BACKGROUND:The majority of metastatic melanoma patients initially do not respond or acquire resistance to anti-programmed cell death 1 (PD-1) immunotherapy. Liquid biopsy biomarkers might provide useful early response information and allow for personalized treatment decisions.METHODS:We prospectively assessed circulating cell-free SHOX2 DNA methylation (SHOX2 ccfDNAm) levels and their dynamic changes in blood plasma of melanoma patients by quantitative methylation-specific polymerase chain reaction. Patients were treated with either palliative (n = 42) or adjuvant (n = 55) anti-PD-1 immunotherapy. Moreover, we included n = 126 control patients without evidence of malignant disease. We analyzed SHOX2 ccfDNAm status prior to and 4 weeks after palliative treatment initiation with regard to outcome [objective response, progression-free survival (PFS), and overall survival (OS)]. In the adjuvant setting, we associated longitudinal SHOX2 ccfDNAm status with disease recurrence.RESULTS:Sensitivity was 60% with 25/42 melanoma patients showing increased SHOX2 ccfDNAm levels, whereas specificity was 98% with 123/126 (P < 0.001) control patients having SHOX2 ccfDNAm levels below cut-off. Pretreatment SHOX2 ccfDNAm status did not correlate with outcome; however, SHOX2 ccfDNAm negativity 4 weeks after palliative treatment initiation was strongly associated with improved survival [PFS: hazard ratio (HR) = 0.25, P = 0.002; OS: HR = 0.12, P = 0.007]. Pretreatment positive patients who reached SHOX2 ccfDNAm clearance after 4 weeks of immunotherapy showed an exceptionally beneficial outcome. SHOX2 ccfDNAm testing allowed for an early detection of distant metastases in adjuvant-treated melanoma patients.CONCLUSIONS:Our study suggests SHOX2 ccfDNAm to be an early predictor of outcome in anti-PD-1 treated melanoma patients. SHOX2 ccfDNAm testing may aid individualized treatment decision-making.

Uveal melanoma represents an aggressive tumor that responds mostly poorly to established melanoma treatments. Comprehensive methylation profiling of the next-generation immunotherapeutic target genes, for example, members of the tumor necrosis factor receptor superfamily, might allow for the development of companion predictive biomarkers. We have analyzed CpG sites within the immune checkpoint genes GITR, OX40, 4-1BB, CD27, and CD40 probed by the Illumina Infinium HumanMethylation450 BeadChip in N = 80 uveal melanomas included in The Cancer Genome Atlas with regard to BAP1 aberrancy, mRNA expression, and overall survival. In all analyzed immune checkpoint genes, BAP1 aberrancy was associated with decreased CpG methylation levels. We identified specific CpG sites that significantly correlated with BAP1 aberrancy, mRNA expression levels, and overall survival. Our results suggest epigenetic regulation of the analyzed immune checkpoint genes via DNA methylation in uveal melanoma and provide rationale for methylation testing in biomarker programs in clinical trials.

Background The majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint gene CTLA4 with regard to its predictive value. Methods We analyzed CTLA4 promoter methylation in tumors of HNSCC patients ( N = 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort ( N = 138) of patients that did not receive ICB with regard to CTLA4 promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine. Results Lower CTLA4 promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression. CTLA4 promoter methylation inversely correlated with infiltrates of CD3 + , CD4 + , CD8 + , and CD45 + immune cells. CTLA4 methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreased CTLA4 methylation and an induction of CTLA4 mRNA and CTLA-4 protein expression in HNSCC cell lines. Conclusions Our results indicate that CTLA4 DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value of CTLA4 DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.

IntroductionThe anti-programmed cell death protein 1 (PD-1) antibody cemiplimab has shown promising results in the treatment of unresectable or metastatic squamous cell carcinoma, however, frequently leads to immune-related adverse events limiting therapy efficacy. Although cutaneous side effects are common, only very few cases of cutaneous lupus erythematosus have been reported under anti-PD-1 immunotherapy. So far, no case of cutaneous lupus has been described under treatment with cemiplimab.Case reportFor the first time, we report the case of a patient with advanced squamous cell carcinoma, who developed clinical and histological findings in sun-exposed skin that were consistent with anti-SS-A/Ro antibody-positive subacute cutaneous lupus erythematosus (SCLE) under treatment with cemiplimab. Additionally, laboratory chemical analyses revealed a severe immune-related hepatitis without clinical symptoms. Both, the SCLE and the hepatitis, resolved after the administration of topical and systemic steroids and the discontinuation of anti-PD-1 therapy.ConclusionTreatment with cemiplimab can be associated with the appearance of cutaneous lupus erythematosus in sun-exposed areas. Application of topical and systemic glucocorticoids can lead to a rapid resolution of the skin eruptions. Moreover, our case illustrates the possibility of simultaneously occurring severe immune-related adverse events. This highlights the importance of additional diagnostics to avoid overlooking additional immune-related adverse events.

BackgroundInducible T cell costimulator ICOS is an emerging target in immuno-oncology. The aim of this study was to investigate the epigenetic regulation of ICOS in melanoma by DNA methylation.MethodsWe comprehensively investigate ICOS DNA methylation of specific CpG sites and expression pattern within the melanoma microenvironment with regard to immune correlates, differentiation, clinical outcomes, and immune checkpoint blockade (ICB) response.ResultsOur study revealed a sequence-contextual CpG methylation pattern consistent with an epigenetically regulated gene. We found a cell type-specific methylation pattern and locus-specific correlations and associations of CpG methylation with ICOS mRNA expression, immune infiltration, melanoma differentiation, prognosis, and response to ICB. High ICOS mRNA expression was identified as a surrogate for enriched immune cell infiltration and was associated with favorable overall survival (OS) in non-ICB-treated patients and predicted response and a prolonged progression-free survival (PFS) following ICB therapy initiation. ICOS hypomethylation, however, significantly correlated with poor OS in non-ICB patients but predicted higher response and prolonged PFS and OS in ICB-treated patients. Moreover, we observed cytoplasmic and sporadically nuclear tumor cell-intrinsic ICOS protein expression. Tumor cell-intrinsic ICOS protein and mRNA expression was inducible by pharmacological demethylation with decitabine.ConclusionOur study identified ICOS DNA methylation and mRNA expression as promising prognostic and predictive biomarkers for immunotherapy in melanoma and points towards a hitherto undescribed role of ICOS in tumor cells.

e18835 Background: Longitudinal data assessing seroconversion after different doses of SARS-CoV-2 vaccines and the role of personal protective measures to prevent COVID-19 infections remain scarce. This is the first study to evaluate the association between SARS-CoV-2 vaccination, tumor subgroups, cancer treatment, and personal protective measures with antibody titers as well as COVID-19 infections in a real-world cohort of cancer patients. Methods: This retrospective cohort study enrolled 633 adult patients all treated at a large oncology clinic in Hamburg (Germany) from January 2020 to July 2022. Data about demographics, cancer treatment, vaccinations, SARS-CoV-2-IgG antibody titers, personal protective measures, COVID-19 infections and infection-associated symptoms were reported in the patients’ health records. Primary outcomes were: (i) patients’ SARS-CoV-2-IgG antibody concentrations and (ii) COVID-19 infections. A multivariate regression model, reporting adjusted odds ratios (AOR), evaluated the association between collected variables and seroconversion rates after SARS-CoV-2 vaccination. A time-varying Kaplan-Meier survival analysis explored the association between the collected variables and the risk of infection. Results: Of 633 cancer patients, 52% had solid tumors and 48 % hematologic malignancies. Seroconversion was significantly associated with the number of vaccine doses, prior infections (AOR: 1.91, 95%CI: 1.01-3.60, p = .045), hematologic cancers (AOR: 0.58, 95%CI: 0.40-0.85, p = .005), and treatment with CD20/38 inhibitors or immune checkpoint inhibitors (ICI) (AOR: 0.58, 95%CI: 0.35-0.95, p = .030). 120 patients (19%) had a COVID-19 infection with 77% experiencing exhaustion, 67% fever, 62% rhinitis, 62% headache, 56% sore throat, and 48% a non-productive cough. Patients on anti-cancer treatments had a 63% (HR: 1.63, 95%CI: 1.04-2.57, p = .034) higher risk of infection. The risk of infection was 43% (AOR: 0.57, 95%CI: 0.38-0.85, p = .006) and 87% (AOR: 0.23, 95%CI: 0.11-0.47, p < .001) lower among patients with 2 and ≥3 vaccine doses, respectively. Wearing FFP2 masks (AOR: 0.74, 95%CI: 0.56-0.99, p = .046) and reducing social contacts (AOR: 0.81, 95%CI: 0.65-0.99, p = .042) were associated with a lower risk of infection, but this was not the case for wearing surgical masks or using a “Corona warning” app. Conclusions: In this study, SARS-CoV-2 vaccination was associated with lower infection rates in cancer patients. However, certain subgroups remained at an increased risk of infection despite vaccination, e.g. patients receiving CD20/38 or ICI anti-cancer therapy with hematologic cancers. More than two vaccine doses, FFP2 masks, and social distancing led to lower infection rates. These findings are instrumental in guiding recommendations for preventing COVID-19 infections in cancer patients.

