SIMULTANEOUS EXPRESSION OF T-CELL ACTIVATING ANTIGENS IN RENAL CELL CARCINOMA: IMPLICATIONS FOR SPECIFIC IMMUNOTHERAPY

0022-5347/04/1716-2456/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 171, 2456 –2460, June 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000118383.86684.38

SIMULTANEOUS EXPRESSION OF T-CELL ACTIVATING ANTIGENS IN RENAL CELL CARCINOMA: IMPLICATIONS FOR SPECIFIC IMMUNOTHERAPY ¨ LLER, ARIANE SCHENK, HANSPETER KIRSCHE, MARK RINGHOFFER, CARLHEINZ R. MU ¨ MICHAEL SCHMITT, JOCHEN GREINER AND JURGEN E. GSCHWEND* From the Departments of Internal Medicine III (MR, AS, HK, MS, JG) and Urology (JEG), University Hospital and German National Bone Marrow Donor Registry (CRM), Ulm, Germany

ABSTRACT

Purpose: The activation of antigen specific T cells by tumor associated antigens (TAA) might be a promising treatment strategy for patients with renal cell carcinoma (RCC). We analyzed TAA expression in patients with RCC as well as the prevalence of fitting HLA phenotypes and calculated the percent of patients eligible for peptide vaccination trials. Materials and Methods: A total of 41 RCC samples from primary tumors were analyzed for TAA expression by reverse transcriptase-polymerase chain reaction. Genes of interest were MAGE-1, MAGE-3, G250 and PRAME since peptides derived from these genes have been shown to activate antigen specific cytotoxic T lymphocytes. Results were combined with data on the HLA gene and haplotype frequencies in the German population as an example of a white population. Results: Tumor specific expression of at least 1 T-cell activating antigen was observed in all patients. Of the patients 80% expressed 2 or more TAAs simultaneously. HLA molecules suitable for presentation of the respective antigens were calculated to be expressed in 51% to 85% of white German patients. These results mirror with only minor variations most of the white populations in Europe and North America. Conclusions: We noted that T-cell activating tumor associated antigens are frequently expressed in patients with RCC. Based on HLA expression analysis in a white population at least 30% of patients with RCC are eligible for monovalent specific immunotherapy and 41% are eligible for polyvalent specific immunotherapy. These data are a rational basis for future prospective vaccination trials in patients with RCC. KEY WORDS: kidney; carcinoma, renal cell; antigens; T-lymphocytes; vaccination

During the last decade several tumor associated antigens (TAAs) have been identified, of which some are able to elicit tumor specific immune responses. Screening experiments using cytotoxic T lymphocytes (CTLs) or the serological screening of recombinant expression libraries (SEREX approach) led to the identification of multiple T-cell epitopes. Furthermore, the use of prediction algorithms (that is the SYFPEITHI, BIMAS or PaProC algorithm) that integrate HLA related peptide motifs and proteasomal cleavage rules have been shown to be powerful tools for the prediction of the definite epitope that might be presented by antigen presenting cells to T cells.1–3 Peptide antigens entered clinical vaccination trials, that is MAGE-3, tyrosinase and Melan A/MART1 in melanoma or prostate specific antigen and prostate specific membrane antigen in prostate cancer. For all of these antigens the enhancement of specific T-cell reactivity could be observed and in some cases clinical responses could be achieved.4 – 6 In renal cell carcinoma (RCC) the yield for truly immunogenic and clinically applicable antigens has remained low. Although many identified antigens have been shown to elicit cellular immune responses, some have only limited therapeutic value due to low frequency of expression7 or their unfavorable expression pattern in tumor and normal tissues.

A basic problem accompanying peptide vaccination strategies, especially in heterogeneous solid tumors, is the selection of tumor cells caused by antigen loss. In the melanoma model it was demonstrated that metastases that developed during successful peptide vaccination lost the immunogenic antigen.8 To overcome this problem it is desirable to establish a polyvalent vaccination strategy that combines several peptide antigens and, therefore, minimizes the risk of immune escape.9 Compared with alternative strategies, such as vaccination with tumor cell lysates10 or RNA11 as a source of antigen, peptide vaccination offers the opportunity to assess the T-cell response accurately with sophisticated techniques, such as ELISpot assays or tetramer staining. Beside TAA expression the second prerequisite for the successful induction of a T-cell response is the presence of an HLA class I molecule on the surface of antigen presenting cells. The interaction between antigenic peptides and the HLA molecule relies on the peptide motif and the molecular structure of the antigen binding site. Since this interaction is rather specific, an antigenic peptide can only be present on specific HLA molecules.12 Therefore, an estimation of the eligibility of patients with RCC for clinical peptide vaccination must consider TAA expression as well as HLA allele distribution. The current study contributes comprehensive data to estimate the number of patients with RCC who would be eligible for an individualized monovalent or polyvalent peptide vaccination trial.

