B7-H4 expression in renal cell carcinoma and tumor vasculature: Associations with cancer progression and survival

Abstract

B7-H4 is a recently described B7 family coregulatory ligand that has been implicated as an inhibitor of T cell-mediated immunity. Although expression of B7-H4 is typically limited to lymphoid cells, aberrant B7-H4 expression has also been reported in several human malignancies. To date, associations of B7-H4 with clinical outcomes for cancer patients are lacking. Therefore, we examined B7-H4 expression in fresh-frozen tumor specimens from 259 renal cell carcinoma (RCC) patients treated with nephrectomy between 2000 and 2003 and performed correlative outcome analyses. We report that 153 (59.1%) RCC tumor specimens exhibited B7-H4 staining and that tumor cell B7-H4 expression was associated with adverse clinical and pathologic features, including constitutional symptoms, tumor necrosis, and advanced tumor size, stage, and grade. Patients with tumors expressing B7-H4 were also three times more likely to die from RCC compared with patients lacking B7-H4 (risk ratio = 3.05; 95% confidence interval = 1.51–6.14; P = 0.002). Additionally, 211 (81.5%) specimens exhibited tumor vasculature endothelial B7-H4 expression, whereas only 6.5% of normal adjacent renal tissue vessels exhibited endothelial B7-H4 staining. Based on these findings, we conclude that B7-H4 has the potential to be a useful prognostic marker for patients with RCC. In addition, B7-H4 represents a target for attacking tumor cells as well as tumor neovasculature to facilitate immunotherapeutic treatment of RCC tumors. Last, we demonstrate that patients with RCC tumors expressing both B7-H4 and B7-H1 are at an even greater risk of death from RCC.

It is well established that members of the B7 family of coregulatory ligands play a central role in the positive and negative regulation of antigen-specific T cell-mediated immune responses (1). B7-H4 (also referred to as B7x or B7S1) was discovered by the laboratories of Lieping Chen, Chen Dong, and James Allison in 2003 (2–4). B7-H4 is a ligand within the B7 family that has been implicated as a negative regulator of T cell-mediated immunity (2–4). Robust B7-H4 protein expression is primarily restricted to activated T cells, B cells, monocytes, and dendritic cells. Weak or sporadic expression of B7-H4 has also been observed in some peripheral tissues, presumably surveyed from human autopsy specimens or tissues that were removed because of pathologic conditions not directly involving the organ being examined (5). Human cancers of the lung, breast, and ovary have also been shown to aberrantly overexpress the B7-H4 protein ligand (4, 6, 7).

Although the receptor for B7-H4 has yet to be identified, in vitro studies using B7-H4 transduced murine tumor cells (EL4) or B7-H4 Ig fusion protein suggest that B7-H4 may deliver an inhibitory signal to T cells, thereby abrogating CD4⁺ and CD8⁺ T cell proliferation, cell-cycle progression, and IL-2 production (2–4). Blockade of B7-H4 has also been shown to enhance the activity of cytotoxic T lymphocytes recovered from the spleens of mice after alloantigenic immunization (4). Collectively, these findings suggest that B7-H4 may function as a negative regulator of immune responses; however, these early investigations have used relatively artificial experimental conditions. Hence, the current understanding of the actual role of B7-H4 under normal and pathophysiologic conditions, especially in the clinical setting, still remains somewhat rudimentary. Further mechanistic investigations of B7-H4 are certainly warranted.

Compelling evidence that tumor-associated B7-H4 influences clinical cancer progression and patient outcome has been lacking. To date, only one study has attempted to correlate aberrant tumor expression of B7-H4 with clinical outcome. Tringler et al. (8) recently evaluated expression of B7-H4 in various forms of ovarian tumors (i.e., serous, endometrioid, clear cell, and mucinous carcinomas) and found that cytoplasmic and membranous patterns of B7-H4 staining were observed only in invasive carcinomas, not in benign lesions or tumors of low malignant potential. However, owing to the relatively limited number of patient samples investigated for each tumor subtype, tumor B7-H4 expression could not be correlated with ovarian tumor grade, stage, cancer recurrence, or survival (8).

