Abstract
Purpose: To study the effect of autologous tumor cell vaccinations on the presence and numbers of circulating CD8+ T cells specific for tumor-associated antigens (TAA) in metastatic melanoma patients. To investigate the correlation between the presence of tumor-infiltrating lymphocytes (TIL) and circulating TAA-specific CD8+ T cells before and after autologous tumor cell vaccination with overall survival. Experimental design: Twenty-five stage III and resected stage IV metastatic melanoma patients were adjuvantly treated with a series of intracutaneously injected autologous tumor cell vaccinations, of which the first two contained BCG as an immunostimulatory adjuvant. Tumor samples and blood samples obtained before and after vaccination of these patients were studied for the presence of TAA-specific T cells using HLA-tetramers and results were correlated with survival. Results: In 5 of 17 (29%) melanoma patients, circulating TAA-specific T cells were detectable prior to immunizations. No significant changes in the frequency and specificity were found during the treatment period in all patients. Presence of circulating TAA-specific T cells was not correlated with survival (log rank, P=0.215). Inside melanoma tissue, TAA-specific TIL could be detected in 75% of 16 available tumor samples. In case of detectable TAA-specific TIL, median survival was 22.5 months compared to median survival of 4.5 months in case of absence of TAA-specific T cells (log rank, P=0.0094). In none of the patients, TAA-specific T cells were found both in tumor tissue and blood at the same time. Conclusions: These data suggest that the presence of TAA-specific TILs forms a prognostic factor, predicting improved survival in advanced-stage melanoma patients. The absence of TAA-specific T cells in the circulation suggests that homing of the tumor-specific T cell population to the tumor site contributes to the effectiveness of antitumor immunity.
Keywords: Immunotherapy, Melanoma, HLA-tetramers, Autologous tumor cell vaccine, Tumor-infiltrating lymphocytes (TIL)
Introduction
Specific immunotherapy for solid tumors, such as peptide, dendritic cell or autologous or allogeneic tumor cell vaccinations, is unlikely to be successful in disease situations with a massive tumor burden (Reviewed in [1]). Probably the preferable clinical setting for new immunological therapies in humans is a minimal residual disease situation as in resected stage III or IV disease. However, in these disease states, the efficacy of the treatment cannot be evaluated by measuring responses according response evaluation criteria in solid tumors (RECIST), but only by measuring time-to-progression, disease-free survival or overall survival [2]. To show a benefit of adjuvant treatment, elaborate randomized clinical trials with large numbers of patients and long follow-up periods are required. Therefore, the development of tests that accurately predict the response to immunotherapy would be of great value. Available tests at present are measurement of immunotherapy-induced specific antitumor T cell responses by MHC tetramer analysis and/or ELISPOT assays and delayed type hypersensitivity (DTH) reactions to the vaccine. In most ongoing immunotherapy trials, immunomonitoring is being performed with these tests [3, 4]. The Cancer Research Institute has launched the Cancer Vaccine Collaborative (http://www.cancerresearch.org/cvc2002/index.html) for NY-eso-1 directed clinical immunotherapy strategies that all use exactly the same immunomonitoring tests to allow comparison of efficacy between trials. This initiative underscores the importance of standardized tests for evaluating immunological responses to immunotherapy.
Previously we have shown that DTH reactions to autologous tumor cell vaccines were highly predictive for long-term survival [5]. Here, in the same set of patients, we set out to study the presence of melanoma-specific T cells in peripheral blood before and during immunotherapy using validated HLA tetramers. In addition, we had the opportunity to study the presence of melanoma-specific tumor-infiltrating lymphocytes (TIL) in metastases that were used for vaccine preparation. It has been demonstrated that melanoma-specific T cells are most often directed against tumor-associated antigens (TAA) MART-1/melanA, tyrosinase and gp100 [6–12]. The majority of the HLA tetramers used for the analysis in this study contained an HLA-A2 restricted epitope from one of these melanosomal proteins.
Although several studies have shown that TAA-specific T cells are detectable in peripheral blood samples from many stage III and stage IV melanoma patients [13–15], the presence of circulating melanoma-specific CD8+ T cells is not correlated with prolonged survival [16]. Here, we show that the presence of TAA-specific T lymphocytes in the tumor infiltrating subset of the T cell compartment does appear to be predictive of prolonged survival in a cohort of resected stage III and IV melanoma patients treated with active specific immunotherapy .
Materials and methods
Patients
The patient population included in this study was selected from patients with metastatic melanoma referred to the Department of Medical Oncology of the Free University Medical Center for participation in our Active Specific Immunotherapy (ASI) trial between 1994 and 1999. All patients included were treated with autologous tumor cell vaccinations after having signed informed consent. Results and techniques that were used have been reported elsewhere [5]. For the current work, stage III and stage IV metastatic melanoma patients were selected that all had undergone a radical metastasectomy and were thereby macroscopically rendered free of tumor.
Vaccine preparation and administration
Single tumor cell suspensions were generated by enzymatic digestion of resected metastatic tumor fragments using DNAse and Collagenase. Cells were stored in liquid nitrogen and thawed and irradiated (200 Gy) on the day of vaccination. Each vaccine contained 107 viable tumor cells and to the first two autologous vaccines 107 life BCG organisms were added as an immune stimulatory adjuvant. One month after surgery, the vaccination procedure was initiated with three weekly intradermal injections in the upper legs and arms. Up to ten booster vaccinations were administered with 3-monthly intervals provided that cells were available and disease-progression absent. Forty-eight hours after each non-BCG containing injection, DTH skin reactions were measured.
Blood/sera/tumor samples for investigation
In the hour before the first tumor cell vaccination, blood was taken from all patients. From immunization three onward blood samples were obtained 48 h after each consecutive tumor cell injection. Serum was obtained by centrifugation of whole heparinized blood samples and stored at −20°C. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll-Hypaque gradient (Pharmacia, Sweden) and subsequently cryopreserved in liquid nitrogen until analysis. Tumor samples were obtained and treated as those for vaccine preparation (vide supra), except for BCG addition and irradiation, and cryopreserved.
Preparation of HLA tetramers
HLA class I tetrameric complexes were prepared as previously described with some modifications [17]. Peptide complexes were generated with the following antigenic peptides: HLA-A2.1: MART −126–35, ELAGIGILTV (A27L variant) [7, 18]; tyrosinase368–376, YMDGTMSQV (N370D variant) [11, 19, 20], gp100280–288, YLEPGPVTA [8, 12] and gp100154–162 (KTWGQYWQV) [6]. HLA-A3: MAGE-A196–104 [21]. In brief, cDNA encoding amino acid 1–276 of the HLA-A2.1 or HLA-A3 heavy chain was cloned in the expression plasmid pET3a upstream of a sequence containing the BirA enzymatic biotinylation site. HLA class I heavy chain and β 2-microglobulin (β2m) were separately expressed in Escherichia coli and were isolated as inclusion bodies. The inclusion bodies were first dissolved in 100 mM Tris (pH 8), 8 M urea. β2m was refolded by dialysis against 20 mM Tris (p H 7), 150 mM NaCl. Heavy chain and peptide were refolded together with β2m by dilution in 100 mM Tris. (pH 8), 400 mM L-arginine HCL, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 mM EDTA and Protease inhibitors. HLA class complexes were subsequently purified, biotinylated by BirA, converted to tetramers by the addition of PE labeled or APC-labeled streptavidin (Molecular Probes) and stored at −20°C. All MHC tetramers have been validated and titrated using specific patient-derived T cell lines [16, 21]. In case of MART-1 tetramers we chose to use the 10-mer modified version (ELAGIGILTV) in stead of the wild-type (EAAGIGILTV), because it was shown that the first is able to detect low-avidity MART-1-specific CD8+ T cells [22].
HLA-tetramer staining of PBMC and tumor cells
First, dead cells were selected and sorted out by means of propidium iodide staining. Second, for investigation of PBMC, 0.5×106 T-cells per sample were incubated for 15 min with 2 μg/ml APC- or PE-labeled MHC tetramer in combination with FITC-labeled anti CD8 mAb at 37°C [23].
For investigation of tumor material, vials containing 5.0×106 dissociated metastatic tumor cells (i.e., nonirradiated vaccines) were analyzed for presence of TAA-specific CTL using the same technique as described above for PBMC. No in vitro T cell expansion was performed, to be able to compare percentages of TAA-specific TIL with TAA-specific CTL in the circulation.
Samples were analyzed by flow cytometry using a FACS calibur and Cell Quest software (Becton Dickinson). Forward and side scatter parameters were used to define lymphocyte populations. For every analysis, at least 250.000 cells within the lymphocyte gate were required. Results were considered negative in case the frequency of MHC tetramer-positive cells was below 0.02% of CD8+ T cells or in case of high background in the CD8-negative population.
Histological assessment of TAA-expression
In case histological slides were available (n=13), expression of the TAAs gp100 and MART-1 was assessed using the monoclonal antibodies HMB45 (DAKO, 1:100) and MelanA (Novocastra, 1:50), respectively.
Statistical analysis
Survival curves were estimated by the nonparametric Kaplan–Meier method. The log-rank test was used for determining differences between survival curves. Univariate and multivariate tests according to Cox proportional hazards model were used to study the relationship between DTH and survival as well as the presence of TAA-specific TIL and survival. Pearson’s exact test was used to study the relationship between DTH and presence of TAA-specific TIL. Statistical analyses were performed with SPSS software version 9.0 for Windows.
Results
Blood and/or tumor samples of 33 resected stage III and stage IV melanoma patients were available. Twenty-five patients were typed positive for HLA-A2 and/or HLA-A3, enabling HLA-tetramer analyses. The median age was 48.3 years (range 21.1–73.3 years), and the sex distribution was 14 males and 11 females. Eighteen patients had surgically treated stage III metastatic melanoma; seven had stage IV disease and had undergone radical metastasectomies (lung 3×, soft tissue 4×) (Table 1). No patient had previously received any form of antimelanoma treatment, apart from surgery.
Table 1.
Patient characteristics
| Patient | Sex | Age | Disease stage | Site-resected metastasis | Expression of TAA on histological slides | Size DTH after third vaccination (mm) | TTP (months) | OS (months) | Clinical status |
|---|---|---|---|---|---|---|---|---|---|
| 1 | M | 64 | IV | Lung | MART-1/gp100 | 10 | 3.7 | 48.2 | D |
| 2 | F | 53 | III | LN | MART-1/gp100 | 12 | 1.4 | 11.5 | D |
| 3 | M | 23 | III | LN | MART-1/gp100 | 13 | 6.0 | 14.9 | D |
| 4 | M | 48 | III | LN | MART-1/gp100 | 28 | 72.2 | 72.2 | NED |
| 5 | F | 57 | III | LN | No expression | 31 | 2.2 | 18.3 | D |
| 6 | M | 24 | III | LN | – | 11 | 15.7 | 22.5 | D |
| 7 | M | 52 | IV | Skin | No expression | 17 | 0.2 | 2.8 | D |
| 8 | M | 51 | III | LN | MART-1/gp100 | 13 | 9.8 | 17.6 | D |
| 9 | M | 46 | III | LN | MART-1/gp100 | 37 | 84.4 | 84.4 | NED |
| 10 | F | 43 | III | LN | gp100 | 15 | 98.4 | 98.4 | NED |
| 11 | M | 21 | III | LN | MART-1/gp100 | 28 | 8.0 | 18.6 | D |
| 12 | F | 31 | III | LN | – | 28 | 92.1 | 92.1 | NED |
| 13 | F | 72 | III | LN | – | 17 | 21.0 | 62.1 | D |
| 14 | M | 44 | III | LN | No expression | 21 | 9.4 | 92.6 | AWD |
| 15 | M | 62 | III | LN | – | 35 | 33.0 | 33.0 | NED |
| 16 | F | 22 | III | LN | No expression | 13 | 5.6 | 29.7 | D |
| 17 | M | 56 | III | LN | – | 30 | 39.9 | 39.9 | NED |
| 18 | M | 73 | III | LN | – | 12 | 20.6 | 22.0 | D |
| 19 | F | 28 | III | LN | – | 30 | 75.7 | 75.7 | NED |
| 20 | M | 28 | IV | Skin | – | 22 | 23.3 | 113.0 | AWD |
| 21 | F | 61 | IV | Lung | MART-1/gp100 | 18 | 20.7 | 66.7 | D |
| 22 | M | 58 | IV | Lung | – | 18 | 4.0 | 87.2 | D |
| 23 | M | 37 | IV | Soft tissue | – | 10 | 1.7 | 16.6 | D |
| 24 | F | 25 | IV | Soft tissue | – | 15 | 3.5 | 4.5 | D |
| 25 | M | 59 | III | LN | – | 12 | 16.5 | 19.5 | D |
TAA tumour-associated antigen; DTH delayed type hypersensitivity reaction; LN lymph node; NED no evidence of disease; AWD alive with disease; TTP time to progression; OS overall survival; D died; – no histological slides available
From 17 out of 25 patients, blood samples could be collected both before the start of vaccination therapy as well as 48 h after consecutive autologous tumor cell vaccinations. Dissociated tumor cell vaccines that had not been used for vaccination therapy were available from 16 patients. From eight of these patients both tumor tissue and blood samples were present.
