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Costimulatory molecule B7-H1 in primary and metastatic clear cell renal cell carcinoma
Article first published online: 5 OCT 2005
Copyright © 2005 American Cancer Society
Volume 104, Issue 10, pages 2084–2091, 15 November 2005
How to Cite
Thompson, R. H., Gillett, M. D., Cheville, J. C., Lohse, C. M., Dong, H., Webster, W. S., Chen, L., Zincke, H., Blute, M. L., Leibovich, B. C. and Kwon, E. D. (2005), Costimulatory molecule B7-H1 in primary and metastatic clear cell renal cell carcinoma. Cancer, 104: 2084–2091. doi: 10.1002/cncr.21470
- Issue published online: 31 OCT 2005
- Article first published online: 5 OCT 2005
- Manuscript Accepted: 9 JUN 2005
- Manuscript Revised: 28 APR 2005
- Manuscript Received: 22 FEB 2005
- renal cell carcinoma;
- neoplasm metastasis;
- T lymphocyte;
- kidney neoplasms;
- tumor biomarker
Cancer cell expression of costimulatory molecule B7-H1 has been implicated as a potent inhibitor of T-cell–mediated antitumoral immunity. The authors recently reported that B7-H1 is aberrantly expressed in primary renal cell carcinoma (RCC). Blockade of B7-H1, as demonstrated in several murine cancer models, now represents a promising therapeutic target in RCC. However, the potential expression of B7-H1 in metastatic RCC has not been investigated. In the current study, the authors updated their primary RCC results with additional follow-up and investigated the potential role of B7-H1 in metastatic RCC.
Between 2000 and 2004, 196 patients underwent nephrectomy and 26 patients had resection of RCC metastases for clear cell RCC. Immunohistochemical analysis was performed on tumor cryosections using a B7-H1 monoclonal antibody (clone 5H1). A urologic pathologist quantified the percentage of B7-H1–positive tumor cells and lymphocytes.
Variable levels of B7-H1 were expressed on primary RCC tumor cells (n = 130 [66.3%]) and primary tumor-infiltrating lymphocytes (n = 115 [58.7%]). Patients with high expression of B7-H1 on primary tumor cells and/or lymphocytes were significantly more likely to die of RCC compared with patients with low B7-H1 expression (risk ratio [RR] = 4.17; 95% confidence interval [95% CI], 1.97–8.84; P < 0.001) and this risk persisted in multivariate analysis after adjusting for the Mayo Clinic stage, size, grade, and necrosis score (RR = 2.63; 96% CI, 1.23–5.64; P = 0.013). Of the 26 metastatic specimens, cancer cell and lymphocyte B7-H1 expression were demonstrated in 17 (65.4%) and 18 (69.2%) specimens, respectively. In total, 14 (54.3%) metastatic specimens had high aggregate B7-H1 levels compared with 44.4% in primary RCC specimens.
Patients with RCC with high B7-H1 expression were significantly more likely to die even after multivariate analysis. The authors also demonstrated that a high percentage of RCC metastases similarly harbored B7-H1. The authors surmised that B7-H1 blockade may augment current immunotherapy, including patients treated for metastases after cytoreductive nephrectomy. Cancer 2005. © 2005 American Cancer Society.
Costimulatory molecules deliver positive and negative signals to modulate the threshold of T-cell activation. Related to this, costimulatory molecule B7-H1 has recently been implicated as a potent negative regulator of T-cell–mediated immunity, functioning in the periphery to inhibit antitumoral immune responses. In humans, cell surface B7-H1 expression is normally restricted to monocyte-derived cells, although aberrant expression has been described in a number of human malignancies.1–3 Tumor cell expression of B7-H1 has been shown to enhance apoptosis of activated tumor-specific T cells in vitro.1 Similarly, it has been demonstrated that induced B7-H1 expression on activated T cells impairs both T-cell function and survival,4 and expression of B7-H1 on myeloid dendritic cells associated with ovarian carcinoma has been found to suppress T-cell activation.5 Furthermore, in vivo monoclonal antibody (MoAb) blockade of B7-H1 has been shown to potentiate antitumoral responses in several murine cancer models.2, 6–8 Thus, B7-H1, along with other negative regulators of T-cell activation, may contribute to the profile of immunosuppression observed in some tumors and represent promising oncologic immunotherapeutic targets.
