Biomarker-directed, pembrolizumab-based combination therapy in non-small cell lung cancer: phase 2 KEYNOTE-495/KeyImPaCT trial interim results.

Although pembrolizumab confers clinical benefit in non-small cell lung cancer (NSCLC), only a subset of patients will respond due to a heterogenous tumor microenvironment. KEYNOTE-495/KeyImPaCT is an ongoing biomarker-directed, adaptively randomized phase 2 study investigating first-line pembrolizumab (200 mg every 3 weeks) + lenvatinib (20 mg daily), anti-CTLA-4 quavonlimab (25 mg every 6 weeks) or anti-LAG-3 favezelimab (200 mg or 800 mg every 3 weeks) in advanced NSCLC. Patients were categorized by T-cell-inflamed gene expression profile (TcellinfGEP) and tumor mutational burden (TMB) status and randomly assigned 1:1:1 to receive pembrolizumab + lenvatinib, pembrolizumab + quavonlimab or pembrolizumab + favezelimab. The primary outcome was investigator-assessed objective response rate (ORR) per Response Evaluation Criteria in Solid Tumors version 1.1 using pre-specified efficacy thresholds for each biomarker-defined subgroup (>5% (TcellinfGEPlowTMBnon-high (group I)), >20% (TcellinfGEPlowTMBhigh (group II) and TcellinfGEPnon-lowTMBnon-high (group III)) and >45% (TcellinfGEPnon-lowTMBhigh (group IV))). Secondary outcomes were progression-free survival, overall survival and safety. At data cutoff, ORR ranges were 0-12.0% in group I, 27.3-33.3% in group II, 13.6-40.9% in group III and 50.0-60.0% in group IV. ORR with pembrolizumab + lenvatinib in group III met the pre-specified efficacy threshold. The safety profile of each treatment arm was consistent with the known safety profile of each combination. These data demonstrate the feasibility of prospective TcellinfGEP and TMB assessment to study the clinical activity of first-line pembrolizumab-based combination therapies in advanced NSCLC. ClinicalTrials.gov registration: NCT03516981 .

Hallmarks of Resistance to Immune-Checkpoint Inhibitors.

Immune-checkpoint inhibitors (ICI), although revolutionary in improving long-term survival outcomes, are mostly effective in patients with immune-responsive tumors. Most patients with cancer either do not respond to ICIs at all or experience disease progression after an initial period of response. Treatment resistance to ICIs remains a major challenge and defines the biggest unmet medical need in oncology worldwide. In a collaborative workshop, thought leaders from academic, biopharma, and nonprofit sectors convened to outline a resistance framework to support and guide future immune-resistance research. Here, we explore the initial part of our effort by collating seminal discoveries through the lens of known biological processes. We highlight eight biological processes and refer to them as immune resistance nodes. We examine the seminal discoveries that define each immune resistance node and pose critical questions, which, if answered, would greatly expand our notion of immune resistance. Ultimately, the expansion and application of this work calls for the integration of multiomic high-dimensional analyses from patient-level data to produce a map of resistance phenotypes that can be utilized to guide effective drug development and improved patient outcomes.

Current strategies for intratumoural immunotherapy - Beyond immune checkpoint inhibition.

Immunotherapy has revolutionised cancer treatment through restoration of host antitumour immune response. Immune checkpoint inhibitors (ICIs) confer durable responses in only a subset of patients. Mechanisms of ICI resistance to improve durable response rates and overall survival are an area of intense clinical research. Robust clinical development is ongoing to evaluate novel combination therapies to overcome ICI resistance, including targeting immunoregulatory pathways in the tumour microenvironment. Intratumoural (IT) immunotherapies such as toll-like receptor agonists, stimulator of interferon-induced gene agonists, retinoic-inducible gene I-like receptor agonists and oncolytic viruses may represent potential combination treatment options to overcome ICI resistance. Use of IT immunotherapies in combination with ICIs may alter the tumour microenvironment to address resistance mechanisms and improve antitumour response. Optimisation of IT immunotherapy clinical trials will elucidate resistance mechanisms, facilitate clinical trial design, define pharmacodynamic predictors that identify patients who may most benefit and inform clinical development of combination immunotherapy regimens. Here we provide an overview of IT immunotherapy principles, mechanisms of action, categories of IT immunotherapeutics, emerging data, clinical development strategies, response assessment, dose and schedule determination, clinical trial design and translational study design.