TAA-specific CD8+ cells in blood
Percentages of tetramer-positive T cells in peripheral blood samples were generally very low. Samples were considered to be positive in case a distinct population of ≥0.02% of CD8+ T cells was stained with HLA tetramers.
In prevaccination blood samples from 5 out of 17 patients (29%) one or more populations of tetramer-positive T-cells could be discerned, all with a high expression of CD8. Of all tetramers tested, only T cells specific for MART26–35 and tyrosinase368–376 were detected. MHC tetramer measurements after consecutive vaccinations revealed that over time frequencies of melanoma-specific T cells were more or less constant (Table 2). Compared with tetramer-negative patients, no difference could be found for patients with tetramer-positive T cells concerning tumor stage, site of initial metastases, age, disease-free survival (P=0.227), site of recurrent disease or overall survival (P=0.215) (Fig 1).
Table 2.
HLA-tetramer analyses on PBMC before and after vaccination therapy
| Patient | Preceding vaccination therapy | Following third tumor cell vaccination | ||
|---|---|---|---|---|
| TAA-specific CTL | % | TAA-specific CTL | % | |
| 1 | MART26–35 | 0.07 | MART26–35 | 0.05 |
| 2 | All negative | 0 | All negative | 0 |
| 3 | All negative | 0 | All negative | 0 |
| 4 | All negative | 0 | All negative | 0 |
| 5 | All negative | 0 | All negative | 0 |
| 6 | All negative | 0 | All negative | 0 |
| 7 | All negative | 0 | All negative | 0 |
| 8 | All negative | 0 | All negative | 0 |
| 9 | MART26–35 + Tyrosinase368–376 | 0.02, 2.10 | MART26–35 + Tyrosinase368–376 | 0.03, 1.56 |
| 10 | MART26–35 | 0.11 | MART26–35 | 0.16 |
| 11 | MART26–35 | 0.05 | MART26–35 | 0.04 |
| 12 | MART26–35 | 0.44 | MART26–35 | 0.39 |
| 13 | All negative | 0 | All negative | 0 |
| 14 | All negative | 0 | All negative | 0 |
| 15 | All negative | 0 | All negative | 0 |
| 16 | All negative | 0 | All negative | 0 |
| 17 | All negative | 0 | All negative | 0 |
The table shows percentages of circulating TAA-specific CTL before and after vaccination therapy, as compared to the total number of circulating CTL. It also shows that percentages of circulating TAA-specific CTL remain virtually unchanged, despite tumor cell vaccinations
PBMC peripheral blood mononuclear cells; TAA-specific CTL tumor-associated-antigen-specific cytotoxic T lymphocytes
Fig. 1.
Overall survival of advanced-stage melanoma patients with and without circulating tumor-associated-antigen (TAA)-specific cytotoxic T-lymphocytes (CTL)
TAA-specific CD8+ cells in tumor tissue
CD8+ tumor-infiltrating lymphocytes were detected in all patients, but the variability between patients was high. As a percentage of all viable cells in the tumor tissue, tumor-infiltrating CD8+ lymphocytes ranged from 5.4% to 27.9% (mean: 13.5%). For stage III patients, the median percentage of CD8+ lymphocytes inside tumor tissue was 14.2%, while for stage IV patients, this was 9.9% (P=0.06). No correlation was observed between the percentage of CD8+ TIL and clinical outcome.
Using HLA tetramers, TAA-specific T cells were identified in 12 of 16 (75%) available tumor samples. T cells recognizing the HLA-A2 epitopes MART26–35 (6/15), gp100154–162 (3/15), gp100280–288 (3/15) and tyrosinase368–376 (1/15) were encountered, while T cells specific for the HLA-A3 epitope MAGE-A196–104(0/4) could not be detected. In general, percentages of TAA-specific CTL were much higher in tumor tissue than in peripheral blood (Table 3).
Table 3.
