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Whereas TLR9 agonists are recognized as powerful stimulators of antitumor immunity, GM-CSF has had mixed reviews. In previously reported randomized trials we assessed the effects of local immune modulation in early-stage melanoma with CpG-B alone or with GM-CSF. Here we discuss the added value of GM-CSF and show sex-related differences. [ABSTRACT FROM AUTHOR]

A response by Mari F.C.M. van den Hout and colleagues to a letter to the editor about their article "Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation," in a 2011 issue of the jurnal is presented.

Melanoma exerts immune-suppressive effects to facilitate tumor progression and metastatic spread. We studied these effects on dendritic cell (DC) and T-cell subsets in 36 melanoma sentinel lymph node (SLN) from 28 stage I-III melanoma patients and determined their clinical significance. Four conventional DC subsets, plasmacytoid DCs, and CD4 + , CD8 + , and regulatory T cells (Tregs), were analyzed by flow cytometry. We correlated these data to clinical parameters and determined their effect on local and distant melanoma recurrence, with a median follow-up of 75 months. In stage I and II melanoma, increased Breslow thickness (i.e., invasion depth of the primary melanoma) was associated with progressive suppression of skin-derived migratory CD1a + DC subsets. In contrast, LN-resident DC subsets and T cells were only affected once metastasis to the SLN had occurred. In stage III patients, increased CD4:CD8 ratios in concert with the accumulation of Tregs resulted in decreased CD8:Treg ratios. On follow-up, lower frequencies of migratory DC subsets proved related to local melanoma recurrence, whereas reduced maturation of LN-resident DC subsets was associated with distant recurrence and melanoma-specific survival. In conclusion, melanoma-mediated suppression of migratory DC subsets in the SLN precedes local spread, whereas suppression of LN-resident DC subsets follows regional spread and precedes further melanoma dissemination to distant sites. This study offers a rationale to target migratory as well as LN-resident DC subsets for early immunotherapeutic interventions to prevent melanoma recurrence and spread. Cancer Immunol Res; 5(11); 969-77. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Although risk of recurrence after surgical removal of clinical stage I-II melanoma is considerable, there is no adjuvant therapy with proven efficacy. Here, we provide clinical evidence that a local conditioning regimen, aimed at immunologic arming of the tumor-draining lymph nodes, may provide durable protection against disease recurrence (median follow-up, 88.8 months). Experimental Design: In two randomized phase II trials, patients, diagnosed with stage I-II melanoma after excision of the primary tumor, received local injections at the primary tumor excision site within 7 days preceding re-excision and sentinel lymph node (SLN) biopsy of either a saline placebo ( n = 22) or low-dose CpG type B (CpG-B) with ( n = 9) or without ( n = 21) low-dose GM-CSF. Results: CpG-B treatment was shown to be safe, to boost locoregional and systemic immunity, to be associated with lower rates of tumor-involved SLN (10% vs. 36% in controls, P = 0.04), and, at a median follow-up of 88.8 months, to profoundly improve recurrence-free survival ( P = 0.008), even for patients with histologically confirmed (i.e., pathologic) stage I-II disease ( P = 0.02). Conclusions: Potentially offering durable protection, local low-dose CpG-B administration in early-stage melanoma provides an adjuvant treatment option for a large group of patients currently going untreated despite being at considerable risk for disease recurrence. Once validated in a larger randomized phase III trial, this nontoxic immunopotentiating regimen may prove clinically transformative. Clin Cancer Res; 23(19); 5679-86. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Melanoma-induced suppression of dendritic cells (DC) in the sentinel lymph node (SLN) interferes with the generation of protective antitumor immunity. In an effort to strengthen immune defense against metastatic spread, we performed a three-arm phase II study comprising 28 patients with stage I-II melanoma randomized to receive intradermal injections around the primary tumor excision site of saline or low-dose CpG-B, alone or combined with GM-CSF, before excision of the SLNs. After pathologic examination, 5 patients were diagnosed with stage III melanoma based on the presence of tumor cells in the SLNs. Combined CpG/GM-CSF administration resulted in enhanced maturation of all identifiable conventional (cDC) and plasmacytoid (pDC) DC subsets and selectively induced increased frequencies of SLN-resident BDCA3/CD141(+) cDC subsets that also expressed the C-type lectin receptor CLEC9A. Correlative in vivo analyses and in vitro studies provided evidence that these subsets were derived from BDCA3(+) cDC precursors in the blood that were recruited to the SLNs in a type I IFN-dependent manner and subsequently matured under the combined influence of CpG and GM-CSF. In line with their reported functional abilities, frequencies of in vivo CpG/GM-CSF-induced BDCA3/CD141(+) DCs correlated with increased ex vivo cross-presenting capacity of SLN suspensions. Combined local CpG/GM-CSF delivery thus supports protective antimelanoma immunity through concerted activation of pDC and cDC subsets and recruitment of BDCA3(+) cDC subsets with T cell-stimulatory and cross-priming abilities., (©2015 American Association for Cancer Research.)