Renal cell carcinoma (RCC) tumors frequently harbor high levels of tumor-infiltrating lymphocytes.9, 10 In addition, favorable treatment responses have been demonstrated in subsets of patients receiving cytokine-based immunotherapy.11 Taken together, these observations implicate RCC as an immunogenic form of cancer that is amenable to immune-based therapy. However, the potential mechanism whereby RCC impairs host immunity facilitating subsequent tumor progression is poorly understood. We recently demonstrated that B7-H1 is aberrantly expressed in human RCC.12 In addition, we found that high levels of B7-H1 are associated with adverse pathologic features and a diminished cancer-specific survival in patients with RCC.12 However, the risk of death was not significant in multivariate analysis after adjusting for the Mayo Clinic SSIGN (stage, size, grade, and necrosis) score. We now report that with additional follow-up, patients with RCC harboring high B7-H1 levels are indeed at significantly increased risk of death even after multivariate adjustment.
The potential association of B7-H1 in metastatic RCC, however, remains unknown. Most patients receiving immunotherapy for metastatic RCC have undergone previous cytoreductive nephrectomy. Although we have shown that B7-H1 is aberrantly expressed in primary RCC, we hypothesized that B7-H1 expressed in metastatic deposits may play a critical role in facilitating tumor progression by impairing immune recognition. We now report that metastatic RCC deposits express B7-H1 similar to primary RCC tumors and are therefore potentially amenable to anti–B7-H1 immunotherapy after primary tumor extirpation.
MATERIALS AND METHODS
After approval was obtained from the Mayo Clinic institutional review board, we identified 196 patients treated with radical nephrectomy or nephron-sparing surgery for unilateral, sporadic clear cell RCC between 2000 and 2002. In addition, 26 patients underwent surgical resection of clear cell RCC metastatic deposits between 2000 and 2004. All patients had fresh-frozen tissue sections available as we have previously demonstrated that the human B7-H1–specific MoAb, 5H1, can reproducibly stain fresh-frozen but not paraffin-embedded tissue sections during immunohistochemical analysis.1
The pathologic features evaluated included histologic subtype, tumor size, the 2002 primary tumor classification, regional lymph node involvement and distant metastases at nephrectomy, nuclear grade, and histologic coagulative tumor necrosis. The microscopic slides from all specimens were reviewed by a urologic pathologist (J.C.C.) without previous knowledge of patient outcome. Histologic subtype was classified according to the International Union Against Cancer, the American Joint Committee on Cancer, and Heidelberg guidelines.13, 14 Nuclear grade was assigned using standardized criteria as previously described.15 Tumor necrosis was defined as the presence of any microscopic coagulative tumor necrosis. Degenerative changes such as hyalinization, hemorrhage, and fibrosis were not considered necrosis.
Immunohistochemical Staining of Tumor Specimens
Cryosections generated from RCC tumors and metastatic RCC deposits were mounted on Superfrost Plus slides, air dried, and fixed in ice-cold acetone. Specimens were stained with the mouse anti-human B7-H1–specific MoAb 5H1 as previously described.12
Quantification of B7-H1 Expression
The percentage of B7-H1 expression on both tumor cells and lymphocytes was quantified in 5–10% increments. The extent of lymphocytic infiltration was assessed and recorded as absent, focal, moderate, or marked. An adjusted score representing lymphocyte B7-H1 expression was calculated as the percentage of lymphocytes that stained positive for B7-H1 multiplied by the extent of lymphocytic infiltration (0, absent; 1, focal; 2, moderate; and 3, marked).
