Influence of microbiota-associated metabolic reprogramming on clinical outcome in patients with melanoma from the randomized adjuvant dendritic cell-based MIND-DC trial.

Tumor immunosurveillance plays a major role in melanoma, prompting the development of immunotherapy strategies. The gut microbiota composition, influencing peripheral and tumoral immune tonus, earned its credentials among predictors of survival in melanoma. The MIND-DC phase III trial (NCT02993315) randomized (2:1 ratio) 148 patients with stage IIIB/C melanoma to adjuvant treatment with autologous natural dendritic cell (nDC) or placebo (PL). Overall, 144 patients collected serum and stool samples before and after 2 bimonthly injections to perform metabolomics (MB) and metagenomics (MG) as prespecified exploratory analysis. Clinical outcomes are reported separately. Here we show that different microbes were associated with prognosis, with the health-related Faecalibacterium prausnitzii standing out as the main beneficial taxon for no recurrence at 2 years (p = 0.008 at baseline, nDC arm). Therapy coincided with major MB perturbations (acylcarnitines, carboxylic and fatty acids). Despite randomization, nDC arm exhibited MG and MB bias at baseline: relative under-representation of F. prausnitzii, and perturbations of primary biliary acids (BA). F. prausnitzii anticorrelated with BA, medium- and long-chain acylcarnitines. Combined, these MG and MB biomarkers markedly determined prognosis. Altogether, the host-microbial interaction may play a role in localized melanoma. We value systematic MG and MB profiling in randomized trials to avoid baseline differences attributed to host-microbe interactions.

Adjuvant dendritic cell therapy in stage IIIB/C melanoma: the MIND-DC randomized phase III trial.

Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88-1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73-2.38; p = 0.44). Grade 3-4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p < 0.001). In conclusion, while adjuvant nDC treatment in stage IIIB/C melanoma patients generated specific immune responses and was well tolerated, no benefit in RFS was observed.

Case Report: A severe case of immunosuppressant-refractory immune checkpoint inhibitor-mediated colitis rescued by tofacitinib.

Immune checkpoint inhibitor therapy for cancer treatment can give rise to a variety of adverse events. Here we report a male patient with metastatic melanoma who experienced life-threatening colitis and duodenitis following treatment with ipilimumab and nivolumab. The patient did not respond to the first three lines of immunosuppressive therapy (corticosteroids, infliximab, and vedolizumab), but recovered well after administration of tofacitinib, a JAK inhibitor. Cellular and transcriptional data on colon and duodenum biopsies shows significant inflammation in the tissue, characterized by a large number of CD8 T cells and high expression of PD-L1. While cellular numbers do decrease during three lines of immunosuppressive therapy, CD8 T cells remain relatively high in the epithelium, along with PD-L1 expression in the involved tissue and expression of colitis-associated genes, indicating an ongoing colitis at that moment. Despite all immunosuppressive treatments, the patient has an ongoing tumor response with no evidence of disease. Tofacitinib might be a good candidate to consider more often for ipilimumab/nivolumab-induced colitis.

The safety risk of information overload and bureaucracy in oncology clinical trial conduct.

Performance of clinical trials has led to major therapeutic developments and substantial improvements in the field of medical oncology. To ensure patient's safety, regulatory aspects for proper clinical trial conduct have been increased over the past two decades but seem to cause information overload and ineffective bureaucracy, possibly even impacting patient safety. To put this in perspective, after the implementation of Directive 2001/20/EC in the European Union, a 90 per cent increase in trial launching time, a 25 per cent decrease in patient participation and a 98 per cent rise in administrative trial costs were reported. The time to initiate a clinical trial has increased from a few months to several years in the past three decades. Moreover, there is a serious risk that information overload with relatively unimportant data endangers the decision-making processes and distracts from essential patient safety information. It is now a critical moment in time to improve efficient clinical trial conduct for our future patients diagnosed with cancer. We are convinced that a reduction of the administrative regulations, information overload, and simplification of the procedures for trial conductance may improve patient safety. In this Current Perspective, we give insight in the current regulatory aspects of clinical research, evaluate the practical consequences of these regulations, and propose specific improvements for optimal clinical trial conduct.

Immunological responses to adjuvant vaccination with combined CD1c+ myeloid and plasmacytoid dendritic cells in stage III melanoma patients.