At initial diagnosis, 30% of renal cell carcinoma (RCC) patients present with metastatic forms of disease. Another 25–30% of RCC patients appear with localized tumors that ultimately disseminate after surgical extirpation (9–11). Unfortunately, conventional radiation and chemotherapy have shown little ability to extend the 6- to 10-month median survival for patients with advanced RCC (12), with the exception of sorafenib (BAY 43-9006), a tyrosine kinase inhibitor approved recently by the Food and Drug Administration.**

RCC is an immunogenic tumor, frequently harboring high levels of tumor-infiltrating lymphocytes and occasionally exhibiting spontaneous regression of metastases after primary tumor removal (13–15). Despite these findings, only 5–10% of advanced RCC patients respond to cytokine-based immunotherapy with a durable response (16, 17). Thus, there has been significant interest in elucidating possible mechanisms whereby RCC tumors evade host antitumoral immunity.

Previous observations by our group suggest that RCC tumor aggressiveness as well as risk for cancer-specific death is substantially enhanced by aberrant tumor cell expression of negative T cell costimulatory ligands (18). Specifically, we have demonstrated that aberrant RCC expression of the negative costimulatory ligand B7-H1 has been associated with increased disease progression and decreased cancer-specific survival (18, 19). To date, however, studies pertaining to RCC tumor expression of B7-H4 have not been performed.

Herein, we report that B7-H4 is not only aberrantly expressed on RCC tumors but also preferentially expressed on the endothelium of RCC tumor vasculature (but not on normal renal tissue vessels). We also report that patients with B7-H4-positive RCC tumors exhibit more aggressive tumors and are at an increased risk of death from RCC. Furthermore, we demonstrate that patients with RCC tumors expressing both B7-H4 and B7-H1 are at an even greater risk of death from RCC. These observations collectively support the notion that RCC tumors, as well as tumor-feeding vessels, may exploit negative costimulatory molecules that collaborate to undermine host antitumoral immunity.

Results

Patients With and Without Fresh-Frozen Tissue.

Of the 531 patients who were eligible for study, 259 (49%) had fresh-frozen tissue available for laboratory investigation. None of the clinical or pathologic features studied was significantly different between patients with and without fresh-frozen tissue available for study. Furthermore, there was not a statistically significant difference in overall survival (P = 0.739) or cancer-specific survival (P = 0.780) between the two groups.

Patient Outcome.

At last follow-up, 63 of the 259 patients studied had died, including 47 patients who died from RCC at a median of 1.2 years after surgery (range of 0–4.4 years). Among the 196 patients remaining, the median duration of follow-up was 2.6 years (range of 0–5.6 years). Overall survival rates at 1, 2, and 3 years after surgery were as follows (hereafter, SE and number still at risk are shown in parentheses after the survival rate): 90.3% (1.9%, 226), 79.7% (2.7%, 148), and 73.9% (3.1%, 88), respectively. Cancer-specific survival rates at the same time points were 92.1% (1.7%, 226), 83.5% (2.5%, 148), and 79.3% (2.9%, 88), respectively. Among the subset of 215 patients with clinically localized RCC at surgery (i.e., pNX/pN0, pM0), 36 progressed to distant metastases at a median of 1.1 years after surgery (range of 0–4.9 years). Progression-free survival rates at 1, 2, and 3 years after surgery were 91.9% (1.9%, 187), 84.8% (2.6%, 125), and 81.5% (3.0%, 74), respectively.

Tumor B7-H4 Expression.

One hundred and fifty-three (59.1%) patient specimens exhibited positive tumor B7-H4 staining (Fig. 1A) with a median level of staining of 20% (range of 5–90%). A comparison of clinical and pathologic features by tumor B7-H4 expression is shown in Table 1. Positive tumor B7-H4 expression was associated with adverse clinical and pathologic features, including the presence of constitutional symptoms, larger tumor size, higher tumor stage and grade, coagulative tumor necrosis, and lymphocytic infiltration.

Fig. 1.