Comparisons among pathologic features and B7-H1 expression were evaluated using the chi-square and the Fisher exact tests. Cancer-specific survival was estimated using the Kaplan–Meier method. Cause of death was determined from the death certificate or physician correspondence and deaths of causes other than RCC were censored. Scatter plots of the percentage of cells that stained positive for B7-H1 versus the difference in observed survival and the survival expected from a Cox proportional hazards regression model (formally known as a martingale residual) were used to identify potential cut points for B7-H1 expression. The associations of these cut points with death from RCC were evaluated using Cox proportional hazards regression models univariately and after adjusting for the pathologic features, one at a time, in multivariate models. The few deaths from RCC observed during the course of the study precluded a multivariate model that included both B7-H1 expression and all of the pathologic features of interest simultaneously. However, the association of B7-H1 expression with death from RCC was also evaluated after adjusting for the Mayo Clinic SSIGN score, a prognostic composite score specifically developed for patients with clear cell RCC that combines the information from important pathologic features into a single score.16 Statistical analyses were performed using the SAS software package (SAS Institute Inc., Cary, NC) and P < 0.05 was considered to be statistically significant.
Follow-Up for the 196 Patients with Primary RCC
At last follow-up, 45 of the 196 patients studied had died, including 37 patients who died of clear cell RCC at a median of 1.2 years after nephrectomy (range, 0–3.5 yrs). Among the 151 patients who were still alive at the time of last follow-up, the median duration of follow-up was 2.7 years (range, 0–4.4 yrs). Compared with our previous report on primary RCC,12 this represents nearly 1 additional year of follow-up, resulting in 7 subsequent cancer-specific deaths. The estimated cancer-specific survival rates (standard error, number still at risk) at 1, 2, and 3 years after nephrectomy were 91.7% (2.0%, 174), 83.2% (2.8%, 115), and 77.5% (3.5%, 53), respectively.
Primary Tumor-Associated B7-H1 Expression
Among the primary RCC specimens, 130 (66.3%) demonstrated aberrant tumor-associated B7-H1 expression (Fig. 1). A scatter plot of tumor B7-H1 expression versus the expected risk of death suggested that a cut point of 10% would be appropriate, and 73 (37.2%) specimens had ≥ 10% tumor B7-H1 expression. The association of tumor B7-H1 expression with death from RCC is shown in Table 1. Univariately, patients with specimens that had ≥ 10% tumor B7-H1 expression were significantly more likely to die of RCC compared with patients with specimens that had < 10% expression (risk ratio [RR] = 2.87; 95% confidence interval [95% CI], 1.47–5.57; P = 0.002) (Fig. 2). In multivariate analyses, patients with specimens that had ≥ 10% tumor B7-H1 expression remained significantly more likely to die of RCC even after adjusting for tumor size, primary tumor classification, or distant metastases. In addition, patients with ≥ 10% tumor B7-H1 expression were nearly twice as likely to die of RCC even after adjusting for the Mayo Clinic SSIGN score, although this did not attain statistical significance (RR = 1.94; 95% CI, 0.99–3.81; P = 0.055).
