Distribution Patterns of Dendritic Cells and T Cells in Diffuse Large B-Cell

Imaging, Diagnosis, Prognosis

Distribution Patterns of Dendritic Cells and T Cells in Diffuse Large B-Cell Lymphomas Correlate with Prognoses Kung-Chao Chang,1, 2 Guan-Cheng Huang,3 DanJones,5 and Ya-Hui Lin4

Abstract Purpose: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin’s lymphomas, accounts for 30% to 40% of all lymphoma cases. However, long-term survival by current chemotherapy was achieved in only 40% of patients, warranting the development of novel therapeutic strategies including T-cell immunotherapy. However, the level of baseline immune activation in DLBCL is unclear. Experimental Design: The density and distribution of dendritic cells and Tcells in 48 cases of primary DLBCL was evaluated by immunohistochemistry. Results: Increased numbers of intratumoral CD1a+ dendritic cells and increased S100+ cells and CD45RO+ T cells around the edges of the tumors were seen in 10 of 48 (21%), 9 of 48 (19%), and 10 of 48 (21%) cases and these were correlated with a favorable prognosis (P =0.015; P =0.070,andP = 0.017, respectively), along with increased granzyme B+ T cells in tumor beds (P = 0.013). Increased peritumoral T cells were correlated with tumor expression of HL A-DR (r = 0.446; P = 0.002). Extranodal lymphomas showed fewer tumor-associated CD45RO+ Tcells (r =-0.407;P = 0.001) and less conspicuous dendritic cell infiltrates. Conclusions: In DLBCL, the presence of baseline antitumor immune response is associated with favorable clinical outcome, and thus adjuvant T-cell immunotherapy may further boost treatment responses.

The most common subtype of non-Hodgkin’s lymphoma is In animal models, the mice, which were immunized with diffuse large B-cell lymphoma (DLBCL; ref. 1). The mainstay irradiated tumor cells and challenged with viable cells of the strategy for treatment of DLBCL is multidrug chemotherapy. same tumor, seemed to reject a normally lethal dose of that However, long-time survival was achieved in only f40% of tumor (3). That finding led to the discovery of the tumor patients, warranting the development of novel therapeutic rejection antigen, which is usually specific for an individual strategies such as monoclonal antibodies (2) and tumor tumor. In DLBCL, the tumor-specific antigen is the unique vaccines by pulsing autologous dendritic cells with tumor idiotype antigen derived from the B-cell receptor expressed by antigens (3, 4). This approach had been extended to equipment the neoplastic clone (8). Through the presentation to T cells by of tumor cells with costimulatory molecules or cytokines that self MHC molecules, this idiotype antigen could be potentially activate dendritic cells (3, 5, 6). Furthermore, the use of recognized as a target by host immunity. Given that the idiotype autogeneic T cells to eradicate lymphoma cells seems to be antigen is so unique and different among individual cases of effective in animal experiments (7). However, little is known DLBCL, the expression of MHC molecules in DLBCL cells can be about the effect of tumor immunity on DLBCL in human. a surrogate marker for lymphoma-specific antigens. It has been found that loss of HLA-DR expression in DLBCL correlated with immune evasion and poor patient survival (9, 10). Authors’ Affiliations: 1Department of Pathology and 2Institute of Clinical Medicine, Medical College, National Cheng Kung University; 3Division of Hemato- Dendritic cells are the most potent antigen-presenting cells. By Oncology, Department of Internal Medicine, Chi-Mei Medical Center; 4Division of expressing high levels of MHC and costimulatory molecules, Clinical Research, National Health Research Institute, Tainan, Taiwan; and dendritic cells can present tumor-associated antigens to elicit 5Department of Hematopathology,The University of Texas M.D. Anderson Cancer T-cell–mediated tumor destruction (11). Dendritic cells are Center, Houston,Texas heterogeneous in human. They are divided into myeloid (mDC/ Received 2/28/07; revised 8/11/07; accepted 8/16/07. Grant support:Taiwan National Science Council grant NSC 94-2320-B-006-051 DC1) and plasmacytoid (pDC/DC2) dendritic cells by lineage and and National Cheng Kung University Hospital, Taiwan, grant NCKUH-96-004 their ability to promote T helper subset 1or subset 2 development, (K.C. Chang). respectively. Dendritic cells also undergo progressive maturation The costs of publication of this article were defrayed in part by the payment of page and have been divided into immature [CD1a+/CD83low/dendritic charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cell–specific lysosome-associated membrane protein negative - Note: None of this work has been submitted for publication elsewhere. (DC-LAMP )/dendritic cell–specific intercellular adhesion mole- Requests for reprints: Kung-Chao Chang, Department of Pathology, National cule-3 grabbing nonintegrin positive (DC-SIGN+)/cytoplasmic Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan 704, Taiwan. Phone: MHC II molecule] versus mature [CD1a+/-/CD83high/DC-LAMP+/ 886-6-235-3535, ext. 2636; Fax: 886-6-276-6195; E-mail: changkc@mail. - ncku.edu.tw. DC-SIGN /surface MHC II molecule] phenotypes (12–15). F 2007 American Association for Cancer Research. The basis of antitumor cellular immunotherapy is that tumor doi:10.1158/1078-0432.CCR-07-0504 cell antigens can be phagocytized by immature dendritic cells,

