Patient selection
The present study included patients (n = 2456) with a pathologically confirmed DLBCL diagnosis who were treated in the First Hospital of China Medical University, Fourth Hospital of China Medical University, or Liaoning Province Cancer Hospital between January 1, 2001, and December 31, 2007. At the time of the analysis, 39% of the slides were available for pathologic review, and 967 patients were considered to have DLBCL (centroblastic, immunoblastic, or anaplastic).
Disease dissemination was evaluated before treatment by physical examination, bone marrow (BM) biopsy, and computed tomography of the chest and abdomen. Patients were staged according to the Ann Arbor system. The number of extranodal sites and larger tumor mass diameters were also determined. Performance status was assessed according to the Eastern Cooperative Oncology Group scale: 0, patient had no symptoms; 1, patient had symptoms but was ambulatory; 2, patient was bedridden for less than half of the day; 3, patient was bedridden for half of the day or longer; and 4, patient was chronically bedridden and required assistance with activities of daily living. Performance status was then classified as 0–1 (the patient was ambulatory) or 2–4 (the patient was not ambulatory).
All the patients provided written informed consent, and the study protocol and the sample collection were approved by the Ethics Committee of China Medical University.
Assessment of response
The primary endpoint was overall survival. Response to therapy was evaluated after the initiation of treatment. CR was defined as the disappearance of all clinical evidence of disease and normalization of all laboratory values, radiographs, computed tomography scans, and BM biopsy findings.
Histologic and immunophenotypic study
The histologic diagnosis of DLBCL was independently determined by 3 pathologists. The diagnosis was based on morphologic examination of slides from routinely processed paraffin-embedded samples stained with hematoxylin-eosin, Giemsa, and Gordon–Sweet stains and on immunophenotyping results. The immunohistochemistry panel consisted of antibodies against CD20, CD10, CD3, CD5, BCL2, BCL6, IRF4/MUM1, human leukocyte antigen (HLA)–DR, and Ki-67.
Staining for CD20, CD3, CD10, and HLA-DR was scored as positive or negative. Each individual case was unanimously scored as negative or positive by the 3 independent investigators, scored using the 2 matching scores when the third investigator did not agree, or recorded as “not evaluable” for a given antigen when there was no agreement between the investigators.
Staining for CD5, BCL2, IRF4/MUM1, and BCL6 was scored in a semiquantitative manner, from 1 to 5, indicating the percentage of positive tumor cells: 1, no staining; 2, 5%–25%; 3, 26%–50%; 4, 51%–75%; and 5, >75%. For Ki-67 (MIB1), a score of 5 was defined as 76%–90%, and an additional score of 6 was introduced (>90%). Whenever individual cores for a given case showed nonconcordant results, the score of the core with the highest number of positive cells was recorded.
Patient selection for SPRR1A expression
SPRR1A expression in DLBCL was analyzed when there were 4 unstained slides available for that case. Only formalin-fixed specimens were selected, while specimens fixed in Bouin’s fluid were excluded. To avoid bias related to treatment, only patients treated with CHOP were included in the study. Overall, 967 cases were studied for SPRR1A expression by immunohistochemical analysis.
Immunohistochemical analysis
Thin slices of tumor tissue for all cases were fixed in 4% formaldehyde solution (pH 7.0) for a duration that did not exceed 24 hours. The tissues were processed in a routine manner for paraffin embedding, and 4-μm thick sections were cut and placed on glass slides coated with 3-aminopropyl triethoxysilane for immunohistochemical analysis. The sections were mounted on microscope slides, air dried, and then fixed in a mixture of 50% acetone and 50% methanol. The sections were then de-waxed with xylene, gradually hydrated with gradient alcohol, and washed with phosphate-buffered saline (PBS). Sections were then incubated for 60 minutes with the primary antibody. After repeated washing with PBS, the sections were incubated for 30 minutes with the secondary biotinylated antibody (Multilink Swine anti-goat/mouse/rabbit immunoglobulin; Dako, Inc.). Thereafter, the avidin-biotin complex (1:1000 dilution; Vector Laboratories, Ltd.) was applied to the sections for 30–60 minutes at room temperature. The immunoreactive products were visualized by catalysis of 3,3′-diaminobenzidine with horseradish peroxidase in the presence of H2O2, after extensive washing. Sections were then counterstained in Gill’s Hematoxylin and dehydrated in ascending grades of methanol, prior to clearing with xylene and mounting under a coverslip.
To identify immunopositive staining for SPRR1A, SPRR1A expression was first classified semiquantitatively according to the following criteria: 0, <1% of cells discretely expressed SPRR1A; 1+, ≥1 and <10% of cells discretely expressed SPRR1A; and, 2+, ≥10% of cells discretely expressed SPRR1A. Samples scored as 1+ or 2+ were considered positive.
Statistical analysis
Patient characteristics were compared using the Chi-square test. Overall survival was analyzed using the Kaplan–Meier method. The log-rank test was used to analyze survival differences. Multivariate analysis was conducted to adjust the effect of SPRR1A expression for potential independent prognostic factors (age, sex, extranodal sites, performance status, clinical stage, bulky disease [_____ >10 cm], evolution, lactate dehydrogenase level, and SPRR1A expression) using the Cox proportional hazards model with forward stepwise selection. A P value of <0.05 was considered statistically significant. All data were analyzed using SPSS (Version 17.0; SPSS Inc., Chicago, IL, USA).