|Characteristics||Risk ratio (95% CI)||P value|
|Tumor B7-H1 expression ≥ 10%|
|Univariate model||2.87 (1.47–5.57)||0.002|
|Multivariate model adjusted for:|
|2002 primary tumor classification (T)||3.01 (1.54–5.87)||0.001|
|Regional lymph node involvement (N)||1.90 (0.91–3.97)||0.090|
|Distant metastases (M)||2.46 (1.26–4.82)||0.008|
|Primary tumor size||2.67 (1.37–5.20)||0.004|
|Nuclear grade||1.74 (0.89–3.40)||0.104|
|Coagulative tumor necrosis||1.88 (0.95–3.72)||0.071|
|Clear cell SSIGN score||1.94 (0.99–3.81)||0.055|
|Lymphocyte B7-H1 expression ≥ 100|
|Univariate model||3.35 (1.75–6.43)||< 0.001|
|Multivariate model adjusted for:|
|2002 primary tumor classification (T)||3.25 (1.69–6.26)||< 0.001|
|Regional lymph node involvement (N)||3.75 (1.94–7.24)||< 0.001|
|Distant metastases (M)||2.07 (1.05–4.07)||0.035|
|Primary tumor size||2.41 (1.25–4.65)||0.009|
|Nuclear grade||2.37 (1.23–4.56)||0.010|
|Coagulative tumor necrosis||2.61 (1.35–5.04)||0.004|
|Clear cell SSIGN score||2.19 (1.13–4.24)||0.020|
|High-aggregate intratumoral B7-H1-expression|
|Univariate model||4.17 (1.97–8.84)||< 0.001|
|Multivariate model adjusted for:|
|2002 primary tumor classification (T)||3.93 (1.85–8.34)||< 0.001|
|Regional lymph node involvement (N)||3.04 (1.37–6.70)||0.006|
|Distant metastases (M)||3.18 (1.49–6.82)||0.003|
|Primary tumor size||3.56 (1.68–7.56)||< 0.001|
|Nuclear grade||2.24 (1.05–4.78)||0.038|
|Coagulative tumor necrosis||2.65 (1.22–5.73)||0.014|
|Clear cell SSIGN score||2.63 (1.23–5.64)||0.013|
Primary Lymphocyte-Associated B7-H1 Expression
Among the primary RCC specimens, 115 (58.6%) patients had tumor-infiltrating lymphocytes. All patients with tumor-infiltrating lymphocytes had lymphocyte-associated B7-H1 expression (Fig. 3). There were 40 (20.4%) specimens with an adjusted lymphocyte B7-H1 score of ≥ 100, which appeared to be a reasonable cut point to evaluate the association of this feature with patient outcome. Univariately, patients with specimens that had an adjusted lymphocyte B7-H1 score ≥ 100 were significantly more likely to die of RCC compared with patients who had specimens with scores < 100 (RR = 3.35; 95% CI, 1.75–6.43; P < 0.001) (Fig. 4). In multivariate analysis, patients with specimens that demonstrated high levels of lymphocyte B7-H1 expression were significantly more likely to die of RCC even after adjusting for other pathologic features predictive of outcome (Table 1). In addition, patients with an adjusted lymphocyte B7-H1 score ≥ 100 remained at significant risk of death from RCC even after adjusting for the Mayo Clinic SSIGN score (RR = 2.19; 95% CI, 1.13–4.24; P = 0.020).
Primary Tumor and Lymphocyte-Associated B7-H1 Expression
There were 87 (44.4%) specimens that had either ≥ 10% tumor B7-H1 expression or an adjusted lymphocyte score ≥ 100 (high aggregate B7-H1 expression). Both features were present in 26 (13.3%) specimens. Patients with high aggregate B7-H1 expression were significantly more likely to have regional lymph node involvement, distant metastases, advanced nuclear grade, and tumor necrosis compared with those with < 10% tumor expression and < 100 lymphocyte expression of B7-H1 (Table 2). Univariately, patients with high aggregate B7-H1 expression were greater than 4 times more likely to die of RCC compared with patients with specimens that had both < 10% tumor expression and < 100 lymphocyte expression (RR = 4.17; 95% CI, 1.97–8.84; P < 0.001) (Fig. 5). In multivariate analysis, patients with specimens that demonstrated high aggregate B7-H1 expression were significantly more likely to die of RCC even after adjusting for other pathologic features predictive of outcome (Table 1). In addition, the significant association with death persisted after adjusting for the Mayo Clinic SSIGN score (RR = 2.63; 95% CI, 1.23–5.64; P = 0.013). Furthermore, patients with high aggregate B7-H1 expression were significantly more likely to die of any cause when compared with patients with specimens that had both < 10% tumor expression and < 100 lymphocyte expression (RR = 3.72; 95% CI, 1.92–7.21; P < 0.001).