While the syndrome of inadequate antidiuresis (SIAD) is still the most common cause of hyponatremia in cancer patients, the rise in endocrine immune-related adverse events (irAEs) owing to immune checkpoint inhibitors (ICI) considerably shaped the differential diagnosis of electrolyte disorders in cancer patients. We report here 3 cases of different endocrine irAEs, first manifesting with new-onset hyponatremia under ICI therapy for malignant melanoma: one with primary adrenal insufficiency, one with hypophysitis, and one with autoimmune type 1 diabetes. Early diagnosis of endocrine toxicities can save lives but may be challenging and essentially delayed by subtle or nonspecific clinical presentation and a lack of readily available endocrinological laboratory evaluation in the primary care setting. This exemplary case series demonstrates the broad spectrum of endocrinopathies that physicians should be aware of under ICI therapy and emphasizes new-onset hyponatremia as a possibly early, simple, and low-cost biomarker of irAEs, which may be considered as a red flag in patients receiving checkpoint blockade. As ICI-induced endocrinopathies are still under-represented in clinical practice guidelines, we here propose an updated algorithm for diagnosis of cancer-related hyponatremia, highlighting the important diagnostic steps to be considered before making the diagnosis of SIAD.

Background TIGIT is an immune checkpoint under investigation as therapeutic target. Understanding the regulation of TIGIT on an epigenetic level might support the development of companion biomarkers. Methods We correlated TIGIT DNA methylation of single CpG sites with gene expression, signatures of immune infiltrates and interferon-γ, and survival in melanoma. We further analyzed methylation levels in immune cell subsets, melanocyte and melanoma cell lines. TIGIT expression patterns within components of the melanoma microenvironment were analyzed by single cell sequencing. We used quantitative methylation-specific PCR, flow cytometry, and immunohistochemistry for correlations between expression and methylation and to assess the effect of pharmacological demethylation of melanoma cells treated with 5‐aza‐2‐deoxycytidine (decitabine). Finally, we investigated the association of patients’ survival with TIGIT mRNA and methylation. Results Depending on the sequence context of the analyzed CpG site, we found a cell type-specific TIGIT gene locus methylation pattern and significant correlations of TIGIT methylation with mRNA expression, an interferon γ signature, and distinct immune cell infiltrates, including TIGIT + lymphocytes. We detected a melanoma cell-intrinsic TIGIT protein expression. Pharmacological demethylation of the A375 melanoma cell line led to a constitutive TIGIT expression. Low promoter flank methylation and high mRNA expression was associated with patients’ prognosis and predicted progression-free survival in patients treated with anti-PD-1 immunotherapy. A high TIGIT + lymphocyte score was associated with better progression-free survival under anti-PD-1 immunotherapy. Conclusions Our data demonstrate an epigenetic regulation of TIGIT expression via DNA methylation within the melanoma microenvironment. TIGIT DNA methylation and expression may serve as predictive biomarkers in the context of immunotherapies in melanoma.

