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BackgroundWe previously reported CpG-B injection at the primary tumor excision site prior to re-excision and sentinel node biopsy to result in immune activation of the sentinel lymph node (SLN), increased melanoma-specific CD8+ T cell rates in peripheral blood, and prolonged recurrence-free survival. Here, we assessed recruitment and activation of antigen-presenting cell (APC) subsets in the SLN and at the injection site in relation to T cell infiltration.MethodsRe-excision skin specimens from patients with clinical stage I-II melanoma, collected 7 days after intradermal injection of either saline (n=10) or 8 mg CpG-B (CPG7909, n=12), were examined by immunohistochemistry, quantifying immune subsets in the epidermis, papillary, and reticular dermis. Counts were related to flow cytometric data from matched SLN samples. Additional in vitro cultures and transcriptional analyses on peripheral blood mononuclear cells (PBMCs) were performed to ascertain CpG-induced APC activation and chemokine profiles.ResultsSignificant increases in CD83+, CD14+, CD68+, and CD123+ APC were observed in the reticular dermis of CpG-B-injected skin samples. Fluorescent double/triple staining revealed recruitment of both CD123+BDCA2+ plasmacytoid dendritic cells (DCs) and BDCA3/CD141+CLEC9A+ type-1 conventional DC (cDC1), of which only the cDC1 showed considerable levels of CD83 expression. Simultaneous CpG-B-induced increases in T cell infiltration were strongly correlated with both cDC1 and CD14 counts. Moreover, cDC1 and CD14+ APC rates in the reticular dermis and matched SLN suspensions were positively correlated. Flow cytometric, transcriptional, and chemokine release analyses of PBMC, on in vitro or in vivo exposure to CpG-B, indicate a role for the activation and recruitment of both cDC1 and CD14+ monocyte-derived APCs in the release of CXCL10 and subsequent T cell infiltration.ConclusionThe CpG-B-induced concerted recruitment of cDC1 and CD14+ APC to the injection site and its draining lymph nodes may allow for both the (cross-)priming of T cells and their subsequent homing to effector sites.

The identification of gene fusions has become an integral part of soft tissue and bone tumour diagnosis. We investigated the added value of targeted RNA‐based sequencing (targeted RNA‐seq, Archer FusionPlex) to our current molecular diagnostic workflow of these tumours, which is based on fluorescence in situ hybridisation (FISH) for the detection of gene fusions using 25 probes. In a series of 131 diagnostic samples targeted RNA‐seq identified a gene fusion, BCOR internal tandem duplication or ALK deletion in 47 cases (35.9%). For 74 cases, encompassing 137 FISH analyses, concordance between FISH and targeted RNA‐seq was evaluated. A positive or negative FISH result was confirmed by targeted RNA‐seq in 27 out of 49 (55.1%) and 81 out of 88 (92.0%) analyses, respectively. While negative concordance was high, targeted RNA‐seq identified a canonical gene fusion in seven cases despite a negative FISH result. The 22 discordant FISH‐positive analyses showed a lower percentage of rearrangement‐positive nuclei (range 15–41%) compared to the concordant FISH‐positive analyses (>41% of nuclei in 88.9% of cases). Six FISH analyses (in four cases) were finally considered false positive based on histological and targeted RNA‐seq findings. For the EWSR1 FISH probe, we observed a gene‐dependent disparity (p = 0.0020), with 8 out of 35 cases showing a discordance between FISH and targeted RNA‐seq (22.9%). This study demonstrates an added value of targeted RNA‐seq to our current diagnostic workflow of soft tissue and bone tumours in 19 out of 131 cases (14.5%), which we categorised as altered diagnosis (3 cases), added precision (6 cases), or augmented spectrum (10 cases). In the latter subgroup, four novel fusion transcripts were found for which the clinical relevance remains unclear: NAB2::NCOA2, YAP1::NUTM2B, HSPA8::BRAF, and PDE2A::PLAG1. Overall, targeted RNA‐seq has proven extremely valuable in the diagnostic workflow of soft tissue and bone tumours.

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 treated clinically stage I-II melanoma patients with 1) GM-CSF (3 μg/kg), 2) CpG-B (PF-3512676/CpG7909, 8 mg), and 3) CpG-B, alone or combined with GM-CSF (2 mg and 100 μg, respectively), through 1-4 intradermal injections, directly adjacent to the primary melanoma excision scar, within a week of re-excision and sentinel lymph node (SLN) biopsy[1–3]. The trials were IRB-approved and written informed consent was obtained from all participating patients. Injections were well tolerated with only transient and mild flu-like symptoms and mild-to-moderate fevers after administration. Primary endpoint of the trials was biological efficacy; immune monitoring revealed recruitment and activation of dendritic cell subsets in the SLN as well as increased intra-SLN and systemic rates of melanoma antigen reactive CD8+ T cells. Here, we present clinical follow-up of all patients (n=62) who participated in these 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/GM-CSF administered groups (3/36 versus 9/26 in the saline control arms, p=0.020), suggesting a rapidly induced antitumor response. Follow-up further provided evidence of systemic tumor protection: in the CpG/GM-CSF-treated groups only two recurrences occurred, both within 26 months of SLN biopsy, whereas nine recurrences were observed in the saline group (range 5-113 months). In post-hoc analysis, at a median follow-up of 79 months, this translated into a significantly longer recurrence-free survival (RFS) in the CpG-treated group (p=0.010). Even for patients with pathologically confirmed and prognostically favorable stage I-II, this difference held true (p=0.017). Disease-specific survival rates at this time were 76.9% in the saline vs. 94.5% in the CpG-B/GM-CSF group.We conclude that intradermally administered CpG-B/GM-CSF 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.

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I–II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3 + DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8 + T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p  = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs.