Berbel J.R. Sluijter

Intradermal CpG-B activates both plasmacytoid and myeloid dendritic cells in the sentinel lymph node of melanoma patients.

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 IFNα 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 CD11chiCD123+CD83+TRAIL+ mature SLN-MDC subset, an increased release of a variety of inflammatory cytokines, and lower frequencies of CD4+CD25hiCTLA-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.

Local Administration of PF-3512676 CpG-B Instigates Tumor-Specific CD8 + T-Cell Reactivity in Melanoma Patients

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-γ 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.

Favorable outcome in clinically stage II melanoma patients is associated with the presence of activated tumor infiltrating T-lymphocytes and preserved MHC class I antigen expression

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.

Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation

To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a(+) subsets were identified as most likely skin-derived CD11c(int) Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11c(hi) dermal DCs with variable expression of langerin. Two other CD1a(-) LN-residing cDC subsets were characterized as CD14(-)BDCA3(hi)CD103(-) and CD14(+)BDCA3(lo)CD103(+), respectively. Whereas the CD1a(+) skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a(-) cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies.

Non-Radical Diagnostic Biopsies Do Not Negatively Influence Melanoma Patient Survival

BackgroundIn fair-skinned Caucasian populations both the incidence and mortality rates of cutaneous melanoma have been increasing over the past decades. With adjuvant therapies still being under investigation, early detection is the only way to improve melanoma patient survival. The influence of incisional biopsies on melanoma patient survival has been discussed for many years. This study investigates both the influence of diagnostic biopsy type and the presence of residual tumor cells in the re-excision specimen on disease free and overall survival.MethodsAfter (partial) removal of a pigmented skin lesion 471 patients were diagnosed with stage I/II melanoma and underwent re-excision and a sentinel node biopsy. All patients were followed prospectively, mean follow up >5 years. Patients were divided according to their diagnostic biopsy type (wide excision biopsy, narrow excision biopsy, excision biopsy with positive margins and incisional biopsy) and the presence of residual tumor cells in their re-excision specimen. Survival analysis was done using Cox’s proportional hazard model adjusted for eight important confounders of melanoma patient survival.ResultsThe diagnostic biopsy was wide in 279 patients, narrow in 109 patients, 52 patients underwent an excision biopsy with positive margins and 31 patients an incisional biopsy. In 41 patients residual tumor cells were present in the re-excision specimen. Both the diagnostic biopsy type and the presence of tumor cells in the re-excision specimen did not influence disease free and overall survival of melanoma patients.ConclusionsNon-radical diagnostic biopsies do not negatively influence melanoma patient survival.

Arming the Melanoma Sentinel Lymph Node through Local Administration of CpG-B and GM-CSF: Recruitment and Activation of BDCA3/CD141+ Dendritic Cells and Enhanced Cross-Presentation

Sluijter and colleagues report that intradermal injection of combined CpG/GM-CSF at the primary melanoma excision site prior to removal of sentinel lymph nodes (SLN) led to recruitment of BDCA3+ conventional dendritic cell (cDC) precursors from blood and enhanced DC maturation with selective increase of SLN-resident CLEC9A/BDCA3/CD141+ cDCs. 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. Cancer Immunol Res; 3(5); 495–505. ©2015 AACR.

4-1BB-mediated expansion affords superior detection of in vivo primed effector memory CD8+ T cells from melanoma sentinel lymph nodes.

We have been studying the re-activation of tumor-associated antigen (TAA)-specific CD8(+) T cells in sentinel lymph nodes (SLN) of melanoma patients upon intradermal administration of the CpG-B oligodeoxynucleotide PF-3512676. To facilitate functional testing of T cells from small SLN samples, high-efficiency polyclonal T cell expansion is required. In this study, SLN cells were expanded via classic methodologies with plate- or bead-bound anti-CD3/CD28 antibodies and with the K562/CD32/4-1BBL artificial APC system (K32/4-1BBL aAPC) and analyzed for responsiveness to common recall or TAA-derived peptides. K32/4-1BBL-expanded T cell populations contained significantly more effector/memory CD8(+) T cells. Moreover, recall and melanoma antigen-specific CD8(+) T cells were more frequently detected in K32/4-1BBL-expanded samples as compared with anti-CD3/CD28-expanded samples. We conclude that K32/4-1BBL aAPC are superior to anti-CD3/CD28 antibodies for the expansion of in vivo-primed specific CD8(+) T cells and that their use facilitates the sensitive monitoring of functional anti-tumor T cell immunity in SLN.

Local Adjuvant Treatment with Low-Dose CpG-B Offers Durable Protection against Disease Recurrence in Clinical Stage I–II Melanoma: Data from Two Randomized Phase II Trials

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.

Absence of Granzyme B positive tumour-infiltrating lymphocytes in primary melanoma excisional biopsies is strongly associated with the presence of sentinel lymph node metastasis

Background: Sentinel Lymph Node (SLN) status is strongly related to clinical outcome in melanoma patients. In this study we investigated the possible association between the presence of activated and/or suppressive Tumour Infiltrating Lymphocytes (TILs) and SLN status in clinically stage I/II melanoma patients. Methods: Diagnostic primary melanoma samples from 20 patients with a sentinel lymph node metastasis were compared to melanoma samples from 20 patients with a negative sentinel lymph node, who were matched for gender, age and Breslow thickness. Presence of activated Granzyme B positive (GrB+) TILs, presence of suppressive (FoxP3+) TILs and MHC class I antigen expression on tumour cells were analysed by immunohistochemistry. Results: FoxP3 and MHC-I expression had no direct bearing on the presence of melanoma metastases in the SLN. Whereas the presence of activated GrB+ TILs in the primary melanoma had no predictive value for SLN status either, their absence was strongly associated with the presence of metastasis in the SLN (p=0.001). While both GrB+ and FoxP3+ TILs could be detected in SLN metastases, a majority did not display MHC-I expression. Conclusions: These data support a role for cytotoxic T cells in the prevention of early metastasis of melanoma to the draining lymph nodes.

Melanoma Sequentially Suppresses Different DC Subsets in the Sentinel Lymph Node, Affecting Disease Spread and Recurrence

Immunological events accompanying local and regional melanoma progression offer a rationale for the therapeutic targeting ofmigratory and LN-resident DC subsets to prevent melanoma recurrence. Immunotherapeutic interventions in early-stage melanoma could alter the clinical course of disease. 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.