HLA-tetramer analyses of tumor-infiltrating lymphocytes (TIL)
| Patient | TAA-specific TIL | TIL as a percentage of live cells in melanoma tissue | TAA-specific TIL as a percentage of all TIL in melanoma tissue |
|---|---|---|---|
| 1 | gp100280–288 | 10.7 | 1.5 |
| 2 | MART26–35 | 10.2 | 6.6 |
| 3 | Tyrosinase368–376 | 19.0 | 0.2 |
| 4 | MART26–35 + gp100154–162 | 13.9 | 1.7, 0.8 |
| 5 | MART26–35 + gp100154–162 | 18.7 | 0.3, 4.1 |
| 6 | gp100154–162 | 13.3 | 3.5 |
| 7 | All negative | 7.5 | – |
| 8 | All negative | 5.4 | – |
| 18 | MART26–35 + gp100154–162 + gp100280–288 | 14.2 | 1.4, 2.8, 3.7 |
| 19 | MART26–35 | 21.6 | 4.2 |
| 20 | MART26–35 | 9.9 | 0.9 |
| 21 | gp100154–162 + gp100280–288 | 16.5 | 0.8, 0.9 |
| 22 | gp100280–288 | 8.2 | 1.6 |
| 23 | gp100280–288 | 11.6 | 1.7 |
| 24 | All negative | 7.8 | – |
| 25 | All negative | 27.9 | – |
The third column shows the percentage of TIL compared to the total number of cells present inside the melanoma tissue and the fourth column shows the percentage of tumor-associated antigen-specific TIL (TAA-specific TIL) compared to the total number of TIL
Presence of TAA-specific TILs correlated with overall survival (P=0.0094); (Fig 2). In case TAA-specific TIL were present, the median OS was 22.5 months, while in case of their absence this was only 4.5 months. We did not find correlations between the presence of TAA-specific TIL in tumor and stage of disease, patient age, nor with delayed type hypersensitivity reactions to autologous tumor cells. We could not find a statistically significant relationship between the presence of TAA-specific TIL and time to recurrence of disease (P=0.208). This may be due to an artifact of small numbers.
Fig. 2.
Overall survival of advanced-stage melanoma patients with and without the presence of TAA-specific tumor-infiltrating T-lymphocytes (TIL) inside melanoma metastases
For eight patients, both blood and tumor tissue were available for investigating the presence of TAA-specific CTL. For six of these patients, TAA-specific CTL were either found in blood or in tumor, but in none of these patients, T cells could be detected for the same epitope in both blood and tumor tissue simultaneously (Table 4).
Table 4.
Comparison of TAA-specific CTL in blood and tumor
| Patient | Circulating TAA-specific CTL preceding vaccination | TAA-specific TIL |
|---|---|---|
| 1 | MART26–35 | gp100154–162 |
| 2 | All negative | MART26–35 |
| 3 | All negative | Tyrosinase368–376 |
| 4 | All negative | MART26–35 + gp100154–162 |
| 5 | All negative | MART26–35 + gp100154–162 |
| 6 | All negative | gp100154–162 |
| 7 | All negative | All negative |
| 8 | All negative | All negative |
The table shows results of tetramer analyses in patients of which both blood and tumor tissue were available. Preceding vaccination therapy, in no patient CTL were found for the same TAA in both blood and tumor tissue
TAA-specific CTL tumor-associated-antigen-specific cytotoxic T lymphocytes
As reported earlier, DTH reactions to non-BCG containing vaccinations correlated with TTP (P=0.037) and OS (P=0.051) [5]. In a multivariate Cox regression analysis, TAA-specific TIL (P=0.069) and DTH (P=0.099) appeared to have independent prognostic value for overall survival, although statistical significance was not reached.
Discussion
Here we show that the presence of melanoma-specificTILs may correlate with the clinical outcome of patients with resected metastatic melanoma. In this cohort of 16 metastatic melanoma patients of which tumor material was still available for HLA-tetramer analyses, we found that the presence of intratumoral tumor-associated antigen (i.e., MART-1 and gp100) -specific T cells was associated with a fivefold increase in median survival compared to patients with no TAA specific TIL. Although this survival difference was statistically significant, the study is small and needs confirmation from larger studies.
