Published OnlineFirst August 20, 2012; DOI: 10.1158/1078-0432.CCR-12-0596 Clinical Cancer Imaging, Diagnosis, Prognosis Research Gene Expression Signature–Based Prognostic Risk Score in Patients with Primary Central Nervous System Lymphoma Atsushi Kawaguchi1, Yasuo Iwadate2, Yoshihiro Komohara3, Masakazu Sano4, Koji Kajiwara5, Naoki Yajima4, Naoto Tsuchiya4, Jumpei Homma4, Hiroshi Aoki4, Tsutomu Kobayashi4, Yuko Sakai7, Hiroaki Hondoh6, Yukihiko Fujii4, Tatsuyuki Kakuma1, and Ryuya Yamanaka7 Abstract Purpose: Better understanding of the underlying biology of primary central nervous system lymphomas (PCNSL) is critical for the development of early detection strategies, molecular markers, and new therapeutics. This study aimed to define genes associated with survival of patients with PCNSL. Experimental Design: Expression profiling was conducted on 32 PCNSLs. A gene classifier was developed using the random survival forests model. On the basis of this, prognosis prediction score (PPS) using immunohistochemical analysis is also developed and validated in another data set with 43 PCNSLs. Results: We identified 23 genes in which expressions were strongly and consistently related to patient survival. A PPS was developed for overall survival (OS) using a univariate Cox model. Survival analyses using the selected 23-gene classifiers revealed a prognostic value for high-dose methotrexate (HD-MTX) and HD- MTX–containing polychemotherapy regimen–treated patients. Patients predicted to have good outcomes by the PPS showed significantly longer survival than those with poor predicted outcomes (P < 0.0001). PPS using immunohistochemical analysis is also significant in test (P ¼ 0.0004) and validation data set (P ¼ 0.0281). The gene-based predictor was an independent prognostic factor in a multivariate model that included clinical risk stratification (P < 0.0001). Among the genes, BRCA1 protein expressions were most strongly associated with patient survival. Conclusion: We have identified gene expression signatures that can accurately predict survival in patients with PCNSL. These predictive genes should be useful as molecular biomarkers and they could provide novel targets for therapeutic interventions. Clin Cancer Res; 18(20); 5672–81. Ó2012 AACR. Introduction tion is often diffuse and multifocal, and most frequently A primary central nervous system lymphoma (PCNSL) is affects the supratentorial brain parenchyma, with periven- an extranodal form of non-Hodgkin lymphoma arising in tricular lesions involving the corpus callosum, basal the craniospinal axis. For many years, PCNSLs were ganglia, or thalamus. The absence of systemic lymphade- reported to represent 3% to 5% of all primary central nopathies and other extracranial localizations of disease nervous system (CNS) tumors (1). However, PCNSL seems should be confirmed. Most PCNSLs belong to the diffuse to be increasing in incidence (2–4). The tumor manifesta- large B-cell lymphomas (DLBCL) but differ from systemic DLBCLs by their less favorable prognosis. The systemic use of high-dose methotrexate (HD-MTX)– 1 Authors' Affiliations: Biostatistics Center, Kurume University, Kurume, based chemotherapy with radiotherapy for newly diag- Fukuoka; 2Department of Neurosurgery, Graduate School of Medical Sciences, Chiba University, Chiba; 3Department of Cell Pathology, Grad- nosed PCNSL has improved the median overall survival uate School of Medical Sciences, Kumamoto University, Kumamoto; (OS) from 20 to 36 months (5–8). However, there are still 4 Department of Neurosurgery, Brain Research Institute, Niigata University, many individual variations within the diagnostic and prog- Niigata; 5Department of Neurosurgery, Graduate School of Medical Sciences, Yamaguchi University, Ube, Yamaguchi; 6Department of Neu- nostic categories, resulting in a need for additional biomar- rosurgery, Toyama Prefectural Central Hospital, Toyama; and 7Graduate kers, partly because of the inability to recognize these School for Health Care Science, Kyoto Prefectural University of Medicine, Kyoto, Japan patients prospectively. Although, the clinical scoring model using age, Karnofsky performance status (KPS), and lactate Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). dehydrogenase (LDH) level has prognostic value for PCNSL (9–11), it has not been used successfully to stratify patients Corresponding Author: Ryuya Yamanaka, Kyoto Prefectural University of Medicine, Graduate School for Health Care Science, 465 Kajii-cho, Kami- for therapeutic trials. Molecular markers could improve the gyoku, Kyoto 602-8566, Japan. Phone: 81-75-212-5429; Fax: 81-75-212- outcome prediction, discover potential targets for therapeu- 5423; E-mail: [email protected] tic intervention, and elucidate mechanisms that result in doi: 10.1158/1078-0432.CCR-12-0596 resistance to chemotherapy. A comprehensive molecular Ó2012 American Association for Cancer Research. approach to predict the prognosis is awaited. In the present 5672 Clin Cancer Res; 18(20) October 15, 2012 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst August 20, 2012; DOI: 10.1158/1078-0432.CCR-12-0596 Biomarker Study for PCNSL These tissues contained more than 95% tumor cells. The Translational Relevance quality of the obtained RNA was verified using a Bioanalyzer In this study, we report the development and valida- System (Agilent Technologies) and RNA Pico Chips (Agilent tion of a risk-score model based on the expression of 23 Technologies). Subsequently, 1 mg of RNA was processed for genes. This 23-gene risk score is highly associated with hybridization to a GeneChip Human Genome U133 Plus the outcome of patients with newly diagnosed primary 2.0 Expression Array (Affymetrix Inc.), which contained central nervous system lymphoma (PCNSL). These approximately 47,000 genes. After hybridization, the chips results suggest the importance of this multimarker panel were processed using a Fluidics Station 450, High-Resolu- as a stratification factor for the design of future compar- tion Microarray Scanner 3000, and GCOS Workstation ative therapeutic trials. Version 1.3 (Affymetrix Inc.). Validation of differential expression by real-time qPCR Quantitative PCR (qPCR) was conducted using a Ste- study, we carried out an expression profiling analysis in pOne Real-Time PCR System (Applied Biosystems) and patients with PCNSL for the identification of genes that are TaqMan Universal PCR Master Mix (Applied Biosystems) predictive of OS. according to the manufacturer’s protocol. The Assays-on- Demand probe/primer sets (Applied Biosystems) used Materials and Methods were as follows: ATAD1, Hs00907773_g1; BRCA1, Samples and study population Hs01556193_m1; FANCA, Hs01116668_m1; GAPDH, Patients were diagnosed and treated at Niigata University Hs99999905_m1; GGH, Hs00914163_m1; GNASAS, Hospital (Niigata, Japan), Chiba University Hospital (Chiba, Hs00294858_m1; PGAM1, Hs01652468_g1; PPP3R1, Japan), Yamaguchi University Hospital (Ube, Yamaguchi, Hs01547793_m1; RBBP8, Hs00161222_m1; ROCK1, Japan), and Toyama Prefectural Central Hospital (Toyama, Hs01127699_m1; STIL, Hs00161700_m1; TRMT6, Japan) between 2000 and 2010. Clinical data were obtained Hs00210942_m1; and ZNF681, Hs01862022_s1. Total through a registered database and chart review. Inclusion RNA (1 mg) was reverse-transcribed into cDNA using Super- criteria were a histology-proven CNS lymphoma without the Script II (Invitrogen), and 1 mL of the resulting cDNA was evidence of systemic lymphoma, and no evidence of HIV-1 used for qPCR. Validation was conducted on a subset of infection, opportunistic infections, or other immunodefi- tumors that were part of the original tumor data set assessed. ciency. Patients were selected on the basis of the availability Assays were carried out in duplicate. The raw data produced of tumor specimens without regard to the clinical outcome. by qPCR referred to the number of cycles required for All patients underwent brain imaging with either computed reactions to reach the exponential phase. Expression of tomography (CT) or magnetic resonance imaging (MRI). glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was After the diagnostic biopsy, detailed history and physical used for normalization of the qPCR data. The mean expres- examination, complete blood count, screening blood tests of sion fold change differences between tumor groups were ÀDDC hepatic and renal function, serum protein electrophoresis, calculated using the 2 T method (12). and chest radiographs were obtained. The CT or MRI of the thorax, abdomen, and pelvis were conducted for all patients. Immunohistochemistry Ophthalmologic consultations and slit-lamp examinations Three antibodies for immunophenotype determination were used to rule out ocular involvement. Bone marrow and 5 commercially available antibodies for proteins biopsy wasnot routinely conducted unless CNS involvement encoded by genes associated with patient survival were was part of a systemic lymphoma. Lumbar puncture for selected for immunohistochemistry (IHC). Sections (5 mm) cerebrospinal fluid (CSF) evaluation was routinely con- of the formalin-fixed, paraffin-embedded tissue specimens ducted. Tissues were snap-frozen in liquid nitrogen within were evaluated. The primary antibodies recognized BCL6 5 minutes of harvesting, and stored at À80 C thereafter. All (DAKO; 1:200 dilution), BRCA1 (Abcam; 1:200 dilution), specimens were