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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patients with metastatic renal cell carcinoma (mRCC) have a limited life expectancy but still a subset of these patients develop immune and clinical responses after immunotherapy including dendritic cell (DC) vaccination. In a recently published phase I/II trials, fourteen HLA-A2 negative patients with progressive mRCC were vaccinated with autologous DC pulsed with allogeneic tumour lysate. Low-dose IL-2 administered subcutaneously was given concomitantly. In this study, we analysed lysate specific proliferation of PBMCs from these patients together with the TH1/TH2 balance of the responding T cells. Also, serum concentrations of IL-10, IL-12, IL-15, IL-17 and IL-18 from these patients and additional thirteen HLA-A2 positive mRCC patients treated with autologous DC pulsed with survivin and telomerase peptides were analysed during vaccination to identify systemic immune responses and potential response biomarkers. In HLA-A2 negative mRCC patients a spontaneous predominance of TH1 secreting tumour lysate specific T cells was observed prior to vaccination in patients attaining stable disease (SD) during treatment whereas patients with continued progressive disease (PD) had a mixed TH1/TH2 response. The TH1/TH2 balance was unchanged during vaccination also when tumour lysate specific T cell responses increased. An increase in IL-12, IL-17 and IL-18 serum concentrations was observed during vaccination but no difference between patients with SD and PD was observed. IL-10 or IL-15 was not measurable in serum.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Renal cell carcinoma (RCC) is a relatively rare disease, comprising approximately 2% of all malignancies. There is a great variability in the clinical behaviour of RCC but up to 50% of diagnosed patients develop metastatic disease with a poor prognosis and a median survival time of <1 year [1]. The disease is highly resistant to conventional therapies such as radiation and chemotherapy with response rates below 10%, a short duration of response and no effect on overall survival. It is well documented that treatment with IFN-α and IL-2 based immunotherapy gives moderate response rates but some of the patients achieve complete responses and become long term survivors [1]. This, together with the prognostic importance of lymphocyte infiltration in RCC [2, 3] has made RCC an obvious candidate for new immunotherapeutic strategies.

In the recent years, therapeutic cancer vaccination based on DC has been tested in numerous clinical trials, most notably in patients with malignant melanoma and renal cell carcinoma [4–10]. The goal of the treatment is to eliminate cancer cells by a specific activation of the patients’ immune system with induction of an immune response against tumour cells. DC are potent antigen presenting cells with capacity to capture and process antigens by phago- or pinocytosis followed by targeting to HLA class I and II molecules and antigen presentation to the T cells [11]. DC for vaccination can be generated by isolation of CD14+ mononuclear cells from peripheral blood, culture of these cells with GM-CSF and IL-4, maturation and antigen loading. Synthetic HLA binding peptides are commonly used as tumour antigens [9]. However, most identified peptides are HLA-A*0201 restricted which is the most common allele in the Caucasian population where it is expressed in approximately 50% of individuals. An alternative and HLA independent approach is the use of tumour lysate derived from autologous tumour tissue or allogeneic tumour cell lines which also has the advantage of inducing a polyclonal immune response against multiple epitopes on the cancer cells [12, 13].

We have recently carried out a clinical phase I/II trial using dendritic cell based vaccination of patients with mRCC [8]. In this trial, HLA-A2 positive patients were treated with autologous DC pulsed with telomerase and survivin peptides while HLA-A2 negative patients were treated with autologous DC pulsed with allogeneic tumour lysate generated from RCC allogeneic tumour cell lines. Safety and clinical response was monitored in all patients and immune reactivity was tested in HLA-A2 positive patients. In the present study, we analysed the immune response in HLA-A2 negative patients against tumour lysates and demonstrated a dominant tumour lysate specific TH1 response prior to and during vaccination in patients with stable disease (SD) and a mixed TH1/TH2 response in patients with progressive disease (PD). IL-10, IL-12, IL-15, IL-17 and IL-18 were measured in serum from both HLA-A2 positive and negative patients and we observed a small increase in IL-12, IL-17 and IL-18 serum concentration during vaccination but no significant differences were observed between patients with SD or PD.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patients.  In total, 27 patients with progressive mRCC were treated with DC vaccination as reported previously [8]. Fourteen of the patients enrolled in this phase I/II trial were HLA-A2 negative, and sufficient material for immune monitoring was present in 11 of these patients. All eligible patients were scheduled for at least six vaccinations with 1 × 107 mature antigen pulsed DC. Vaccinations 1–4 were given weekly and vaccinations 5–10 were given biweekly, followed by monthly vaccination until disease progression. HLA-A2 negative patients were immunized with autologous DC pulsed with tumour lysate and Keyhole Limpet Haemocyanine. From the 2nd vaccine, concomitantly IL-2 was administered at dosage 2 MIU Interleukin 2 (Proleukin; Swedish Orphan, Denmark) subcutaneously from day 2–6 after each vaccination. Disease stabilization as determined by the RECIST-criteria was seen in 7/11 patients.