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Immunohistochemical staining. Immunohistochemical staining was Table 1. Antibodies used in this study done on deparaffinized tissue sections of formalin-fixed material following microwave-enhanced epitope retrieval. After blocking of Antibodies Cells Clone Source Titer endogenous activity, sections were then incubated with primary stained name antibodies at room temperature for 2 h. Detection was done with + Bcl-6 Tumor cells PG-B6p DAKO 1:10 streptavidin-biotinylated peroxidase-conjugated reagents (LSAB kit, CD10 Tumor cells 56C6 Novocastra 1:50 DAKO) with 3-amino-9-ethylcarbazole as the chromogen and hema- CD1a Immature MTB1 Novocastra 1:10 toxylin for counterstain. The selected primary antibodies, which can be dendritic cells used in formalin-fixed tissues, are summarized in Table 1. The germinal CD83 Mature 1H4b Novocastra 1:10 center immunophenotype was determined by the combined expression dendritic cells of CD10 and bcl-6, as described in our previous study (19). Images DC-LAMP Mature 104.G4 Immunotech 1:10 were photographed using an Olympus DP12 Digital Microscope dendritic cells S100 Dendritic cells* Polyclonal DAKO 1:400 Camera (Olympus Co.) and processed with Adobe Photoshop version HLA-DR Tumor cells LN3 Novocastra 1:20 7.0 software (Adobe Systems, Inc.). CD45RO T cellsc UCHL1 DAKO 1:100 Granzyme B+ T cells GrB-7 DAKO 1: 50 Table 2. Summary of clinicopathologic features of *Dendritic cells and monocytic forms. c high DLBCL cases Effector or memory T cells are CD45RO ;na¨Ive T cells are CD45ROabsent/dim (3). Characteristics n (%)