|Feature||High aggregate intratumoral B7-H1 expression (%)||P value|
|No (n = 109)||Yes (n = 87)|
|2002 primary tumor classification|
|pT1 and pT2||88 (80.7)||62 (71.3)||0.120|
|pT3 and pT4||21 (19.3)||25 (28.7)|
|Regional lymph node involvement|
|pNx and pN0||108 (99.1)||76 (87.4)||< 0.001|
|pN1 and pN2||1 (0.9)||11 (12.6)|
|pM0||99 (90.8)||69 (79.3)||0.022|
|pM1||10 (9.2)||18 (20.7)|
|Primary tumor size|
|< 5 cm||46 (42.2)||25 (28.7)||0.051|
|≥ 5 cm||63 (57.8)||62 (71.3)|
|1 and 2||69 (63.3)||23 (26.4)||< 0.001|
|3||36 (33.0)||50 (57.5)|
|4||4 (3.7)||14 (16.1)|
|Coagulative tumor necrosis|
|Absent||94 (86.2)||55 (63.2)||< 0.001|
|Present||15 (13.8)||32 (36.8)|
Metastatic RCC B7-H1 Expression
Twenty-six patients had surgical resection of metastatic clear cell RCC from 14 different sites, most commonly from the adrenal gland (5), followed by bone (4), and lung (3). Other areas of metastatic resection included liver, pancreas, stomach, testicle, spinal cord, and skin. A total of 17 (65.4%) specimens exhibited cancer cell expression of B7-H1, including 4 of the 5 adrenal metastases. Lymphocytic infiltration was demonstrated in 18 specimens, with all 18 exhibiting lymphocyte-associated B7-H1 expression. Similar to primary RCC, 100% of tumors associated with lymphocytic infiltration also demonstrated lymphocytic B7-H1 expression. One patient was included in both the primary and metastatic cohorts with tumor-associated B7-H1 present in 80% and 90% of their primary and metastatic (lung) resection, respectively. In total, 14 (53.9%) metastatic specimens had high aggregate B7-H1 levels expressed by cancer cells or lymphocytes (Fig. 6), comparable to the 44.4% demonstrated in primary RCC.
To our knowledge, B7-H1 is the first T-cell costimulatory molecule associated with a diminished cancer-specific survival not only in patients with RCC, but in any solid malignancy. Moreover, we present novel evidence that patients with RCC harboring high levels of B7-H1 are at a significantly increased risk of death even after multivariate analysis. In addition, the current study provides previously undescribed evidence that B7-H1 is present clinically in both primary and metastatic RCC, potentially fostering cancer progression through impairment of host T-cell–mediated immunity. We believe that B7-H1 may be utilized as a biomarker to identify subsets of patients with RCC most likely to derive benefit from immunotherapy, perhaps when integrated with other pathologic indices such as TNM classification, tumor grade, and coagulative tumor necrosis. We also surmise that B7-H1 blockade represents a promising immunotherapeutic target in the multimodal treatment of patients with RCC.
Discovered by Dong et al. in 1999, B7-H1 is a cell surface glycoprotein belonging to the B7 family of costimulatory molecules and participates in the activation of naive T cells and deletion of activated T cells.17, 18 In humans, cell surface B7-H1 expression is normally restricted to a fraction of macrophage-lineage cells and is not present in the normal human kidney.1 In contrast, several different human cancers have now been reported to aberrantly express B7-H1.1–3 The mechanism for tumor cell expression is believed to be related to impaired gene regulation at the posttranscriptional level.8, 19 Tumor-associated B7-H1 has been shown to inhibit tumor-specific T-cell–mediated immunity, through binding to the T-cell PD-1 (or a putative non–PD-1) receptor, inducing T-cell apoptosis, impairing cytokine production, and diminishing the cytotoxicity of activated T cells.1, 7, 20, 21 Similarly, it has been shown that activated T cells also express B7-H1, functioning to downregulate primed T-cell responses through the induction of apoptosis or inhibition of T-cell clonal expansion.4 Consistent with these observations, blockade of tumor-associated B7-H1 has been shown to potentiate antitumoral T-cell responses directed against both artificially transfected and endogenously expressed B7-H1–positive tumors in mice.1, 2, 6–8 Thus, based on its recognized ability to impair the function and survival of activated tumor-specific T cells, B7-H1 may play a critical role in fostering tumor progression in a setting of impaired host immune surveillance.
