Abstract
Background: We examined the expression of CD200, a ligand of immune tolerance, in transitional cell carcinoma of the human bladder (TCC). Materials and Methods: CD200 was analyzed by immunohistochemistry (IHC) in 90 patients with suspected TCC lesions of the bladder. Expression of CD200 was exemplarily validated by quantitative reverse transcription polymerase chain reaction and western blot analysis. Results: CD200 was detectable at mRNA and protein levels in TCC homogenate and TCC cell lines (T24, UMUC3). TCC tissues showed significantly higher CD200 expression (p<0.005) than normal bladder tissues. CD200 signals were also higher in metastasized compared to localized TCC (p<0.05). CD200 was significantly correlated to tumor grading (p<0.001) and was strongest in the subgroup with high-grade G2 TCC (vs. low-grade G2 p<0.05). Conclusion: This is the first report of CD200 expression in patients with TCC. The significant correlation between CD200 expression and tumor grading may suggest CD200 as a potential target and marker for immunotherapeutic approaches.
Despite different treatment options, bladder cancer is still associated with high morbidity and mortality rates (1). Furthermore, long-term survival is limited; e.g. in Germany, median 10-year survival rates for tumor stages overall ranged between 44 and 52% (1, 2). To date, various markers have been described for prognosis and therapy monitoring but due to their low sensitivity and high costs, none of them have attained application in clinical routine (3-6). With a trend towards individually tailored concepts for patients with cancer, it is essential to decode the pathways of bladder cancer in order to optimize patient outcome, find relevant biomarkers, prognosticators, and specific therapeutic agents.
In the past 10 years a joint endeavor to understand the basics of cancer development bore fruit by introducing new individual therapies and prognostic indicators (7). An innovative approach in cancer treatment is inhibition of immune checkpoints, so far mainly applied to patients with renal cell cancer, and melanoma (8, 9). As localized bladder cancer is one of the first types of malignant tumor treated by non-specific immunotherapy with bacillus Calmette-Guérin (BCG), first described in 1976 by Morales and co-workers, particular bladder cancer subgroups may be especially susceptible to new therapeutic approaches with specific immunotherapy (10, 11). In 2017, the immune checkpoint inhibitors atezolizumab, pembrolizumab and nivolumab were approved by the European Medicines Agency for the treatment of metastasized or locally advanced bladder cancer after platinum-based chemotherapy; other new substances are currently under clinical evaluation in phase III clinical trials (12-15).
CD200, a type 1a glycoprotein, is a promising biomarker, immune checkpoint and target for specific immunotherapy. CD200 is expressed in critical tissue e.g. brain and bone marrow, testis, and placenta, as well as certain leukocytes including T-and B-cells as well as macrophages (16, 17). The short transmembrane domain lacks a canonical signaling motif but its receptors CD200R1-R4 are found on a wide variety of bone marrow-derived cells such as dendritic cells, macrophages, and T-cells (18). Its major role seems to be the protection of immune-privileged sites and the promotion of peripheral tolerance (19). In addition, CD200 seems to play a crucial role in tissue repair, autoimmune disease, and graft rejection (20-25). There is strong evidence that tumor cells and viruses use the CD200 pathway for immune evasion (25, 26). In our study, we analyzed the expression of CD200 in human bladder cancer cell lines and in tissue of patients suffering from different stages of bladder cancer.
Materials and Methods
Patients. The study included 90 patients with suspected TCC bladder lesions (60 male, 30 female; mean age=72 years, range=25-93 years). The study was approved by the local Institutional Review Board (AZ 52/14). All patients were treated with transurethral tumor resection (TUR-BT). Resected tissue was collected in a prospective manner. Representative specimens of the formalin-fixed paraffin-embedded primary tumor were selected and evaluated according to the Union international contre le cancer (UICC) classification (27). Patients with non-muscle invasive bladder cancer (NMIBC) were followed-up for tumor recurrence using a clinical database system.
