HLA-B8 association with late-stage melanoma – an immunological lesson?
© Fensterle et al; licensee BioMed Central Ltd. 2006
Received: 25 October 2005
Accepted: 13 March 2006
Published: 13 March 2006
Differences in HLA allele frequencies between the diseased and healthy populations may signify efficient immune responses, a notion that has been successfully tested for infectious diseases or for association with genetic elements involved in a distinct type of immunity. This retrospective study is intended to detect differences in MHC class I carrier frequencies of advanced melanoma patients compared to healthy bone marrow donors.
The HLA-A and -B carrier frequencies of 748 stage IV melanoma patients retrieved from serotyping at 6 different centers in Germany were compared using a chi-square test to 13,386 fully HLA typed bone marrow donors registered in the German national bone marrow donor registry.
The comparison of HLA carrier frequencies in advanced cancer patients with healthy bone marrow donors revealed a significant decrease in HLA-B8 carrier frequencies, which was also apparent in patients with advanced disease compared to patients with loco-regional disease.
The data suggest that protective immune responses restricted to distinct MHC class I molecules may be operational in a subset of melanoma patients, which is the prerequisite for a large scale screen for the corresponding epitopes. Alternatively, the known association of the ancestral haplotype HLA-A1, -B8 and -DR3 with genetic elements such as distinct TNF-α alleles might have a protective effect on disease progression. In any case, identification of the cause of protection within this patient subset might lead to a significant improvement in the efficacy of current immunotherapeutic approaches.
Active immunization against solid tumors is amongst the most promising therapeutic approaches for such diseases. Different immunization schedules are currently being explored in numerous clinical trials, including vaccination with MHC class I restricted T-cell epitopes derived from tumor associated antigens (TAA) . To date, oncogenes have been intensively studied as targets for immunotherapy to minimize the risk of immune escape. As an example, we have recently identified B-Raf V600E (previously designated B-Raf V599E) as a potential TAA for immunotherapies against melanoma [2, 3]. The tumor antigens deployed are characterized by their ability to elicit immune responses in patients, and the choice of peptide epitopes derived from these antigens is mainly dictated by the frequency of HLA alleles in the normal population . However, this strategy for identifying targets for therapy might actually miss the most valuable epitopes.
As early as 1991, it was proposed that disease-associated imbalances in HLA allele frequencies between diseased and healthy populations may signify efficient immune responses . Although this approach was successfully adapted for the malaria system , transfer to cancer research has not yet been envisioned. In theory, any epitope derived from TAA that elicits an effective immune response able to eradicate the tumor should impact on the frequency of the respective HLA allele in this patient population. Assuming that the antigen is reasonably common within the patient cohort, the frequency of the HLA allele in the context of which the epitope is presented should be substantially reduced in patients with progressive disease, i.e. those who failed to mount an effective immune response. In practice, it is difficult to validate this edifice of ideas, as several prerequisites usually remain unknown: (i) the nature and (ii) the grade of expression of the corresponding TAA, and (iii) the percentage of patients mounting a protective immune response against this antigen. The fact that the HLA phenotype is known for a large number of patients suffering from advanced stages of immunogenic tumors such as melanoma allows at least one consequence of this intellectual experiment to be verified or falsified: viz., whether the frequency of certain HLA class I alleles is reduced in patients in whom the immune system failed to control the disease.
However, HLA imbalances can also be explained by linkage to genetic factors influencing disease progression. In this respect, it should be noted that many non-MHC genes encoding immunologically relevant molecules such as TNF-α or components of the complement system are located in the HLA locus, and that some alleles of these non-MHC genes are genetically linked to certain HLA alleles.
Data acquisition and HLA serotyping
The results of the serological HLA-A and -B typing of 748 AJCC stage IV melanoma patients performed in the University Hospitals of Erlangen (267 patients), Berlin (217 patients), Würzburg (141 patients), Mannheim (82 patients), Mainz (26 patients) and Münster (15 patients) were collected. To compare stage I-III with stage IV patients, the serotyping of 302 stage I-III patients performed in Berlin was also taken into account. The scientific workup of the HLA phenotyping results was approved by the local ethics committees, provided the patient gave informed consent.
Data comparison and statistical analysis
The data compiled from this patient cohort were compared to those from 13,386 fully HLA-typed bone marrow donors registered in the German national bone marrow donor registry . Data analysis was performed using the Graph Pad Prism software package and MS Excel 2003. Stage IV patients were compared to healthy bone marrow donors using a two sided chi-square test; stage I-III patients were compared to stage IV patients from Berlin, and to the whole cohort, using a two sided Fisher's exact test. An observation was regarded as significant if p < 0.01. The ratio between stage IV and stage I-III carrier frequencies was calculated to compare the relative decrease between different HLA alleles; the mean ratio comprises all HLA alleles with carrier frequencies above 15% (n = 11) +/- standard deviation.
