Mari F.C.M. van den Hout

Prognosis in Patients With Sentinel Node–Positive Melanoma Is Accurately Defined by the Combined Rotterdam Tumor Load and Dewar Topography Criteria

PURPOSE Prognosis in patients with sentinel node (SN)-positive melanoma correlates with several characteristics of the metastases in the SN such as size and site. These factors reflect biologic behavior and may separate out patients who may or may not need additional locoregional and/or systemic therapy. PATIENTS AND METHODS Between 1993 and 2008, 1,080 patients (509 women and 571 men) were diagnosed with tumor burden in the SN in nine European Organisation for Research and Treatment of Cancer (EORTC) melanoma group centers. In total, 1,009 patients (93%) underwent completion lymph node dissection (CLND). Median Breslow thickness was 3.00 mm. The median follow-up time was 37 months. Tumor load and tumor site were reclassified in all nodes by the Rotterdam criteria for size and in 88% by the Dewar criteria for topography. RESULTS Patients with submicrometastases (< 0.1 mm in diameter) were shown to have an estimated 5-year overall survival rate of 91% and a low nonsentinel node (NSN) positivity rate of 9%. This is comparable to the rate in SN-negative patients. The strongest predictive parameter for NSN positivity and prognostic parameter for survival was the Rotterdam-Dewar Combined (RDC) criteria. Patients with submicrometastases that were present in the subcapsular area only, had an NSN positivity rate of 2% and an estimated 5- and 10-year melanoma-specific survival (MSS) of 95%. CONCLUSION Patients with metastases < 0.1 mm, especially when present in the subcapsular area only, may be overtreated by a routine CLND and have an MSS that is indistinguishable from that of SN-negative patients. Thus the RDC criteria provide a rational basis for decision making in the absence of conclusions provided by randomized controlled trials.

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.

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.

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.

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.

Abstract 4692: Local adjuvant treatment of clinical stage I-II melanoma with low dose CpG-B and/or GM-CSF: Long-term follow-up of three randomized controlled phase II trials

Introduction: Currently, there is no widely used adjuvant treatment available to improve survival after surgical excision of localized melanoma. Here, we present the clinical outcome of patients who participated in three randomized phase-II trials and received low-dose local immunotherapy, which was shown to be safe and to boost loco-regional and systemic anti-melanoma T cell immunity. Patients and Methods: In three single-center, single-blinded, randomized and placebo (saline) controlled phase-II clinical trials, patients with early stage melanoma were treated with 1) Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF), 2) unmethylated CpG type-B oligodeoxynucleotide CpG7909 (CpG-B), and 3) CpG-B, alone or combined with GM-CSF, through 1-4 intradermal injections at the site of the primary melanoma excision scar, within 7 days preceding re-excision and sentinel lymph node (SLN) biopsy. For clinical follow-up analysis, all treated patients were grouped together (treated group n=36) as were the patients who received saline (saline group n=28). Results: 10-year recurrence-free survival rate in the treated group was 94% (95% CI 78-98) versus 48% (95% CI 21-71) in the saline group (P=0.005), hazard ratio (HR) for recurrence was 0.15 (95% CI 0.06-0.60) for the treated group. This apparent antitumor efficacy was in line with the observation upon pathological examination of less tumor positive SLN in the treated group (P=0.05). The 10-year distant recurrence-free survival rate in treated patients was also higher (94%, 78-98 versus 59%, 28-81, HR 0.22, 0.07-0.93; P=0.04). Conclusion: Local low-dose immunotherapy in patients with early-stage melanoma may offer durable protection against distant recurrences. These findings warrant further clinical exploration of this local non-toxic immune potentiating regimen. Citation Format: Bas D. Koster, Mari F. van den Hout, Berbel J. Sluijter, Barbara G. Molenkamp, Ronald J. Vuylsteke, Arnold Baars, Paul A. van Leeuwen, Rik J. Scheper, Monique P. van den Tol, Alfons J. van den Eertwegh, Tanja D. de Gruijl. Local adjuvant treatment of clinical stage I-II melanoma with low dose CpG-B and/or GM-CSF: Long-term follow-up of three randomized controlled phase II trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4692. doi:10.1158/1538-7445.AM2017-4692

Abstract A050: Local administration of CpG-B increases recurrence-free survival in early-stage melanoma patients: Long-term follow-up of two randomized clinical trials evaluating adjuvant treatment

Although 5-year overall survival rates in patients with Stage I-II melanoma range from 95% to as low as 45%, currently there is no adjuvant treatment available that has been shown to reduce the chances of recurrence after surgical excision of localized melanoma. Between 2004 and 2007 we conducted two randomized and placebo controlled phase II clinical trials in which we treated clinically stage I-II melanoma patients with intradermally injected CpG-B (PF-3512676/CpG7909 at 8 or 2 mg, n=30) or saline (n=22) directly adjacent to the primary melanoma excision scar, seven and two days before re-excision and sentinel lymph node (SLN) biopsy. CpG-B injections were well tolerated by all patients with only transient and mild flu-like symptoms and mild-to-moderate fevers after administration. Immune monitoring revealed recruitment and activation of dendritic cell subsets in the SLN with cross-priming ability upon CpG-B administration as well as increased intra-SLN and systemic rates of melanoma antigen reactive CD8+ T cells. Here, we present clinical follow-up of all 52 patients that participated in these two trials. Upon pathological examination of the SLN, a remarkable difference in numbers of pathologically stage III patients was revealed, with significantly less tumor positive SLN in the CpG-B administered group (3/30 versus 8/22 in the saline control arm, p=0.037), indicating a rapid CpG-induced antitumor effect. Follow-up further provided evidence of systemic tumor protection: in the CpG-treated group only two recurrences occurred, both within 26 months of SLN biopsy, whereas nine recurrences were observed in the saline group (range 9-99 months). In a post-hoc analysis at a median follow-up of 76.5 months, this translated into a significantly longer recurrence free survival (RFS) in the CpG-treated group (p=0.019). Even for patients with pathologically confirmed and prognostically favorable stage I-II, this difference held true, although at borderline significance (p=0.068). Disease-specific survival rates at this time were 77.2% in the saline vs. 93.3% in the CpG-B group. We conclude that intradermally administered CpG-B is safe and may prolong RFS. Consistent with findings from immune monitoring, apparent down-staging and prolonged RFS may result from the respective boosting of local and systemic antitumor immunity through local conditioning of the SLN. These exciting findings warrant further clinical exploration of local non-toxic immune potentiation in early-stage melanoma to prevent disease recurrence and metastatic spread. Citation Format: Tanja D. de Gruijl, Bas D. Koster, Mari F.C.M. van den Hout, Berbel J.R. Sluijter, Barbara G. Molenkamp, Paul A.M. van Leeuwen, Rik J. Scheper, M. Petrousjka van den Tol, Alfons J.M. van den Eertwegh. Local administration of CpG-B increases recurrence-free survival in early-stage melanoma patients: Long-term follow-up of two randomized clinical trials evaluating adjuvant treatment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A050.

Adjuvant treatment of early-stage melanoma by local i.d. administration of low-dose CpG-B and GM-CSF increases recurrence-free survival: long-term follow-up of three randomized clinical trials

Meeting abstracts Currently, there is no adjuvant treatment available that has been shown to reduce the chances of recurrence after surgical excision of localized melanoma. Between 2002 and 2007 we conducted three randomized and placebo (saline) controlled Phase II clinical trials in which we

Pre-operative intradermal administration of CpG-B ± GM-CSF in stage I-III melanoma patients arms the sentinel lymph node: evidence for reduced tumor spread

