B7-H4 expression in renal cell carcinomaand tumor vasculature: Associations withcancer progression and survivalAmy E. Krambeck*, R. Houston Thompson*, Haidong Dong†, Christine M. Lohse‡, Eugene S. Park*, Susan M. Kuntz†,Bradley C. Leibovich*, Michael L. Blute*, John C. Cheville§¶, and Eugene D. Kwon*†¶�

Departments of *Urology, †Immunology, ‡Health Sciences Research, and §Laboratory Medicine and Pathology, Mayo Medical School, Mayo Clinic,Rochester, MN 55905

Edited by James P. Allison, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved May 19, 2006 (received for review February 3, 2006)

B7-H4 is a recently described B7 family coregulatory ligand that hasbeen 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 severalhuman malignancies. To date, associations of B7-H4 with clinicaloutcomes for cancer patients are lacking. Therefore, we examinedB7-H4 expression in fresh-frozen tumor specimens from 259 renalcell carcinoma (RCC) patients treated with nephrectomy between2000 and 2003 and performed correlative outcome analyses. Wereport that 153 (59.1%) RCC tumor specimens exhibited B7-H4staining and that tumor cell B7-H4 expression was associated withadverse clinical and pathologic features, including constitutionalsymptoms, tumor necrosis, and advanced tumor size, stage, andgrade. Patients with tumors expressing B7-H4 were also threetimes more likely to die from RCC compared with patients lackingB7-H4 (risk ratio � 3.05; 95% confidence interval � 1.51–6.14; P �0.002). Additionally, 211 (81.5%) specimens exhibited tumor vas-culature endothelial B7-H4 expression, whereas only 6.5% ofnormal adjacent renal tissue vessels exhibited endothelial B7-H4staining. Based on these findings, we conclude that B7-H4 has thepotential to be a useful prognostic marker for patients with RCC.In addition, B7-H4 represents a target for attacking tumor cells aswell as tumor neovasculature to facilitate immunotherapeutictreatment of RCC tumors. Last, we demonstrate that patients withRCC tumors expressing both B7-H4 and B7-H1 are at an evengreater risk of death from RCC.

B7-H1 � costimulation � tumor biomarker � immunotherapy �kidney neoplasms

I t is well established that members of the B7 family of coregu-latory 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 bythe laboratories of Lieping Chen, Chen Dong, and James Allisonin 2003 (2–4). B7-H4 is a ligand within the B7 family that hasbeen implicated as a negative regulator of T cell-mediatedimmunity (2–4). Robust B7-H4 protein expression is primarilyrestricted to activated T cells, B cells, monocytes, and dendriticcells. Weak or sporadic expression of B7-H4 has also beenobserved in some peripheral tissues, presumably surveyed fromhuman autopsy specimens or tissues that were removed becauseof pathologic conditions not directly involving the organ beingexamined (5). Human cancers of the lung, breast, and ovary havealso been shown to aberrantly overexpress the B7-H4 proteinligand (4, 6, 7).

Although the receptor for B7-H4 has yet to be identified, invitro studies using B7-H4 transduced murine tumor cells (EL4)or B7-H4 Ig fusion protein suggest that B7-H4 may deliver aninhibitory 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 theactivity of cytotoxic T lymphocytes recovered from the spleens

of mice after alloantigenic immunization (4). Collectively, thesefindings suggest that B7-H4 may function as a negative regulatorof immune responses; however, these early investigations haveused relatively artificial experimental conditions. Hence, thecurrent understanding of the actual role of B7-H4 under normaland pathophysiologic conditions, especially in the clinical setting,still remains somewhat rudimentary. Further mechanistic inves-tigations of B7-H4 are certainly warranted.