Translational Biomarkers and Rationale Strategies to Overcome Resistance to Immune Checkpoint Inhibitors in Solid Tumors.

Immune checkpoint inhibitors (ICIs) targeting the programed cell-death protein 1 (PD-1) or its ligand PD-L1 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) pathways have improved the survival for patients with solid tumors. Unfortunately, durable clinical responses are seen in only 10-40% of patients at the cost of potential immune-related adverse events. In the tumor microenvironment (TME), tumor cells can influence the microenvironment by releasing extracellular signals and generating peripheral immune tolerance, while the immune cells can affect the initiation, growth, proliferation, and evolution of cancer cells. Currently, translational biomarkers that predict responses to ICIs include high PD-L1 tumor proportion score, defective DNA mismatch repair, high microsatellite instability, and possibly high tumor mutational burden. Characterization of immune cells in the TME, such as tumor-infiltrating lymphocytes, T-cell gene expression profile, T-cell receptor sequencing, and peripheral blood biomarkers are being explored as promising biomarkers. Recent neoadjuvant studies have integrated the real-time assessment of both molecular and immune biomarkers using the tissue and blood specimens simultaneously and longitudinally. This review summarizes the current knowledge and progress in developing translational biomarkers and rational combinational strategies to improve the efficacy of ICIs tailored to individual cancer patients.

Absolute lymphocyte count as a prognostic biomarker for overall survival in patients with advanced melanoma treated with ipilimumab.

Biomarkers are needed to estimate which patients benefit most from combination ipilimumab and nivolumab immunotherapy. Rigorous biomarker analyses from prior ipilimumab randomized studies without nivolumab are likely to inform which biomarker analyses should be prioritized when examining patients treated with the combination. For the first time, the current analyses investigate absolute lymphocyte count (ALC) in randomized, controlled trials of ipilimumab without nivolumab to assess whether ALC is prognostic or predictive of ipilimumab treatment benefit. Data included patients (n = 1136) treated in the two randomized, controlled phase III studies MDX010-20 and CA184-024. ALC was measured at pretreatment baseline and every 3 weeks for up to 12 weeks, before each dose of ipilimumab. Cox proportional hazards models were used to estimate and test associations between ALC measures and overall survival (OS). In both randomized studies, baseline ALC and ALC halfway through induction (at week 6) were associated with OS not only in ipilimumab-treated patients but also in patients treated with non-ipilimumab control treatments. ALC increased in patients receiving ipilimumab, but this degree of change was not predictive of ipilimumab treatment benefit. Using data from randomized, controlled studies, we were able to conclude for the first time that baseline ALC, ALC halfway through induction (week 6) and the degree of ALC change from baseline to week 6 are prognostic biomarkers in melanoma patients, and do not appear to be predictive of ipilimumab treatment benefit. This more comprehensive understanding of ALC as a biomarker from ipilimumab trials will inform subsequent biomarker investigations in ongoing ipilimumab combination studies such as ipilimumab in combination with nivolumab.

Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade.

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8+ and CD4+ T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy. Cancer Immunol Res; 6(2); 189-200. ©2018 AACR.

Immunotherapy of cancers comes of age.

INTRODUCTION: Cancer immunotherapy has evolved and is aimed at generating the efficacious therapeutic modality to enhance the specificity and power of the immune system to combat tumors. Areas covered: Current efforts in cancer immunotherapy fall into three main approaches. One approach is through the blockade of immune checkpoints, another approach is through adoptive cellular therapy, and the last approach is through vaccination. The goal of this review is to summarize the current understanding and status of cancer immunotherapy in these three categories. Expert commentary: We foresee the development of therapeutic protocols combining these approaches with each other or conventional therapies to achieve the most appropriate guideline for management of cancer.

Immunotherapy biomarkers 2016: overcoming the barriers.

This report summarizes the symposium, 'Immunotherapy Biomarkers 2016: Overcoming the Barriers', which was held on April 1, 2016 at the National Institutes of Health in Bethesda, Maryland. The symposium, cosponsored by the Society for Immunotherapy of Cancer (SITC) and the National Cancer Institute (NCI), focused on emerging immunotherapy biomarkers, new technologies, current hurdles to further progress, and recommendations for advancing the field of biomarker development.