BCR-ABL negative myeloproliferative neoplasms (MPNs) consist of essential thrombocythemia, polycythemia vera, and myelofibrosis. The majority of patients harbor the JAK2-activating mutation V617F. JAK2 inhibitors were shown to reduce symptom burden and splenomegaly in MPN patients. However, treatment options are limited after failure of JAK2 inhibitors. AXL, a member of the TAM family of receptor tyrosine kinases, mediates survival and therapy resistance of different myeloid cancers including acute myeloid leukemia and chronic myeloid leukemia. We studied the relevance of AXL as a target in MPN using primary patient cells and preclinical disease models. We found that AXL is abundantly activated in MPN cells and that its ligand growth arrest-specific gene 6 is upregulated in MPN patients. Pharmacologic and genetic blockade of AXL impaired viability, decreased proliferation and increased apoptosis of MPN cells. Interestingly, ruxolitinib treatment induced increased phosphorylation of AXL indicating that activation of AXL might mediate resistance to ruxolitinib. Consistently, the AXL inhibitor bemcentinib exerted additive effects with ruxolitinib via impaired STAT3, STAT5, and AKT signaling. Both agents had activity when employed alone and exerted an additive effect on survival and splenomegaly in vivo. Moreover, bemcentinib treatment normalized red blood cell count and hemoglobin levels in vivo. Thus, our data indicate that AXL inhibition represents a novel treatment option in MPN warranting clinical investigation.

Background Upregulation of the histone methyltransferase enzyme EZH2 and its histone modification H3K27me3 has been linked to melanoma progression, metastasis, and resistance to immune checkpoint blockade (ICB). In clinical trials, EZH2 inhibitors are currently tested to overcome resistance to ICB. The aim of this study is to evaluate expression patterns and the predictive value of H3K27me3 and EZH2 in metastatic melanoma samples prior to ICB. As H3K27me3 expression has been associated with a dedifferentiated, invasive melanoma phenotype, we also investigated the prognostic value of H3K27me3 expression in primary melanomas. Results H3K27me3 and EZH2 expression were evaluated in a cohort of 44 metastatic melanoma samples before ICB using immunohistochemistry (IHC). 29/44 (66%) of melanomas showed H3K27me3 expression, and 6/44 (14%) showed EZH2 expression. No predictive value for therapeutic response to anti-PD-1 therapy could be found for H3K27me3 or EZH2 expression on melanoma cells. To investigate the prognostic significance of H3K27me3, we analyzed H3K27me3 expression in a representative cohort of 136 primary melanomas with known sentinel lymph node status. H3K27me3 expression is associated with increased tumor thickness and nodal involvement. Melanoma metastases showed a higher expression of H3K27me3 in comparison to primary melanomas. In human melanoma cell lines, TNFα and INFγ could not induce H3K27me3 expression. Conclusion Our study shows that H3K27me3 expression is more frequent than EZH2 and is associated with a more invasive and metastatic melanoma cell phenotype. We suggest that H3K27me3 expression by IHC might be a suitable method to evaluate the activity of EZH2 inhibitors in clinical trials.