We now provide evidence that both primary and metastatic clear cell carcinomas of the kidney are capable of expressing B7-H1. We previously reported that patients with high aggregate B7-H1 in primary RCC were at significant risk of death, although statistical significance was not maintained after adjusting for the Mayo Clinic SSIGN score.12 The SSIGN score is a composite index developed specifically for patients with clear cell RCC utilizing pathologic features predictive of death in a multivariate model.16 It encompasses TNM classification, tumor size, nuclear grade, and coagulative tumor necrosis to generate a prognostic score for prognosis and follow-up surveillance. The significance of these pathologic features was recently internationally validated by Ficarra et al.22 in a multivariate model. With additional deaths and longer follow-up, we now report that patients with high aggregate B7-H1 in primary RCC remain at a significantly increased risk of death from RCC even after adjusting for the Mayo Clinic SSIGN score (RR = 2.63; 95% CI, 1.23–5.64; P = 0.013). Although it is possible that with additional follow-up B7-H1 may no longer be significantly associated with death, we believe our data represent compelling evidence that B7-H1 may be functioning at the clinical level, promoting cancer progression through impairment of host T-cell–mediated immunity.
Our observation that intratumoral B7-H1 potentially facilitates tumor progression in primary RCC may have important implications to the immunotherapeutic treatment of RCC tumors. Currently, cytokine-based immunotherapy, adoptive transfer of tumor-specific activated T cells, and autologous tumor cell vaccinations have been extensively studied in RCC, albeit with limited clinical success. Consistent with these observations, several studies have reported a defective antitumoral immunity in patients with RCC.23–29 In addition, a paradoxical relation between increased levels of tumor-infiltrating T cells and diminished survival for patients with RCC has recently been reported.9, 10 Taken together, these observations implicate an in vivo microenvironment of immune resistance present in subsets of patients with RCC. However, the mechanisms by which RCC evades immune surveillance are only beginning to be understood. Our study provides a potential explanation for the profile of immunosuppression observed in some patients with RCC and may help explain how primary RCC tumor cells confer resistance to both innate and augmented immunotherapeutic attacks.
Currently, most patients with metastatic RCC undergo cytoreductive nephrectomy before initiation of immunotherapy, essentially eliminating previously reported areas of aberrant B7-H1 expression. We now report that RCC metastatic deposits similarly express B7-H1 on both tumor cells and infiltrating lymphocytes. Although the clinical effect of B7-H1 expression in metastatic RCC remains to be determined, we speculate that similar to primary RCC, B7-H1 modulates the threshold of T-cell activation. In addition, we surmise that B7-H1 in metastatic RCC potentially abrogates nonspecific stimulatory immunotherapy, contributing to the modest (15–20%) complete and partial responses with interleukin-2 and interferon-alpha. Importantly, B7-H1 is amenable to MoAb blockade. As such, blockade of B7-H1 may augment the efficacy of established immunotherapy and may provide appropriate protection for effector lymphocytes in patients with RCC, including those who have undergone previous nephrectomy.
Currently, high expression of B7-H1 in primary RCC is associated with aggressive tumors that are at a significantly increased risk of death even after multivariate adjustments. In addition, B7-H1 is expressed in metastatic RCC deposits similar to primary RCC. Based on these observations, we surmise that B7-H1 expressed within RCC tumors may serve to facilitate tumor progression by undermining host antitumoral T-cell–mediated immunity. Thus, B7-H1 may represent a novel target for antitumoral immunotherapy and a valuable prognostic marker to predict outcome and treatment responses.