Peripheral CD8 effector-memory type 1 T-cells correlate with outcome in ipilimumab-treated stage IV melanoma patients.

The role of the assessment of peripheral T-cell phenotypes in predicting overall survival (OS) after ipilimumab treatment is unclear. Here, we analysed mononuclear cells in the blood before and at different time points during treatment with ipilimumab in 137 late-stage melanoma patients. The proportions of baseline naïve and memory T-cells were measured by flow cytometry and correlated with OS, with an emphasis on PD-1 expression. High frequencies (>13%) of CD8 effector-memory type 1 (EM1) T-cells at baseline correlated with longer OS (p = 0.029) and higher clinical response rates (p = 0.01). The frequency of these EM1 cells and the M category had independent impacts on OS (hazard ratio = 1.5, p = 0.033; and hazard ratio = 1.9, p = 0.007). In contrast, high baseline frequencies of late stage-differentiated effector memory CD8 cells (>23.8%) were negatively associated with OS (p = 0.034) but did not correlate with clinical response. Following treatment, a decrease of CD8 cells from baseline to the time of the second drug dose and at later time points was strongly and consistently correlated with a high clinical response rate. Our observations thus suggest an important predictive role of baseline CD8 EM1 cells and changes in CD8 cells for clinical response of ipilimumab. Further validation of these biomarker candidates is warranted.

Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 106 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR.

A Pilot Study of Preoperative Single-Dose Ipilimumab and/or Cryoablation in Women with Early-Stage Breast Cancer with Comprehensive Immune Profiling.

PURPOSE: To assess the safety and tolerability of preoperative cryoablation-mediated tumor antigen presentation and/or ipilimumab-mediated immune modulation in women with operable breast cancer. EXPERIMENTAL DESIGN: In this pilot study, 19 women with breast cancer for whom mastectomy was planned were treated with preoperative tumor cryoablation (n = 7), single-dose ipilimumab at 10 mg/kg (n = 6), or both (n = 6). The primary outcome for this pilot study was safety/tolerability as defined as freedom from delays in pre-planned, curative-intent mastectomy. Exploratory studies of immune activation were performed on peripheral blood and tumor. RESULTS: Preoperative cryoablation and/or ipilimumab were safe and tolerable, with no delays in pre-planned surgery. Grade III toxicity was seen in 1 of 19 (unrelated rash after ipilimumab). Combination therapy was associated with sustained peripheral elevations in: Th1-type cytokines, activated (ICOS+) and proliferating (Ki67+) CD4+ and CD8+ T cells, and posttreatment proliferative T-effector cells relative to T-regulatory cells within tumor. CONCLUSIONS: Preoperative cryoablation and single-dose ipilimumab are safe alone or in combination with no surgical delays incurred. Potentially favorable intratumoral and systemic immunologic effects were observed with the combination, suggesting the possibility for induced and synergistic antitumor immunity with this strategy. Clin Cancer Res; 22(23); 5729-37. ©2016 AACR.

Circulating protein and antibody biomarker for personalized cancer immunotherapy.

Immune checkpoint blockade therapies are revolutionizing standard cancer treatments. Immune checkpoint inhibitors likely function to enhance the tumor specific antigen response in order to achieve favorable clinical outcomes. Thus, continuous efforts to identify the common tumor-specific antigens are essential for the broad clinical application of these therapies. Several immunoproteomics approaches have been used in order to screen for this specificity. In a recent article from Jhaveri and colleagues published in the February issue of Cancer Immunology Research, antibody biomarkers were screened in pancreatic cancer patients who received allogeneic, granulocyte-macrophage colony stimulating factor-secreting pancreatic cancer vaccine (GVAX) by using a serum antibody-based SILAC immunoprecipitation (SASI) approach. Using this assay, several new tumor antigens (MYPT1, PSMC5 and TRFR) were identified that were found to have significantly different expression in tumors compared with normal tissue. Moreover, patients with detectable antibodies showed improved disease-free survival after GVAX therapy. These targets need to be further validated to determine the full spectrum of tumor antigen immunogencity and their potential clinical application. In addition to antibodies, circulating protein, DNA and RNA in peripheral blood are under clinical investigation as liquid biopsies and have the potential to provide guidance for future personalized cancer immunotherapy.