Results and discussion
HLA-A, -B and -C carrier frequencies of stage IV melanoma patients. HLA-A and B alleles of 739 and 709 stage IV melanoma patients were typed serologically; HLA-C (391 stage IV melanoma patients) was typed by PCR. The frequencies of all alleles included in the analysis are shown. Subtypes are given in italics; the main types that have been partially subtyped are shown in bold. HLA carrier frequencies of the main types that have been partially subtyped encompass the carrier frequencies of the subtypes; the fraction of patients who have been subtyped for a certain allele is given as a percentage.
HLA-A (n = 739)
HLA-B (n = 709)
HLA-C (n = 391)
Comparison of HLA-A and B carrier frequencies between stage IV melanoma patients and healthy bone marrow donors. HLA-A and -B carrier frequencies of stage IV melanoma patients (739 HLA-A; 709 HLA-B) and healthy bone marrow donors (13,386) were compared using a two sided chi-square test with a 95% confidence interval and sorted according to the absolute difference. Results are regarded as significant for p values < 0.01; alleles with significant changes are depicted in bold; results are sorted according to the percentage difference. Only alleles with carrier frequencies > 5% are included. Subtypes are only included if more than 89% of the corresponding allele was subtyped in the melanoma and bone marrow donor group. OR: odds ratio; ci: confidence interval.
0.6184 to 0.9302
95% ci OR
0.6409 to 0.9744
0.9575 to 1.342
0.9561 to 1.356
0.6509 to 1.039
0.5015 to 0.9846
0.6728 to 1.070
0.6133 to 1.053
0.7047 to 1.087
0.5834 to 1.055
0.9504 to 1.591
0.5774 to 1.108
0.5939 to 1.132
0.8930 to 1.266
0.6839 to 1.137
0.8761 to 1.287
0.8856 to 1.228
0.8526 to 1.395
0.8705 to 1.242
0.8666 to 1.238
0.8724 to 1.173
0.8385 to 1.163
0.7598 to 1.249
0.7814 to 1.329
0.7151 to 1.366
It should be noted that several independent reports have already demonstrated a reduction of HLA-B8 and HLA-B35 in melanoma patients [11–13] or a selective loss of HLA alleles in metastasis . Furthermore, HLA-B8- and HLA-B35-restricted melanoma-specific T cell responses have been identified [15, 16]. The recognized epitopes are derived from tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6 and NY-ESO-1 [17–19]; we recently added a HLA-B35-restricted epitope of the inhibitor of apoptosis protein survivin to this list . Thus, it is tempting to speculate that the observed decrease of HLA-B8 carrier frequencies in advanced melanoma is the result of an effective immune response directed against specific TAA-derived epitopes presented by the respective HLA restriction elements, or, conversely, the lack of such response in patients not expressing these restriction elements. However, whether the epitopes already identified are indeed the targets for the hypothetical, protective immune response responsible for the observed decrease in HLA frequencies remains elusive. Such epitopes could only be defined by quantitative and qualitative comparison of the cellular immune responses between patients with early disease, preferentially the primary tumor, and those with advanced disease; in the latter patients, the protective responses should be underrepresented.
An alternative explanation for the observed decrease in HLA frequencies could be the genetic linkage to a tumor suppressor in the broadest sense. HLA-B8 has been shown to be linked genetically to HLA-A1 . This linkage is also obvious in our data set of stage IV melanoma patients, where 80.8% of the patients carrying HLA-B8 are also positive for HLA-A1. Although no gene dosage effect that could translate into a reduced number of homozygous HLA-B8 carriers in the patient group was observed (data not shown), the association of the 8.1 ancestral haplotype with autoimmune diseases or distinct TNF-α or complement alleles might be of special interest, as this might exert an influence on the anti-tumoral immune response [22–24]. In the case of TNF-α, the -308A allele that is linked with 8.1 AH is associated with altered TNF-α production in vitro . Also, 8.1 AH is characterized by a non-functional C4B allele and the lack of the C4A gene, translating into defective complement function [25, 26]. As the complement cascade is involved in inflammation, the clearance of immune complexes, antibody-dependent cellular cytotoxicty and other immunologically relevant processes, a genetic defect in the complement system may indeed affect both innate and adaptive immunity. However, the definitive answer to whether HLA-B8 itself or genetic elements linked to HLA-B8 are responsible for the observed protective phenotype can only be elucidated by further studies, including the study of TNF-α or complement alleles.
Overall, this study suggests a significant decrease of HLA-B8 carrier frequencies in melanoma patients compared to healthy donors. This difference, which was observed when HLA carrier frequencies in advanced cancer patients were compared with healthy bone marrow donors or patients with loco-regional disease, supports the hypothesis that protective immune responses may be operational in a subset of melanoma patients independently of therapy, and that distinct MHC class I restriction elements seem to be preferentially involved in such responses. Alternatively, the association of 8.1 AH with genetic elements such as distinct TNF-α alleles or defective complement components might result in an altered quality of immune response, preventing the progression of the disease. If the observed differences can be verified in a follow-up study, a search for the corresponding T-cell epitopes and/or the associated genetic elements is indicated and might lead to a new generation of effective tumor vaccines.
The authors wish to express their gratitude to E.B. Broecker, A. Enk, S. Grabbe, D. Schadendorf, G. Schuler and A. Tuettenberg for contributing the HLA phenotypes of stage IV melanoma patients, to U.R. Rapp, M. Boeck, S. Klingert, H. Drexler and M. Lau for stimulating discussions and critically reading the manuscript, and to F. Marohn and J.-H. Krannich for help with the statistics.
- Jager D, Jager E, Knuth A: Vaccination for malignant melanoma: recent developments. Oncology. 2001, 60: 1-7. 10.1159/000055289.View ArticlePubMedGoogle Scholar
- Fensterle J, Becker JC, Potapenko T, Heimbach V, Vetter CS, Brocker EB, Rapp UR: B-Raf specific antibody responses in melanoma patients. BMC Cancer. 2004, 4: 62-10.1186/1471-2407-4-62.View ArticlePubMedPubMed CentralGoogle Scholar
- Andersen MH, Fensterle J, Ugurel S, Reker S, Houben R, Guldberg P, Berger TG, Schadendorf D, Trefzer U, Bröcker EB, Straten PT, Rapp UR, Becker JC: Immunogenicity of constitutively active V599EBRaf. Cancer Res. 2004, 64: 5456-5460. 10.1158/0008-5472.CAN-04-0937.View ArticlePubMedGoogle Scholar
- Andersen MH, Pedersen LO, Capeller B, Brocker EB, Becker JC, thor Straten P: Spontaneous cytotoxic T-cell responses against survivin-derived MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients. Cancer Res. 2001, 61: 5964-5968.PubMedGoogle Scholar
- Hill AV, Allsopp CE, Kwiatkowski D, Anstey NM, Twumasi P, Rowe PA, Bennett S, Brewster D, McMichael AJ, Greenwood BM: Common west African HLA antigens are associated with protection from severe malaria. Nature. 1991, 352: 595-600. 10.1038/352595a0.View ArticlePubMedGoogle Scholar
- Hill AV, Elvin J, Willis AC, Aidoo M, Allsopp CE, Gotch FM, Gao XM, Takiguchi M, Greenwood BM, Townsend AR, et al: Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature. 1992, 360: 434-439. 10.1038/360434a0.View ArticlePubMedGoogle Scholar
- Muller CR: Populationsgenetische Parameter der Gewebemerkmale der deutschen Bevölkerung und ihre Anwendungen bei der Suche nach nicht-verwandten Blutstammzellspendern. dissertation. 2000, University of Ulm, Medical FacultyGoogle Scholar
- Svejgaard A, Ryder LP: HLA and disease associations: detecting the strongest association. Tissue Antigens. 1994, 43: 18-27.View ArticlePubMedGoogle Scholar
- Mantel N, Haenszel W: Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst. 1959, 22: 719-748.PubMedGoogle Scholar
- Cao K, Hollenbach J, Shi X, Shi W, Chopek M, Fernandez-Vina MA: Analysis of the frequencies of HLA-A, B, and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol. 2001, 62: 1009-1030. 10.1016/S0198-8859(01)00298-1.View ArticlePubMedGoogle Scholar
- Marincola FM, Shamamian P, Rivoltini L, Salgaller M, Cormier J, Restifo NP, Simonis TB, Venzon D, White DE, Parkinson DR: HLA associations in the antitumor response against malignant melanoma. J Immunother Emphasis Tumor Immunol. 1995, 18: 242-252.View ArticlePubMedPubMed CentralGoogle Scholar
- Geertsen RC, Hofbauer GF, Yue FY, Manolio S, Burg G, Dummer R: Higher frequency of selective losses of HLA-A and -B allospecificities in metastasis than in primary melanoma lesions. J Invest Dermatol. 1998, 111: 497-502. 10.1046/j.1523-1747.1998.00305.x.View ArticlePubMedGoogle Scholar
- Rovini D, Sacchini V, Codazzi V, Vaglini M, Illeni MT: HLA antigen frequencies in malignant melanoma patients. A second study. Tumori. 1984, 70: 29-33.PubMedGoogle Scholar
- Anastassiou G, Rebmann V, Wagner S, Bornfeld N, Grosse-Wilde H: Expression of classic and nonclassic HLA class I antigens in uveal melanoma. Invest Ophthalmol Vis Sci. 2003, 44: 2016-2019. 10.1167/iovs.02-0810.View ArticlePubMedGoogle Scholar
- Yee C, Gilbert MJ, Riddell SR, Brichard VG, Fefer A, Thompson JA, Boon T, Greenberg PD: Isolation of tyrosinase-specific CD8+ and CD4+ T cell clones from the peripheral blood of melanoma patients following in vitro stimulation with recombinant vaccinia virus. J Immunol. 1996, 157: 4079-4086.PubMedGoogle Scholar
- Hom SS, Schwartzentruber DJ, Rosenberg SA, Topalian SL: Specific release of cytokines by lymphocytes infiltrating human melanomas in response to shared melanoma antigens. J Immunother. 1993, 13: 18-30.View ArticleGoogle Scholar
- Benlalam H, Linard B, Guilloux Y, Moreau-Aubry A, Derre L, Diez E, Dreno B, Jotereau F, Labarriere N: Identification of five new HLA-B*3501-restricted epitopes derived from common melanoma-associated antigens, spontaneously recognized by tumor-infiltrating lymphocytes. J Immunol. 2003, 171: 6283-6289.View ArticlePubMedGoogle Scholar
- Schultz ES, Zhang Y, Knowles R, Tine J, Traversari C, Boon T, van der Bruggen P: A MAGE-3 peptide recognized on HLA-B35 and HLA-A1 by cytolytic T lymphocytes. Tissue Antigens. 2001, 57: 103-109. 10.1034/j.1399-0039.2001.057002103.x.View ArticlePubMedGoogle Scholar
- Morel S, Ooms A, Van Pel A, Wolfel T, Brichard VG, van der Bruggen P, Van den Eynde BJ, Degiovanni G: A tyrosinase peptide presented by HLA-B35 is recognized on a human melanoma by autologous cytotoxic T lymphocytes. Int J Cancer. 1999, 83: 755-759. 10.1002/(SICI)1097-0215(19991210)83:6<755::AID-IJC10>3.0.CO;2-S.View ArticlePubMedGoogle Scholar
- Reker S, Becker JC, Svane IM, Ralfkiaer E, Straten PT, Andersen MH: HLA-B35-restricted immune responses against survivin in cancer patients. Int J Cancer. 2004, 108: 937-941. 10.1002/ijc.11634.View ArticlePubMedGoogle Scholar
- Muller CR, Ehninger G, Goldmann SF: Gene and haplotype frequencies for the loci hLA-A, hLA-B, and hLA-DR based on over 13,000 German blood donors. Hum Immunol. 2003, 64: 137-151. 10.1016/S0198-8859(02)00706-1.View ArticlePubMedGoogle Scholar
- Lio D, Candore G, Colombo A, Colonna Romano G, Gervasi F, Marino V, Scola L, Caruso C: A genetically determined high setting of TNF-alpha influences immunologic parameters of HLA-B8, DR3 positive subjects: implications for autoimmunity. Hum Immunol. 2001, 62: 705-713. 10.1016/S0198-8859(01)00264-6.View ArticlePubMedGoogle Scholar
- Wilson AG, de Vries N, Pociot F, di Giovine FS, van der Putte LB, Duff GW: An allelic polymorphism within the human tumor necrosis factor alpha promoter region is strongly associated with HLA A1, B8, and DR3 alleles. J Exp Med. 1993, 177: 557-560. 10.1084/jem.177.2.557.View ArticlePubMedGoogle Scholar
- Candore G, Lio D, Colonna Romano G, Caruso C: Pathogenesis of autoimmune diseases associated with 8.1 ancestral haplotype: effect of multiple gene interactions. Autoimmun Rev. 2002, 1: 29-35. 10.1016/S1568-9972(01)00004-0.View ArticlePubMedGoogle Scholar
- Christiansen FT, Zhang WJ, Griffiths M, Mallal SA, Dawkins RL: Major histocompatibility complex (MHC) complement deficiency, ancestral haplotypes and systemic lupus erythematosus (SLE): C4 deficiency explains some but not all of the influence of the MHC. J Rheumatol. 1991, 18: 1350-1358.PubMedGoogle Scholar
- O'Neill GJ, Nerl CW, Kay PH, Christiansen FT, McCluskey J, Dawkins RL: Complement C4 is a marker for adult rheumatoid arthritis. Lancet. 1982, 2: 214-10.1016/S0140-6736(82)91057-1.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/4/5/prepub
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