We evaluated the immunological responses of lymph-node involved (stage III) melanoma patients to adjuvant dendritic cell vaccination with subsets of naturally occurring dendritic cells (nDCs). Fifteen patients with completely resected stage III melanoma were randomized to receive adjuvant dendritic cell vaccination with CD1c+ myeloid dendritic cells (cDC2s), plasmacytoid dendritic cells (pDCs) or the combination. Immunological response was the primary endpoint and secondary endpoints included safety and survival. In 80% of the patients, antigen-specific CD8+ T cells were detected in skin test-derived T cells and in 55% of patients, antigen-specific CD8+ T cells were detectable in peripheral blood. Functional interferon-γ-producing T cells were found in the skin test of 64% of the patients. Production of nDC vaccines meeting release criteria was feasible for all patients. Vaccination only induced grade 1-2 adverse events, mainly consisting of fatigue. In conclusion, adjuvant dendritic cell vaccination with cDC2s and/or pDCs is feasible, safe and induced immunological responses in the majority of stage III melanoma patients.

Immunological and Genomic Analysis Reveals Clinically Relevant Distinctions between Angiosarcoma Subgroups.

Angiosarcomas (AS) are extremely rare and aggressive vascular malignancies subdivided in de novo primary AS (pAS) and secondary AS (sAS). We hypothesize that the combination of immunological and genomic profiles significantly differs between primary and secondary AS, with potential impact on treatment strategies and a role for immunotherapy. Tumor-infiltrating lymphocytes were analyzed using multiplex immunohistochemistry from 79 pAS and 178 sAS. Median cell density was significantly higher in sAS for CD3+ T-cells (p < 0.001), CD8+ cytotoxic T-cells (p = 0.033), CD4+ T-helper cells (p < 0.001) and FoxP3+ T-regulatory cells (p < 0.001). CD20+ B-cell density was comparable (p = 0.417). Comprehensive genomic profiling was performed in 25 pAS and 25 sAS. A (likely) pathogenic mutation was detected in 80% of pAS vs. 88% of sAS (p = 0.702). Amplifications were found in 15% of pAS vs. 84% of sAS (p < 0.001). DNA damage response (DDR) pathway mutations (p = 0.021) and MYC amplifications (p < 0.001) were predominantly seen in sAS. In conclusion we observed a clear and clinical relevant distinction in immune infiltration and genomic profiles between pAS and sAS. The T-cell infiltrated tumor microenvironment and frequent DDR gene mutations, especially in sAS, warrant clinical trials with immunotherapy.

Trial watch: Dendritic cell (DC)-based immunotherapy for cancer.

Dendritic cell (DC)-based vaccination for cancer treatment has seen considerable development over recent decades. However, this field is currently in a state of flux toward niche-applications, owing to recent paradigm-shifts in immuno-oncology mobilized by T cell-targeting immunotherapies. DC vaccines are typically generated using autologous (patient-derived) DCs exposed to tumor-associated or -specific antigens (TAAs or TSAs), in the presence of immunostimulatory molecules to induce DC maturation, followed by reinfusion into patients. Accordingly, DC vaccines can induce TAA/TSA-specific CD8+/CD4+ T cell responses. Yet, DC vaccination still shows suboptimal anti-tumor efficacy in the clinic. Extensive efforts are ongoing to improve the immunogenicity and efficacy of DC vaccines, often by employing combinatorial chemo-immunotherapy regimens. In this Trial Watch, we summarize the recent preclinical and clinical developments in this field and discuss the ongoing trends and future perspectives of DC-based immunotherapy for oncological indications.

Human pDCs Are Superior to cDC2s in Attracting Cytolytic Lymphocytes in Melanoma Patients Receiving DC Vaccination.

Plasmacytoid dendritic cells (pDCs) and type 2 conventional dendritic cells (cDC2s) are currently under evaluation for use in cancer vaccines. Although both DC subsets can activate adaptive and innate lymphocytes, their capacity to recruit such cells is rarely considered. Here, we show that pDCs and cDC2s display a striking difference in chemokine secretion, which correlates with the recruitment of distinct types of immune effector cells. Activated pDCs express high levels of CXCR3 ligands and attract more CD8+ T cells, CD56+ T cells, and γδ T cells in vitro, compared to cDC2s. Skin from melanoma patients shows an influx of immune effector cells following intradermal vaccination with pDCs or cDC2s, with pDCs inducing the strongest influx of lymphocytes known to possess cytolytic activity. These findings suggest that combining both DC subsets could unite the preferred chemoattractive properties of pDCs with the superior T cell priming properties of cDC2s to ultimately enhance vaccine efficacy.

Autologous monocyte-derived DC vaccination combined with cisplatin in stage III and IV melanoma patients: a prospective, randomized phase 2 trial.

BACKGROUND: Autologous dendritic cell (DC) vaccines can induce tumor-specific T cells, but their effect can be counteracted by immunosuppressive mechanisms. Cisplatin has shown immunomodulatory effects in vivo which may enhance efficacy of DC vaccination. METHODS: This is a prospective, randomized, open-label phase 2 study (NCT02285413) including stage III and IV melanoma patients receiving 3 biweekly vaccinations of gp100 and tyrosinase mRNA-loaded monocyte-derived DCs with or without cisplatin. Primary objectives were to study immunogenicity and feasibility, and secondary objectives were to assess toxicity and survival. RESULTS: Twenty-two stage III and 32 stage IV melanoma patients were analyzed. Antigen-specific CD8+ T cells were found in 44% versus 67% and functional T cell responses in 28% versus 19% of skin-test infiltrating lymphocytes in patients receiving DC vaccination with and without cisplatin, respectively. Four patients stopped cisplatin because of toxicity and continued DC monotherapy. No therapy-related grade 3 or 4 adverse events occurred due to DC monotherapy. During combination therapy, one therapy-related grade 3 adverse event, decompensated heart failure due to fluid overload, occurred. The clinical outcome parameters did not clearly suggest significant differences. CONCLUSIONS: Combination of DC vaccination and cisplatin in melanoma patients is feasible and safe, but does not seem to result in more tumor-specific T cell responses or improved clinical outcome, when compared to DC vaccination monotherapy.

Response and survival of metastatic melanoma patients treated with immune checkpoint inhibition for recurrent disease on adjuvant dendritic cell vaccination.

Vaccination with autologous dendritic cells (DC) loaded ex vivo with melanoma-associated antigens is currently being tested as an adjuvant treatment modality for resected locoregional metastatic (stage III) melanoma. Based on its mechanism of action, DC vaccination might potentiate the clinical efficacy of concurrent or sequential immune checkpoint inhibition (ICI). The purpose of this study was to determine the efficacy of ICI administered following recurrent disease during, or after, adjuvant DC vaccination. To this end, we retrospectively analyzed clinical responses of 51 melanoma patients with either irresectable stage III or stage IV disease treated with first- or second-line ICI following recurrence on adjuvant DC vaccination. Patients were analyzed according to the form of ICI administered: PD-1 inhibition monotherapy (nivolumab or pembrolizumab), ipilimumab monotherapy or combined treatment with ipilimumab and nivolumab. Treatment with first- or second-line PD-1 inhibition monotherapy after recurrence on adjuvant DC vaccination resulted in a response rate of 52%. In patients treated with ipilimumab monotherapy and ipilimumab-nivolumab response rates were 35% and 75%, respectively. In conclusion, ICI is effective in melanoma patients with recurrent disease on adjuvant DC vaccination.

The clinical application of cancer immunotherapy based on naturally circulating dendritic cells.

Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy.

Early Recurrence in Completely Resected IIIB and IIIC Melanoma Warrants Restaging Prior to Adjuvant Therapy.

PURPOSE: To evaluate the results of restaging completely resected stage IIIB/C melanoma prior to start of adjuvant therapy. PATIENTS AND METHODS: One hundred twenty patients with stage IIIB or IIIC (AJCC 2009) melanoma who underwent complete surgical resection were screened for inclusion in our trial investigating adjuvant dendritic cell therapy (NCT02993315). All patients underwent imaging to exclude local relapse or metastasis before entering the trial. The frequency of recurrent disease within 12 weeks after resection and the method of detection were investigated. RESULTS: Sixty-nine (58%) stage IIIB and 51 (43%) stage IIIC melanoma patients were screened. Median age was 54 (range 27-79) years. Twenty-two (18%) of 120 patients with completely resected stage IIIB/C melanoma had evidence of early recurrent disease, despite exclusion thereof by prior imaging. Median interval between resection and detection of relapse was 7.4 (range 4.3-10.7) weeks. Recurrence was asymptomatic in 17 (77%) patients, but metastasis was noticed by the patient or physician in 5 (23%). Eight patients with local relapse received local treatment with curative intent, and one was treated with systemic therapy. The remaining patients had distant metastasis, 1 of whom underwent resection of a solitary liver metastasis while 12 patients received systemic treatment. CONCLUSIONS: Patients with completely resected stage IIIB/C melanoma have high risk of early recurrence before start of adjuvant therapy. Restaging should be considered for high-risk melanoma patients before start of adjuvant therapy.

Dendritic Cell Cancer Therapy: Vaccinating the Right Patient at the Right Time.