Generation of mature antigen loaded DC.  Dendritic cells were generated as previously described in details [8–10]. In brief, autologous peripheral blood mononuclear cells (PBMC) were isolated by leucapheresis, washed and adhered to plastic Nuclon dishes (Nunc, Slangerup, Denmark) for 1 h. Adherent monocytes were then differentiated into DC by addition of 250 U/ml rhIL-4 (CellGenix, Freiburg, Germany and 1000 U/ml rhGM-CSF (Leucomax; Schering Plough, New York, NY, USA) for 7 days. DC were loaded with 100 μg/ml and 50 μg/ml tumour lysate on day 5 and 6 respectively and matured with 1000 U/ml IL-1β, 1000 U/ml TNFα, 1000 U/ml IL-6 (all CellGenix) and 1 μg/ml PGE2 (Pfizer, Brussels, Belgium). Allogeneic tumour lysate was prepared from allogeneic RCC cell lines; A-498, Caki-1 and Caki-2 (American Type Culture Collection, ATCC®) For use in in vitro studies, unpulsed DC maturated with the maturation cocktail described above were frozen. In the in vitro assays either mature unpulsed DC [so-called nude DC (DC-N)] or mature tumour lysate pulsed DC (DC-P) were used.

Blood samples for immune evaluation.  Heparinized blood samples were collected at the following time points: At baseline, after the 4th, 6th, 10th vaccination and hereafter every 3 months until disease progression. PBMC were separated by centrifugation on a Lymphoprep (Nycomed, Oslo, Norway) density gradient using standard procedures. Aliquots of PBMCs were frozen in RPMI with 10% human AB serum and 10% DMSO at −140 °C.

ELISPOT assays.  peripheral blood mononuclear cells from different time-points during vaccination were thawed, and adjusted to 105 cells/well in round bottomed 96-well culture plate. PBMC were co-cultured with 104 cells/well DC-P in a total volume of 200 μl RPMI-1640 supplemented with Glutamax and antibiotics and placed in the incubator (37 °C, 5% CO2). On day 1, 100 IU/ml IL-2 was added to each well, and PBMC were expanded for another 6–8 days. 105 expanded cells were then set up in triplicates in an ELISPOT assay against 104 cells/well DC-N, DC-P, culture medium or phytohemaglutinin (PHA) (10 μg/ml) and the number of IFN-γ, IL-4 and IL-5 spot forming cells was analysed. The ELISPOT assay was performed as previously described [9, 10]. In short, MAHAS4510 plates (Millipore, Molsheim, France) were coated with 7.5 μg/ml anti-human IFN-γ or 5 μg/ml anti-human IL-4 or anti-human IL-5 capture antibody (anti IFN-γ from Endogen USA and others from BD Pharmingen, Rockford, IL, USA), respectively in PBS as 75 μl/well. The plate was stored at 4 °C overnight, washed six times in PBS and blocked for 2 h (37 °C, 5% CO2) with complete medium. Cells were then added as described above and the plate incubated for 20 h (37 °C, 5% CO2). After incubation, cells were discarded and the ELISPOT plate was washed six times in PBS/0.05% Tween 20 and relevant detecting antibody was added. These were 0.75 μg/ml biotinylated anti-human IFN-γ and 2 μg/ml anti-human IL-4 or IL-5 (anti-IFN-γ from Endogen USA and others from BD Pharmingen). After incubation for 2 h at room temperature the plate was washed six times in PBS/ 0.05% Tween and horseradish peroxidase conjugated streptavidin (75 μl/well, 1:500 dilution of P037 from DAKO, Glostrup, Denmark) was added and the plate was incubated for 1 h at room temperature. Then, after a final washing step, 200 μl/well substrate (4-chloro-1-naphtol dissolved in 10 ml methanol and 50 ml tri-ethanol buffered saline together with H2O2) was added and the plate developed for 20–60 min before the reaction was stopped with water. The spots were counted by an automated ELISPOT plate reader (Carl Zeiss Vision, Jena, Germany).