Accepted for publication December 19, 2003. Study received local ethics committee approval. Supported by Institutional Grants P.731 and P.763 from the University of Ulm. * Correspondence: Department of Urology, University of Ulm, MATERIALS AND METHODS Prittwitz-Strasse 43, 89075 Ulm, Germany (telephone: ⫹⫹49 –731Patients samples and cell lines. We prospectively analyzed 500 –27808; FAX: ⫹⫹49 –731-500 –33166; e-mail: juergen.gschwend@ tissue samples from 41 patients with RCC. Patients provided medizin.uni-ulm.de). 2456

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informed consent. About 1 cm3 of a single tumor tissue sample was prepared per patient and cryopreserved immediately after radical nephrectomy. In all cases the diagnosis of RCC was confirmed by pathological findings. Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated from snap frozen tumor samples using Trizol reagent (Gibco, Karlsruhe, Germany). Approximately 2 ␮g of each RNA sample were as subjected to cDNA synthesis using a SuperScript Kit (Gibco) containing oligo(deoxythymidine) primer for reverse transcription. As a control, ␤-actin PCR was performed and monitored by agarose gel electrophoresis. Generally the reaction mixture was composed of a standard PCR buffer (10 mM tris-HCl, pH 8.3 and 50 mM KCl) containing 1.5 mM magnesium chloride, deoxynucleoside triphosphates (250 nM each), primer pairs (10 pmol each) and 1.5 ␮l template cDNA, corresponding to about 1 ng template cDNA, in a final volume of 50 ␮l 2.5 U AmpliTaq (Perkin Elmer, Foster City, California). The reaction was started with an initial denaturation step (95C for 5 minutes), followed by the respective number of amplification cycles and a final elongation step (72C for 10 minutes). A Gene Amp PCR system 2400 device (Perkin Elmer) was used for the amplification procedure. Primer sequences and PCR conditions. Table 1 lists primer sequences and PCR conditions. All primer pairs were located in different exons to avoid the amplification of contaminating genomic DNA. The identity of PCR products was initially verified by direct sequencing with an ABI Prism 373 DNA sequencer (Applied Biosystems, Foster City, California). Discrimination among MAGE-3, MAGE-2 and MAGE-6 was further guaranteed by restriction analysis using the endonuclease Sca I (Boehringer Ingelheim, Mannheim, Germany), which digests MAGE-2 and MAGE-6 but not MAGE-3. For G250 we established RT-PCR with a second primer pair since we found low sensitivity of the already published primer pairs. All negative samples were evaluated again with this second primer pair. Table 2 lists the results of these 2 PCR reactions. Calculation of the eligibility of patients with RCC for vaccination trials. The fraction of patients with a fitting HLA allele was estimated based on the allele and haplotype frequencies of the German population according to Muller et al.13 It was compared with data on the United States National Marrow Donor Program (NMDP).14 The antigen frequency fA2 HLA-A2 can be obtained from the gene gA2 using formula 1, fA2 ⫽ 1 ⫺ (1 ⫺ gA2)2. Due to the linkage disequilibrium between the loci HLA-A and HLA-B the formula for the frequency (fX) of individuals carrying at least 1 of the antigens A1, A2, A24 and B35 is more complex. First, the gene frequencies gA1, gA2, gA24 and gB35, and the 2