Dear Editor, Immune checkpoint blockade (ICB) for metastasized melanoma in patients with pre-existing autoimmune disorders is challenging, as case series suggest that programmed death 1 (PD-1) antibodies may flare autoimmune diseases.1, 2 Furthermore, immunosuppressive premedication of the patients may hamper the desired therapeutic T-cell activation. Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system but only a few reports exist dealing with treatment of metastasized melanoma in patients with MS. A retrospective analysis indicates that anti-PD-1 or anti-cytotoxic T-lymphocyte-associated protein-4 agents in monotherapy in MS patients may be associated with rapid worsening of neurological condition and death.3 No information is currently available on dual ICB with ipilimumab and nivolumab in MS patients. We report the case of a 41-year-old man with highly active MS who presented with an aggressive course of melanoma. Shortly after surgical treatment for a high-risk melanoma of the calf with lymph node metastases, the patient relapsed with irresectable cutaneous and nodal disease. Anti-melanoma treatment was initiated with BRAF/mitogen-activated protein kinase kinase inhibition (dabrafenib/trametinib) followed by talimogene laherparepvec while the relapsing–remitting MS presenting with paraparesis was treated with peginterferon-β and intermittent high-dose steroids. When the patient developed multiple cerebral metastases, dual ICB was considered, knowing full well that it might aggravate the highly active MS. To hold MS activity at bay and to warrant T-cell function at the same time, we resorted to the anti-CD-20 antibody ocrelizumab for MS stabilization prior to ICB. Other established anti-MS therapeutics (e.g. natalizumab, alemtuzumab) were dismissed because of their suppression of cytotoxic T-cell function. The administration of ocrelizumab followed by dual ICB was considered as the ultima ratio choice of treatment to allow for quality of life regarding MS symptoms while enabling ICB to treat melanoma metastases. We speculated that ocrelizumab could not only ameliorate MS symptoms but might also exert a beneficial effect on melanoma metastases by diminishing immunosuppressive B-cell subpopulations that were reported to attenuate local antitumor immune responses.4 There are also reports that targeting of CD20+ melanoma stem cells may eradicate established melanoma lesions.5 In our patient, treatment with ocrelizumab improved MS symptoms but had no effect on cutaneous and nodal metastases. When ICB with nivolumab and ipilimumab was administrated 3 months later, it was well tolerated without severe toxicities or worsening of MS. Unfortunately, the intracerebral metastases progressed even after radiotherapy and the patient succumbed to progressively growing brain metastases. We report for the first time that dual ICB is feasible in patients with metastasized melanoma and active MS. Interdisciplinary treatment, careful monitoring as well as sensible patient selection (e.g. patients with cerebral metastases) are necessary. Depletion of B cells by an anti-CD20 antibody could be a possible solution to contain MS under ICB. Still, it is unclear to what extent B cells play a role in anti-melanoma responses. We cannot exclude that treatment with ocrelizumab accelerated the course of melanoma disease. Also the long-term effects of ICB on MS are unknown. More clinical data are needed. F. H. and A. F. have received speakers' honoraria or travel expense reimbursements from the following companies: Roche, BMS, Pierre Fabre, Novartis and MSD. J. S. received speaker's honoraria, travel expense reimbursements and has participated in advisory boards from Novartis, BMS, MSD and Roche. U. H. is a consultant/advisory board member for BMS, Bayer, Jansen, Novocure and Karyopharm, and has received speaker's honoraria from Novartis and Medac. J. L. is a consultant/ advisory board member of Bristol-Myers Squibb, Merck, Novartis and Roche. No potential conflicts of interest were declared by the other authors.

Treatment of choice for nodular basal-cell carcinomas (BCCs) is complete excision, implying that small lesions are of minor concern. Metastasis is very rare (< 1%). However, locally advanced basal-cell carcinomas (laBCCs), which are ineligible for surgery or radiation, are a therapeutic challenge. First-generation Smoothened (SMO) inhibitors (vismodegib, sonidegib) have been approved for treatment, but common side effects limit their use. Numerous new compounds are being investigated in clinical trials as potential therapeutic alternatives, among them second-generation SMO inhibitors, other Hedgehog signaling pathway inhibitors, immune-checkpoint inhibitors and intralesional modalities such as oncolytic viruses. Neoadjuvant treatment regimens open another field. This article deals with the clinical management of laBCCs, based on the description of an illustrative case from our department featuring multiple extensive lesions of the scalp. In this short review we will discuss therapeutic options and implications for the future. Some of the new strategies might potentially evolve as alternatives in the management of genodermatoses such as basal-cell carcinoma syndrome, if proven effective and safe.

