Leukemia (2003) 17, 196–202 2003 Nature Publishing Group All rights reserved 0887-6924/03 $25.00 www.nature.com/leu Clinical significance of nm23-H1 proteins expressed on cell surface in non-Hodgkin’s lymphoma N Niitsu1, Y Honma2, K Iijima3, T Takagi4, M Higashihara1, U Sawada5 and J Okabe-Kado2 1Department of Hematology and Internal Medicine IV, Kitasato University School of Medicine, Kanagawa, Japan; 2Saitama Cancer Center Research Institute, Saitama, Japan; 3First Department of Internal Medicine, Toho University School of Medicine, Tokyo, Japan; 4Hematology- Oncology Division, Chiba, Cancer Center Hospital, Chiba, Japan; and 5First Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan The nm23 gene was isolated as a metastasis suppressor gene in both aggressive NHL and AML.7–9 In a collaborative study that exhibits low expression in high-level metastatic cancer between many institutions involving a large number of cells. Its gene is related to the prognosis of acute myelogenous leukemia (AML) and non-Hodgkin’s lymphoma (NHL). In this patients, we found that serum nm23-H1 was an important study, we examined the expression of nm23-H1 protein on the independent prognostic factor in NHL for diffuse large B cell lymphoma cell surface of NHL. In 28 of 108 cases (25.9%), we lymphoma (DLBCL) and peripheral T cell lymphoma (PTCL).8 observed Ն20% of cell surface nm23-H1 protein expression We also examined four distinct risk groups defined by the and expression was especially high in peripheral T cell lym- widely used clinical international prognostic index (IPI) and phomas and extranodal NK/T cell lymphomas. We also demonstrated that when serum nm23-H1 protein levels are observed a significant correlation between serum nm23-H1 level and cell surface nm23-H1 expression levels. In patients high, prognosis is poor in all four risk groups. Given these with high levels of cell surface nm23-H1 expression, overall and results, measurement of nm23-H1 protein may be useful in progression-free survival rates were significantly lower than the treatment method selection process. Whether such serum those in patients with low surface nm23-H1 expression levels. nm23-H1 protein is directly produced by lymphoma cells is When surface nm23-H1 and serum nm23-H1 were combined, unclear, but its production appears to reflect the prognosis of patients with high levels of both exhibited a poorer prognosis lymphoma. In this study, to examine the clinical significance than patients with a high level of one or the other. These results indicate that in addition to serum nm23-H1, cell surface nm23- of nm23-H1 protein on the surface of lymphoma cells, we H1 may be used as a prognostic factor in planning a treatment studied the relationships of surface nm23-H1 to serum nm23- strategy. The nm23-H1 protein appears to be intimately related H1 and other cell surface markers by flow cytometry measure- to biological aggressiveness of lymphoma and, therefore, ment and tracked the subsequent survival rates. might be a molecular target of NHL treatment. Leukemia (2003) 17, 196–202. doi:10.1038/sj.leu.2402699 Keywords: nm23-H1 proteins; cell surface; non-Hodgkin’s lym- phoma; prognostic factor Materials and methods Patients Introduction We measured cell surface nm23-H1 expression from February 1999 to March 2000 in a consecutive series of 108 patients The first nm23 gene was isolated on the basis of its reduced with untreated NHL (62 men and 46 women; median age, 68 expression in highly metastatic murine melanoma cell lines, years; age range, 22–78 years) whose clinical course was as compared with their nonmetastatic counterparts, and has being managed by the lymphoma treatment study group in therefore been proposed as a metastatic suppressor gene.1 Japan. The cases were classified morphologically according Subsequently, high homology between nm23 proteins and to the REAL schema10 as defined elsewhere with or without nucleoside diphosphate kinases (NDPK) has been identified in adjunctive immunohistochemical and molecular studies. Of a number of species.2 We found that a differentiation inhibi- these 108 patients with NHL, 79 presented with B cell neo- tory factor purified from conditioned medium of a differen- plasms and 29 with T cell neoplasms. Of the 79 patients with tiation-resistant mouse myeloid leukemia cell line was ident- B cell neoplasms, three had B cell chronic lymphocytic leuke- ical to the nm23 protein.3,4 In humans, eight nm23 isotypes mia (CLL), three had mucosa-associated lymphoid tissue (nm23-H1, nm23-H2, DR-nm23, nm23-H4, nm23-H5, nm23- (MALT), five had mantle cell lymphoma (MCL), 13 had follicu- H6, nm23-H7 and nm23-H8) have been identified to date.5 lar lymphoma (FL), and 55 had DLBCL. Of the 29 patients Immunohistochemical studies have reported that patients with with T cell neoplasms, 18 had PTCL, two had anaplastic large high-grade non-Hodgkin’s lymphoma (NHL) and Hodgkin’s cell lymphoma (ALCL), six had extranodal NK/T cell lym- lymphoma exhibit