Compelling evidence that tumor-associated B7-H4 influencesclinical cancer progression and patient outcome has been lacking.To date, only one study has attempted to correlate aberrant tumorexpression of B7-H4 with clinical outcome. Tringler et al. (8)recently evaluated expression of B7-H4 in various forms of ovariantumors (i.e., serous, endometrioid, clear cell, and mucinous carci-nomas) and found that cytoplasmic and membranous patterns ofB7-H4 staining were observed only in invasive carcinomas, not inbenign lesions or tumors of low malignant potential. However,owing to the relatively limited number of patient samples investi-gated for each tumor subtype, tumor B7-H4 expression could notbe 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. Another25–30% of RCC patients appear with localized tumors thatultimately disseminate after surgical extirpation (9–11). Unfor-tunately, conventional radiation and chemotherapy have shownlittle ability to extend the 6- to 10-month median survival forpatients with advanced RCC (12), with the exception of sor-afenib (BAY 43-9006), a tyrosine kinase inhibitor approvedrecently by the Food and Drug Administration.**

RCC is an immunogenic tumor, frequently harboring highlevels of tumor-infiltrating lymphocytes and occasionally exhib-iting spontaneous regression of metastases after primary tumorremoval (13–15). Despite these findings, only 5–10% of ad-vanced RCC patients respond to cytokine-based immunotherapywith a durable response (16, 17). Thus, there has been significantinterest in elucidating possible mechanisms whereby RCC tu-mors evade host antitumoral immunity.

Previous observations by our group suggest that RCC tumoraggressiveness as well as risk for cancer-specific death is sub-

Conflict of interest statement: The authors and institution have a potential financial con-flict of interest in that they have applied for patents pertaining to B7-H4 as a biomarker forcancer therapy.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: RCC, renal cell carcinoma; C.I., confidence interval.

¶J.C.C. and E.D.K. contributed equally to this work.

�To whom correspondence should be addressed at: Departments of Urology and Immunology,Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: kwon.eugene@mayo.edu.

**Escudier, B., Szczylik, C., Eisen, T., Stadler, W. M., Schwartz, B., Shan, M. & Bukowski, R. M.(2005) American Society of Clinical Oncologists Annual Meeting, May 13–17, 2005,Orlando, FL, abstr. 4510.

© 2006 by The National Academy of Sciences of the USA

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stantially enhanced by aberrant tumor cell expression of negativeT cell costimulatory ligands (18). Specifically, we have demon-strated that aberrant RCC expression of the negative costimu-latory ligand B7-H1 has been associated with increased diseaseprogression and decreased cancer-specific survival (18, 19). Todate, however, studies pertaining to RCC tumor expression ofB7-H4 have not been performed.

Herein, we report that B7-H4 is not only aberrantly expressedon RCC tumors but also preferentially expressed on the endo-thelium of RCC tumor vasculature (but not on normal renaltissue vessels). We also report that patients with B7-H4-positiveRCC tumors exhibit more aggressive tumors and are at anincreased risk of death from RCC. Furthermore, we demon-strate that patients with RCC tumors expressing both B7-H4 andB7-H1 are at an even greater risk of death from RCC. Theseobservations collectively support the notion that RCC tumors, aswell as tumor-feeding vessels, may exploit negative costimula-tory molecules that collaborate to undermine host antitumoralimmunity.

ResultsPatients With and Without Fresh-Frozen Tissue. Of the 531 patientswho were eligible for study, 259 (49%) had fresh-frozen tissueavailable for laboratory investigation. None of the clinical orpathologic features studied was significantly different betweenpatients with and without fresh-frozen tissue available for study.Furthermore, there was not a statistically significant differencein 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 studiedhad died, including 47 patients who died from RCC at a medianof 1.2 years after surgery (range of 0–4.4 years). Among the 196patients remaining, the median duration of follow-up was 2.6years (range of 0–5.6 years). Overall survival rates at 1, 2, and3 years after surgery were as follows (hereafter, SE and numberstill 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 timepoints were 92.1% (1.7%, 226), 83.5% (2.5%, 148), and 79.3%(2.9%, 88), respectively. Among the subset of 215 patients withclinically localized RCC at surgery (i.e., pNX�pN0, pM0), 36progressed to distant metastases at a median of 1.1 years aftersurgery (range of 0–4.9 years). Progression-free survival rates at1, 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%). Acomparison of clinical and pathologic features by tumor B7-H4expression is shown in Table 1. Positive tumor B7-H4 expressionwas associated with adverse clinical and pathologic features,including the presence of constitutional symptoms, larger tumorsize, higher tumor stage and grade, coagulative tumor necrosis,and lymphocytic infiltration.