Responses were considered positive when >20 spots/well were counted and when significantly increased compared with response from PBMC without stimulating DC.

Proliferation assay.  Peripheral blood mononuclear cells were set up with DC-N, DC-P, medium or PHA as described in the ELISPOT assay and co-cultured in round-bottomed 96-well plates. Cells were incubated for 4–5 days before addition of 3H-Thymidine with a concentration of 25 μCi/ml, giving a total concentration of 0.5 μCi/well. After incubation for 18 h (37 °C, 5% CO2), cells were harvested onto 96 well filter-plates (Unifilter GF/C from Perkin Elmer, Waltham, MA, USA). Plates were analysed in a scintillation counter after adding 20 μl/well scintillation fluid.

Cytokine measurements.  Serum samples were collected at baseline, after the 4th, 6th vaccine and stored at −80 °C until analysis. Serum IL-10, IL-12, IL-15 and IL-17 were analysed using a 4-Plex assay kit from Biosource (Biosource, Invitrogen, Carlsbad, CA, USA Cat No LHC0101, LHC0121, LHC0151 and LHC0171 respectively) according to the manufactures protocol. Measurement was carried out using the Luminex 100 IS (Luminexcorp, Austin, TX, USA). More than 100 events were acquired per bead set. StarStation ver. 2.0 software (Applied Cytometry Systems, Sheffield, UK) was used for cytokine quantification analysis. IL-18 was measured with a commercial ELISA kit (Biosource, Cat. No KHC0181).

Flowcytometry.  Cells from co-cultures of DC and PBMC were harvested and stained for 0.5 h in PBS with 0.25% BSA, using fluorescein isothiocyanate (FITC) or phycoerythrin (PE) coupled antibodies against CD4, CD8, CD45RA and CD45RO (all from DAKO). Subsequently, cells were washed twice and cells were analysed on a FACSCalibur (BD , Franklin Lakes, NJ, USA) and data were analysed using BD CellQuest software. At least 10,000 cells were collected, using a live gate based on forward scatter/side scatter properties.

Statistics.  Significant differences between logaritmitized sample means from SD and PD patients were determined with the two-tailed paired Student’s t-test, and results were considered significant when P < 0.05. For determination of differences over time a linear mixed-effect model was used. A two-way anova was used to determine significant differences in proliferation assays.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patient characteristics and clinical response

Detailed data on patient characteristics, safety and clinical response have been published elsewhere [8]. In summary, 14 out of the 27 renal carcinoma patients enrolled in the trial were HLA-A2 negative, and sufficient material was present in 11 of these for use in immune monitoring. Seven of eleven patients had stable disease and 4/11 patients had progressive disease after six vaccinations as determined by the RECIST criteria (see Table 1).

Table 1.   Patient characteristics.
Patient NoClinical response Age/sexNo. meta-static sitesTotal no. vaccinesPFS (mo)Survival after 1. vaccine (mo)
  1. SD, stable disease; PD, progressive disease.