Age (y) which then present tumor antigens to tumor-specific T cells <60 19 (39.6) + + >60 29 (60.4) following maturation. Both CD4 T cells and cytotoxic CD8 Sex T cells need to participate in this antitumor effect (3, 12). Male 27 (56.3) Because dendritic cells elicit both tumor-specific CD4+ and Female 21 (43.8) CD8+ T cells, and both types of T cells are indispensable for Site + Nodal 26 (54.2) lymphoma immunity (10, 16, 17), assessment of CD45RO Extranodal 22 (45.8) effector or memory T cells may reflect the final common Stage pathway of cell-mediated antitumor response. The presence of I 11 (22.9) dense infiltrating CD45RO+ T cells in colorectal cancers has II 18 (37.5) been found to correlate with increased survival (18). III 10 (20.8) IV 9 (18.8) In this study, by immunohistochemical staining, we showed LDH level (IU/L) + for the first time that the presence of CD1a dendritic cells and <200 21 (43.8) increased granzyme B+ T cells within tumors and the patterns of >200 27 (56.3) denser S100+ cells and CD45RO+ T cells around the tumor edge Germinal center phenotype Positive 8 (16.7) correlated with a favorable prognosis. The latter T-cell Negative 40 (83.3) distribution pattern was associated with tumor expression of EBV status HLA-DR. These findings indicate that in f20% of DLBCL there Positive 8 (16.7) is existence of antitumor immune response, and enhancing this Negative 40 (83.3) effect could be a target for DLBCL immunotherapy. HLA-DR Positive 18 (37.5) Negative 30 (62.5) Materials and Methods CD1a dendritic cells Positive 10 (20.8) Negative 38 (79.2) Tissue samples. Forty-eight cases of primary pretreated DLBCL were CD83 dendritic cells recruited from the archival files at the National Cheng Kung University Positive 22 (45.8) Hospital from February 1995 to December 1998. The specimens were Negative 26 (54.2) fixed in 10% neutral formalin and embedded in paraffin. Part of DC-LAMP dendritic cells the cases were studied previously (19). Studies were carried under a Positive 5 (10.4) Negative 43 (89.6) laboratory protocol approved by the institutional review board and No. S100 cells in tumor were in accordance with the Helsinki Declaration of 1975, which was <23 30 (62.5) revised in 1983. Primary nodal and extranodal lymphomas were >23 18 (37.5) previously defined (19, 20). Peritumoral S100 pattern Clinical data were obtained by chart review, including sex, age, serum Positive 9 (18.8) level of lactate dehydrogenase (LDH), tumor site, Ann Arbor stage, Negative 39 (81.3) treatment modality, and overall survival in months. There were 27 male No. CD45RO+ T cells in tumor and 21female patients with a mean age of 60.6 years (range, 26- <31 30 (62.5) 86 years). All the patients with available information were followed >31 18 (37.5) Peritumoral T-cell pattern up and the duration ranged from 1to 87 months. All the lymphoma Positive 10 (20.8) patients were typically treated with a curative cyclophosphamide, Negative 38 (79.2) doxorubicin, vincristine, and prednisone regimen, as described in our No. granzyme B+ T cells in tumor previous study (19). For selected patients, surgical intervention and/or <5 27 (56.3) radiotherapy preceded chemotherapy. None of the patients has received >5 21 (43.8) rituximab administration.

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Measurements. Quantitative evaluation of cells positive for CD1a, were few in number in most DLBCL tissues. The distribution of CD83, and DC-LAMP was assessed by counting at three high-power these cells was mainly aggregated around lymphatics or blood Â fields (HPF; 400 magnifications) on higher density areas, as previously vessels and scarce in tumor beds. Most of the intratumoral described (15, 21). Because most of the cases showed few positive cells, we dendritic cells showed mDC-type morphology (Fig. 1). The also used S100 to highlight dendritic cells and monocytic forms (22–24). CD1a+, CD83+, and DC-LAMP+ dendritic cell numbers in the Only the cells with dendritic cytoplasmic processes were counted at 10 HPFs. For cases showing difference in cell density between intratumoral tumor bed ranged from 0 to 11 (mean, 0.4; median, 0), 0 to 25 and peritumoral areas, both areas were evaluated. Staining results were (mean, 3.4; median, 0), and 0 to 5 cells (mean, 0.2; median, 0) Â evaluated and semiquantified for HLA-DR, CD10, and bcl-6. For HLA-DR, per HPF ( 400) and the positive rates were 21% (10 of 48), 46% unequivocal membrane staining in z30% of tumor cells was graded as (22 of 48), and 10% (5 of 48), respectively, based on the criteria positive (10). The cutoff value was 10% for CD10 and bcl-6 (19). Intra- of unequivocally positive cell(s) in 2 or more separate HPFs or tumoral CD45RO+ and granzyme B+ T cells were counted at 10 fields under z2 positive cells in one HPF. On the contrary, S100+ cell oil immersion (Â1,000). For cases also showing difference in cell density numbers ranged from 0 to 79 cells (mean, 23; median, 16) per between intratumoral and peritumoral areas, both areas were evaluated. HPF. Notably, there were 9 of 48 (19%) cases showing a in situ In situ EBV hybridization. hybridization study was done to characteristic pattern of denser S100+ cells around the tumor detect EBV-encoded RNA 1using a PCR-derived digoxigenin-labeled edge than in the tumor bed (Fig. 2A). The difference in this DNA probe (25, 26). The test for nucleotide integrity was done by the P use of the RNA-positive control probe (Ventana Medical System, Inc.). rimming pattern was statistically significant ( < 0.05, paired t Statistical analysis. Appropriate statistical tests were used to test). This increased peritumoral accentuation was also noted + examine the relationships and correlations between variables, including for CD45RO T cells in 10 of 48 (21%) cases (Fig. 2B; P < 0.001, Student’s t tests and Pearson or Kendall’s s correlation. The overall paired t test). There were five cases showing peritumoral survival was measured from initial diagnosis to death from any cause, accentuation for both S100+ and CD45RO+ cells. In addition, with follow-up data of surviving patients assessed at the last contact there was positive correlation between the density of S100+ cells date. Estimates of overall survival distribution were calculated using the and that of CD45RO+ T cells within the tumor (Pearson corre- method of Kaplan and Meier (27). Time-to-event distributions were lation coefficient r = 0.296; P = 0.041). The numbers of compared using the log-rank test (28). Cox proportional hazard model CD45RO+ and granzyme B+ T cells (Fig. 2C) in the tumor bed was used to test the simultaneous influence on overall survival of all ranged from 2 to 99 (mean, 31; median, 25) and 0 to 76 (mean, covariates found to be significant in the univariate analysis (29). The Â P value referred to is two sided. The analyses were carried using SPSS 14; median, 5) per HPF ( 1,000) and were positively correlated r P 13.0 statistical software (SPSS, Inc.). (Pearson correlation coefficient = 0.353; = 0.016). No pattern of peritumoral accentuation was noted for granzyme B stain. Results HLA-DR staining pattern in DLBCL. Tumor cells showed predominantly membranous and occasional cytoplasmic stain- The clinicopathologic features of 48 lymphoma cases are ing for HLA-DR in 18 of 48 (38%) cases (Fig. 2D). The HLA- + summarized in Table 2. DR tumor cells were diffuse in distribution in most cases. Staining patterns of dendritic cells and T cells in Correlation of dendritic cells with T cells and clinical + DLBCL. Dendritic cells expressing CD1a, CD83, or DC-LAMP features. The rimming pattern of denser CD45RO T cells