Univariately, patients with B7-H4-positive tumors were morethan twice as likely to die from any cause compared with patientswith B7-H4-negative tumors [risk ratio � 2.51; 95% confidenceinterval (C.I.) � 1.42–4.45; P � 0.002]. The overall survival rateat 3 years after surgery for patients with B7-H4-positive tumorswas 66.1% (4.5%, 43) compared with 84.5% (3.9%, 45) forpatients with B7-H4-negative tumors. Patients with B7-H4-positive tumors were also significantly more likely to die fromRCC (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 withB7-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 aslikely to die from RCC, but this difference did not attainstatistical significance (risk ratio � 1.78; 95% C.I. � 0.88–3.63;P � 0.112). Among the subset of 215 patients with clinicallylocalized RCC at surgery, patients with B7-H4-positive tumorswere three times more likely to progress compared with patientswith B7-H4-negative tumors (risk ratio � 2.99; 95% C.I. �1.36–6.57; P � 0.006). The 3-year progression-free survival ratefor 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.

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. Tu-mors 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 positivetumor B7-H4 expression (risk ratio � 2.21; 95% C.I. � 1.24–3.93; P � 0.007) were independently associated with death fromany cause. This finding was also true for the associations ofpositive B7-H1 expression (risk ratio � 3.95; 95% C.I. �1.76–8.85; P � 0.001) and positive B7-H4 expression (riskratio � 2.57; 95% C.I. � 1.27–5.20; P � 0.009) with death fromRCC. After adjusting for the SSIGN (stage, size, grade, andnecrosis) score, the risk ratios for the associations of positivetumor B7-H1 and B7-H4 expression with death from RCC were4.61 (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-specificsurvival 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-positiveand B7-H4-positive tumors were 94.0%, 92.3%, 86.6%, and

Fig. 1. B7-H4 expression in normal kidney and RCC. (A) Representative RCCtumor specimen with strong membranous tumor cell and vascular B7-H4immunohistochemical staining. (B) RCC tumor specimen with negative tumorcell 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 stain-ing of the distal convoluted renal tubules (indicated by arrows). (D) Normaltumor-adjacent kidney specimen with no B7-H4 staining. Magnification for allphotomicrographs is �400.

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60.9%, respectively. Cancer-specific survival rates did not differsignificantly among patients in the first three groups (P � 0.308).However, cancer-specific survival was significantly lower forpatients with B7-H1-positive and B7-H4-positive tumors com-pared with patients in the other three groups (P � 0.001; Fig. 3).Patients with B7-H1-positive and B7-H4-positive tumors weremore than four times more likely to die from RCC comparedwith 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 whichpersisted 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 47patients who died from RCC had tumors that were positive forboth B7-H1 and B7-H4. Among the subset of 215 patients withclinically localized RCC at surgery, patients with B7-H1-positiveand B7-H4-positive tumors were significantly more likely toprogress to distant metastases compared with patients withnegative or singly positive tumors (risk ratio � 2.58; 95% C.I. �1.34–4.99; P � 0.005).

A comparison of clinical and pathologic features by thecombination of tumor B7-H1 and B7-H4 expression is shown inTable 2. Patients with B7-H1-positive and B7-H4-positive tu-mors were significantly more likely to exhibit adverse clinical andpathologic features, including symptoms at presentation, largertumor size, higher tumor stage and grade, tumor necrosis,sarcomatoid differentiation, and lymphocytic infiltration.

Table 1. Clinical and pathologic features by tumorB7-H4 expression

Feature

Tumor B7-H4expression, n (%)

PNegative,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)

SexFemale 40 (37.7) 45 (29.4) 0.161Male 66 (62.3) 108 (70.6)

Symptoms at presentation 49 (46.2) 86 (56.2) 0.114Constitutional symptoms at

presentation9 (8.5) 31 (20.3) 0.010

Primary tumor size, cm�5 54 (50.9) 48 (31.4) �0.0015 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 tumorclassification

pT1a 41 (38.7) 40 (26.1) 0.012pT1b 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 nodeinvolvement

pNX�pN0 105 (99.1) 139 (90.9) 0.005pN1�pN2 1 (0.9) 14 (9.1)

Distant metastases atnephrectomy

pM0 91 (85.9) 128 (83.7) 0.632pM1 15 (14.1) 25 (16.3)

2002 TNM stage groupingsI 69 (65.1) 68 (44.4) 0.006II 10 (9.4) 20 (13.1)III 12 (11.3) 39 (25.5)IV 15 (14.2) 26 (17.0)

Nuclear grade1 7 (6.6) 6 (3.9) �0.0012 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) �0.001Sarcomatoid differentiation 1 (0.9) 7 (4.6) 0.094Lymphocytic infiltration