B7-H4 expression in normal kidney and RCC. (A) Representative RCC tumor specimen with strong membranous tumor cell and vascular B7-H4 immunohistochemical staining. (B) RCC tumor specimen with negative tumor cell but positive vascular B7-H4 endothelial staining. VL, vascular lumen. (Inset) Low-power, wide-view photomicrograph of the same tissue section. (C) Normal tumor-adjacent kidney specimen with focal and sporadic B7-H4 staining of the distal convoluted renal tubules (indicated by arrows). (D) Normal tumor-adjacent kidney specimen with no B7-H4 staining. Magnification for all photomicrographs is ×400.

Table 1.

Clinical and pathologic features by tumor B7-H4 expression

Feature	Tumor B7-H4 expression, n (%)		P
	Negative, n = 106	Positive, n = 153
Age at surgery, years
<65	55 (51.9)	81 (52.9)	0.867
≥65	51 (48.1)	72 (47.1)
Sex
Female	40 (37.7)	45 (29.4)	0.161
Male	66 (62.3)	108 (70.6)
Symptoms at presentation	49 (46.2)	86 (56.2)	0.114
Constitutional symptoms at     presentation	9 (8.5)	31 (20.3)	0.010
Primary tumor size, cm
<5	54 (50.9)	48 (31.4)	<0.001
5 to <7	25 (23.6)	28 (18.3)
7 to <10	12 (11.3)	35 (22.9)
≥10	15 (14.2)	42 (27.5)
2002 primary tumor     classification
pT1a	41 (38.7)	40 (26.1)	0.012
pT1b	32 (30.2)	29 (19.0)
pT2	11 (10.4)	28 (18.3)
pT3a	10 (9.4)	18 (11.8)
pT3b	11 (10.4)	32 (20.9)
pT3c	1 (0.9)	4 (2.6)
pT4	0 (0.0)	2 (1.3)
Regional lymph node     involvement
pNX/pN0	105 (99.1)	139 (90.9)	0.005
pN1/pN2	1 (0.9)	14 (9.1)
Distant metastases at     nephrectomy
pM0	91 (85.9)	128 (83.7)	0.632
pM1	15 (14.1)	25 (16.3)
2002 TNM stage groupings
I	69 (65.1)	68 (44.4)	0.006
II	10 (9.4)	20 (13.1)
III	12 (11.3)	39 (25.5)
IV	15 (14.2)	26 (17.0)
Nuclear grade
1	7 (6.6)	6 (3.9)	<0.001
2	53 (50.0)	33 (21.6)
3	42 (39.6)	89 (58.2)
4	4 (3.8)	25 (16.3)
Coagulative tumor necrosis	16 (15.1)	57 (37.3)	<0.001
Sarcomatoid differentiation	1 (0.9)	7 (4.6)	0.094
Lymphocytic infiltration
Absent	55 (51.9)	44 (28.8)	<0.001
Focal	37 (34.9)	47 (30.7)
Moderate	11 (10.4)	43 (28.1)
Marked	3 (2.8)	19 (12.4)

Univariately, patients with B7-H4-positive tumors were more than twice as likely to die from any cause compared with patients with B7-H4-negative tumors [risk ratio = 2.51; 95% confidence interval (C.I.) = 1.42–4.45; P = 0.002]. The overall survival rate at 3 years after surgery for patients with B7-H4-positive tumors was 66.1% (4.5%, 43) compared with 84.5% (3.9%, 45) for patients with B7-H4-negative tumors. Patients with B7-H4-positive tumors were also significantly more likely to die from RCC (risk ratio = 3.05; 95% C.I. = 1.51–6.14; P = 0.002; Fig. 2). The 3-year cancer-specific survival rates for patients with B7-H4-positive and B7-H4-negative tumors were 71.2% (4.4%, 43) and 90.5% (3.0%, 45), respectively. After adjusting for the Mayo Clinic SSIGN (stage, size, grade, and necrosis) score, patients with B7-H4-positive tumors were still nearly twice as likely to die from RCC, but this difference did not attain statistical significance (risk ratio = 1.78; 95% C.I. = 0.88–3.63; P = 0.112). Among the subset of 215 patients with clinically localized RCC at surgery, patients with B7-H4-positive tumors were three times more likely to progress compared with patients with B7-H4-negative tumors (risk ratio = 2.99; 95% C.I. = 1.36–6.57; P = 0.006). The 3-year progression-free survival rate for patients with B7-H4-positive tumors was 74.1% (4.5%, 34) compared with 91.2% (3.2%, 40) for patients with B7-H4-negative tumors.

