AHighTumor-AssociatedMacrophage Content Predicts Favorable Outcome in Follicular Lymphoma PatientsTreated with Rituximab and Cyclophosphamide-Doxorubicin-Vincristine-Prednisone MinnaTaskinen,

Purpose:Tumor-associated macrophage (TAM) content predicts survival in follicular lymphoma (FL) patients treated with chemotherapy. The aim of this study was to determine how combination of rituximab with chemotherapy influencesTAM-associated clinical outcome. Experimental Design: Expressionof amacrophagemarker, CD68,was determined immunohistochemically from FL samples of 96 patients treated with rituximab and cyclophosphamideAdriamycin-vincristine-prednisone regimen. Of them, 71received therapy at diagnosis and 25 at relapse. Neutrophil and CD3+ lymphocyte counts were also measured. The median follow-up time for the cohort was 54 months. Fourty-five patients previously treated with chemotherapy served as a control group. Results: Consistent with previous studies, high TAM amount was associated with adverse outcome in chemotherapy-treated patients (P = 0.026). In contrast, after rituximab and cyclophosphamide-doxorubicin-vincristine-prednisone regimen, high TAM content correlated with longer survival rates. According to Kaplan Meier estimates, the median progression free survival (PFS) was not reached for patients with high TAM content compared with 45 months for patients with low TAM scores (P = 0.006). A trend toward a better overall survival (OS) at 5 years was also observed for patients with high TAM content (OS, 97% versus 90%, P = 0.116). The positive prognostic value of TAMs was seen both for the patients treated at diagnosis and at relapse. In multivariate analyses,TAM content remained an independent prognostic factor for OS and PFS. Neutrophil and CD3+ lymphocyte counts did not correlate with outcome. Conclusions:The data suggest that highTAM score is associated with a favorable prognosis in FL patients treated with immunochemotherapy. Follicular lymphoma (FL) is the second most common subtype of all non–Hodgkin lymphomas accounting for f20% of all adult cases in the western world. FL arises from B cells in the germinal center of lymphoid tissue. It is an indolent and a chemotherapy-sensitive disease, which is, however, considered rarely curable due to its propensity to relapse. Recently, however, a significant improvement of the outcome of patients has been obtained by combining a monoclonal anti-CD20 antibody, rituximab, with induction chemotherapy (1–3), or by prolonging the remission with rituximab maintenance therapy (4, 5). Despite the advances, response to treatment varies substantially among individual patients and outcome is often unpredictable. Treatment is also costly. These facts raise the need to identify more accurately the patients who benefit from immunochemotherapy. In FL, a specific Follicular Lymphoma International Prognostic Index (FLIPI) has been proposed (6). Although FLIPI was developed before rituximab was established in the treatment of FL, it was recently shown to be a useful predictor of the outcome also in response to rituximab and cyclophosphamideAdriamycin-vincristine-prednisone (R-CHOP) regimen (7). In estimating the prognosis of FL, FLIPI seems to distribute the patients into risk groups better than the International Prognostic Index for aggressive non–Hodgkin lymphomas (8) and is likely to be a helpful tool in selecting the most appropriate treatment for individual FL patients. However, five clinical characteristics of FLIPI do not provide information on the biological and molecular features of the FL. Differences in the tumor microenvironment have been shown to associate with clinical course and outcome of FL patients treated with chemotherapy (9). In particular, tumorinfiltrating cytotoxic and regulatory T cells and macrophages have prognostic effect in FL (10–16). However, all biological Imaging, Diagnosis, Prognosis Authors’ Affiliations: Department of Oncology, Helsinki University Central Hospital, Molecular Cancer Biology Program, Biomedicum Helsinki, and Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland Received 4/3/07; revised 7/12/07; accepted 7/19/07. Grant support: Finnish Medical Foundation, Finnish Cancer Societies, University of Helsinki, and Helsinki University Central Hospital. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Sirpa Leppa« , Department of Oncology, Helsinki University Central Hospital, P.O. Box 180, FIN-00029 Helsinki, Finland. Phone: 358-407558293; E-mail: [email protected]. F2007 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-07-0778 www.aacrjournals.org Clin Cancer Res 2007;13(19) October1, 2007 5784 Research. on July 15, 2017. © 2007 American Association for Cancer clincancerres.aacrjournals.org Downloaded from data has been produced before rituximab has been adapted into clinical use, and there are currently no biomarkers that have been revalidated and shown to be prognostic in patients treated with rituximab-containing regimens. Our recent microarray and immunohistochemical pilot study suggest that gene expression by nonmalignant tumor cells has prognostic effect also in R-CHOP–treated FL patients (17). Taking into account these previous observations, we evaluated the predictive value of tumor-infiltrating inflammatory cells and especially macrophages in the era of R-CHOP treatment. Materials andMethods Patients. A total of 141 FL patients treated at the Helsinki University Central Hospital were included in the study. Of these, 96 FL patients received R-CHOP in front-line (n = 71) or relapse (n = 25) (postrituximab era), whereas 45 patients treated with chemotherapy or radiotherapy before rituximab was adapted into clinical routine served as a control group (pre-rituximab era). All patients in the postrituximab group received rituximab for the first time. The relapsed patients had received various treatments front-line, including chlorambucil, combination chemotherapy, and local irradiation. All tissue samples were taken before treatment. Lymphoma classifications, including histopathology and immunophenotyping, were done at the Department of Pathology at Helsinki University Central Hospital Laboratory Diagnostics according to the WHO classification. Treatment records of all patients were reviewed to confirm the appropriate treatment protocols and to document clinical characteristics, prognostic factors, and long-term follow-up. The protocol and sampling were approved by Institutional Review Board and Finnish National Authority for Medicolegal Affairs. Immunohistochemistry. Immunohistochemistry was done on formalinfixed, paraffin-embedded tissue sections either on individual slides or as a part of a tissue microarray (TMA) as described earlier (17). The sections were stained with anti-CD68 (1:2,000; clone KP1, Dako), anti-CD3 (1:100; Novocastra Laboratories Ltd.), or with Leder stain (18) to detect macrophages, T lymphocytes, and neutrophils,