Background: The co-receptor lymphocyte-activation gene-3 (LAG3, LAG-3, CD223) is a potential target for immune checkpoint inhibition immunotherapies. However, little is known about the biological and clinical significance of LAG3 DNA methylation in melanoma and its microenvironment. Methods: We evaluated LAG3 promoter and gene body methylation in a cohort of N = 470 melanoma patients obtained from The Cancer Genome Atlas (TCGA cohort), an independent cohort of N = 120 patients from the University Hospital Bonn, and in subsets of peripheral blood leukocytes, melanocytes, and melanoma cell lines. We validated the association of LAG3 methylation with mRNA expression in vitro in the melanoma cell line A375 treated with the hypomethylating agent 5-azacytidine and stimulated with interferon-γ. Finally, we investigated correlations between LAG3 methylation and progression-free survival in patients treated with immune checkpoint blockade (ICB cohort, N = 118). Findings: Depending on the analysed locus (promoter, gene body) we found region-dependent significant LAG3 methylation differences between monocytes, B cells, CD8+ and CD4+ T cells, regulatory T cells, melanocytes, and melanoma cell lines. In tumor tissues, methylation correlated significantly with LAG3 mRNA expression, immune cell infiltrates (histopathologic lymphocyte score and RNA-Seq signatures of distinct immune infiltrates), and an interferon-γ signature. Finally, LAG3 methylation was associated with overall survival in the TCGA cohort and progression-free survival in the ICB cohort. We detected basal LAG3 mRNA expression in the melanoma cell A375 and an interferon-γ inducible expression after demethylation with 5-azacytidine. Interpretation: Our study points towards an epigenetic regulation of LAG3 via promoter methylation and suggests a prognostic and predictive significance of LAG3 methylation in melanoma. Our results give insight in the tumor cell-intrinsic transcriptional regulation of LAG3 in melanoma. In perspective, our results might pave the way for investigating LAG3 methylation as a predictive biomarker for response to anti-LAG3 immune checkpoint blockage. Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Sponges are commonly known as general nutrient providers for the marine ecosystem, recycling organic matter into various forms of bioavailable nutrients such as ammonium and nitrate. In this study we challenge this view. We show that nutrient removal through microbial denitrification is a common feature in six cold-water sponge species from boreal and Arctic sponge grounds. Denitrification rates were quantified by incubating sponge tissue sections with 15NO3--amended oxygen-saturated seawater, mimicking conditions in pumping sponges, and de-oxygenated seawater, mimicking non-pumping sponges. It was not possible to detect any rates of anaerobic ammonium oxidation (anammox) using incubations with 15NH4+. Denitrification rates of the different sponge species ranged from below detection to 97 nmol N cm−3 sponge d−1 under oxic conditions, and from 24 to 279 nmol N cm−3 sponge d−1 under anoxic conditions. A positive relationship between the highest potential rates of denitrification (in the absence of oxygen) and the species-specific abundances of nirS and nirK genes encoding nitrite reductase, a key enzyme for denitrification, suggests that the denitrifying community in these sponge species is active and prepared for denitrification. The lack of a lag phase in the linear accumulation of the 15N-labelled N2 gas in any of our tissue incubations is another indicator for an active community of denitrifiers in the investigated sponge species. Low rates for coupled nitrification–denitrification indicate that also under oxic conditions, the nitrate used to fuel denitrification rates was derived rather from the ambient seawater than from sponge nitrification. The lack of nifH genes encoding nitrogenase, the key enzyme for nitrogen fixation, shows that the nitrogen cycle is not closed in the sponge grounds. The denitrified nitrogen, no matter its origin, is then no longer available as a nutrient for the marine ecosystem. These results suggest a high potential denitrification capacity of deep-sea sponge grounds based on typical sponge biomass on boreal and Arctic sponge grounds, with areal denitrification rates of 0.6 mmol N m−2 d−1 assuming non-pumping sponges and still 0.3 mmol N m−2 d−1 assuming pumping sponges. This is well within the range of denitrification rates of continental shelf sediments. Anthropogenic impact and global change processes affecting the sponge redox state may thus lead to deep-sea sponge grounds changing their role in marine ecosystem from being mainly nutrient sources to becoming mainly nutrient sinks.