 4SD57/M210 3.929.5+
 6SD79/F213 7.625
 8SD61/M210 3.7 7.5
16SD60/M2 6NA 5
17SD38/M413 6.716.5
18SD61/M12115.220+
24SD78/M213 7.811
28SD66/F4231716+
 3PD59/M3 61.96.5
10PD60/M3 62.1 3
21PD54/M3 62.6 5
26PD42/M2 62.810.5
29PD70/M2 61.8 4.5
30PD59/M3 61.913+

Prevaccination lysate specific TH1/TH2 deviation

To test lysate specific T cell responses, PBMC collected pretreatment were stimulated with autologous unpulsed (DC-N) or tumour lysate pulsed (DC-P) dendritic cells in an ELISPOT assay. Because dendritic cells were used as stimulator cells we generally observed a high background. Also, as an allogeneic lysate was used, we observed lysate specific T cell responses before vaccination (Fig. 1). Compared with unpulsed DC, tumour lysate pulsed DC induced significant numbers of IFN-γ secreting T cells (P = 0.03) in prevaccination samples from patients with SD, whereas no lysate specific IL-4 or IL-5 secretion was observed (P = 0.3 and 0.2 respectively) (Fig. 1). In contrast, both significant lysate specific IFN-γ (P = 0.03), IL-4 (P = 0.01) and IL-5 (P = 0.004) secretion was observed in samples from patients with PD, thus indicating that patients with SD had a predominant TH1 response, whereas patients with PD had a mixed TH1/TH2 response.

image

Figure 1.  Spontaneous tumour lysate specific T cell responses in HLA-A2 negative mRCC patients. Tumour lysate pulsed DC were co-cultured with PBMC obtained prior to vaccination and responsive cells were rechallenged with mature tumour lysate pulsed DC (DC-P) or unpulsed DC (DC-N) in the ELISPOT assay. The mean number of SFC’s producing IFN-γ, IL-4 and IL-5 prevaccination was analysed in SD and PD patients. Data are presented as the mean ± SEM of more than nine individual patients. *P < 0.05.

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Vaccine induced immune responses

We next analysed the vaccine induced lysate specific IFN-γ, IL-4 and IL-5 secretion from T cells in an ELISPOT assay together with total lysate specific proliferation of PBMC at the indicated timepoints. In patients with PD, a significant level of lysate specific IFN-γ as well as IL-4 and IL-5 secreting cells were demonstrable throughout the vaccination period, indicating a mixed TH1/TH2 response in this group of patients (Fig. 2). Also, lysate specific responses seem to increase over time, suggesting a vaccine induced combined TH1/TH2 response. In patients with SD we found a markedly increase in IFN-γ secretion after the 13th vaccination whereas a less marked increase in IL-4 and IL-5 is seen over time (Fig. 2). Thus, although a TH2 response is induced, the T cell response still seems predominantly TH1 like also at late time points of vaccination. Finally, analysis of lysate specific PBMC proliferation demonstrated a rapid increase in patients with SD, whereas less increase in lysate specific proliferation over time was seen in patients with PD (Fig. 2). Also to be noticed is the difference in the level of proliferation in the two groups where SD patients exhibited initial cpm around 2000 whereas PD patients exhibited cpm 10 times higher.

image

Figure 2.  Tumour lysate specific T cell responses in HLA-A2 negative mRCC patients during vaccination. Tumour lysate pulsed DC were co-cultured with PBMC obtained before and during vaccination and reactive cells were rechallenged with mature tumour lysate pulsed DC (DC-P) or unpulsed DC (DC-N) in the ELISPOT assay. In addition proliferation of primary cultures was measured by 3H-thymidine incorporation. The mean number of SFC’s producing IFN-γ, IL-4 and IL-5 or lysate specific proliferation was analysed. Data are presented as the mean ± SEM of up to six individual patients. *P < 0.05.

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Phenotype of lysate specific T cells

From co-cultures of DC and PBMC we analysed the phenotype of reactive T cells using flowcytometry. In experiments where lysate-pulsed dendritic cells were co-cultured with PBMC obtained at different time-points of vaccination, we observed that primarily CD4 T cells where expressing CD45RO indicating that mostly CD4 + T cells and not CD8 + T cells were activated (Fig. 3). However, we were not able to identify lysate specific changes in T cell phenotype and the observed changes were also present in samples before vaccination (data not shown).

image

Figure 3.  Phenotype of tumour lysate specific T cells in HLA-A2 negative mRCC patients during vaccination. Tumour lysate pulsed DC were co-cultured with PBMC obtained before and during vaccination and reactive cells were analysed for expression of CD4, CD8, CD45RA and CD45RO using flowcytometry. Data are presented from one out three patients analysed.

