Increases in serum TARC/CCL17 levels are associated with progression-free survival in advanced melanoma patients in response to dendritic cell-based immunotherapy.

INTRODUCTION: Changes in the levels of serum cytokines and growth factors are associated with response to therapy. We examined cytokine, chemokine, and growth factor levels in serum collected from normal volunteers or metastatic melanoma patients receiving dendritic cell-based immunotherapy. MATERIALS AND METHODS: Using an array for 42 cytokines, chemokines, or growth factors, sera from normal controls and metastatic melanoma patients at baseline and week 4 were analyzed for qualitative changes. Quantitative determination of the levels of the chemokine thymus and activation-regulated chemokine (TARC/CCL17) was determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Significant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (P = 0.026) and a significant increase in TARC/CCLl7 (P = 0.008) from week 0 to week 4 which was associated with improved overall survival (P = 0.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (P = 0.005). Receiver-operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.

Monitoring response to treatment in melanoma patients: potential of a serum glycomic marker.

A mechanistic marker correlating with tumor progression and clinical response is useful for assessing therapeutic response and determining the course of therapy. Since serum-total-ganglioside (sTG) and antiganglioside-IgM antibody levels reflected tumor progression, the feasibility of utilizing sTG for assessing the response to immunotherapy of metastatic-melanoma was tested. Patients (n = 34) were immunized with dendritic cells cocultured with irradiated, IFN gamma-treated autologous tumor cells admixed with GM-CSF. Levels of sTG and antiganglioside-IgM antibody titers were measured in sera of vaccine recipients at 0, 4 and 24 weeks of treatment. Based on sTG-level, whether lower (L) or higher (H) than the mean + 1 SD of normal and healthy volunteers on weeks 0, 4 and 24, patients were categorized into cohorts-I (LLL, n = 16), II (HHL/HLL, n = 4), III (LLH/LHH/LHL, n = 7) and IV (HHH/HLH, n = 7). The cohorts were regrouped as sTG- downregulators (sTG-DR; n = 20) and upregulators (sTG-UR; n = 14). These two cohorts differed significantly in their overall (p < 0.012) and progression-free (p = 0.0001) survival post-treatment. 43% sTG-UR died within 39 months, with a median survival of 39 months, whereas 61% of the sTG-DR survived for 48 months. Both endogenous and vaccine-induced antiganglioside-IgM antibodies appeared to regulate sTG levels. Nonresponders had increased sTG with no or low IgM antibody response. The sTG level is regulated within 24 weeks post-treatment and therefore, may serve as an ideal biomarker for assessing therapeutic responses in patients. Clinical correlations of sTG indicate that sTG-downregulating therapy may be an effective treatment strategy for melanoma.

Characterization of interferon-γ-treated melanoma tumor cells for use in dendritic cell-based immunotherapy.

Efficient delivery of tumor-associated antigens to professional antigen-presenting cells is important for inducing a response in patients receiving cancer immunotherapy. Interferon-gamma (IFN-γ) is used by the immune system to combat viral and fungal infections by restricting cell proliferation and, in some cases, inducing apoptosis. Using IFN-γ to activate target tumor cells prior to antigen loading of dendritic cells (DCs) may enhance the beneficial qualities of whole-cell tumor vaccines. The incubation of melanoma cell cultures with IFN-γ resulted in an increase in the expression of major histocompatibility complex molecules and ICAM-1 but generally decreased the expression of melanoma-associated tumor antigens. Additionally, important immune-stimulating molecules (heat-shock proteins, high-mobility group box-1 protein, and calreticulin) were also present but differentially regulated by IFN-γ. Loading of DCs with IFN-γ-treated tumor cells resulted in a small but significant increase in the expression of CD83-positive DCs, indicating the initiation of DC maturation (p=0.019). IFN-γ treatment of melanoma cell lines prior to antigen loading of DCs may aid in antigen processing and presentation.

Establishment of stable cell lines for personalized melanoma cell vaccine.

Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patient's own tumor within a short duration and with less passages but with optimized expression of tumor-associated antigens (TAAs). Its feasibility is established by comparing pure cell lines generated from fresh and cryopreserved tissues (n=164) of patients with lymph node (LN) and distant metastases. Stable cell lines (from 67% of specimens) are subcultured after cryopreserving them. Pure cell lines established after eliminating fibroblasts (from 96% of the cell lines) include those from LN (69%), soft tissues including cutaneous (60%), liver (64%), lung (75%), bone (80%), brain (75%), and other sites (73%). Within 3.5 months, stable cell lines (> or =50 million cells) are established from initiating the cell culture. For LN metastases, the duration differs significantly (P2<0.05) between fresh (1.4-3.4 months) and cryopreserved (2.4-4.7 months) tissues. The expression of TAAs varies as follows: Tyrosinase (81%) >Melan-A (80%) >HMB45/gp-100 (75%) >Mel-5/TRP-1 (65%) >MAGE-1 (47%) > S-100 (28%). The number of TAAs per cell line differs between early (<7) and late (>7) passages. Among late passage cell lines, lesser percentage of cell lines express three to six antigens pointing out that early passage (<7) cell lines may be needed for antigen-targeted immunotherapy. This study provides a protocol for establishing cell lines within 2-5 months for personalized vaccine therapy for nodal and organ metastatic melanoma patients.