Absent 55 (51.9) 44 (28.8) �0.001Focal 37 (34.9) 47 (30.7)Moderate 11 (10.4) 43 (28.1)Marked 3 (2.8) 19 (12.4)

Fig. 2. Association of tumor B7-H4 expression with cancer-specific survivalfor 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), and71.2% (4.4%, 43), respectively, for patients with B7-H4-positive tumors com-pared with 97.1% (1.6%, 99), 90.5% (3.0%, 65), and 90.5% (3.0%, 45), respec-tively, for patients with B7-H4-negative tumors (P � 0.002).

Fig. 3. Association of combined tumor B7-H1 and B7-H4 expression withcancer-specific survival for 259 patients with clear cell RCC. The cancer-specificsurvival 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-H1and B7-H4 negative tumors and 94.1% (2.3%, 95), 89.5% (3.2%, 64), and89.5% (3.2%, 42), respectively, for patients with singly positive tumors.

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Tumor and Normal Vasculature B7-H4 and B7-H1 Expression. Wefound that 211 (81.5%) cases exhibited B7-H4 endothelialexpression within the tumor vasculature (Fig. 1B), with a medianlevel of expression of 50% (range of 5–100%). Almost all(148�153, 96.7%) patients with B7-H4-positive tumors alsoexhibited B7-H4 endothelial staining within the tumor vascula-ture. Of the 106 patients with B7-H4-negative tumors, 63(59.4%) exhibited B7-H4 endothelial staining within the tumorvasculature. None of the specimens demonstrated B7-H1 stain-ing within the tumor vasculature.

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

DiscussionThis report provides evidence of B7-H4 expression by clear cellcarcinoma of the kidney, which represents the most commonform of renal malignancy (20). We demonstrate that B7-H4expression by RCC tumors is associated with aggressive tumorbehavior. The main goal of our study was to evaluate theassociation of B7-H4 with death from RCC, and, in fact, wedemonstrate a diminished patient survival in patients withtumors expressing B7-H4. We also show that two independentbut related potential T cell inhibitory molecules, B7-H4 andB7-H1, collaborate as predictive markers for the assessment ofcancer progression and death from RCC. Moreover, we provideevidence that B7-H4 is preferentially expressed by the endothe-lium of tumor vasculature but not by vessels in normal renaltissues. We believe that, with further investigation, B7-H4 couldbecome an established clinical prognostic marker and a targetfor immunotherapeutic treatment of RCC tumors.

Although B7-H4 mRNA has been noted in a number of non-lymphoid organs (3), it was originally believed that protein expres-sion was restricted to activated lymphoid cells (4, 6, 7). Recentreports, however, indicate focal and infrequent protein expressionin certain somatic tissues, including distal convoluted tubules of thekidney, ductal and acinar cells of the pancreas, endometrial glands,

transitional epithelia of the ureter and bladder, bronchial epithe-lium of the lung, and columnar epithelium of the gallbladder (5).Additionally, B7-H4 has been found to be aberrantly expressed athigh levels in human serous, endometrioid, and clear cell ovariancarcinoma; ductal and lobular breast cancer; and lung cancer (5, 6,21). This overexpression of B7-H4 by malignant tissues may renderB7-H4 a particularly useful target for facilitating antitumoral im-munotherapeutic responses.

Despite the lack of a known receptor, early investigations,although limited in scope, suggest that B7-H4 may act as a

Fig. 4. Association of combined tumor B7-H1 and B7-H4 expression withcancer-specific survival for 259 patients with clear cell RCC. The cancer-specificsurvival 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 neg-ative or singly positive tumors (P � 0.001).

