Shipping blood to a central laboratory in multicenter clinical trials: effect of ambient temperature on specimen temperature, and effects of temperature on mononuclear cell yield, viability and immunologic function.

BACKGROUND: Clinical trials of immunologic therapies provide opportunities to study the cellular and molecular effects of those therapies and may permit identification of biomarkers of response. When the trials are performed at multiple centers, transport and storage of clinical specimens become important variables that may affect lymphocyte viability and function in blood and tissue specimens. The effect of temperature during storage and shipment of peripheral blood on subsequent processing, recovery, and function of lymphocytes is understudied and represents the focus of this study. METHODS: Peripheral blood samples (n = 285) from patients enrolled in 2 clinical trials of a melanoma vaccine were shipped from clinical centers 250 or 1100 miles to a central laboratory at the sponsoring institution. The yield of peripheral blood mononuclear cells (PBMC) collected before and after cryostorage was correlated with temperatures encountered during shipment. Also, to simulate shipping of whole blood, heparinized blood from healthy donors was collected and stored at 15 °C, 22 °C, 30 °C, or 40 °C, for varied intervals before isolation of PBMC. Specimen integrity was assessed by measures of yield, recovery, viability, and function of isolated lymphocytes. Several packaging systems were also evaluated during simulated shipping for the ability to maintain the internal temperature in adverse temperatures over time. RESULTS: Blood specimen containers experienced temperatures during shipment ranging from -1 to 35 °C. Exposure to temperatures above room temperature (22 °C) resulted in greater yields of PBMC. Reduced cell recovery following cryo-preservation as well as decreased viability and immune function were observed in specimens exposed to 15 °C or 40 °C for greater than 8 hours when compared to storage at 22 °C. There was a trend toward improved preservation of blood specimen integrity stored at 30 °C prior to processing for all time points tested. Internal temperatures of blood shipping containers were maintained longer in an acceptable range when warm packs were included. CONCLUSIONS: Blood packages shipped overnight by commercial carrier may encounter extreme seasonal temperatures. Therefore, considerations in the design of shipping containers should include protecting against extreme ambient temperature deviations and maintaining specimen temperature above 22 °C or preferably near 30 °C.

Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting.

PURPOSE: Human melanoma cells express shared antigens recognized by CD8(+) T lymphocytes, the most common of which are melanocytic differentiation proteins and cancer-testis antigens. However, peptide vaccines for melanoma usually target only one or two MHC class I-associated peptide antigens. Because melanomas commonly evade immune recognition by selective antigen loss, optimization of melanoma vaccines may require development of more complex multipeptide vaccines. EXPERIMENTAL DESIGN: In a prospective randomized clinical trial, we have evaluated the safety and immunogenicity of a vaccine containing a mixture of 12 peptides from melanocytic differentiation proteins and cancer-testis antigens, designed for human leukocyte antigen types that represent 80% of the melanoma patient population. This was compared with a four-peptide vaccine with only melanocytic differentiation peptides. Immune responses were assessed in peripheral blood and in vaccine-draining lymph nodes. RESULTS: These data show that (a) the 12-peptide mixture is immunogenic in all treated patients; (b) immunogenicity of individual peptides is maintained despite competition with additional peptides for binding to MHC molecules; (c) a broader and more robust immune response is induced by vaccination with the more complex 12-peptide mixture; and (d) clinical outcome in this peptide vaccine trial correlates with immune responses measured in the peripheral blood lymphocytes. CONCLUSIONS: These data support continued investigation of complex multipeptide vaccines for melanoma.

Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens.

PURPOSE: A phase I/II trial was performed to evaluate the safety and immunogenicity of a novel melanoma vaccine comprising six melanoma-associated peptides defined as antigenic targets for melanoma-reactive helper T cells. Source proteins for these peptides include MAGE proteins, MART-1/MelanA, gp100, and tyrosinase. PATIENTS AND METHODS: Thirty-nine patients with stage IIIB to IV melanoma were vaccinated with this six-peptide mixture weekly at three dose levels, with a preceding phase I dose escalation and subsequent random assignment among the dose levels. Helper T-lymphocyte responses were assessed by in vitro proliferation assay and delayed-type hypersensitivity skin testing. Patients with measurable disease were evaluated for objective clinical response by Response Evaluation Criteria in Solid Tumors. RESULTS: Vaccination with the helper peptide vaccine was well tolerated. Proliferation assays revealed induction of T-cell responses to the melanoma helper peptides in 81% of patients. Among 17 patients with measurable disease, objective clinical responses were observed in two patients (12%), with response durations of 1 and 3.9+ years. Durable stable disease was observed in two additional patients for periods of 1.8 and 4.6+ years. CONCLUSION: Results of this study support the safety and immunogenicity of a vaccine comprised of six melanoma helper peptides. There is also early evidence of clinical activity.

MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma.

Twelve peptides derived from melanocyte differentiation proteins and cancer-testis Ags were combined and administered in a single mixture to patients with resected stage IIB, III, or IV melanoma. Five of the 12 peptides included in this mixture had not previously been evaluated for their immunogenicity in vivo following vaccination. We report in this study that at least three of these five peptides (MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622)) are immunogenic when administered with GM-CSF in Montanide ISA-51 adjuvant. T cells secreting IFN-gamma in response to peptide-pulsed target cells were detected in peripheral blood and in the sentinel immunized node, the node draining a vaccine site, after three weekly injections. The magnitude of response typically reached a maximum after two vaccines, and though sometimes diminished thereafter, those responses typically were still detectable 6 wks after the last vaccines. Most importantly, tumor cell lines expressing the appropriate HLA-A restriction element and MAGE-A1, MAGE-A10, or gp100 proteins were lysed by corresponding CTL. This report supports the continued use of the MAGE-A1(96-104), MAGE-A10(254-262), and gp100(614-622) epitopes in peptide-based melanoma vaccines and thus expands the list of immunogenic peptide Ags available for human use. Cancer-testis Ags are expressed in multiple types of cancer; thus the MAGE-A1(96-104) and MAGE-A10(254-262) peptides may be considered for inclusion in vaccines against cancers of other histologic types, in addition to melanoma.