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Serum cytokine profile

We analysed concentrations of IL-10, IL-12, IL-15, IL-17 and IL-18 in serum of 27 mRCC patients during vaccination. Measurable concentrations were obtained for IL-12, IL-17 and IL-18 (Fig. 3) whereas IL-10 and IL-15 was not detectable in serum. IL-12 and IL-17 was evaluable in all 27 mRCC patients whereas IL-18 data were only obtained from 12 patients. We observed an increase in IL-12 after four and six vaccinations (P < 0.0001 and P = 0.009 respectively), a small increase in IL-17 after six vaccinations (P = 0.045) and a small increase in IL-18 after four vaccinations (P = 0.004) as compared with pretreatment values. No significant difference was measured in patients with PD as compared with SD patients before or after vaccination (Fig. 4).

image

Figure 4.  Cytokine serum concentrations during vaccination in mRCC patients. IL-12, IL-17 and IL-18 was analysed from serum before and during vaccination. Data are presented as median (solid black line), the 25th and 75th percentiles (boxed) and the maximum and minimum value (error bars) of up to 14 patients with SD or PD. *P < 0.05, **P < 0.001 for all patients (both SD and PD) as compared with pretreatment values.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

We have previously performed a clinical phase I/II trials in patients with mRCC [8] to test safety, immunogenicity and clinical efficacy of vaccination with tumour antigen pulsed DC. In this trial patients were allocated to treatment with DC pulsed with either HLA-A*0201 binding tumour antigen derived peptides (HLA-A2 positive patients) or DC loaded with allogeneic tumour lysate (HLA-A2 negative patients). In the present study, we analyse the tumour lysate specific T cell responses in HLA-A2 negative patients. Tumour lysate specific T cell IFN-γ, IL-4 and IL-5 responses were measured in an ELISPOT assay based on co-culture of PBMC and lysate pulsed DC at various time points before and during vaccination. In addition, serum concentrations of IL-10, IL-12, IL-15, IL-17 and IL-18 were measured in both HLA-A2 positive and negative patients before and during vaccination.

Vaccines based on DC pulsing with allogeneic tumour lysate were well tolerated as previously described [8]. The primary side effect was fatigue and flu-like symptoms with fever, chills and muscle pain because of IL-2 administration. Indeed, several clinical trials using vaccination with DC pulsed with tumour lysate have now established that obtained side effects are minimal [14–16]. In the present study, all patients had verified disease progression when entering the trial and 13/27 patients achieved SD after six vaccinations. None of the patients achieved an objective treatment response. In comparison, a few objective clinical responses according to RECIST criteria have been observed in both metastatic MM and mRCC in some analogous trials [16–18]. Also, DC-based vaccination of patients with e.g. glioblastoma multiforme, B-cell chronic lymphocytic leukaemia, colorectal cancer or thyroid cancer has demonstrated induction of tumour specific immune responses and in some studies clinical efficacy [14, 19–21].

Antigen loading of DC with tumour lysates have previously been tested, and it has been shown that lysate pulsed DC can be maturated with cytokines and cryopreserved without loss of CCR7 expression and ability to induce tumour lysate specific T cell responses including CTL responses [12, 13]. As, DC are potent stimulators and allogeneic lysates were used as antigen, we observed a spontaneous tumour lysate specific response in PBMC already prior to vaccination. This allowed us to investigate differences in pre-existing immune responses in the patients. We were able to identify significant differences in patients obtaining treatment related SD compared with PD. Thus, patients with SD demonstrated a predominant tumour lysate specific IFN-γ secretion from T cells whereas patients with PD reacted with secretion of both IL-4 and IL-5 in addition to IFN-γ. IFN-γ is a pro-inflammatory cytokine which suggests a predominant TH1 response present in the SD patients. IL-4 and IL-5 are involved in TH2 responses [22] pointing towards a mixed TH1/TH2 response in the PD patients prior to vaccination. Similarily, potent TH1 responses have been shown to be induced when patients are co-treated with e.g. tetanus toxoid pulsed DC [16]. Furthermore, murine experimental models of immunotherapy have clearly demonstrated that tumour antigen specific TH1 cells can lyse tumour cells via FasL–Fas interaction, thus complementing the cytolytic functions of CTL, whereas TH2 cells in contrast can promote tumour progression by promoting proliferation and cytokine production from tumour cells [23]. Similarly, a predominant increase in IFN-γ secretion during treatment was observed in patients with SD whereas in patients with PD IL-4 and IL-5 secretion increased as well. In particular, tumour lysate specific IFN-γ has been shown to correlate to objective changes in tumour burden in some [15] but not all studies [16].