Immune checkpoint inhibitors propelled the field of oncology with clinical responses in many different tumor types. Superior overall survival over chemotherapy has been reported in various metastatic cancers. Furthermore, prolonged disease-free and overall survival have been reported in the adjuvant treatment of stage III melanoma. Unfortunately, a substantial portion of patients do not obtain a durable response. Therefore, additional strategies for the treatment of cancer are still warranted. One of the numerous options is dendritic cell vaccination, which employs the central role of dendritic cells in activating the innate and adaptive immune system. Over the years, dendritic cell vaccination was shown to be able to induce an immunologic response, to increase the number of tumor infiltrating lymphocytes and to provide overall survival benefit for at least a selection of patients in phase II studies. However, with the success of immune checkpoint inhibition in several malignancies and considering the plethora of other treatment modalities being developed, it is of utmost importance to delineate the position of dendritic cell therapy in the treatment landscape of cancer. In this review, we address some key questions regarding the integration of dendritic cell vaccination in future cancer treatment paradigms.

Direct inhibition of STAT signaling by platinum drugs contributes to their anti-cancer activity.

Platinum-based chemotherapeutics are amongst the most powerful anti-cancer drugs. Although their exact mechanism of action is not well understood, it is thought to be mediated through covalent DNA binding. We investigated the effect of platinum-based chemotherapeutics on signaling through signal transducer and activator of transcription (STAT) proteins, which are involved in many oncogenic signaling pathways. We performed in vitro experiments in various cancer cell lines, investigating the effects of platinum chemotherapeutics on STAT phosphorylation and nuclear translocation, the expression of STAT-modulating proteins and downstream signaling pathways. Direct binding of platinum to STAT proteins was assessed using an AlphaScreen assay. Nuclear STAT3 expression was determined by immunohistochemistry and correlated with disease-free survival in retrospective cohorts of head and neck squamous cell carcinoma (HNSCC) patients treated with cisplatin-based chemoradiotherapy (n= 65) or with radiotherapy alone (n = 32). At clinically relevant concentrations, platinum compounds inhibited STAT phosphorylation, resulting in loss of constitutively activated STAT proteins in multiple distinct cancer cell lines. Platinum drugs specifically inhibited phospho-tyrosine binding to SH2 domains, thereby blocking STAT activation, and subsequently downregulating pro-survival- and anti-apoptotic- target genes. Importantly, we found that active STAT3 in tumors directly correlated with response to cisplatin-based chemoradiotherapy in HNSCC patients (p = 0.006). These findings provide insight into a novel, non-DNA-targeted mechanism of action of platinum drugs, and could be leveraged into the use of STAT expression as predictive biomarker for cisplatin chemotherapy and to potentiate other therapeutic strategies such as immunotherapy.

Adjuvant dendritic cell vaccination induces tumor-specific immune responses in the majority of stage III melanoma patients.

PURPOSE: To determine the effectiveness of adjuvant dendritic cell (DC) vaccination to induce tumor-specific immunological responses in stage III melanoma patients. EXPERIMENTAL DESIGN: Retrospective analysis of stage III melanoma patients, vaccinated with autologous monocyte-derived DC loaded with tumor-associated antigens (TAA) gp100 and tyrosinase after radical lymph node dissection. Skin-test infiltrating lymphocytes (SKILs) obtained from delayed-type hypersensitivity skin-test biopsies were analyzed for the presence of TAA-specific CD8(+) T cells by tetrameric MHC-peptide complexes and by functional TAA-specific T cell assays, defined by peptide-recognition (T2 cells) and/or tumor-recognition (BLM and/or MEL624) with specific production of Th1 cytokines and no Th2 cytokines. RESULTS: Ninety-seven patients were analyzed: 21 with stage IIIA, 34 with stage IIIB, and 42 had stage IIIC disease. Tetramer-positive CD8(+) T cells were present in 68 patients (70%), and 24 of them showed a response against all 3 epitopes tested (gp100:154-162, gp100:280-288, and tyrosinase:369-377) at any point during vaccinations. A functional T cell response was found in 62 patients (64%). Rates of peptide-recognition of gp100:154-162, gp100:280-288, and tyrosinase:369-377 were 40%, 29%, and 45%, respectively. Median recurrence-free survival and distant metastasis-free survival of the whole study population were 23.0 mo and 36.8 mo, respectively. CONCLUSIONS: DC vaccination induces a functional TAA-specific T cell response in the majority of stage III melanoma patients, indicating it is more effective in stage III than in stage IV melanoma patients. Furthermore, performing multiple cycles of vaccinations enhances the chance of a broader immune response.

Ipilimumab administered to metastatic melanoma patients who progressed after dendritic cell vaccination.