locus haplotype frequencies gA1-B35, gA2-B35 and gA24-B35 are used to calculate the cumulative frequency (hC) of all HLAA-B haplotypes carrying at least 1 gene for A1, A2, A24 and B35 using the formula 2, hc ⫽ gA1 ⫽ gA2 ⫹ gA24 ⫽ (gB35 ⫺ hA1-B35 ⫺ hA2-B35 ⫺ hA24-B35), where the term in parentheses corresponds to the frequency of all B35 haplotypes without A1, A2 and A24. The desired frequency can then be calculated in the same way as formula 1 using the equation 3, fc ⫽ 1 ⫺ (1 ⫺ hc)2. To determine the percent of patients eligible for vaccination with a single peptide the appropriate values of HLA frequency and the mRNA expression rate of a certain TAA had to be multiplied. Thus, the eligibility rates eG, eM and eP for the 3 peptides G250 (G), MAGE-3 (M) and PRAME (P), respectively, were calculated using formulas 4A to C, eG ⫽ fA2 ⫻ fG, eM ⫽ fc ⫻ fM and eP ⫽ fA2 ⫻ fP, respectively, using the antigen frequency of HLA-A2 for G 250 and PRAME, the combined frequencies of A1, A2, A24, B35 for MAGE-3, and the respective expressions rates fG, fM and fP. When calculating the eligibility rates for monovalent (emono), polyvalent (fpoly) and overall (ftotal) peptide vaccination, one must consider the dependency of the HLA aspect of the peptide presentation. Overall eligibility can be calculated as the sum of the eligibility rate contributed by individuals with the antigen HLA-A2, where the contributions of the 3 peptide accumulate like independent variables and of individuals with HLA-A1, HLA-A24 or HLA-B35 but without HLA-A2, which are able to present MAGE-3. The 2 groups correspond to the 2 terms in formula 4, etotal ⫽ (1 ⫺ (1 ⫺ fG) ⫻ (1 ⫺ fM) ⫻ (1 ⫺ fP)) ⫻ fA2 ⫹ fM ⫻ (fC ⫺ fA2) ⫽ (fG ⫹ fM ⫹ fP ⫺ fG ⫻ fM ⫺ fM ⫻ fP ⫺ fP ⫻ fG ⫹ fG ⫻ fM ⫻ fP) ⫻ fA2 ⫹ fM ⫻ (fC ⫺ fA2). Here polyvalent vaccination is only feasible in individuals with the antigen HLA-A2. The eligibility frequency (epoly) can be calculated by formula 5, epoly ⫽ (fG ⫻ fM ⫹ fM ⫻ fP ⫹ fP ⫻ fG ⫺ 2fG ⫻ fM ⫻ fP) ⫻ fA2. As a consequence, the difference is the fraction of individuals eligible for a monovalent vaccination, as calculated by formula 6, emono ⫽ etotal ⫺ epoly ⫽ (fG ⫹ fM ⫹ fP ⫺ 2 ⫻ fG ⫻ fM ⫺ 2 ⫻ fP ⫻ fG ⫹ 3 ⫻ fG ⫻ fM ⫻ fP) ⫻ fA2 ⫹ fM ⫻ (fC ⫺ fA2). These calculations are based on the observation that TAA expressions are independent of each other. In parallel the calculations were performed using the observed fractions of expression of only 1 or more than 1 TAA. Statistical analysis. We used 2 ⫻ 2 contingency tables and the 2-sided Fisher exact test to obtain R values and significance levels concerning the co-expression of the different TAAs.15 The chi-square test was used to evaluate the relationships of TAA expression to the features tumor size, grading and histology.

TABLE 1. Primer sequences and RT-PCR conditions Gene of Interest (sequence) G 250 a: 5⬘ ACT GCT GCT TCT GAT GCC TGT 3⬘ 5⬘ AGT TCT GGG AGC GGC GGG A 3⬘ G 250 b: 5⬘ ACT TCA GCC GCT ACT TCC AA 3⬘ 5⬘ TCT CAT CTG CAC AAG GAA CG 3⬘ MAGE-1: 5⬘ CGG CCG AAG GAA CCT GAC CCA G 3⬘ 5⬘ GCT GGA ACC CTC ACT GGG TTG CC 3⬘ MAGE-3: 5⬘ ACC AAG GAG AAG ATC TGC CAG TGG GTC TC 3⬘ 5⬘ ACA GTC GCC CTC TTT TGC GAT TAT GG 3⬘ PRAME: 5⬘ GTC CTG AGG CCA GCC TAA GT 3⬘ 5⬘ GGA GAG GAG GAG TCT ACG CA 3⬘ ␤-Actin: 5⬘ GCA TCG TGA TGG ACT CCG 3⬘ 5⬘ GCT GGA AGG TGG ACA GCG A 3⬘

Annealing Temperature (°C)

Product Length (bp)

Denaturation/Annealing/ Elongation (mins)

Genbank Accession No.

68

494

2/2/1

NM_001216.1

58

370

1/1/1

NM_001216.1

64

421

1/1/1

NM_004988.2

72

722

1/2/2

NM_005362.2

64

822

1/1/1

NM_006115

68

613

1/1/1

NM_001101.2

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Patients and tumor characteristics. A total of 41 patients with RCC, including 27 males and 14 females, were studied. Median age of our patients was 66 years (range 34 to 79). RCC histology and grading were classified according to Thoenes et al.16 Table 2 shows an overview of the total data. Figure 1 shows the distribution of carcinomas among the subgroups according to the 1997 TNM classification, grading and histology. Frequency of mRNA TAA expression in RCC samples. Antigen mRNA expression was found in 25 of 41 patients (61%) for MAGE-3, in 25 of 41 (61%) for PRAME and in 38 of 41 (93%) for G250 on RT-PCR. Of 30 patients with a clear cell histology 29 (97%) had positive G250 mRNA. Although a strong signal was obtained in the myeloid cell line K562, which served as a positive control, none of the 18 RCC samples expressed MAGE 1. Figure 2 shows an ethidium bromide stained agarose gel with the PCR product showing all investigated antigens. Each patient expressed at least 1 TAA. A total of 33 samples (80%) showed a polyvalent antigen mRNA expression pattern (fig. 3). We used the chi-square test to evaluate if there was any relationship between TAA mRNA expression and tumor stage, grading or histology. We could not detect significant differences among these subgroups (data not shown) except for a correlation of G250 expression and a chromophilic or clear cell phenotype (p ⫽ 0.02). On further analysis we investigated if there was any correlation of the expression of the antigens under investigations. The R value for the co-expression of each pair of the 3 TAAs investigated was between – 0.13 and 0.16 (2-sided

FIG. 1. Tumor characteristics of patient cohort

Fisher exact test p ⱖ 0.6). As a consequence, we assumed an independent expression in formulas 4 to 6. Calculating the number of patients eligible for specific immunotherapy based on tumor antigen expression and HLA molecule distribution. Specific immunotherapy requires a peptide consisting of an amino acid sequence with a distinct HLA anchor motif. Therefore, the number of patients suit-

TABLE 2. Patient characteristics and corresponding TAA expression pattern Code UL-RCC-2 UL-RCC-3 UL-RCC-4 UL-RCC-5 UL-RCC-6 UL-RCC-9 UL-RCC-10 UL-RCC-11 UL-RCC-12 UL-RCC-13 UL-RCC-14 UL-RCC-15 UL-RCC-16 UL-RCC-17 UL-RCC-18 UL-RCC-19 UL-RCC-20 UL-RCC-21 UL-RCC-22 UL-RCC-23 UL-RCC-25 UL-RCC-26 UL-RCC-27 UL-RCC-28 UL-RCC-29 UL-RCC-33 UL-RCC-34 UL-RCC-35 UL-RCC-38 UL-RCC-39 UL-RCC-40 UL-RCC-41 UL-RCC-42 UL-RCC-43 UL-RCC-44 UL-RCC-45 UL-RCC-46 UL-RCC-47 UL-RCC-48 UL-RCC-49 UL-RCC-53

Pt Sex — Age F — 54 M — 76 M — 60 F — 77 F — 66 M — 74 M — 61 M — 67 M — 59 M — 76 F — 62 M — 69 F — 79 M — 59 M — 63 M — 78 M — 67 F — 60 F — 79 M — 76 F — 74 M — 72 F — 62 F — 46 M — 61 F — 79 M — 63 M — 72 M — 69 F — 75 M — 74 M — 65 F — 78 M — 73 M — 57 M — 34 M — 50 M — 65 M — 66 M — 65 F — 61

TNM Stage

Grade

␤-Actin

pT1N0M0 pT1N0M0 pT1pN0M0 pT3aNxM0 pT2pN0M0 pT3aNxM0 pT3bpN0M0 pT3bN0M0 pT3bN0M0 pT3aN0M0 pT1N0M0 pT1N0M0 pT3bN0M0 pT2pN0M0 pT3bN0M0 pT1pN0M0 pT2pN0M0 pT3pN0M0 pT3bpNxM0 pT3pN0M0 pT3N0Mx pT3pN1Mx pT2NxMx pT2NxM0 pT2NxMx pT3bpN0M0 pT3apN0M0 pT3apNxM0 pT1pN0M0 pT1N0M0 pT3bpN0M0 pT1pN0M0 pT1pN0M0 pT3apNxM0 pT3pN0M1 pT2pN0M0 pT1pNxM0 pT3pN0M0 pT1N0M0 pT1pN0M0 pT1pN0M0