Table 2. Clinical and pathologic features by combined tumorB7-H1 and B7-H4 expression

Feature

B7-H1-positive and B7-H4-positive, n (%)

PNo, 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)

SexFemale 54 (32.7) 31 (33.0) 0.967Male 111 (67.3) 63 (67.0)

Symptoms at presentation 78 (47.3) 57 (60.6) 0.038Constitutional symptoms at

presentation17 (10.3) 23 (24.5) 0.002

Primary tumor size, cm�5 76 (46.1) 26 (27.7) �0.0015 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 tumorclassification

pT1a 62 (37.6) 19 (20.2) �0.001pT1b 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 nodeinvolvement

pNX�pN0 161 (97.6) 83 (88.3) 0.002pN1�pN2 4 (2.4) 11 (11.7)

Distant metastases atnephrectomy

pM0 143 (86.7) 76 (80.9) 0.213pM1 22 (13.3) 18 (19.1)

2002 TNM stage groupingsI 103 (62.4) 34 (36.2) �0.001II 14 (8.5) 16 (17.0)III 26 (15.8) 25 (26.6)IV 22 (13.3) 19 (20.2)

Nuclear grade1 12 (7.3) 1 (1.1) �0.0012 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) �0.001Sarcomatoid differentiation 2 (1.2) 6 (6.4) 0.029Lymphocytic infiltration

Absent 77 (46.7) 22 (23.4) �0.001Focal 64 (38.8) 20 (21.3)Moderate 19 (11.5) 35 (37.2)Marked 5 (3.0) 17 (18.1)

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negative regulator of T cell responses (2–4). Separate studieshave indicated a direct role for B7-H4 in preventing tumor cellapoptosis. Specifically, overexpression of B7-H4 in human ovar-ian cancer cell lines has been reported to enhance tumorformation in SCID (severe combined immunodeficiency) mice.Conversely, short interfering RNA-mediated knockdown ofB7-H4 mRNA and protein expression in the SKBR3 breastcancer cell line enhanced intracellular caspase activity, leadingto acceleration of tumor cell apoptosis (7). Thus, B7-H4 mayfunction through two distinct mechanisms: (i) as a negativeregulator of T cell-mediated antitumoral immunity and (ii) byrendering tumor cells refractory to apoptosis. Both of thesemechanisms would promote aggressive tumor activity and facil-itate disease progression. Clearly, however, the current under-standing of B7-H4’s precise role in regulating cell survival as wellas T cell-mediated responses remains in its relative infancy,especially within the context of normal physiologic or pathologicconditions in the clinical setting.

Our current study demonstrates that clear cell RCC tumors ofthe kidney express B7-H4. Sporadic expression of B7-H4 wasalso noted in some distal convoluted tubules of the kidney, aspreviously reported (5). In contrast, proximal tubules of thekidney, from which clear cell RCC tumors originate, were devoidof B7-H4 staining (22–24). Tumor B7-H4 expression was alsoassociated with lymphocytic infiltration, which portends a poorerclinical outcome for patients with RCC (25). In addition, weobserved that B7-H4 is associated with an increased risk of deathfrom RCC and a higher risk for disease progression in patientswith pathologically localized renal tumors in univariate analysis.The combined expression of B7-H4 and B7-H1 [a secondnegative coregulatory molecule previously found to be associ-ated with RCC death (18)] within the same tumor was associatedwith an increased risk of death from RCC beyond the risk ofeither molecule alone. This risk persisted in a multivariatesetting. Taken together, our clinical observations suggest thatB7-H4 acts to inhibit antitumoral immunity or extend tumor cellsurvival, which is consistent with the existing literature pertain-ing to what is known about B7-H4 function.

As in our prior B7-H1 studies (18, 19), the association of tumorexpression of B7-H4 with RCC progression and patient survivalmay have important implications for future therapy. SystemicIL-2 immunotherapy was the first Food and Drug Administra-tion-approved treatment for metastatic RCC. This form oftherapy confers a palliative benefit to only 15–20% of patientswith advanced RCC (24, 26). Because it appears that both B7-H1and B7-H4 impair T cell function [B7-H4 by limiting T cellproliferation (8) and B7-H1 by induction of T cell apoptosis(27–30)], one might infer that the presence of these two Tcell-inhibitory ligands within RCC tumors could act to constrainresponses to IL-2 therapy, which is thought to nonspecificallystimulate antitumoral immunity. As a corollary to this, wesurmise that assessment of combined B7-H1 and B7-H4 expres-sion within RCC tumors may be useful in discriminating betweenpatients who are most likely to benefit from immunotherapyversus alternate forms of systemic therapy.