Fig. 1. Dendritic cells are sparse within DLBCL tumor infiltrates (Â400). A, CD1a; B, CD83; C, DC-LAMP; D, S10 0. The cells positive for CD1a, CD83, and DC-LAMP are fewer in number than S100+ cells. Most of the positive-stained dendritic cells show monocytoid (myeloid) appearances.

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Fig. 2. Increased peritumoral infiltration of dendritic cells and CD45RO+ Tcells is a feature of 20% of DLBCL. A, S100 + cells show accumulation at the tumoral edge (bottom left) and are fewer in the tumor bed (Â40).These S100+ cells possess dendritic cytoplasmic processes (inset ; Â400). B, CD45RO+ Tcells are denser around the peritumoral area (bottom left)andfewerin the tumor bed (Â40).The positive cells are highlighted in inset (Â400). C, intratumoral granzyme B+ Tcells show cytoplasmic granular staining (Â400). D, HL A-DR is highly expressed in the DLBCL shown above (Â400).

around the tumor edge and fewer in the tumor bed was T cells and dendritic cells, although the decrease of dendritic correlated with tumor expression of HLA-DR (Kendall’s cells was not statistically significant. Presence of CD1a+ s correlation coefficient r = 0.446; P = 0.002), presence of dendritic cells was associated with increased granzyme B+ CD83+ dendritic cells (r = 0.341; P = 0.016) and CD1a+ T cells in tumor beds (r = 0.327; P = 0.037). For clinical dendritic cells (r = 0.59; P < 0.001) in tumor beds, and the features, tumor stage was positively correlated with LDH level rimming pattern of S100+ cells (r = 0.419; P = 0.003). The latter (r = 0.349; P = 0.002). S100+ pattern was also correlated with presence of DC-LAMP+ Correlation of survival with clinicopathologic factors in DLBCL dendritic cells (r = 0.363; P = 0.009) and CD1a+ dendritic cells patients. The clinicopathologic features affecting patient sur- (r = 0.418; P = 0.002) in tumor beds. Notably, there was inverse vival are summarized in Table 3. In the univariate analysis, the correlation between extranodal lymphoma and CD45RO+ significant parameters related to a favorable prognosis included T-cell density (r = -0.407; P = 0.001) and between LDH level low-stage tumors (stage I/II), low LDH level (<200 IU/L), and presence of CD1a+ dendritic cells (r = -0.301; P = 0.012). expression of HLA-DR by tumor cells, the presence of CD1a+ That is, in extranodal lymphomas there were fewer CD45RO+ dendritic cells within tumors, the rimming pattern of S100+