Proportions of blood-borne Vδ1+ and Vδ2+ T-cells are associated with overall survival of melanoma patients treated with ipilimumab.

Human γδ T-cells possess regulatory and cytotoxic capabilities, and could potentially influence the efficacy of immunotherapies. We analysed the frequencies of peripheral γδ T-cells, including their most prominent subsets (Vδ1+ and Vδ2+ cells) and differentiation states in 109 melanoma patients and 109 healthy controls. We additionally analysed the impact of γδ T-cells on overall survival (OS) calculated from the first dose of ipilimumab in melanoma patients. Higher median frequencies of Vδ1+ cells and lower median frequencies of Vδ2+ cells were identified in patients compared to healthy subjects (Vδ1+: 30% versus 15%, Vδ2+: 39% versus 64%, both p < 0.001). Patients with higher frequencies of Vδ1+ cells (≥30%) had poorer OS (p = 0.043) and a Vδ1+ differentiation signature dominated by late-differentiated phenotypes. In contrast, higher frequencies of Vδ2+ cells (≥39%) were associated with longer survival (p = 0.031) independent of the M category or lactate dehydrogenase level. Patients with decreasing frequencies of Vδ2+ cells under ipilimumab treatment had worse OS and a lower rate of clinical benefit than patients without such decreases. Therefore, we suggest frequencies of both Vδ1+ and Vδ2+ cells as candidate biomarkers for outcome in melanoma patients following ipilimumab. Further studies are needed to validate these results and to clarify whether they represent prognostic associations or whether γδ T-cells are specifically and/or functionally linked to the mode of action of ipilimumab.

Increases in Absolute Lymphocytes and Circulating CD4+ and CD8+ T Cells Are Associated with Positive Clinical Outcome of Melanoma Patients Treated with Ipilimumab.

PURPOSE: To investigate changes of peripheral blood biomarkers and their impact on clinical outcome following treatment with ipilimumab in advanced melanoma patients. EXPERIMENTAL DESIGN: Changes in blood counts and the frequency of circulating immune cell populations analyzed by flow cytometry were investigated in 82 patients to compare baseline values with different time-points after starting ipilimumab. Endpoints were overall survival (OS) and best clinical response. Statistical calculations were done by Wilcoxon-matched pairs tests, Fisher exact test, Kaplan-Meier analysis, and Cox regression analysis. RESULTS: Increases in absolute lymphocyte counts (ALC) 2 to 8 weeks (P = 0.003) and in percentages of CD4+ and CD8+ T cells 8 to 14 weeks (P = 0.001 and P = 0.02) after the first dose of ipilimumab were correlated with improved survival. These associations did not meet significance criteria, when conservatively adjusted for multiple testing, but were additionally correlated with clinical responses (all P < 0.05). However, validation is required. Increases in all three factors were observed in 36% of patients, who had a favorable outcome and survival probabilities of 93.3% and 63.8% at 12 and 24 months, respectively. A partial or complete response was observed in 71% of these patients compared with only 8% in patients with decreases in ≥1 of the 3 factors, respectively. Changes of regulatory T cells or myeloid-derived suppressor cells were not associated with OS. CONCLUSIONS: Increases of ALC observed 2 to 8 weeks after initiation of ipilimumab and delayed increases in CD4+ and CD8+ T cells reflect changes associated with positive outcome. These changes represent surrogate marker candidates and warrant further validation. Clin Cancer Res; 22(19); 4848-58. ©2016 AACR.

Current status and perspectives in translational biomarker research for PD-1/PD-L1 immune checkpoint blockade therapy.