Additionally, our studies suggest that B7-H4 may contribute toRCC tumorogenesis by acting at sites somewhat distant to thetumor cells themselves. Specifically, 81.5% of tumors examinedin this series expressed B7-H4 within the endothelium of thetumor vasculature. In contrast, 6.5% of tumor-adjacent ‘‘nor-mal’’ renal vessels were observed to express B7-H4, and suchvessels may in fact represent either efferent or afferent vesselsfeeding the tumor. This observation suggests a potential role forB7-H4 in facilitating or maintaining RCC tumor neovascular-ization, but the mechanism whereby this promotion might occurremains unknown. Regardless, our studies suggest that B7-H4may encompass a unique vascular therapeutic target for facili-tating the treatment of RCC and, perhaps, other tumors.

We acknowledge several limitations to our current study. Wehave not yet developed a reliable method to immunohistochemi-cally stain formalin-fixed, paraffin-embedded tissues for B7-H4expression, which limited our immunohistochemical analyses tofresh-frozen RCC specimens that were collected at our institu-tion since January 2000. Consequently, patient follow-up in ourpresent study was somewhat attenuated, with a maximum of 5years. With only 47 deaths from RCC, the statistical power of ourstudy is limited. Nevertheless, we did observe that patients withB7-H4-positive RCC tumors were three times as likely to diefrom RCC. Although this association was not statistically sig-nificant in multivariate analysis, we anticipate that with addi-tional patients and longer follow-up, statistical significance willbe achieved. Despite the limited scope of our study, we didobserve a nearly 4-fold increase in the risk of death from RCCfor patients whose tumors stained positive for both B7-H1 andB7-H4. This association remained statistically significant evenafter multivariate analysis. As such, it appears that negative Tcell coregulatory ligands such as B7-H1 and B7-H4 may collab-orate to impair antitumoral immune function, thereby promotingaggressive tumor progression in patients with RCC.

ConclusionB7-H4 expression in RCC tumors of the kidney represents apredictor of tumor aggressiveness and risk of death from RCC.With further study, B7-H4 may prove useful as an independentpredictor of clinical RCC outcome. A collaborative effect wasalso noted when both B7-H4 and B7-H1 expression was presentin the same tumor. The basis for these associations may representinhibition of T cell immunity by B7-H4 and B7-H1 leading to lessrestricted tumor progression. Furthermore, we noted B7-H4staining of tumor vasculature, which represents a promisingtarget for future antitumoral immunotherapy.

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

Clinical and Pathologic Features. The clinical features studiedincluded age, sex, and symptoms. Patients with a palpable flankor abdominal mass, discomfort, gross hematuria, acute-onsetvaricocele, or constitutional symptoms (including rash, sweats,weight loss, fatigue, early satiety, and anorexia) were consideredsymptomatic at presentation. The pathologic features studiedincluded histologic subtype, tumor size, the 2002 primary tumorclassification (T), regional lymph node involvement (N), distantmetastases at nephrectomy (M), the 2002 TNM stage groupings,nuclear grade, coagulative tumor necrosis, sarcomatoid differ-entiation, and lymphocytic infiltration (assessed as absent, focal,moderate, or markedly present). Histologic subtype was classi-fied according to the Union Internationale Contre le Cancer,American Joint Committee on Cancer, and Heidelberg guide-lines (32, 33). These features were obtained by a review of themicroscopic slides from all nephrectomy specimens by a urologicpathologist (J.C.C.) without knowledge of patient outcome.

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B7-H4 Immunohistochemical Staining. Cryosections from RCC tu-mors (5 �m thick, �20°C) were mounted on Superfrost Plusslides, air-dried, and fixed in ice-cold acetone. Sections werestained by using the DAKO Autostainer and DAKO CytomationCSA II kit. Slides were blocked with H2O2 for 5 min and thenincubated with the antibody applied for 30 min at room tem-perature. Anti-mouse Ig-horseradish peroxidase (HRP) wasthen applied at room temperature for 15 min, followed byincubation with amplification reagent for 15 min. Next, slideswere incubated for 15 min with anti-f luorescein-HRP and visu-alized with diaminobenzidine substrate for 8 min. Finally, sec-tions were counterstained for 1 min with hematoxylin. Theantibody used for this protocol was mouse anti-human B7-H4monoclonal antibody (clone hH4.1). Human ovarian cancertissue was used as a positive control. Irrelevant isotype-matchedantibodies were used to control for nonspecific staining.

B7-H1 Immunohistochemical Staining. B7-H1 immunohistochemi-cal staining was performed as described in ref. 18.