Background PD-L1 (programmed cell death 1 ligand 1) expression in melanoma has been associated with a better response to anti-PD-1 (programmed cell death 1) therapy. However, patients with PD-L1-negative melanomas can respond to anti-PD-1 blockade, suggesting that the other PD-1 ligand, PD-L2 (programmed cell death 1 ligand 2), might also be relevant for efficacy of PD-1 inhibition. We investigated PD-L2 expression and methylation as a prognostic and predictive biomarker in melanoma. Methods DNA methylation at five CpG loci and gene expression of PD-L2 were evaluated with regard to survival in 470 melanomas from The Cancer Genome Atlas. PD-L2 promoter methylation in correlation with PD-L2 mRNA and protein expression was analyzed in human melanoma cell lines. Prognostic and predictive value of PD-L2 methylation was validated using quantitative methylation-specific PCR in a multicenter cohort of 129 melanoma patients receiving anti-PD-1 therapy. mRNA sequencing data of 121 melanoma patients receiving anti-PD-1 therapy provided by Liu et al. were analyzed for PD-L2 mRNA expression. Results We found significant correlations between PD-L2 methylation and mRNA expression levels in melanoma tissues and cell lines. Interferon-γ inducible PD-L2 protein expression correlated with PD-L2 promoter methylation in melanoma cells. PD-L2 DNA promoter hypomethylation and high mRNA expression were found to be strong predictors of prolonged overall survival. In pre-treatment melanoma samples from patients receiving anti-PD-1 therapy, low PD-L2 DNA methylation and high PD-L2 mRNA expression predicted longer progression-free survival. Conclusion PD-L2 expression seems to be regulated via DNA promoter methylation. PD-L2 DNA methylation and mRNA expression may predict progression-free survival in melanoma patients receiving anti-PD-1 immunotherapy. Assessment of PD-L2 should be included in further clinical trials with anti-PD-1 antibodies.

Abstract. Sponges are commonly known as general nutrient providers for the marine ecosystem, recycling organic matter into various forms of bio-available nutrients such as ammonium and nitrate. In this study we challenge this view. We show that nutrient removal through microbial denitrification is a common feature in six cold-water sponge species from boreal and Arctic sponge grounds. Denitrification rates were quantified by incubating sponge tissue sections with 15NO3- – amended oxygen saturated seawater, mimicking conditions in pumping sponges, and de-oxygenated seawater, mimicking non-pumping sponges. Rates of anaerobic ammonium oxidation (anammox) using incubations with 15NH4+ could not be detected. Denitrification rates of the different sponge species ranged from 0 to 114 nmol N cm-3 sponge day-1 under oxic conditions, and from 47 to 342 nmol N cm-3 sponge day-1 under anoxic conditions. An exponential relationship between the highest potential rates of denitrification (in the absence of oxygen) and the species-specific abundances of nirS and nirK genes encoding nitrite reductase, a key enzyme for denitrification, suggests that the denitrifying community in these sponge species is both prepared and optimized for denitrification. The lack of a lag phase in the linear accumulation of the 15N labelled N2 gas in any of our tissue incubations is another indicator for an active community of denitrifiers in the investigated sponge species. High rates for coupled nitrification-denitrification (up to 89 % of nitrate reduction in the presence of oxygen) shows that under these conditions, the NO3- reduced in denitrification was primarily derived from nitrification within the sponge, directly coupling organic matter degradation and nitrification to denitrification in sponge tissues. Under anoxic condition when nitrification was not possible, nitrate to fuel the much higher denitrification rates had to be retrieved directly from the seawater. The lack of nifH genes encoding nitrogenase, the key enzyme for nitrogen fixation, shows that the nitrogen cycle is not closed in the sponge grounds. The denitrified nitrogen, no matter of its origin, is then no longer available as a nutrient for the marine ecosystem. Considering average sponge biomasses on typical boreal and Arctic sponge grounds, our sponge denitrification rates reveal areal denitrification rates of 0.8 mmol N m-2 day-1 assuming non-pumping sponges and still 0.3 mmol N m-2 day-1 assuming pumping sponges. This is well within the range of denitrification rates of continental shelf sediments. For the most densely populated boreal sponge grounds we calculated denitrification rates of up to 2 mmol N m-2 day-1, which is comparable to rates in coastal sediments. Increased future impact of sponge grounds by anthropogenic stressors reducing sponge pumping activity and further stimulating sponge anaerobic processes may thus lead to that deep-sea sponge grounds change their role in the marine ecosystem from nutrient sources to nutrient sinks.