G2 G2 G2 G1–2 G1 G1–2 G2—3 G2–3 G2 G2 G1 G1 G2 G2 G1 G2–4 G2 G1 G2 G2 G2–3 G1–3 G1 G1 G2 G2 G2 G1–3 G2–4 G3 G2 G1 G2 G2 G2 G2 G2 G2 G1 G1 G1

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

MAGE-1

Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not Not

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined determined

MAGE-3

Prame

G250

Histology

Neg Pos Pos Pos Pos Pos Neg Pos Pos Neg Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg Neg Pos Neg Neg Neg Neg Neg Neg Pos Pos Neg Neg Pos

Pos Pos Pos Neg Neg Neg Pos Pos Pos Pos Pos Neg Pos Neg Pos Pos Neg Neg Pos Neg Pos Neg Pos Pos Pos Neg Pos Neg Pos Neg Pos Pos Neg Pos Pos Neg Pos Pos Pos Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Pos Pos Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Clear cell Clear cell Clear cell Clear cell Clear cell Clear cell Chromophilic/papillary Clear cell Chromophilic/papillary Chromophobe Chromophobe Clear cell Clear cell Clear cell Clear cell Chromophilic/papillary Clear cell Clear cell Clear cell Clear cell Clear cell Clear cell Chromophobe Clear cell Clear cell Chromophilic/papillary Chromophilic/papillary Chromophilic/papillary Chromophilic/papillary Clear cell Clear cell Clear cell Clear cell Clear cell Clear cell Clear cell Clear cell Chromophilic/papillary Clear cell Clear cell Clear cell

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RENAL CELL CANCER AND PEPTIDE VACCINATION TRIALS TABLE 4. Subsequent estimated total eligibility rate Observed/Calculated %

Monovalent Polyvalent Totals Total of 41 patients.

FIG. 2. Ethidium bromide staining of 1.5% weight per volume agarose gels with all investigated antigens (I). Samples or cell lines serving as positive control were MAGE-1 (K562) (a), MAGE-3 (K562) (b), PRAME (K562) (c), G250a (d) and G250b (UL-RCC-46) (e). G250 PCRs and corresponding ␤-actin PCR were run on different gels. II, ␤-actin control for integrity and presence of amplifiable cDNA. M ⫽ marker.

FIG. 3. Simultaneous TAA expression in RCC samples. Y axis indicates actual number of TAA expressing samples.

able for such a treatment strategy depends on the HLA gene distribution in a given population. To calculate the frequency of individuals carrying at least 1 suitable HLA antigen (A1, A2, A24 and B35) we founded on the gene haplotype frequencies of a German reference population, as described. Comparing our German HLA data with a similar study of American population data from the United States NMDP14 revealed extremely similar gene frequencies for their white population (HLA-A1 15.2% vs 15.7%, HLA-A2 28.7% vs 29.9%, HLA A 24 9.3% vs 9.2% and HLA-B35 9.7% vs 9.7%). Since the NMDP database only gives the frequency of the haplotype HLA-A2B35, we cannot give exact calculations for all 5 ethnic subgroups of the United States. However, the eligibility for a peptide vaccination would be almost identical for white Americans and white Germans, and it should be greater than 60% for the Asian, Latin and native American populations. For black Americans a substantially lower eligibility rate of about 45% is expected due their lower frequency of HLA-A2. Tables 3 and 4 show the detailed calculation for the German population.

TAA Expression (No. pts)

Cumulative Eligibility

19.5/17.5 (8) 80.5/81.3 (33) 100/98.8 (41)