Quantification of B7-H1 and B7-H4 Expression. The percentages oftumor cells that stained positive for B7-H1 and B7-H4 werequantified in 5% increments by a urologic pathologist (J.C.C.).The tumor was considered positive if there was histologicevidence of cell-surface membrane staining. Tumor staining wasgenerally moderate to strong; cases with �5% tumor stainingwere considered negative.

Statistical Methods. Comparisons of B7-H4 and B7-H1 tumorexpression with clinical and pathologic features were evaluatedby using �2 and Fisher’s exact tests. Overall, cancer-specific, andprogression-free survivals were estimated by using the Kaplan–Meier method. The duration of follow-up was calculated fromthe date of surgery to the date of cancer progression (i.e., distantmetastases), death, or last known follow-up. Cause of death wasdetermined from the death certificate or physician correspon-dence. The associations of B7-H1 and B7-H4 tumor expressionwith death from any cause, death from RCC, and cancerprogression were evaluated by using Cox proportional hazardsregression models. Associations with death from RCC werefurther adjusted for the SSIGN (stage, size, grade, and necrosis)score, a prognostic composite score specifically developed forpatients with clear cell RCC (34). These associations weresummarized by using risk ratios and 95% C.I.s. Statisticalanalyses were performed by using the SAS software package(SAS Institute, Cary, NC). All tests were two-sided, and P � 0.05was considered statistically significant.

We thank Dr. Lieping Chen (The Johns Hopkins University School ofMedicine, Baltimore) for providing the B7-H4 monoclonal antibody(clone hH4.1). This work was supported in part by generous gifts fromthe Richard M. Schulze Family Foundation and the CommonwealthFoundation for Cancer Research and by donations from the Helen andMartin Kimmel Foundation.

1. Carreno, B. M. & Collins, M. (2003) Trends Immunol. 24, 524–527.2. Zang, X., Loke, P., Kim, J., Murphy, K., Waitz, R. & Allison, J. P. (2003) Proc.

Natl. Acad. Sci. USA 100, 10388–10392.3. Prasad, D. V., Richards, S., Mai, X. M. & Dong, C. (2003) Immunity 18, 863–873.4. Sica, G. L., Choi, I. H., Zhu, G., Tamada, K., Wang, S. D., Tamura, H.,

Chapoval, A. I., Flies, D. B., Bajorath, J. & Chen, L. (2003) Immunity 18,849–861.

5. Tringler, B., Zhuo, S., Pilkington, G., Torkko, K. C., Singh, M., Lucia, M. S., Heinz,D. E., Papkoff, J. & Shroyer, K. R. (2005) Clin. Cancer Res. 11, 1842–1848.

6. Choi, I. H., Zhu, G., Sica, G. L., Strome, S. E., Cheville, J. C., Lau, J. S., Zhu,Y., Flies, D. B., Tamada, K. & Chen, L. (2003) J. Immunol. 171, 4650–4654.

7. Salceda, S., Tang, T., Kmet, M., Munteanu, A., Ghosh, M., Macina, R., Liu, W.,Pilkington, G. & Papkoff, J. (2005) Exp. Cell Res. 306, 128–141.

8. Tringler, B., Liu, W., Corral, L., Torkko, K. C., Enomoto, T., Davidson, S.,Lucia, M. S., Heinz, D. E., Papkoff, J. & Shroyer, K. R. (2006) Gynecol. Oncol.100, 44–52.

9. Curti, B. D. (2004) J. Am. Med. Assoc. 292, 97–100.10. Levy, D. A., Slaton, J. W., Swanson, D. A. & Dinney, C. P. (1998) J. Urol. 159,

1163–1167.11. Ljungberg, B., Alamdari, F. I., Rasmuson, T. & Roos, G. (1999) BJU Int. 84,

405–411.12. Figlin, R. A., Pierce, W. C., Kaboo, R., Tso, C. L., Molawer, N., Gitlitz, B.,

deKernion, J. & Belldegrun, A. (1997) J. Urol. 158, 740–750.13. Bromwich, E. J., McArdle, P. A., Canna, K., McMillan, D. C., McNicol, A. M.,

Brown, M. & Aitchison, M. (2003) Br. J. Cancer 89, 1906–1908.14. Nakano, O., Sato, M., Naito, Y., Suzuki, K., Orikasa, S., Aizawa, M., Suzuki,