To meet the increasing global energy demand, expanding exploration for oil and gas reserves as well as associated drilling activities are expected in the Arctic-boreal region where sponge aggregations contribute to up to 90% of benthic biomass. These deep-water sponges along with their microbial endobionts play key roles in the nitrogen cycling in Arctic-boreal ecosystems. This study aimed to investigate the effects of drilling discharges and associated sediment resuspension events on net fluxes of oxygen, ammonium, nitrate and nitrite in three common deep-water sponge species in the form of explants. Sponges were exposed to suspended bentonite and barite, the primary particulate compounds in drilling waste, as well as suspended natural sediment particles for a period of 33 days (on average 10 mg L-1 for 12 h day(-1)). The exposure period was followed by a pollution abatement period for a further 33 days. No sponge mortality was observed during the experiment. However, exposure to these particles, especially to barite, led to reduced oxygen consumption by up to 33% that was linearly correlated with reduced nitrite nitrate release by the sponges. The changes in net fluxes were accompanied by decreased tissue oxygenation by up to 54% within the sponges. These findings reveal the effects of fine particles on sponge metabolic processes by reducing aerobic respiration and microbial nitrification, and possibly by favouring anaerobic processes such as microbial denitrification. Most of the sponge responses recovered to their control levels upon the pollution abatement period, but the effects caused by barite may not be reversible. Our findings provide the first insight into the ecological consequences of oil and gas drilling activities on sponge-mediated nitrogen cycling in the Arctic-boreal region. (C) 2017 Elsevier Ltd. All rights reserved.

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5′-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge’s sulfur cycle.

After disinfection of ballast water, it is crucial to detect organisms and determine their vitality to assess the performance of the chosen treatment technique. Ultraviolet (UV) irradiation is a treatment technology commonly used for water disinfection. In this study, the phytoplankter Tetraselmis suecica was UV irradiated and subsequently stained with both 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and SYTOX Blue, staining metabolically active and membrane-permeable cells, respectively. This dual staining protocol can be used to assess samples during type approval of UV-based treatment systems. Non-irradiated and UV-irradiated samples were incubated in darkness, to simulate a ballast water transport, after which the vitality and viability T. suecica were monitored regularly over a period of 15 d. Flow cytometry (FCM) analysis separated the cells into 4 FCM populations (=single cells grouped together based on their fluorescence signals) according to differences in esterase activity and membrane integrity. UV-irradiated samples followed a different staining pattern compared to non-irradiated samples, where 1 specific FCM population of cells expressed esterase activity, but at the same time gave signals for disrupted membranes. This is useful as a sign of future death and is interpreted as an ‘early warning’ FCM population. FCM results were also compared to corresponding plate count results, differentiating vital, viable cells from vital, non-viable cells. We argue that dual staining with SYTOX Blue and CFDA-AM facilitates and improves FCM analysis when evaluating the performance of UV-based water treatment systems.