Table 3. Prognostic factors affecting overall survival of DLBCL patients

Parameters Favorable factor Univariate analysis Multivariate analysis PP(HR, 95% CI)

Age <60 y 0.218 — Site (nodal vs extranodal) Nodal 0.115 — Stage (I/II vs III/IV) Low stage (I/II) 0.083 0.306 LDH level <200 IU/L 0.031 0.093 Germinal center phenotype Positive 0.213 — EBV association Negative 0.252 — HLA-DR expression Positive 0.029 0.089 CD1a dendritic cells in tumor beds Positive 0.015 0.016 (10.9, 1.4-84.1) CD83 dendritic cells in tumor beds Positive 0.114 — DC-LAMP dendritic cells in tumor beds Positive 0.202 — No. S100 cells in tumor beds >23 cells/Â400 0.413 — Peritumoral S100 pattern Positive 0.070 0.064 No. CD45RO+ T cells in tumor beds >31 cells/Â1,000 0.503 — Peritumoral T-cell pattern Positive 0.017 0.028 (3.0, 1.1-8.1) No. granzyme B+ T cells in tumor beds >5 cells/Â1,000 0.013 0.023 (3.5, 1.0-12.2)

Abbreviations: HR, hazard ratio; 95% CI, 95% confidence interval.

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Fig. 3. Kaplan-Meier curves for overall survival according to the tumor stage (A), LDH level (B), HL A-DR expression (C), the presence of CD1a+ dendritic cells (DC) within tumors (D), and the patterns of peritumoral denser S100+ cells (E)and CD45RO+ Tcells(F).The better prognostic factors include low stage (stage I/II), low LDH level (<200 IU/L), expression of HLA-DR, the presence of CD1a+ dendritic cellswithintumors,andthepatternsof peritumoral denser S100+ cells and CD45RO+ T cells (duration in months).

cells, the rimming pattern of CD45RO+ T cells, and the little is known about the antilymphoma immunity in increased granzyme B+ T cells within tumors (Fig. 3). In human. In this study, we showed that the presence of multivariate analysis, the factors significantly associated with CD1a+ dendritic cells and increased granzyme B+ T cells in better outcome were the presence of CD1a+ dendritic cells tumor beds and the rimming pattern of denser S100+ cells within tumors (hazard ratio, 10.9; 95% confidence interval, and CD45RO+ T cells around the tumor edge were 1.4-84.1), the rimming pattern of CD45RO+ T cells (hazard correlated with a favorable prognosis for DLBCL patients. ratio, 3.0; 95% confidence interval, 1.1-8.1), and the increased Extranodal lymphoma elicited fewer CD45RO+ T-cell and granzyme B+ T cells within tumors (hazard ratio, 3.5; 95% dendritic cell infiltrates. confidence interval, 1.0-12.2). Neither higher number of S100+ There are several studies drawing the issue about dendritic cells nor denser CD45RO+ T cells in tumor beds was related to cells and T cells in B-cell lymphomas (22, 33, 34). However, we a favorable prognosis. found for the first time that the peritumoral distribution of S100+ cells and CD45RO+ T cells was correlated with a Discussion favorable prognosis in DLBCL. This rimming pattern can be a prognostic indicator and, most importantly, a target for immu- With the advent of tumor immunobiology, vaccination for notherapy. Our study further clarified that increased recruit- lymphoma treatment was developed (30–32). However, ment of S100+ cells and T cells in the tumor bed was not