significantly higher levels of nm23-H1 phoma (NK/T), and three had adult T cell leukemia/lymphoma expression than do those with low-grade NHL.6 (ATLL). Clinical staging was performed according to the Ann Recently, we established an enzyme-linked immunosorbent Arbor classification system.11 Evaluation included a complete assay (ELISA) technique to determine the serum level of nm23- history and physical examination, chest roentgenography, H1 protein. We previously reported that serum levels of bone marrow aspiration and biopsy, computed tomography nm23-H1 in aggressive NHL and acute myelogenous leuke- of the chest, abdomen and pelvis, hemogram and differential mia (AML) were significantly higher than those in controls, counts and routine biochemistry tests. Laparotomy was not and that high nm23-H1 levels correlated with poor prognosis performed for staging. The IPI was determined in all NHL cases.12 Patients with aggressive NHL and disseminated dis- ease were treated with cyclophosphamide, vincristine, predni- Correspondence: N Niitsu, Department of Hematology and Internal Medicine IV, Kitasato University School of Medicine, 1-15-1 Kitasato, solone, bleomycin, doxorubicin and procarbazine (COP- 13 Sagamihara-shi, Kanagawa 228-8555, Japan; Fax: +81-42-778-9978 BLAM), cyclophosphamide, doxorubicin, vincristine and Received 4 February 2002; accepted 19 June 2002 prednisolone (CHOP),14 or another anthracycline-containing Cell surface nm23-H1 and NHL N Niitsu et al 197 combination chemotherapy regimen. Indolent lymphoma was strate and an alkaline phosphatase-detecting kit (BioRad Lab). in most cases treated with a single agent or cyclophospham- The reaction was stopped with 50 ␮l of 0.4 N NaOH. ide, vincristine and prednisone (COP). Patients were re-evalu- Absorbance was measured at 405–415 nm with a correction ated every few months, undergoing physical examination, wavelength of 620–630 nm using a microplate reader. hemogram and differential counts, biochemistry tests, and computed tomography of the chest, abdomen and pelvis. The median follow-up time was 21 months (range, 15–25 months), Statistical analysis and the final follow-up examination was carried out in April 2001. Approval of the study protocol was obtained from the The patients’ characteristics were compared using chi-square institutional review board at each participating institute. Infor- tests. Judgment criteria used for the analysis were progression- med consent was obtained from patients according to the free survival (PFS) and overall survival (OS). Progression was Declaration of Helsinki. defined as a progression of the lymphoma in nonresponding patients or partial response patients, a relapse for complete response patients, or death from any cause without pro- Flow cytometry for human cell surface nm23-H1 gression. PFS was calculated as the time from randomization to the first progression. Patients who did not experience a pro- The compound 7-amino-actinomycin D (7AAD) was pur- gression at the time of analysis were censored at the most chased from Sigma Chemical (St Louis, MO, USA). The drug recent date of disease assessment or at the stop date if the was diluted in phosphate-buffered saline (PBS) as a 25 ␮g/ml ° most recent date was later. OS was measured from the date stock solution, which was stored at 4 C. FITC-conjugated of randomization to the date of death, regardless of cause. monoclonal anti-nm23-H1 antibody was purchased from Sei- Patients who were still living at the time of analysis were cen- kagaku Kougyo (Tokyo, Japan). PE-conjugated anti-Leu4 sored at the most recent date of disease assessment or at the (CD3), Leu3a (CD4), Leu2a (CD8), LeuM5 (CD11c), Leu11a stop date if the most recent date was later. Survival analysis (CD16), and Leu12 (CD19) were purchased from Becton was performed according to the Kaplan–Meier method.15 The Dickinson (Cowley, UK) and CD5, CD10 and CD56 were pur- statistical significance of differences in survival was determ- chased from BD PharMingen (San Diego, CA, USA). FITC- and ined by the log-rank and generalized Wilcoxon’s tests.16 Dif- PE-conjugated murine IgG monoclonal antibodies of unre- ferences between groups were evaluated by the Mann–Whit- lated specificities were always used as controls. ney U test (nonparametric analysis),17 and P < 0.05 was taken Fresh tissues were transported immediately in RPMI 1640 to indicate significance. solution to the flow cytometry laboratory. Lymphocytes were disaggregated and released from solid tissue by injection of RPMI 1640 solution. Up to 1 × 106 cells were pelleted, incu- bated with 5 ␮l of 7AAD solution (final 1 ␮g/ml), 50 ␮lof Results FITC-conjugated monoclonal antibody, and PE-conjugated monoclonal antibody for 30 min at 4°C while protected from Correlation between nm23-H1 expression and serum light, and then pelleted by centrifugation. The supernatant was parameters or cell surface markers removed, and the cells were resuspended in Cell FIX solution (Becton Dickinson).