In this study we investigated whether the presence of specific populations of tumor infiltrating lymphocytes (TILs) in diagnostic primary melanoma biopsies are related to outcome in clinically stage II melanoma patients. Moreover, we investigated whether the presence of TILs correlates with expression of MHC class I antigen and MHC class II antigen on tumor cells and/or tumor infiltrating antigen presenting cells. Diagnostic primary melanoma samples of 15 patients with an unfavorable outcome were compared with 20 patients with favorable outcome. Patients were matched for age, gender and Breslow thickness. Biopsies were examined for the presence of granzyme B+, CD8+, CD4+ and CD56+ TILs and for expression of MHC class I antigen and MHC class II antigen on tumor and/or tumor infiltrating cells. A favorable clinical outcome was strongly associated with the presence of GrB+ and CD4+ TILs, with expression of MHC class I antigen on tumor cells and with expression of MHC class II antigen on intratumoral antigen presenting cells. These data strongly support the notion that in melanoma patients the cellular immune response is a major factor in preventing melanoma cell dissemination.

Purpose: Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. Local administration of PF-3512676 (formerly known as CpG 7909) has shown immunostimulatory effects of both dendritic cell and T-cell subsets in the melanoma SLN. Here, we set out to ascertain whether these PF-3512676-induced immunostimulatory effects translate into higher frequencies of melanoma-specific CD8(+) T cells., Experimental Design: Twenty-four stage I to III melanoma patients were randomized to preoperative local administration of either PF-3512676 or saline. CD8(+) T cells from SLN and peripheral blood were tested for reactivity by IFN-gamma ELISPOT assay against several HLA-A1/A2/A3-restricted epitopes derived from various melanoma-associated antigens (MAA) in 21 of 24 enrolled patients. Frequencies of natural killer (NK) cells and frequencies and maturation state of dendritic cell subsets in the SLN were determined by flow cytometry., Results: Melanoma-specific CD8(+) T-cell response rates against >1 MAA epitope in the SLN were 0 of 11 for the saline group versus 5 of 10 for the PF-3512676-administered group (P = 0.012). Of these 5 responding patients, 4 also had a measurable response to >1 MAA epitope in the blood. Increased frequencies in the SLN of both MAA-specific CD8(+) T cells and NK cells correlated to CpG-induced plasmacytoid dendritic cell maturation., Conclusions: These data show an increase in melanoma-specific CD8(+) T-cell frequencies as well as an increased effector NK cell rate after a single dose of PF-3512676 and thus support the utility of local PF-3512676 administration as adjuvant treatment in early-stage melanoma to try and halt metastatic spread.

Purpose: A decrease in the frequency and activation state of dendritic cells in the sentinel lymph node (SLN) has been observed in early stages of melanoma development. This may hinder the generation of effective antitumor T-cell responses and increase the likelihood of metastatic spread. Immunopotentiation of the melanoma SLN may therefore be a valuable adjuvant treatment option. One way to achieve this is through the use of bacterially derived unmethylated cytosine-phosphate-guanine (CpG) DNA sequences that bind Toll-like receptor 9 and activate plasmacytoid dendritic cells (PDC). CpG-activated PDC, in turn, release IFN alpha and may thus boost T-cell and natural killer cell responses as well as activate conventional myeloid dendritic cells (MDC)., Experimental Design: We studied the effects of preoperative local administration of the CpG B-type oligodeoxynucleotide (ODN) PF-3512676 (formerly known as CPG 7909) on dendritic cell and T-cell subsets in the SLN of 23 stage I to III melanoma patients, randomized to receive intradermal injections of either PF-3512676 or saline (NaCl 0.9%)., Results: PF-3512676 administration resulted in bulkier SLN, higher yields of isolated SLN leukocytes, and activation of BDCA-2(+)CD123(+) PDC as well as of CD1a(+) MDC. In addition, PF-3512676 administration was associated with the presence of a newly identified CD11c(hi)CD123(+)CD83(+)TRAIL(+) mature SLN-MDC subset, an increased release of a variety of inflammatory cytokines, and lower frequencies of CD4(+)CD25(hi)CTLA-4(+)FoxP3(+) regulatory T cells in the SLN., Conclusions: These findings point to the possible utility of the conditioning of SLN by PF-3512676 as an adjuvant immunotherapeutic modality for early-stage melanoma.

Cutaneous melanoma is the most aggressive type of skin cancer. Paradoxically, melanoma is also the most immunogenic tumour identified to date: tumour-reactive T cells are detectable both in the blood and in tumour-draining lymph nodes (TDLN) of melanoma patients and their frequency can be increased by specific vaccination. However, early melanoma development is accompanied by impaired immune effector functions in the initial TDLN, the sentinel lymph node (SLN). Most notably, a reduced frequency and activation state of dendritic cells (DC) interferes with the uptake and presentation of tumour-associated antigens (TAA) to specific anti-tumour cytotoxic T-lymphocytes (CTL) and T helper cells (Th). These impaired immune effector functions may contribute to the early metastatic events that are associated with this tumour type. Since complete surgical excision at an early stage remains the only curative treatment option (adjuvant therapy options are limited and show no survival benefits), immunopotentiation of the SLN to jump-start or boost tumour specific immunity in early stage melanoma may be a valuable adjuvant treatment option that can be generally applied with minimal discomfort to the patient. Early clinical studies indicate that local Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) or Cytosine-phosphate-Guanine (CpG) administration leads to activation of different DC subsets and conditions the SLN microenvironment to be more conducive to the generation of T-cell-mediated anti-tumour immunity.