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This finding was interesting but not found to recruitment of immature or mature dendritic cells has been be significant on univariate and multivariate analyses for found in cutaneous melanomas and breast carcinomas outcome. The reason may be likened to that higher T-cell (15, 21). However, in those studies there were no survival data infiltrates in tumor beds did not necessarily correlate with a to highlight the clinical significance of the dendritic cells. To better outcome. In addition, it seems that nodal lymphomas the best of our knowledge, this is the first study drawing the have a different pattern of T-cell infiltration. The inclusion of prognostic implication of dendritic cells on the DLBCL extranodal cases, which typically have far fewer infiltrating patients. T cells at some tissue sites, may be a confounding variable in Previous studies have shown that tumors prevent dendritic the multivariate analysis. In contrast to no role of intratumoral cell maturation and increased numbers of immature dendritic CD45RO+ T-cell infiltrates, granzyme B+ T-cell infiltrates within cells in the tumors may be associated with worse outcomes due tumors were significant for better outcome. to the tolerizing effects of these cells (37, 41). In this study, the It may be of concern that the numbers of cells stained for majority of DLBCL containing CD1a+ immature dendritic cells dendritic cell markers were relatively few and the S100 was not also recruited CD83+ mature dendritic cells in tumors and bore entirely specific as a dendritic cell surrogate. It has been found a favorable outcome, and the numbers of CD83+ dendritic cells that the sensitivity of immunostaining on paraffin-embedded were greater than those of CD1a+ dendritic cells. Taken tissue sections was lower than on frozen tissue sections (36). together, it suggests that maturation and differentiation of Furthermore, the dendritic cell numbers in this study were intratumoral dendritic cells can be achieved in a subset of approximate to those in previous studies (15, 37). Dendritic DLBCL and might serve as a therapeutic target. On the other cells are heterogeneous in human. Given the shifts in intensity hand, the different findings of the correlation of immature in dendritic cell marker expression during dendritic cell dendritic cells with tumor prognosis may result from the differentiation and maturation during nodal transit, there is heterogeneity and diversity of dendritic cells as well as the no one marker that detects all dendritic cell stages reliably in different markers used for detection. paraffin sections. Therefore, we have included several different Lost or low expression of HLA-DR on DLBCL cells has been markers and found that S100, which was counted only in found to be correlated with poor patient survival (10). The dendritic forms, can give an independent and perhaps broader decreased tumor immunosurveillance was considered as a measure of the dendritic cell number at all maturation stages. result of diminished tumor-infiltrating CD8+ T cells. In parallel, Tumor stage and LDH level are two well-recognized factors we also found a similar result for HLA-DR to correlate with for prognosis (1). As shown in univariate analysis (Table 3), we a better outcome and with rimming pattern of CD45RO+ further found HLA-DR, CD1a+ dendritic cells, granzyme T cells. B+ T cells, and the rimming patterns of S100+ cells and In conclusion, we showed that in f20% of DLBCL cases, the CD45RO+ T cells to be relevant for prognosis, although only presence of CD1a+ dendritic cells within tumors and the rim- CD1a+ dendritic cells, the rimming pattern of CD45RO+ T cells, ming effects of S100+ cells and CD45RO+ T cells were correlated and intratumoral granzyme B+ T cells were significant in with a favorable prognosis. The latter T-cell distribution pattern multivariate analysis. This was likely related to the general was associated with tumor expression of HLA-DR. Taken association of HLA-DR and the S100 rimming pattern with the together, it may imply that adjuvant dendritic cell and T-cell presence of CD1a+ dendritic cells in tumors. Furthermore, the immunotherapy may further boost treatment responses. majority of cases (70%) containing CD1a+ immature dendritic cells also recruited CD83+ mature dendritic cells in tumors. Acknowledgments Because neoplasia is a chronic process and prolonged activation of mature dendritic cells induces their ‘‘exhaustion’’ or We thank Mi-Chia Ma (Associate Professor, Department of Statistics, National ‘‘paralysis’’ (14, 38, 39), the finding of CD1a+ dendritic cells Cheng Kung University,Tainan,Taiwan) for advice with statistical interpretation.

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Clin Cancer Res 2007;13(22) November 15, 2007 6672 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Distribution Patterns of Dendritic Cells and T Cells in Diffuse Large B-Cell Lymphomas Correlate with Prognoses

Kung-Chao Chang, Guan-Cheng Huang, Dan Jones, et al.

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