Twelve-year survival and immune correlates in dendritic cell-vaccinated melanoma patients

Abstract

Background: Reports on long-term (≥10 years) effects of cancer vaccines are missing. Therefore, in 2002, we initiated a phase I/II trial in cutaneous melanoma patients to further explore the immunogenicity of our DC vaccine and to establish its long-term toxicity and clinical benefit after a planned 10-year followup.

Methods: Monocyte-derived DCs matured by TNFα, IL-1β, IL-6, and PGE2 and then loaded with 4 HLA class I and 6 class II-restricted tumor peptides were injected intradermally in high doses over 2 years. We performed serial immunomonitoring in all 53 evaluable patients.

Results: Vaccine-specific immune responses including high-affinity, IFNγ-producing CD4+ and lytic polyfunctional CD8+ T cells were de novo induced or boosted in most patients. Exposure of mature DCs to trimeric soluble CD40 ligand, unexpectedly, did not further enhance such immune responses, while keyhole limpet hemocyanin (KLH) pulsing to provide unspecific CD4+ help promoted CD8+ T cell responses - notably, their longevity. An unexpected 19% of nonresectable metastatic melanoma patients are still alive after 11 years, a survival rate similar to that observed in ipilimumab-treated patients and achieved without any major (>grade 2) toxicity. Survival correlated significantly with the development of intense vaccine injection site reactions, and with blood eosinophilia after the first series of vaccinations, suggesting that prolonged survival was a consequence of DC vaccination.

Conclusions: Long-term survival in advanced melanoma patients undergoing DC vaccination is similar to ipilimumab-treated patients and occurs upon induction of tumor-specific T cells, blood eosinophilia, and strong vaccine injection site reactions occurring after the initial vaccinations.

Trial registration: ClinicalTrials.gov NCT00053391.

Funding: European Community, Sixth Framework Programme (Cancerimmunotherapy LSHC-CT-2006-518234; DC-THERA LSHB-CT-2004-512074), and German Research Foundation (CRC 643, C1, Z2).

Keywords: Clinical Trials; Vaccines.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Study scheme.

(A) Leukaphereses were performed before trial start and then repeated after 3 months, 1 year, and 2 years. Vaccinations were given in increasing intervals of 2 weeks to 6 months. Clinical evaluations were performed every 3 months. (B) CONSORT 2010 flow chart describing patient numbers for enrollment, induction, and maintenance phase of the trial, as well as trial completion.

Figure 2. Overview of immune responses.

Immune responses of all patients to vaccine peptides before and during induction and maintenance phase of DC vaccination as measured by in vitro–stimulated IFNγ ELIspot are shown. Data from several time points are combined (1 or 2 before and 2–6 time points for induction and maintenance phase, respectively). Plots show the means with whiskers approximating the 95% CI. (A) Cumulated responses of all patients to the different tumor peptides used. (B) Immune responses in each cohort combined for HLA-A1–, HLA-A2–, and the class II–restricted vaccine peptides. For comparison of KLH-DC– and CD40L-DC–induced responses, an unpaired 2-tailed t test with the assumption of inconsistent SD among samples and a false discovery rate approach of 1% was used.

Figure 3. Ex vivo cell analysis.

(A) Ex vivo multiplexed tetramer stainings were performed in 49 patients. Ex vivo–detectable frequencies of vaccine-specific CD8+ T cells are shown as % of total CD8+ T cells. Note the logarithmic scaling. Values below the detection limit are shown with gray background. Black lines and dots represent patients with a response that met the response definition criteria (at least 10 events in the tetramer gate and at least 0.01% of CD8+ T cells). For statistical analysis, a ratio-based 2-tailed paired t test with 95% confidence level was used. (B) Downregulation of CD45RA in ex vivo–detectable vaccine-specific T cells in comparison with virus-specific and total CD8+ T cells each before and after vaccination is shown as box plots of CD45RA mean fluorescence intensity (MFI) means with 95% CI. (C) Ex vivo class II tetramer staining (Tyro-DR4) is shown for patients 07, 50, and 62 at leukapheresis time points in combination with staining for FoxP3 and CD127. In the right panel, total CD4+ T cells are overlayed with tetramer-positive CD4+ T cells (red).

Figure 4. Overview and breadth of DC-vaccination–induced immune responses.

Shown are the numbers of positive immune responses (possible maximum of 4 responses to class I and 6 to class II tumor peptides ) detected by the different methods and according to the following response criteria: ELIspot, at least 2.5-fold above background and at least 10 spots after substraction of background at any time point; ex vivo tetramer, at least 0.01% of tetramer-positve cells among vital CD8+ T cells and at least 10 events in the tetramer gate; and MLPC, at least 1 well of the limiting dilution assay wells needs to be present with sufficient tetramer-positive cells.

Figure 5. Local vaccine reaction and rash.

Massive DTH reaction with formation of very large indurated areas (up to 20 cm) at the intradermal injection sites (photos of 1 limb only); simultaneous development of a rash (face, trunk, arms in patient 29 and in patient 36).

Figure 6. Overall survival.

Kaplan-Meier analysis of overall survival of fully evaluable metastatic (stage III + IV) melanoma patients (for characteristics, see Figure 4), either tumor-free or nonresectable. Two patients were censored after 87 and 117 months due to death unrelated to melanoma. None of the tumor-bearing patients still alive underwent therapy with kinase inhibitors. Only 1 patient underwent therapy with checkpoint blockade antibodies (patient 36 received 2 infusions of ipilimumab — 7 years after trial start in September 2011 — that had to be stopped due to autoimmune colitis. DC vaccination using a different method of tumor antigen loading, namely transfection with autologous tumor mRNA, was started in April 2012 and was kept up until recently).

Figure 7. Correlation of overall survival with vaccine injection site reactions and with development of eosinophilia upon vaccination.

(A) Kaplan-Meier analysis of overall survival according to differences in local reactions at vaccine injection sites. Reactions were either absent (n = 4), grade 1 (redness, n = 13), or grade 2 (redness and edema, n = 36) according to CTC criteria v 4.0. We subcategorized CTC grade 2 further in grade 2A (redness and induration) and grade 2B (redness and induration surrounded by a white margin due to very strong edema). Two patients censored after 87 and 117 months due to death unrelated to melanoma. For comparison of curves, a log rank (Mantel-Cox) test was used. The stronger the DTH reaction, the better the survival. If grade 0 + 1 were compared with grade 2 (i.e., grade 2A and grade 2B pooled), the P value was P < 0.0001. (B) Kaplan-Meier analysis of overall survival according to maximal absolute eosinophil counts (count per 100 μl of blood) after vaccination (time points from vacc #2 until vacc #10) in tumor-free and tumor-bearing patients. Two patients censored after 87 and 117 months due to death unrelated to melanoma death. For comparison of curves, a log rank (Mantel-Cox) test was used.