On the molecular level, melanoma is associated with several genetic changes, including mutations or transcriptional variations in tumor suppressors like p53, CDKN2A/p16, CDKN1A/p21 or in oncogenes like N-Ras [4]. Recently, it has been discovered by Davies et al. that 66% of melanoma have a mutated BRAF gene which results in higher kinase activity due to a single amino acid exchange (B-Raf V599E) occurring in almost 90% of the mutations [5]. Most interestingly, this mutation is somatic [6,7] and some authors describe the presence of the mutation already in benign nevi [8], whereas others fail to reproduce the high frequencies in early stages and speculate of mutated B-Raf being relevant for progression rather than initiation of melanoma [9]. Nevertheless, the high incidence of the mutations in melanoma qualify the B-Raf protein as a potential target for tumor therapy and preliminary results of phase II clinical trials with Raf kinase inhibitors suggest protective activity [10].

Promising results with new approaches for melanoma therapy have been obtained with active immunizations against tumor associated antigens (TAA) like MAGE-3 [11], MART-1 [12-14] tyrosinase [15] or survivin [16]. Regarding the high incidence of B-Raf mutations and the increased expression level in tumors, B-Raf should be an attractive target for immunotherapy [17] and most recently, independent findings demonstrated mutation specific CD4⁺ T-cell responses in melanoma patients [18]. Moreover, we have recently demonstrated CD8⁺ T-cells in melanoma patients reactive against an HLA B27 restricted B-Raf V599E epitope encompassing the mutation identified using computer assisted algorithms [19]. However, assessing CD4⁺ or CD8⁺ T-cell responses is not suitable for screening larger numbers of patients to estimate responder frequencies.

Comparison between Raf specific antibody responses in sera from melanoma patients and control patients reveals apparent differences (figure ​(figure3).3). Raf specific responses are significantly different from background values for melanoma patients and all Raf variants tested (two tailed Mann-Whitney U test). In contrast, the difference observed within the control group is not significant (Mann-Whitney U test). However, reactivity against C-Raf is rather weak in all sera tested and only 5 of 271 tested sera from melanoma patients reach C-Raf specific titers of 1:300. A comparison between the percentage of patients with B-Raf specific antibody levels above a given titer reveals that sera derived from melanoma patients show consistently higher values compared to the control group (fig. ​(fig.4A).4A). In contrast, the frequency of C-Raf specific responses are only marginally higher in the melanoma population and not detectable at titers above 1:300. If positive response is defined as sera with titers of 1:300 or higher, 2.5% of the control group is positive for B-Raf and 1.68% for B-Raf V599E antibodies (fig. ​(fig.4B).4B). However, 5.41% (P = 0.12, Fisher's exact test) and 8.86% (P = 0.028, Fisher's exact test) of sera derived from melanoma patients were positive for B-Raf V599E and B-Raf respectively (fig. ​(fig.4B).4B). Using this cut off, no control patients could be identified with C-Raf specific antibodies, and only 1.85% (P = 0.329, Fisher's exact test) of the melanoma patients had detectable C-Raf specific antibodies (fig. ​(fig.4B).4B). These results suggest that melanoma is associated with higher rates of patients with detectable B-Raf specific antibody responses. The difference between the values obtained for B-Raf V599E and B-Raf specific antibodies, which is expressed in lower scores for B-Raf V599E, is most probably due to differences in the antigen preparation and is less likely due to different specificity as in most cases the scores against B-Raf V599E are in the same range as the scores against B-Raf (see figure ​figure1B),1B), whereas the C-Raf response consistently shows much lower titers in positive cases and a different shape of the curve (figure ​(figure1B).1B). Taken together, these data strongly suggest that 8.9% of melanoma patients have a B-Raf specific antibody response and 2.5% of the control patients. At this stage, it is far too early to foresee the consequences of this finding for diagnostics or therapy of melanoma. All melanoma patients were in a very advanced, rapidly progressing stage with high tumor loads (stage IV) and consequently the presence of antibodies was not correlated with survival (data not shown). However, at least some patients had low but detectable levels of B-Raf specific IgG antibodies which is in line with the independent finding of B-Raf V599E specific T-cells in melanoma patients [18].

Another interesting point to note is the small, although not significant, number of control patients with detectable levels of Raf specific antibodies. At least two out of 119 control patients showed a very strong B-Raf specific response in the same range as we have observed for positive melanoma patients (figure ​(figure3).3). Due to the design of the study, no information is available for this patient group. Serum was sampled during diagnostic procedures, therefore other underlying diseases as a cause for Raf specific antibodies cannot be excluded, including autoimmune diseases or neoplastic diseases of different origin. Furthermore, as the control group has been sampled in the dermatology department, this group might contain patients with high numbers or irregular melanocytic nevi or even unrecognised melanoma.

For different extracellular tumor antigens, including gangliosides, in melanoma, positive associations between the induction of IgM and IgG antibodies and survival time have been reported [21] and the protective effect was attributed to antibody dependent cellular toxicity [22]. Therefore, the induction of Raf antibodies might be a favourable goal for the design of melanoma vaccines. However it is questionable whether humoral responses against an intracellular antigen like Raf will have an effect. In this respect it is interesting to ask why responses against intracellular antigens like Raf or survivin [23] can be observed at all. The most straightforward explanation, which is particularly likely for advanced stage IV melanoma patients, relies on the fact that the partial necrosis of big tumor masses allows the crosspresentation of intracellular proteins. The longitudinal analysis of our patients, e.g. the one depicted in figure ​figure1,1, illustrates, that the occurrence of humoral immune responses was correlated rather to tumor burden than therapeutic measures, since the conversion occurred during the period when no therapy was applied.

As oncogenic mutation of B-Raf and even transformation by other oncogenic events is frequently accompanied by Raf overexpression, the induction of an antibody response is not necessarily due to the presence of the mutation. Even though a polyclonal IgM response is very unlikely to be specific for a point mutation, this notion would explain the lack of specificity for the mutational epitope. However, it is also possible that a cellular immune response prior to the induction of Raf specific antibodies has occurred. Such a T-cell response directed against Raf or other tumor antigens, would also result in the lysis of tumor cells and the liberation of intracellular antigens. It is important to mention that the lysis leading to the formation of antibodies against intracellular antigens can be caused by T-cells other than the already observed B-Raf V599E specific CD4⁺ or CD8⁺ T-cells. This question still remains to be clarified as up to now only advanced stage patients have been examined. It is obvious that at this stage the occurrence of antibodies is not correlated with prognosis and that it is very unlikely that the antibody response itself will have an influence on the disease. However, as it is rather unlikely that the frequency of a B-Raf specific T-cell response exceeds the frequency of antigen responses, our data can help to set the upper limit for the expected frequency of B-Raf or B-Raf V599E specific T-cells as 8.5% which allows the rational design of search strategies especially for Raf specific CD8⁺ T-cells.