factors in both DLBCLs and peripheral T-cell lymphomas. In the present study, cytoplasmic nm23-H1 expression in H–RS cells was analyzed in 128 patients with CHL, and the relationships between cytoplasmic nm23-H1 expression and clinical and laboratory characteristics and patients’ prognosis were studied. Consequently, it was found that nm23-H1 was frequently expressed in CHL, and NS patients showed a high rate of nm23-H1 expression. Regarding the relationship between clinical characteristics of CHL and nm23-H1 expression, high IPS, presence of bulky mass, presence of B symptoms, and non-CR status were significantly associated with nm23-H1 expression. Since there were no clear correlations between nm23-H1 expression and the seven prognostic factors of the IPS, the above correlations seem to be related to the tumor volume and treatment responsiveness rather than spread of the lymphoma. Next, we examined the relationship between nm23-H1 expression and PFS. Patients with cytoplasmic nm23-H1-positive CHL had significantly shorter PFS than those with nm23-H1-negative CHL. We found a significant difference in PFS between the nm23-H1-positive CHL and nm23-H1-negative CHL groups, especially in the MC group. Multivariate analysis also suggested that cytoplasmic nm23-H1 may be a significant prognostic factor in CHL. We previously reported that the serum nm23-H1 level, the measurement of which requires only a small amount of serum, can be used as a prognostic factor for lymphoma. The present study documents that the patient group with nm23-H1 protein expression, which had been determined by immunohistochemistry, had a poor prognosis, and multivariate analysis demonstrated that nm23-H1 expression was an independent prognostic factor in CHL. Cytoplasmic nm23-H1 can be more easily examined at the time of conventional phenotypic examinations for diagnosis of HL, but quantitative evaluation of immunohistochemical staining is difficult. Thus, the methods of determination of the serum nm23-H1 level and cytoplasmic nm23-H1 expression have their own merits and demerits.
In conclusion, we demonstrated that patients with CHL with nm23-H1 expression in H–RS cells have a significantly poorer prognosis than patients without nm23-H1 expression. Examination of nm23-H1 expression in H–RS cells in CHL patients is recommended as a routine pathologic practice. In addition, it seemed that patients with high cytoplasmic nm23-H1 expression in H–RS cells require a more aggressive treatment strategy.
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Ministry of Health and Welfare and Grants-in-Aid for Scientific Research (C) and Cancer Research from The Ministry of Education, Culture, Sports, Science, and Technology of Japan (17591008).
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Acknowledgements |
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The authors thank Dr S Nakamura, Dr Oshima, Dr N Nakamura, and Dr Yoshino for performing pathological diagnosis.
Received for publication April 23, 2008. Revision received June 2, 2008. Accepted for publication June 5, 2008.
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