29.9 /30.9 41.3 /40.9 71.2 /71.8

DISCUSSION

In the current study we evaluated the expression pattern of proven T cell activating TAAs that might be involved in the induction of T-cell specific immunity. An important finding was the frequent expression of MAGE-3 in RCC. This is of particular interest since MAGE-3 has been successfully involved in peptide vaccination trials in patients with melanoma,17 demonstrating the induction of MAGE-3 specific CTLs. To date MAGE-3 derived CTL activating peptides have been identified for a wide range of HLA molecules, which renders a vaccination feasible for a wide range of patients. Meanwhile, several T-helper cell activating epitopes derived from the MAGE-3 gene have been described.18 This offers the opportunity of the simultaneous recruitment of CD4⫹ and CD8⫹ T cells through stimulation with HLA classes I and II binding peptides, which may result in a probably more efficient antitumor response. The intriguing importance of MAGE-3 was most recently demonstrated in melanoma cases since only the induction of cytotoxic responses against this antigen correlated with a clinical benefit.19 In the current study the PRAME gene was expressed at a higher percent (61% vs 40%) than previously described.20 Since HLA-A2 matched CTL activating peptides derived from the PRAME gene have been described only recently,21 this is obviously another interesting target structure. In accordance with the data contributed by Neumann et al,20 we failed to detect MAGE-1 expression in our samples. The expression rate of G250 was in the expected range in our series. For the G250 antigen a CD4⫹ helper T-cell stimulating epitope has also been described.22 The importance of a polyvalent strategy for cellular immunotherapy was shown in a recent study. A total of 18 patients with stage IV melanoma were immunized with a polyvalent vaccine using dendritic cells pulsed with up to 4 melanoma peptide antigens. Regression of more than 1 tumor lesion was observed in 7 of 10 patients who showed enhanced T-cell responses to more than 2 antigens. The overall immunity to melanoma antigens was significantly associated with the clinical outcome.9 To evaluate the relevance of our findings for clinical purposes we calculated the number of patients suitable for such a peptide vaccination trial based on data on HLA allele and haplotype frequencies in Germany, and compared it to NMDP results.13, 14 We focused on the most frequent genotypes for which tumor associated antigens have been de-

TABLE 3. T-cell activating peptide motifs and calculation of eligible patients for vaccination trials Gene of Interest (HLA type)

Peptide Motif

% HLA Expression

G 250 A2 HLSTAFARV 50.8 MAGE-3: 85.2 A1 EVDPIGHLY A2 FLWGPRALV A24 IMPKAGLLI, TFPDLESEF B35 EVDPIGHLY PRAME 50.8 A2 VLDGLDVLL, SLYSFPEPEA, ALYVDSLFFL, SLLQHLIGL Total of 41 patients. Peptides have T-cell immunogenicity and peptides are shown as single letter code for each amino acid.

% TAA Expression (No. pts)

% Eligible for Vaccination

92.7 (38) 61.0 (25)

47.1 52.0

61.0 (25)

31.0

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scribed. Our data show that about 70% of patients with RCC would be eligible for a peptide vaccination trial. In more than half of them a polyvalent approach seems to be feasible. This observation is based on the assumption that there is no linkage between the HLA phenotype and the incidence of RCC or the expression of a certain TAA. Our calculation algorithm may be easily adapted to further HLA genotypes, for example if affinity dependent binding assays23 may reveal new relevant TAA/HLA molecule combinations. We are aware that the current analysis has certain limitations. Clearly our analysis is a best case scenario that does not consider that some patients have down-regulated antigen or HLA molecule expression. Also, we cannot state the antigen expression on the protein/peptide level and how strong those signals must be to elicit a sufficient immune response. According to differences in the efficacy of antigen presentation and differences in the T-cell receptor repertoire these factors may considerably vary among patients. Finally, some patients with metastatic disease undergo immune selection due to the heterogeneity of solid tumors, resulting in altered antigen expression in the primary tumor, which we analyzed, and metastatic lesions. However, some of these events would only appear during therapy and our results may hold true at least for the adjuvant situation. The optimal approach to immunize these patients remains to be determined but the prerequisite is always the presence of immunogenic antigens on the surface of the tumor cell. The current study focused on the eligibility of patients with RCC for clinical vaccination trials but is clearly not able to predict the outcome of such trials. For a clinical study in the emerging field of tumor immunization the therapeutic strategy must be efficient and at the same time safe and easy to monitor. Immunization with mere peptides like MAGE-3, PRAME and G250 or with peptide pulsed dendritic cells would most probably fulfill these criteria because the acting antigens are known and the immune response can be measured by ELISpot assay or tetramer staining. As we noted, the majority of patients with RCC in the adjuvant or palliative setting may be eligible for a clinical trial of peptide vaccination. CONCLUSIONS

About 71% of patients with RCC are eligible for tumor specific immunotherapy and in 41% of patients with RCC even a polyvalent strategy is feasible. The estimation is based on the expression frequencies of proven T-cell activating TAA and the frequencies of the appropriate HLA class I molecule(s) that are necessary for the presentation of the antigenic determinants toward CTLs. These data have a direct impact on the design of new phase I RCC studies. S. Braun, K. Singer and A. Szmaragowska provided technical assistance. Dr. Philipp Dahm, Duke University, North Carolina critically read the manuscript. REFERENCES

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