RNA isolation, quantitative reverse transcription polymerase chain reaction (qRT-PCR), protein isolation and western-blot analysis. T24 (HTB-4) and UMUC3 (CRL-1749) cell lines derived from transitional cell carcinoma (ATCC, Bioresource Center, Manassas, VA, USA) were cultured in complete growth medium (RPMI 1640 supplemented with 10% fetal calf serum, PAN-Biotech, Aidenbach, Germany). Total RNA from tumor tissue or cultured cells was extracted by TriFast procedure according to the RNA isolation protocol (PEQLAB Biotechnologie, Erlangen, Germany). RNA (0.5 μg) was submitted to DNAse I treatment and cDNA synthesis was performed with random hexamer primers and M-MLV reverse transcriptase in 20 μl volume. cDNA solution (1 μl) was submitted to SYBR green-based (Thermo Fisher Scientific, Waltham, MA, USA) qRT-PCR (IQ5, Biorad, München, Germany). Cycling conditions used were: 95°C for 7.5 min followed by 60 cycles at 95°C for 15 s; 58°C for 30 s; 72°C for 30 s. CD200 mRNA levels are displayed as Delta Ct (ΔCt) difference to β-actin as reference mRNA. The custom-made primer sets (Biomers, Ulm, Germany) were deduced from Genbank sequences (Acc. No. NM_001101 and NM_005944, respectively): CD200+: ACC AGC ATC CTC CAT ATC AAA GAC C, CD200-: CCG GTG ACG TTT CCA GTA CAG TAA G, amplicon length 204 bp; β-actin+: TAT CCA GGC TGT GCT ATC CCT GTA C; β-actin-TTC ATG AGG TAG TCA GTC AGG TCC C, amplicon length 168 bp.
Total protein from homogenate of one tumor tissue was prepared by TriFast procedure according to the protein isolation protocol (PEQLA Biotechnologie, Erlangen, Germany). Protein of cells was obtained according to the RIPA buffer protocol (Cell Signaling Technology Europe, Frankfurt, Germany). Protein (40 μg/lane) was loaded on a gel and blotted to a nitrocellulose membrane (Biorad, Munich, Germany). After blocking, the membrane was incubated with a CD200 antibody (Catalog Number: AF2724, RD-Systems, Wiesbaden, Germany). Immunoreactions were subsequently detected by incubation with a horseradish peroxidase-coupled secondary antibody and chemiluminescence procedure.
Immunohistochemistry (IHC) staining. The specimens were cut into 3 μm sections mounted on silanized positively charged slides and dried overnight. The antiserum against CD200 was generated by immunization of rabbits with recombinant human CD200 Protein (extracellular domain), following standard protocols. After serum isolation from whole blood, the antibodies were further purified by immunoaffinity-absorption with matrix-coupled antigen and validated by western blot and IHC of human placenta as best practice recommends (28). The staining procedure was performed on a DAKO Autostainer after heat treatment and antigen retrieval with Trilogy™ (Sigma-Aldrich, Taufkirchen, Germany). Dako EnVision™+ Dual Link System-HRP (Agilent, Santa Clara, CA, USA) was used as detection system followed by chromogen reaction and counterstaining with Mayer's hematoxylin. Staining intensity of tumor cells (cytoplasm and membrane) was determined and evaluated in a blinded fashion. For analysis the whole tumor slides were evaluated, first at low magnification and afterwards at high magnification. We assessed general staining intensity and spots of high staining intensity at low magnification. In the case of general strong CD200 expression, at least 1 mm2 of tumor was scanned at high magnification. In the case of a scattered or weak CD200 expression, the whole slide was scanned at high magnification. All staining was graded on a 4-score system as follows (see Figure 1): 0: no staining, 1: only few tumor cells stained with weak staining intensity, 2: many tumor cells stained with weak staining intensity, 3: tumor cells stained with strong staining intensity.
Statistics. All data were computerized by Microsoft Excel® 2013 (Microsoft, Redmond, MA, USA) and analyzed with IBM SPSS® Statistics for Windows Version 22 (Ehningen, Germany) using the Mann–Whitney U-test and the Kruskal–Wallis ANOVA test. A p-value of less than 0.05 was considered statistically significant.
Results
CD200 was detectable at the mRNA and protein levels in TCC tissue homogenates and two human TCC cell lines (T24 and UMUC3). The levels of CD200 mRNA and protein were consistent (Figure 2).
In 20 patients (12 males, eight females, mean age=65 years, range=25-90 years), TUR-BT and subsequent evaluation of the resected tissues exhibited no signs of malignancy. These patients served as a control group.
In 70 patients (48 males, 22 females, mean age=74 years, range=47-93 years), histological examination revealed TCC: 59% (n=41) with NMIBC and 27% (n=19) with muscle-invasive (MIBC) and 14% (n=10) with metastasized disease. High-grade TCC was evident in 39 and low-grade TCC in 31 patients. The guideline-concordant treatment after primary TUR-BT comprised transurethral re-resection as well as radical surgery with urinary diversion depending on the respective tumor stage. All patient characteristics are summarized in Table I.
