Long-term outcomes in patients with metastatic melanoma vaccinated with melanoma peptide-pulsed CD34(+) progenitor-derived dendritic cells

Abstract

Between March 1999 and May 2000, 18 HLA-A*0201(+) patients with metastatic melanoma were enrolled in a phase I trial using a dendritic cell (DC) vaccine generated by culturing CD34(+) hematopoietic progenitors. This vaccine includes Langerhans cells. The DC vaccine was loaded with four melanoma peptides (MART-1/MelanA, tyrosinase, MAGE-3, and gp100), Influenza matrix peptide (Flu-MP), and keyhole limpet hemocyanin (KLH). Ten patients received eight vaccinations, one patient received six vaccinations, one patient received five vaccinations, and six patients received four vaccinations. Peptide-specific immunity was measured by IFN-gamma production and tetramer staining in blood mononuclear cells. The estimated median overall survival was 20 months (range: 2-83), and the median event-free survival was 7 months (range: 2-83). As of August 2005, four patients are alive (three patients had M1a disease and one patient had M1c disease). Three of them have had no additional therapy since trial completion; two of them had solitary lymph node metastasis, and one patient had liver metastasis. Patients who survived longer were those who mounted melanoma peptide-specific immunity to at least two melanoma peptides. The present results therefore justify the design of larger follow-up studies to assess the immunological and clinical outcomes in patients with metastatic melanoma vaccinated with peptide-pulsed CD34-derived DCs.

Fig. 1

Tetramer staining on uncultured cells. a PBMCs, at baseline (left plot) and after four vaccines (right plot) are thawed, stained with MART-1 tetramer and 7AAD, and analyzed by FACS. Gating strategy: live cells were first gated based on low side scatter (SSC) properties and exclusion of 7AAD (viability dye) (R1); next, lymphocytes were gated based on forward scatter (FSC) properties (R2), and high CD8 expression (R3) as we have learned that CD8 medium staining cells are often apoptotic and therefore represent a confounding parameter; finally, high-intensity tetramer-binding cells were measured (R4). b Percentage of MART-1 tetramer+CD8+T cells (ordinate) in melanoma patients and healthy volunteers (HV, green bars) (abscissa). Patient samples (apheresis) were analyzed in at least three different experiments at baseline (black bars) and after four DC vaccinations (red bars). Average and standard deviation are shown for each patient

Fig. 2

Peptide-specific IFN-γ secretion correlates with the frequency of tetramer-binding CD8+T cells. a Percentage of MART-1 tetramer+ and (b) Flu-MP tetramer+CD8+T cells (ordinate) correlates with direct MART-1 or Flu-MP-specific IFN-γ ELISPOT (abscissa, number of spots/2×10⁵ PBMCs). Non-parametric Spearman correlation, axes are log transformed for visualization

Fig. 3

Breadth of melanoma peptide-specific immunity and survival. Overall (a–c) and event-free (d) survival in patients who mounted immunity to none (a), one (b) or more than one (c) melanoma antigens. A positive response to melanoma antigen was considered as at least threefold increase in peptide-specific IFN-γ ELISPOT and ≥10 spots/2×10⁵ PBMC. Logrank test. Survival proportions (ordinate) at indicated time (abscissa), status as of August 2005