The presence of IL-5 alone does not necessary indicate a TH2 response – in one of the few studies where multiple cytokines have been analysed in a few patients, allo specific IL-5 production was actually seen in conjunction with an array of TH1 promoting cytokines without IL-4 secretion [24]. In addition, DC based vaccination against tumour lysates have been shown to increase the number of NK cells during vaccination [15], and it is possible that part of the IFN-γ response seen in our study could be due to activation of these cells.

A major aim of cancer vaccination is the induction of CTL with specificity for tumour antigen derived 9 mer peptides associated with MHC class I molecules. It is likely that such cells are also induced in our study, as other studies of vaccination with tumour lysate pulsed DC have shown that these are induced either directly or as the result of epitope spreading [16]. However, tumour peptide responses are most easily assessed in HLA-A2 positive patients and was not included in these HLA-A2 negative patients.

A large number of preclinical models demonstrate that IL-10 has immunosuppressive actions in cancer, e.g. by suppression of CD8+ T cells, NK and NKT cells and by decreasing MHC expression of tumour cells [25–28]. Thus, as IL-10 was not detectable in serum from these patients, the immunity was not likely to be blunted by this mechanism. In contrast, we observed measurable IL-12 in serum of most patients which was slightly increased during vaccination. This cytokine is known to activate TH1 cells and NK cells, upregulate MHC molecules and to induce IFN-γ secretion of T cells which is important for antitumour effects [29–31]. However, IL-15 which stimulate NK cells and activated CD8 + T cells was not measurable and as this cytokine protects against IL-2 mediated activation induced cell death and demonstrate a preferential activation of memory CD8 + T cells as compared with Treg cells [32, 33] induction of IL-15 secretion during vaccination would be preferable. In contrast, we were able to measure a small increase in IL-18 which is known to promote IFN-γ secretion, TH1 differentiation and NK activation, but at the same time can be involved in tumour progression. Thus, the functions of this cytokine in cancer are more complex, indeed high serum levels of IL-18 are often detected in patients with progressive cancer [34–36]. Finally, we also detected a small increase in IL-17 after six vaccinations. IL-17 has important roles in chronic inflammation and is now known to be secreted from a unique T cell subset known as TH17 cells. However, whether IL-17 is beneficial or not in cancer is still under debate [37, 38]. None of the cytokine concentrations of IL-12, IL-17 or IL-18 was significantly different between patients with SD and PD and could therefore not be used as response biomarkers.

In conclusion, this study shows that treatment with autologous DC pulsed with allogeneic tumour lysate can induce tumour lysate specific T cell responses in patients with metastatic renal cell carcinoma. A predominant TH1 response with tumour lysate specific IFN-γ T cell responses before and during vaccination was shown to correlate to disease stabilization. In contrast, this could not be observed in serum concentrations of cytokines where both SD and PD patients had comparable levels of IL-12, IL-17 and IL-18 which was slightly increased during vaccination in both groups of patients. No patients obtained complete remission. Therefore, the future of DC based cancer vaccines probably lies in a combined regimen with current cancer treatments, depletion of regulatory T cells or expansion of effector T cells, but further data are needed on the effects of such in immune competent patients.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This work was supported by grants from The Danish Cancer Society and ‘Aase og Ejnar Danielsens Fond’. We would like to thank Kirsten Nikolajsen, Eva Gaarsdal and Ane Rulykke for excellent technical performance. We would like to thank Tobias Wirenfeldt Klausen for help with statistical analysis.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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