Melanoma cutis is virtually incurable when it has metastasized to distant organs. Unfortunately, there is no safe and effective adjuvant treatment available for early stage patients that may prevent the development of metastasis. Since early melanoma development is accompanied by impaired immune effector functions in the sentinel lymph node (SLN), there is a strong rationale for therapeutic immune modulation of the SLN aimed at strengthening cellular immune functions. In two placebo controlled trials, the immunological effects of i.d. administration of CpG type-B (PF-3512676, 1 or 8 mg) ± GM-CSF (Leukine, 100 mg) at the tumor site, one week before SLN excision, were investigated. The trials showed that this treatment is clinically safe and flow cytometric and functional analyses of viable immune effector cells isolated from the SLN showed increased frequencies and activation state of LN resident CD141+ cDC subsets as well as activation of pDC and CD1a+ skin derived cDC subsets in the treated groups. This DC activation was accompanied by an increased Th1 cytokine response profile and melanoma-specific CD8+ T cell reactivity. Interestingly, when combining the treated groups of both studies (n=31), we found remarkable clinical differences compared to the combined placebo groups (n=22). Important predictors for recurrence and SLN tumor positivity are Breslow depth and ulceration of the primary tumor. These two clinical parameters were higher (although not significantly so) in the treated group. Nevertheless, the number of tumor positive SLN in this group was significantly lower (3/31 vs. 8/22 p=0.02). At a median follow-up of 71 months we also found a longer recurrence free survival in the treated group (p=0.14). We conclude that local administration of CpG ± GM-CSF is safe, shows very promising immunological efficacy with evidence for clinical impact on lymphatic spread and recurrence free survival and could potentially be used as adjuvant treatment combined with primary excision of suspected melanoma lesions.

Abstract 5618: Arming the melanoma sentinel lymph node against tumor spread: The effects of local administration of combined low-dose CpG-B PF-3512676 and GM-CSF

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The sentinel lymph node (SLN) procedure has proven a useful prognostic tool for the assessment of melanoma relapse and mortality risk, but is also of great value for the assessment of immunological interventions. Since early melanoma development is accompanied by a suppressed immune state of the SLN, there is a strong rationale for therapeutic immune modulation of the SLN in order to strengthen local as well as systemic cell-mediated anti-tumor immunity. Previous Phase II studies showed promising immunostimulatory effects on the melanoma SLN of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) and the CpG-B oligodeoxynucleotide PF-3512676. The present 3-arm Phase II study was designed to determine the effects of combined low-dose GM-CSF+CpG-B administration. Stage I-III melanoma patients (n=28) were randomized to receive i.d. injections around the primary tumor excision site of saline, CpG-B alone (1 mg), or CpG-B combined with GM-CSF (100 μg), 7 and 2 days prior to surgical excision of the SLN. Flowcytometric DC analyses revealed significantly increased maturation of all identifiable cDC and pDC SLN subsets upon administration of CpG-B with or without GM-CSF, but remarkably, the combination resulted in significantly higher levels of co-stimulatory molecules on both cDC and pDC. In addition, a significant CpG-related increase in frequencies of CD1a−CD11chiCD14+/− cDC subsets was observed in the SLN, possibly resulting from DC and/or monocyte mobilisation from blood. Indeed, increased activation of pro-inflammatory 6-sulfo LacNac+slanDC, monocytes, and pDC was observed in peripheral blood upon administration of CpG-B alone, and additionally of CD1c+ cDC when combined with GM-CSF. In the case of slanDC this activation was accompanied by significantly reduced frequencies. Peripheral blood rates of CD14+DRlo myeloid suppressors remained unchanged after saline or combined CpG and GM-CSF administration, but significantly decreased after administration of CpG-B alone. T cell analyses revealed a CpG-related increase in CTLA-4 and FoxP3 levels on Tregs in SLN, without changes in their actual rates. Nevertheless, higher intracellular Th1 cytokine levels were detected in ex vivo expanded T cells of CpG-administered patients than of patients receiving saline or CpG-B+GM-CSF. In keeping with this, also higher frequencies were found in SLN of MAA-specific CD8+ T cells in low dose CpG-B administered patients (n=4) than in saline controls (n=5), as determined by tetramer binding and cytokine release. We conclude that the imunomodulatory effects observed in melanoma SLN upon local administration of combined low-dose GM-CSF and CpG-B are mostly attributable to CpG-B and that they are consistent with the induction of protective cell-mediated anti-tumor immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5618.