BACKGROUND: Ipilimumab has proven to be effective in metastatic melanoma patients. The purpose of this study was to determine the efficacy of ipilimumab in advanced melanoma patients who showed progressive disease upon experimental dendritic cell (DC) vaccination. METHODS: Retrospective analysis of 48 stage IV melanoma patients treated with ipilimumab after progression upon DC vaccination earlier in their treatment. DC vaccination was given either as adjuvant treatment for stage III disease (n = 18) or for stage IV disease (n = 30). Ipilimumab (3 mg/kg) was administered every 3 weeks for up to 4 cycles. RESULTS: Median time between progression upon DC vaccination and first gift of ipilimumab was 5.4 mo. Progression-free survival (PFS) rates for patients that received ipilimumab after adjuvant DC vaccination, and patients that received DC vaccination for stage IV melanoma, were 35% and 7% at 1 y and 35% and 3% at 2 y, while the median PFS was 2.9 mo and 3.1 mo, respectively. Median overall survival of patients pre-treated with adjuvant DC vaccination for stage III melanoma was not reached versus 8.0 mo (95% CI, 5.2-10.9) in the group pre-treated with DC vaccination for stage IV disease (HR of death, 0.36; p = 0.017). Grade 3 immune-related adverse events occurred in 19% of patients and one death (2%) was related to ipilimumab. CONCLUSIONS: Clinical responses to ipilimumab were found in a considerable number of advanced melanoma patients with progression after adjuvant DC vaccination for stage III disease, while the effect was very limited in patients who showed progression after DC vaccination for stage IV disease.

Immune-related Adverse Events of Dendritic Cell Vaccination Correlate With Immunologic and Clinical Outcome in Stage III and IV Melanoma Patients.

The purpose of this study was to determine the toxicity profile of dendritic cell (DC) vaccination in stage III and IV melanoma patients, and to evaluate whether there is a correlation between side effects and immunologic and clinical outcome. This is a retrospective analysis of 82 stage III and 137 stage IV melanoma patients, vaccinated with monocyte-derived or naturally circulating autologous DCs loaded with tumor-associated antigens gp100 and tyrosinase. Median follow-up time was 54.3 months in stage III patients and 12.9 months in stage IV patients. Treatment-related adverse events occurred in 84% of patients; grade 3 toxicity was present in 3% of patients. Most common adverse events were flu-like symptoms (67%) and injection site reactions (50%), and both correlated with the presence of tetramer-positive CD8 T cells (both P<0.001). In stage III melanoma patients experiencing flu-like symptoms, median overall survival (OS) was not reached versus 32.3 months in patients without flu-like symptoms (P=0.009); median OS in patients with an injection site reaction was not reached versus 53.7 months in patients without an injection site reaction (P<0.05). In stage IV melanoma patients (primary uveal and mucosal melanomas excluded), median OS in patients with or without flu-like symptoms was 13.1 versus 8.9 months, respectively (P=0.03); median OS in patients with an injection site reaction was 15.7 months versus 9.8 months in patients without an injection site reaction (P=0.003). In conclusion, DC vaccination is safe and tolerable and the occurrence of the immune-related side effects, such as flu-like symptoms and injection site reactions, correlates with immunologic and clinical outcome.

T-cell Landscape in a Primary Melanoma Predicts the Survival of Patients with Metastatic Disease after Their Treatment with Dendritic Cell Vaccines.

Tumor-infiltrating lymphocytes appear to be a predictor of survival in many cancers, including cutaneous melanoma. We applied automated multispectral imaging to determine whether density and distribution of T cells within primary cutaneous melanoma tissue correlate with survival of metastatic melanoma patients after dendritic cell (DC) vaccination. CD3(+) T cell infiltration in primary tumors from 77 metastatic melanoma patients was quantified using the ratio of intratumoral versus peritumoral T-cell densities (I/P ratio). Patients with longer survival after DC vaccination had stronger T-cell infiltration than patients with shorter survival in a discovery cohort of 19 patients (P = 0.000026) and a validation cohort of 39 patients (P = 0.000016). I/P ratio was the strongest predictor of survival in a multivariate analysis including M substage and serum lactate dehydrogenase level. To evaluate I/P ratio as a predictive biomarker, we analyzed 19 chemotherapy-treated patients. Longer survival times of DC-vaccinated compared with chemotherapy-treated patients was observed for high (P = 0.000566), but not low (P = 0.154) I/P ratios. In conclusion, T-cell infiltration into primary melanoma is a strong predictor of survival after DC vaccination in metastatic melanoma patients who, on average, started this therapy several years after primary tumor resection. The infiltration remains predictive even after adjustment for late-stage prognostic markers. Our findings suggest that the I/P ratio is a potential predictive biomarker for treatment selection. Cancer Res; 76(12); 3496-506. ©2016 AACR.