Fig. 2.

Association of tumor B7-H4 expression with cancer-specific survival for 259 patients with clear cell RCC. The cancer-specific survival rates at 1, 2, and 3 years after nephrectomy were 88.4% (2.6%, 127), 78.5% (3.6%, 83), and 71.2% (4.4%, 43), respectively, for patients with B7-H4-positive tumors compared with 97.1% (1.6%, 99), 90.5% (3.0%, 65), and 90.5% (3.0%, 45), respectively, for patients with B7-H4-negative tumors (P = 0.002).

Combination of Tumor B7-H1 and B7-H4 Expression.

There were 59 (22.8%) tumors that were both B7-H1-negative and B7-H4-negative, 59 (22.8%) that were B7-H1-negative and B7-H4-positive, 47 (18.2%) that were B7-H1-positive and B7-H4-negative, and 94 (36.3%) that were both B7-H1-positive and B7-H4-positive. Tumors that were B7-H1-positive were more likely to be B7-H4-positive compared with tumors that were B7-H1-negative (66.7% versus 50.0%; P = 0.007).

When combined in a model, positive tumor B7-H1 expression (risk ratio = 2.63; 95% C.I. = 1.42–4.86; P = 0.002) and positive tumor B7-H4 expression (risk ratio = 2.21; 95% C.I. = 1.24–3.93; P = 0.007) were independently associated with death from any cause. This finding was also true for the associations of positive B7-H1 expression (risk ratio = 3.95; 95% C.I. = 1.76–8.85; P < 0.001) and positive B7-H4 expression (risk ratio = 2.57; 95% C.I. = 1.27–5.20; P = 0.009) with death from RCC. After adjusting for the SSIGN (stage, size, grade, and necrosis) score, the risk ratios for the associations of positive tumor B7-H1 and B7-H4 expression with death from RCC were 4.61 (95% C.I. = 2.02–10.53; P < 0.001) and 1.59 (95% C.I. = 0.79–3.23; P = 0.196), respectively. The 3-year cancer-specific survival rates for patients with B7-H1-negative and B7-H4-negative tumors, B7-H1-negative and B7-H4-positive tumors, B7-H1-positive and B7-H4-negative tumors, and B7-H1-positive and B7-H4-positive tumors were 94.0%, 92.3%, 86.6%, and 60.9%, respectively. Cancer-specific survival rates did not differ significantly among patients in the first three groups (P = 0.308). However, cancer-specific survival was significantly lower for patients with B7-H1-positive and B7-H4-positive tumors compared with patients in the other three groups (P < 0.001; Fig. 3). Patients with B7-H1-positive and B7-H4-positive tumors were more than four times more likely to die from RCC compared with patients with negative or singly positive tumors (risk ratio = 4.49; 95% C.I. = 2.40–8.39; P < 0.001; Fig. 4), a difference which persisted even after adjusting for the SSIGN score (risk ratio = 3.69; 95% C.I. = 1.95–6.98; P < 0.001). In fact, 33 of the 47 patients who died from RCC had tumors that were positive for both B7-H1 and B7-H4. Among the subset of 215 patients with clinically localized RCC at surgery, patients with B7-H1-positive and B7-H4-positive tumors were significantly more likely to progress to distant metastases compared with patients with negative or singly positive tumors (risk ratio = 2.58; 95% C.I. = 1.34–4.99; P = 0.005).

Fig. 3.

Association of combined tumor B7-H1 and B7-H4 expression with cancer-specific survival for 259 patients with clear cell RCC. The cancer-specific survival rates at 1, 2, and 3 years after nephrectomy were 85.9% (3.6%, 77), 70.9% (5.0%, 52), and 60.9% (5.8%, 27), respectively, for patients with B7-H1-positive and B7-H4-positive tumors compared with 98.3% (1.7%, 54), 94.0% (3.4%, 32), and 94.0% (3.4%, 19), respectively, for patients with B7-H1 and B7-H4 negative tumors and 94.1% (2.3%, 95), 89.5% (3.2%, 64), and 89.5% (3.2%, 42), respectively, for patients with singly positive tumors.

Fig. 4.

Association of combined tumor B7-H1 and B7-H4 expression with cancer-specific survival for 259 patients with clear cell RCC. The cancer-specific survival rates at 1, 2, and 3 years after nephrectomy were 85.9% (3.6%, 77), 70.9% (5.0%, 52), and 60.9% (5.8%, 27), respectively, for patients with B7-H1-positive and B7-H4-positive tumors compared with 95.6% (1.6%, 149), 91.1% (2.4%, 96), and 91.1% (2.4%, 61), respectively, for patients with negative or singly positive tumors (P < 0.001).

A comparison of clinical and pathologic features by the combination of tumor B7-H1 and B7-H4 expression is shown in Table 2. Patients with B7-H1-positive and B7-H4-positive tumors were significantly more likely to exhibit adverse clinical and pathologic features, including symptoms at presentation, larger tumor size, higher tumor stage and grade, tumor necrosis, sarcomatoid differentiation, and lymphocytic infiltration.

Table 2.

Clinical and pathologic features by combined tumor B7-H1 and B7-H4 expression

Feature	B7-H1-positive and B7-H4- positive, n (%)		P
	No, n = 165	Yes, n = 94
Age at surgery, years
<65	91 (55.2)	45 (47.9)	0.259
≥65	74 (44.8)	49 (52.1)
Sex
Female	54 (32.7)	31 (33.0)	0.967
Male	111 (67.3)	63 (67.0)
Symptoms at presentation	78 (47.3)	57 (60.6)	0.038
Constitutional symptoms at     presentation	17 (10.3)	23 (24.5)	0.002
Primary tumor size, cm
<5	76 (46.1)	26 (27.7)	<0.001
5 to <7	41 (24.9)	12 (12.8)
7 to <10	21 (12.7)	26 (27.7)
≥10	27 (16.4)	30 (31.9)
2002 primary tumor     classification
pT1a	62 (37.6)	19 (20.2)	<0.001
pT1b	46 (27.9)	15 (16.0)
pT2	16 (9.7)	23 (24.5)
pT3a	15 (9.1)	13 (13.8)
pT3b	24 (14.6)	19 (20.2)
pT3c	2 (1.2)	3 (3.2)
pT4	0 (0.0)	2 (2.1)
Regional lymph node     involvement
pNX/pN0	161 (97.6)	83 (88.3)	0.002
pN1/pN2	4 (2.4)	11 (11.7)
Distant metastases at     nephrectomy
pM0	143 (86.7)	76 (80.9)	0.213
pM1	22 (13.3)	18 (19.1)
2002 TNM stage groupings
I	103 (62.4)	34 (36.2)	<0.001
II	14 (8.5)	16 (17.0)
III	26 (15.8)	25 (26.6)
IV	22 (13.3)	19 (20.2)
Nuclear grade
1	12 (7.3)	1 (1.1)	<0.001
2	71 (43.0)	15 (16.0)
3	72 (43.6)	59 (62.8)
4	10 (6.1)	19 (20.2)
Coagulative tumor necrosis	32 (19.4)	41 (43.6)	<0.001
Sarcomatoid differentiation	2 (1.2)	6 (6.4)	0.029
Lymphocytic infiltration
Absent	77 (46.7)	22 (23.4)	<0.001
Focal	64 (38.8)	20 (21.3)
Moderate	19 (11.5)	35 (37.2)
Marked	5 (3.0)	17 (18.1)

Tumor and Normal Vasculature B7-H4 and B7-H1 Expression.

We found that 211 (81.5%) cases exhibited B7-H4 endothelial expression within the tumor vasculature (Fig. 1B), with a median level of expression of 50% (range of 5–100%). Almost all (148/153, 96.7%) patients with B7-H4-positive tumors also exhibited B7-H4 endothelial staining within the tumor vasculature. Of the 106 patients with B7-H4-negative tumors, 63 (59.4%) exhibited B7-H4 endothelial staining within the tumor vasculature. None of the specimens demonstrated B7-H1 staining within the tumor vasculature.

We also randomly selected 46 patients from the 259 under study who had available fresh-frozen tissue from normal kidney adjacent to the tumor. Of these, only three (6.5%) specimens exhibited B7-H4 staining in the normal vasculature. Twenty-six (56.5%) specimens exhibited sporadic B7-H4 staining in the distal tubules (Fig. 1C), and the remaining specimens demonstrated no B7-H4 staining (Fig. 1D).

Discussion

This report provides evidence of B7-H4 expression by clear cell carcinoma of the kidney, which represents the most common form of renal malignancy (20). We demonstrate that B7-H4 expression by RCC tumors is associated with aggressive tumor behavior. The main goal of our study was to evaluate the association of B7-H4 with death from RCC, and, in fact, we demonstrate a diminished patient survival in patients with tumors expressing B7-H4. We also show that two independent but related potential T cell inhibitory molecules, B7-H4 and B7-H1, collaborate as predictive markers for the assessment of cancer progression and death from RCC. Moreover, we provide evidence that B7-H4 is preferentially expressed by the endothelium of tumor vasculature but not by vessels in normal renal tissues. We believe that, with further investigation, B7-H4 could become an established clinical prognostic marker and a target for immunotherapeutic treatment of RCC tumors.

Although B7-H4 mRNA has been noted in a number of nonlymphoid organs (3), it was originally believed that protein expression was restricted to activated lymphoid cells (4, 6, 7). Recent reports, however, indicate focal and infrequent protein expression in certain somatic tissues, including distal convoluted tubules of the kidney, ductal and acinar cells of the pancreas, endometrial glands, transitional epithelia of the ureter and bladder, bronchial epithelium of the lung, and columnar epithelium of the gallbladder (5). Additionally, B7-H4 has been found to be aberrantly expressed at high levels in human serous, endometrioid, and clear cell ovarian carcinoma; ductal and lobular breast cancer; and lung cancer (5, 6, 21). This overexpression of B7-H4 by malignant tissues may render B7-H4 a particularly useful target for facilitating antitumoral immunotherapeutic responses.

Despite the lack of a known receptor, early investigations, although limited in scope, suggest that B7-H4 may act as a negative regulator of T cell responses (2–4). Separate studies have indicated a direct role for B7-H4 in preventing tumor cell apoptosis. Specifically, overexpression of B7-H4 in human ovarian cancer cell lines has been reported to enhance tumor formation in SCID (severe combined immunodeficiency) mice. Conversely, short interfering RNA-mediated knockdown of B7-H4 mRNA and protein expression in the SKBR3 breast cancer cell line enhanced intracellular caspase activity, leading to acceleration of tumor cell apoptosis (7). Thus, B7-H4 may function through two distinct mechanisms: (i) as a negative regulator of T cell-mediated antitumoral immunity and (ii) by rendering tumor cells refractory to apoptosis. Both of these mechanisms would promote aggressive tumor activity and facilitate disease progression. Clearly, however, the current understanding of B7-H4’s precise role in regulating cell survival as well as T cell-mediated responses remains in its relative infancy, especially within the context of normal physiologic or pathologic conditions in the clinical setting.

Our current study demonstrates that clear cell RCC tumors of the kidney express B7-H4. Sporadic expression of B7-H4 was also noted in some distal convoluted tubules of the kidney, as previously reported (5). In contrast, proximal tubules of the kidney, from which clear cell RCC tumors originate, were devoid of B7-H4 staining (22–24). Tumor B7-H4 expression was also associated with lymphocytic infiltration, which portends a poorer clinical outcome for patients with RCC (25). In addition, we observed that B7-H4 is associated with an increased risk of death from RCC and a higher risk for disease progression in patients with pathologically localized renal tumors in univariate analysis. The combined expression of B7-H4 and B7-H1 [a second negative coregulatory molecule previously found to be associated with RCC death (18)] within the same tumor was associated with an increased risk of death from RCC beyond the risk of either molecule alone. This risk persisted in a multivariate setting. Taken together, our clinical observations suggest that B7-H4 acts to inhibit antitumoral immunity or extend tumor cell survival, which is consistent with the existing literature pertaining to what is known about B7-H4 function.

As in our prior B7-H1 studies (18, 19), the association of tumor expression of B7-H4 with RCC progression and patient survival may have important implications for future therapy. Systemic IL-2 immunotherapy was the first Food and Drug Administration-approved treatment for metastatic RCC. This form of therapy confers a palliative benefit to only 15–20% of patients with advanced RCC (24, 26). Because it appears that both B7-H1 and B7-H4 impair T cell function [B7-H4 by limiting T cell proliferation (8) and B7-H1 by induction of T cell apoptosis (27–30)], one might infer that the presence of these two T cell-inhibitory ligands within RCC tumors could act to constrain responses to IL-2 therapy, which is thought to nonspecifically stimulate antitumoral immunity. As a corollary to this, we surmise that assessment of combined B7-H1 and B7-H4 expression within RCC tumors may be useful in discriminating between patients who are most likely to benefit from immunotherapy versus alternate forms of systemic therapy.

Additionally, our studies suggest that B7-H4 may contribute to RCC tumorogenesis by acting at sites somewhat distant to the tumor cells themselves. Specifically, 81.5% of tumors examined in this series expressed B7-H4 within the endothelium of the tumor vasculature. In contrast, 6.5% of tumor-adjacent “normal” renal vessels were observed to express B7-H4, and such vessels may in fact represent either efferent or afferent vessels feeding the tumor. This observation suggests a potential role for B7-H4 in facilitating or maintaining RCC tumor neovascularization, but the mechanism whereby this promotion might occur remains unknown. Regardless, our studies suggest that B7-H4 may encompass a unique vascular therapeutic target for facilitating the treatment of RCC and, perhaps, other tumors.

We acknowledge several limitations to our current study. We have not yet developed a reliable method to immunohistochemically stain formalin-fixed, paraffin-embedded tissues for B7-H4 expression, which limited our immunohistochemical analyses to fresh-frozen RCC specimens that were collected at our institution since January 2000. Consequently, patient follow-up in our present study was somewhat attenuated, with a maximum of 5 years. With only 47 deaths from RCC, the statistical power of our study is limited. Nevertheless, we did observe that patients with B7-H4-positive RCC tumors were three times as likely to die from RCC. Although this association was not statistically significant in multivariate analysis, we anticipate that with additional patients and longer follow-up, statistical significance will be achieved. Despite the limited scope of our study, we did observe a nearly 4-fold increase in the risk of death from RCC for patients whose tumors stained positive for both B7-H1 and B7-H4. This association remained statistically significant even after multivariate analysis. As such, it appears that negative T cell coregulatory ligands such as B7-H1 and B7-H4 may collaborate to impair antitumoral immune function, thereby promoting aggressive tumor progression in patients with RCC.

Conclusion

B7-H4 expression in RCC tumors of the kidney represents a predictor of tumor aggressiveness and risk of death from RCC. With further study, B7-H4 may prove useful as an independent predictor of clinical RCC outcome. A collaborative effect was also noted when both B7-H4 and B7-H1 expression was present in the same tumor. The basis for these associations may represent inhibition of T cell immunity by B7-H4 and B7-H1 leading to less restricted tumor progression. Furthermore, we noted B7-H4 staining of tumor vasculature, which represents a promising target for future antitumoral immunotherapy.

Materials and Methods

Patient Selection.

Upon approval from the Mayo Clinic Institutional Review Board, we identified from the Mayo Clinic Nephrectomy Registry 531 patients who had been treated with radical nephrectomy or nephron-sparing surgery for unilateral, sporadic, noncystic clear cell RCC between 2000 and 2003. Because pathologic features and patient outcome differ by RCC subtype, all analyses were restricted to patients with clear cell RCC, the most common of the RCC subtypes (31). In addition, patients were selected based on the availability of fresh-frozen tissue because, to date, the human B7-H4-specific monoclonal antibody (clone hH4.1) has been applied to stain only fresh-frozen tissues, not formalin-fixed, paraffin-embedded tissues, for immunohistochemical analysis.

Clinical and Pathologic Features.

The clinical features studied included age, sex, and symptoms. Patients with a palpable flank or abdominal mass, discomfort, gross hematuria, acute-onset varicocele, or constitutional symptoms (including rash, sweats, weight loss, fatigue, early satiety, and anorexia) were considered symptomatic at presentation. The pathologic features studied included histologic subtype, tumor size, the 2002 primary tumor classification (T), regional lymph node involvement (N), distant metastases at nephrectomy (M), the 2002 TNM stage groupings, nuclear grade, coagulative tumor necrosis, sarcomatoid differentiation, and lymphocytic infiltration (assessed as absent, focal, moderate, or markedly present). Histologic subtype was classified according to the Union Internationale Contre le Cancer, American Joint Committee on Cancer, and Heidelberg guidelines (32, 33). These features were obtained by a review of the microscopic slides from all nephrectomy specimens by a urologic pathologist (J.C.C.) without knowledge of patient outcome.

B7-H4 Immunohistochemical Staining.

Cryosections from RCC tumors (5 μm thick, −20°C) were mounted on Superfrost Plus slides, air-dried, and fixed in ice-cold acetone. Sections were stained by using the DAKO Autostainer and DAKO Cytomation CSA II kit. Slides were blocked with H₂O₂ for 5 min and then incubated with the antibody applied for 30 min at room temperature. Anti-mouse Ig-horseradish peroxidase (HRP) was then applied at room temperature for 15 min, followed by incubation with amplification reagent for 15 min. Next, slides were incubated for 15 min with anti-fluorescein-HRP and visualized with diaminobenzidine substrate for 8 min. Finally, sections were counterstained for 1 min with hematoxylin. The antibody used for this protocol was mouse anti-human B7-H4 monoclonal antibody (clone hH4.1). Human ovarian cancer tissue was used as a positive control. Irrelevant isotype-matched antibodies were used to control for nonspecific staining.

B7-H1 Immunohistochemical Staining.

B7-H1 immunohistochemical staining was performed as described in ref. 18.

Quantification of B7-H1 and B7-H4 Expression.

The percentages of tumor cells that stained positive for B7-H1 and B7-H4 were quantified in 5% increments by a urologic pathologist (J.C.C.). The tumor was considered positive if there was histologic evidence of cell-surface membrane staining. Tumor staining was generally moderate to strong; cases with <5% tumor staining were considered negative.

Statistical Methods.

Comparisons of B7-H4 and B7-H1 tumor expression with clinical and pathologic features were evaluated by using χ² and Fisher’s exact tests. Overall, cancer-specific, and progression-free survivals were estimated by using the Kaplan–Meier method. The duration of follow-up was calculated from the date of surgery to the date of cancer progression (i.e., distant metastases), death, or last known follow-up. Cause of death was determined from the death certificate or physician correspondence. The associations of B7-H1 and B7-H4 tumor expression with death from any cause, death from RCC, and cancer progression were evaluated by using Cox proportional hazards regression models. Associations with death from RCC were further adjusted for the SSIGN (stage, size, grade, and necrosis) score, a prognostic composite score specifically developed for patients with clear cell RCC (34). These associations were summarized by using risk ratios and 95% C.I.s. Statistical analyses were performed by using the sas software package (SAS Institute, Cary, NC). All tests were two-sided, and P < 0.05 was considered statistically significant.

Abbreviations

RCC

renal cell carcinoma

C.I.

confidence interval.