In various other tumor types, including breast, prostate, renal cell, esophageal, colorectal and ovarian cancer, the overall presence of tumor-infiltrating lymphocytes has been correlated with better prognosis [24–29]. This suggests that also in less immunogenic tumors, immune responses may actively participate in the body’s defense against cancer. However, it cannot be excluded that there is no causal link between prognosis and the presence of a lymphocytic infiltrate in these tumors. Here, we describe for the first time that the presence of TAA-specific tumor-infiltrating T cells is correlated with improved survival in vaccinated melanoma patients. Presence of tumor-specific T cell infiltrates in tumor tissue may reflect an ongoing immune response against melanoma. However, a recent study investigating melanoma-infiltrating MART-1 specific CTL showed the majority of antigen-specific TIL not to be able to produce IFN-γ after an antigenic stimulus, representing functional tolerance [15]. Vaccination of patients with immunogenic autologous tumor cells may boost the antimelanoma T cell reactivity, resulting in a better survival. Alternatively, survival may already be longer due to the presence of melanoma-specific TIL notwithstanding vaccination. Since all patients in this study underwent vaccination, its role in the improved survival in the group of patients with melanoma-specific TIL remains uncertain.
In this small-scale study, no correlation was found between overall survival and the absolute number of tumor-infiltrating CTL, while others have reported such relations (for instance [30]). This could indicate that it is rather the quality of the response, i.e., specificity for TAAs, than the quantity that determines the observed correlation between TAA-specific TIL and survival. Since the majority of the HLA tetramers were specific for melanosomal antigens MART-1, tyrosinase and gp100, we cannot exclude the possibility that despite the immunodominance of spontaneous T cell reactivity against these antigens, the outcome would be different with another set of HLA tetramers. Moreover, due to limited availability of tumor tissue from most patients, we cannot rule out that we overlooked TAA-specific CTL populations present at low precursor frequencies.
In this study, we did not find a correlation between the presence and specificity of melanoma-specific T cells in peripheral blood samples and melanoma tissue. Few studies investigated the simultaneous presence of melanoma-specific T cells at metastatic sites and peripheral blood. Disparity in frequencies between melanoma-specific T cells in tumor-involved lymph nodes and peripheral blood have been found [31, 32], although it is unclear whether this is a common phenomenon.
During the course of the vaccination treatment of the melanoma patients we did not observe significant changes in numbers of circulating TAA-specific CTL. To rule out that the absence of a post-vaccination frequency change resulted from a lack of antigen in the vaccines, expression of MART-1 and gp100 on tumor tissue was assessed by immunohistochemistry showing that these antigens were expressed on 8 and 9 of 13 investigated tumor samples, respectively (Table 1).
No correlation was found between the presence of circulating melanoma-specific T cells and clinical outcome which is in line with our recent report describing a cohort of 62 HLA-A2-positive stage IV melanoma patients in which circulating melanoma-specific T cells did not have impact on survival [16]. In contrast, in this small retrospective study in melanoma patients, we show that the presence of MART-1, tyrosinase and gp100-specific tumor-infiltrating lymphocytes in metastatic lesions appears to be predictive of a favorable outcome. One explanation may be that circulating melanoma-specific CTL lack homing capacities that are essential for the establishment of an effective antimelanoma immune response. Unfortunately, the limited availability of patient material did not allow combining MHC tetramer analyses with immunophenotyping (expression of CD62L, CLA, CCR7 etc), so that we could not address this issue properly.
In conclusion, presented data suggest that the presence of TAA-specific TIL predicts improved survival in advanced-stage resected melanoma patients. Secondly, circulating TAA-specific CTL do not reflect the intratumoral situation and therefore peripheral blood appears to be an inadequate source of tissue material to perform immunomonitoring studies. Biopsies from DTH skin reaction to the vaccine, however, may better reflect the intratumoral situation. DTH reactions have been shown to harbor tumor- or vaccine-specific T cells [33]. These T cells are more likely to share properties with TIL because of similar homing capacities and effector functions. Furthermore, since taking biopsies from tumor tissue will not always be feasible, biopsies from DTH skin site may be an alternative tissue source to monitor vaccination trials. Based on our preliminary results, future adjuvant vaccination trials should address surrogate endpoints like the presence of tumor-specific T cells in tumor or DTH site biopsies by incorporating MHC tetramer analysis and ELISPOT assays to confirm its predictive value for survival. In addition, this will allow studying the immunological impact of vaccination on survival as well, providing further incentive for vaccine-based studies.
Footnotes
J.B.A.G. Haanen and A. Baars contributed equally to this work.
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