Y., Shintaku, I., Nagura, H. & Ohtani, H. (2001) Cancer Res. 61, 5132–5136.15. Couillard, D. R. & DeVere-White, R. W. (1993) Urol. Clin. North Am. 20, 263–275.16. Yang, J. C., Sherry, R. M., Steinberg, S. M., Topalian, S. L., Schwarzentruber,

A. J., Hwu, P., Seipp, C. A., Rogers-Freezer, L., Morton, K. E., White, D. E.,et al. (2003) J. Clin. Oncol. 21, 3127–3132.

17. Fyfe, G., Fisher, R. I., Rosenberg, S. A., Sznol, M., Parkinson, D. R. & Louie,A. C. (1995) J. Clin. Oncol. 13, 688–696.

18. Thompson, R. H., Gillett, M. D., Cheville, J. C., Lohse, C. M., Dong, H.,Webster, W. S., Krejci, K. G., Lobo, J. R., Sengupta, S., Chen, L., et al. (2004)Proc. Natl. Acad. Sci. USA 101, 17174–17179.

19. Thompson, T. 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., et al.(2005) Cancer 104, 2084–2091.

20. Pantuck, A. J., Zisman, A. & Belldegrun, A. S. (2001) J. Urol. 166, 1611–1623.21. Greenwald, R. J., Freeman, G. J. & Sharpe, A. H. (2005) Annu. Rev. Immunol.

23, 515–548.22. Bander, N. H., Finstad, C. L., Cordon-Cardo, C., Ramsawak, R. D., Vaughan,

E. D., Jr., Whitmore, W. F., Jr., Oettgen, H. F., Melamed, M. R. & Old, L. J.(1989) Cancer Res. 49, 6774–6780.

23. van den Berg, E., van der Hout, A. H., Oosterhuis, J. W., Storkel, S., Djikhui-zen, T., Dam, A., Zweers, H. M., Mensink, H. J., Buys, C. H. & de Jong, B.(1993) Int. J. Cancer 55, 223–227.

24. Rini, B. I., Winberg, V. & Small, E. J. (2004) J. Urol. 171, 2115–2121.25. Webster, W. S., Lohse, C. M., Thompson, R. H., Dong, H., Frigola, X., Dicks,

D. L., Sengupta, S., Frank, I., Leibovich, B. C., Blute, M. L., et al. (2006)Cancer, in press.

26. Motzer, R. J. (2003) Crit. Rev. Oncol. Hematol. 46S, 33–39.27. Dong, H., Strome, S. E., Salomao, D. R., Tamura, H., Hirano, F., Flies, D. B.,

Roche, P. C., Lu, J., Zhu, G., Tamada, K., et al. (2002) Nat. Med. 8, 793–800.28. Iwai, Y., Ishida, M., Tanaka, Y., Okazaki, T., Honjo, T. & Minato N. (2002)

Proc. Natl. Acad. Sci. USA 99, 12293–12297.29. Wintterle, S., Schreiner, B., Mitsdoerffer, M., Schneider, D., Chen, L., Mey-

ermann, R., Weller, M. & Wiendl, H. (2003) Cancer Res. 63, 7462–7467.30. Blank, C., Brown, I., Peterson, A. C., Spiotto, M., Iwai, Y., Honjo, T. &

Gajewski, T. F. (2004) Cancer Res. 64, 1140–1145.31. Cheville, J. C., Lohse, C. M., Zincke, H., Weaver, A. L. & Blute, M. L. (2003)

Am. J. Surg. Pathol. 27, 612–624.32. Storkel, S., Eble, J. N., Adlakha, K., Amin, M., Blute, M. L., Bostwick, D. G.,

Darson, N., Delahunt, B. & Iczkowski, K. (1997) Cancer 80, 987–989.33. Kovacs, G., Akhtar, M., Beckwith, B. J., Bugert, P., Cooper, C. S., Delahunt,

B., Eble, J. N., Fleming, S., Ljungberg, B., Medeiros, L. J., et al. (1997) J. Pathol.183, 131–133.

34. Frank, I., Blute, M. L., Cheville, J.-C., Lohse, C. M., Weaver, A. L. & Zincke,H. (2002) J. Urol. 168, 2395–2400.

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