Results of CD200 staining of the control and the TCC groups are shown in Table II. Compared to the control group, patients with TCC had significantly higher CD200 expression (p<0.005). We found no correlation between CD200 staining intensity and pT stage (p=0.24). No difference was found between CD200 expression in patients with NMIBC (n=41) and those with MIBC (n=19, p=0.13). In case of metastasized TCC (n=10), there was a significantly higher CD200 expression compared to those with localized disease (p≤0.05). Concerning TCC grading we found significantly elevated CD200 expression with increasing grade of malignancy (high grade vs. low grade, p<0.001).
Comparing the subgroup of patients with high-grade G2 TCC (n=11) and low-grade G2 TCC (n=18), a significantly stronger CD200 expression was observed in high grade G2 TCC (p<0.05, see Table III).
The recurrence rate for patients with NMIBC (median follow-up=18.5 months, range 2-60=months) was 24% (n=10). Statistical analysis showed no correlation between TCC recurrence in NMIBC and primary CD200 expression (p=0.73).
Discussion
TCC is a frequently diagnosed cancer. Due to high morbidity as well as mortality rates with limited overall survival, there is a need for novel prognostic factors and new therapeutic approaches (1, 2, 6). Specific immunotherapy seems to be a promising approach for optimizing the outcome in TCC but the understanding of different pathway factors, their location and interaction, as well as the translation into the clinical setting is still at the beginning (15, 29). CD200 is a type 1a membrane glycoprotein capable of modulating the immune system via its inhibitory receptor CD200R that is expressed on both myeloid and lymphoid cells. After their interaction, immune response is negatively regulated by suppression of T-cell response. Thus, CD200 and its receptor represent promising targets to improve therapy in immune-mediated as well as oncological diseases (30-33).
Here, to our knowledge for the first time, we described CD200 expression both in human TCC cell lines and in TCC tissue showing higher levels in human TCC tissue. CD200 expression in TCC tissue was significantly higher compared to benign bladder tissue. Moreover, in our cohort we observed a highly significant correlation between CD200 expression and grade of malignancy. We found no correlation with common clinical prognosticators such as pT stage, recurrence rate, or subgroups (NMIBC and MIBC), although metastatic TCC showed the highest CD200 expression levels.
Nevertheless, our initial data provide a strong indication for a putative role of CD200 in the immune-evasion of bladder cancer as already noted in other malignancies. Gorczynski et al. demonstrated CD200-dependent immune-evasion in a transgenic mouse model of breast cancer (34-37). Furthermore, in acute lymphocytic leukemia (AML) cell lines and in patients with chronic lymphocytic leukemia, immunomodulation was shown via CD200 signaling at the tumor cell level (19,38).
It is yet unclear to what extent CD200 and its receptor CD200R may be used as a therapeutic target to affect the immune system towards antitumor response. This warrants further investigation. First in vivo and in vitro studies on CD200 blockage are on the way. First experimental data obtained from AML and breast cancer models are promising. Based on our findings, it seems worthwhile to further elucidate the role of CD200 in TCC. There is evidence that targeting the CD200-CD200R interaction, combined with chemotherapy or other immune checkpoint inhibitors, can be of therapeutic benefit in different oncological and non-oncological diseases (30). Furthermore, a clinical trial with the humanized monoclonal antibody to CD200, samalizumab, completed in 2010, showed good tolerability, dose-dependent effects on tumor growth and antitumor immune response (39). The results of an ongoing study for samalizumab in patients with advanced cancer is currently recruiting and the results are eagerly anticipated (40).
Conclusion
For the first time, CD200 expression at RNA and protein levels in different TCC cell lines and in TCC tissue were demonstrated. Immunohistochemical analyses of a clinical cohort of patients with TCC revealed CD200 expression in a wide subset of human bladder cancer with pronounced staining in poorly differentiated tumors. In particular, CD200 correlated with tumor grading and was elevated in metastatic TCC. CD200 staining was not associated with tumor recurrence or tumor stage.
Acknowledgements
The Authors thank Viktoria Wischmann for her excellent technical assistance.
Footnotes
↵* These Authors contributed equally to this study.
- Received February 23, 2018.
- Revision received March 15, 2018.
- Accepted March 22, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved