Edman degradation for quantitative HLA analysis
Although other techniques have in the meantime surpassed Edman chemistry in sensitivity, cost, and use for routine protein characterization, automated Edman degradation remains the most effective tool for obtaining N-terminal amino acid sequence information, and represents a reliable quantitative technique in its routine application [
26]. Therefore we chose Edman degradation with the intention of establishing a method for direct quantitative analysis of HLA molecules immunopurified from RCC. To our knowledge this is the first study comparing HLA yield on tumor, normal tissue and metastases of RCC using direct quantification. Previous studies used indirect methods, such as immunofluorescence, or analysed the HLA class I expression on mRNA level [
20,
27,
28].
Considering intrinsic effects of automated Edman degradation chemistry, such as inefficiencies in the coupling and cleavage reactions as well as different recoveries of PTH amino acids, we sequenced and analyzed the first seven amino acids of the HLA class I α-chain (GSHSMRY), focusing especially on quantitation of histidine in the third cycle. Raw values were neither corrected for repetitive yields (for the instruments operating in most laboratories, a 92-94% overall repetitive yield is considered acceptable [
26]) nor for initial yields or PTH amino acid recoveries [
23]. The first monitoring measurements with known yields of recombinant HLA class I monomers evinced histidine in sequence position 3 as most reliable parameter for the overall HLA amount of the analyzed sample. Other authors reported of large errors in the calculated amount of peptides when several different amino acids were used for repetitive and initial yield calculations [
26,
29].
The aim of this study was not to establish a method for determining the absolute amount of HLA class I molecules on the cell surface, but to obtain relative amounts that are comparable between different types of tissues. A study of the Edman Sequencing Research Group (ESRG) showed that the relative amounts comparing two different samples were highly accurate, the average error being 6.8% [
26].
Thus Edman degradation proved to be an appropriate method for direct quantitative comparison of HLA class I molecules immunopurified from tumor, normal tissue and metastases of RCC patients. We have to emphasize, however, that rather large samples are required with respect to the sensitivity of Edman sequencers. Our study included tissues of far more than 2 g in most cases; the need for a big sample size prevented the analysis of larger cohorts.
Results of quantitative HLA analysis
The presentation of HLA class I antigens on tumor cells represents a major factor in determining the success and clinical outcome of immunotherapeutic concepts aimed at increasing specific anti-tumor activity of CTLs. In order to predict the success of immunotherapeutic approaches for metastatic RCC, it was decided to compare HLA class I presentation on primary tumor, autologous normal kidney tissue and metastases of renal cell carcinoma. We performed this comparison on different levels, including local and distant metastases. To our knowledge this is the first study determining HLA class I yield directly on the protein level from RCCs and metastatic tissue.
The HLA yield was found in most cases to be dependent on the tissue mass (Table
1). Low amounts of HLA class I recovered from bulky tumors with a mass up to 60 g are presumably due to necrotic decay or stromal invasion of these tumors
in vivo.
The overall comparison of all 47 analyzed tissue samples (Figure
1) as well as the direct comparison of 24 autologous pairs of tumor and normal tissue (Figure
2) showed a significantly higher yield of HLA class I from tumor than normal tissue.
It has to be mentioned that tumors represent heterogeneous tissues which contain different subsets of cells. Although RCC is expected to be more homogenous than, for example, colon carcinoma, the tumors analyzed in this study might harbor endothelial cells and - most important - immune cells that could distort HLA quantification because of their high HLA expression. To demonstrate that a greater HLA yield from the tumors was indeed due to a higher HLA class I amount on tumor cells and not to tumor infiltrating lymphocytes (TILs), which could be found in 70% of all renal cell carcinomas (Table
1 and C. Gouttefangeas, unpublished), we compared primary RCCs from which TILs could be cultured with tumors from which no TILs could be isolated (Figure
2). Although we observed a tendency of TIL-containing tumors to yield slightly more HLA class I molecules than the TIL-free samples, the differences were not statistically significant (p = 0.51) and thus much smaller than the differences between tumor and normal tissue. From these data there seems to be no major impact of TILs on the results of quantitative HLA analysis in RCC tumors. On the other hand, we cannot rule out completely that endothelial cells or antigen presenting cells [
20,
21] contribute to the enhanced recovery of HLA molecules from tumor tissue and in particular from lymph node metastases. Immunohistochemical staining of tissues, as exemplarily shown in RCC377, confirms abundant expression of HLA class I on the surface of tumor cells but also endothelial and immune cells. Figure
3 gives an impression of HLA density on the different subsets of cell types. As outlined above, however, immunhistochemistry as a semi-quantitative method gives information on one layer of tissue only.
Recent reports have described a total or partial loss of HLA class I for nearly all types of tumors [
18,
30]. HLA class I downregulation or complete loss was observed in 10 - 50% of melanomas, breast, lung, colorectal, cervix and prostate cancers [
19]. In case of RCC, downregulation and loss of HLA class I has also been described: The amount of HLA negative tumors was determined to be up to 37.8% and was correlated with lower 5-year survival rates [
20]. On the other hand, higher HLA expression in RCC compared to normal kidney tissue has been reported as well [
22,
31]. The individual analysis of our study indicates a slight HLA downregulation on tumor tissue compared to normal tissue for only 5 of 24 tumors and normal tissue pairs (20.8%) (Table
1), whereas for most of the tumor tissues considerably higher HLA class I amounts were quantified. There was no significant difference in HLA presentation on different subtypes of RCCs investigated in this study.
Since T-cell based immunotherapy is not expected to clear bulky tumor masses, many clinical applications focus on the stage of minimal residual disease. To combat micrometastases remaining after dissection of the primary tumor, the metastases must express certain HLA levels and present tumor-associated peptides. In a recent report we confirmed that tumor-associated HLA ligands are indeed shared between primary tumors and metastases [
32]. Here, we performed a quantitative comparison of HLA class I molecules immunopurified from metastases, primary tumors, and normal tissues (Figures
1A, B) and detected significantly higher HLA amounts on metastatic tissue compared to normal kidney tissue. HLA class I yield from metastatic tissue was slightly higher than from tumor tissue, but beyond the level of significance. Contrary to the expectation that lymph node metastases, due to their higher amount of immune cells, should contain more HLA class I molecules, the comparison between lymph node metastases and distant metastases (Figure
1C) revealed a significantly higher amount of HLA on distant metastases than on normal tissue and lymph node metastases. These findings are in contrast to reports of HLA down-regulation or loss even stronger in metastases than in primary tumors [
33]. However, HLA loss rates vary between different tumor entities [
34] and there are only few reports on HLA expression in distant metastases of RCC. Numerous reports have correlated tumor progression with downregulation or complete loss of HLA [
16,
21,
35], for example in metastatic melanoma a high HLA class I amount (on metastases) correlated with regression whereas a low HLA amount correlated with progression under therapy [
16]. These facts and our data hold promise for the success of immunotherapeutic strategies also against metastatic RCC.