Modulating immune inhibitory pathways has been a major recent breakthrough in cancer treatment. Checkpoint blockade antibodies targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programed cell-death protein 1 (PD-1) have demonstrated acceptable toxicity, promising clinical responses, durable disease control, and improved survival in some patients with advanced melanoma, non-small cell lung cancer (NSCLC), and other tumor types. About 20 % of advanced NSCLC patients and 30 % of advanced melanoma patients experience tumor responses from checkpoint blockade monotherapy, with better clinical responses seen with the combination of anti-PD-1 and anti-CTLA-4 antibodies. Given the power of these new therapies, it is important to understand the complex and dynamic nature of host immune responses and the regulation of additional molecules in the tumor microenvironment and normal organs in response to the checkpoint blockade therapies. In this era of precision oncology, there remains a largely unmet need to identify the patients who are most likely to benefit from immunotherapy, to optimize the monitoring assays for tumor-specific immune responses, to develop strategies to improve clinical efficacy, and to identify biomarkers so that immune-related adverse events can be avoided. At this time, PD-L1 immunohistochemistry (IHC) staining using 22C3 antibody is the only FDA-approved companion diagnostic for patients with NSCLC-treated pembrolizumab, but more are expected to come to market. We here summarize the current knowledge, clinical efficacy, potential immune biomarkers, and associated assays for immune checkpoint blockade therapies in advanced solid tumors.

Active opioid use does not attenuate the humoral responses to inactivated influenza vaccine.

BACKGROUND: Influenza vaccination is recommended for vulnerable individuals, including active drug users, to prevent influenza complications and decrease influenza spread. Recent studies suggest that opioids negatively regulate immune responses in experimental models, but the extent to which opioid use will affect the humoral responses to influenza vaccine in humans is unknown. This information is critical in maximizing vaccination efforts. OBJECTIVE: To determine whether there is a difference in antibody response after influenza vaccination in heroin or methadone users compared to control subjects. METHODS: We studied active heroin users, subjects on methadone maintenance treatment (MMT) and subjects that did not use any drugs before and 1 and 4 weeks after vaccination with trivalent influenza vaccine (TIV). We measured hemagglutination inhibition and microneutralization titers, and we compared geometric mean titers (GMT), and rates of seroprotection and seroconversion for each of the vaccine strains among the 3 groups of subjects. RESULTS: Heroin users, subjects on MMT and non-user controls mount a similarly robust serologic response to TIV. GMT and rates of seroprotection and seroconversion were not significantly different among groups. CONCLUSION: Our results suggest that opioid use do not significantly alter antibody responses to influenza vaccine supporting the vaccination effort in these populations.

Chemokine receptor patterns in lymphocytes mirror metastatic spreading in melanoma.

Melanoma prognosis is dictated by tumor-infiltrating lymphocytes, the migratory and functional behavior of which is guided by chemokine or cytokine gradients. Here, we retrospectively analyzed the expression patterns of 9 homing receptors (CCR/CXCR) in naive and memory CD4+ and CD8+ T lymphocytes in 57 patients with metastatic melanoma (MMel) with various sites of metastases to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Homing receptor expression on lymphocytes strongly correlated with MMel dissemination. Loss of CCR6 or CXCR3, but not cutaneous lymphocyte antigen (CLA), on circulating T cell subsets was associated with skin or lymph node metastases, loss of CXCR4, CXCR5, and CCR9 corresponded with lung involvement, and a rise in CCR10 or CD103 was associated with widespread dissemination. High frequencies of CD8+CCR9+ naive T cells correlated with prolonged OS, while neutralizing the CCR9/CCL25 axis in mice stimulated tumor progression. The expansion of CLA-expressing effector memory CD8+ T cells in response to a single administration of CTLA4 blockade predicted disease control at 3 months in 47 patients with MMel. Thus, specific CCR/CXCR expression patterns on circulating T lymphocytes may guide potential diagnostic and therapeutic approaches.

Baseline Peripheral Blood Biomarkers Associated with Clinical Outcome of Advanced Melanoma Patients Treated with Ipilimumab.

PURPOSE: To identify baseline peripheral blood biomarkers associated with clinical outcome following ipilimumab treatment in advanced melanoma patients. EXPERIMENTAL DESIGN: Frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), serum lactate dehydrogenase (LDH), routine blood counts, and clinical characteristics were assessed in 209 patients. Endpoints were overall survival (OS) and best overall response. Statistical calculations were done by Kaplan-Meier and Cox regression analysis, including calibration and discrimination by C-statistics. RESULTS: Low baseline LDH, absolute monocyte counts (AMC), Lin(-)CD14(+)HLA-DR(-/low)-MDSC frequencies, and high absolute eosinophil counts (AEC), relative lymphocyte counts (RLC), and CD4(+)CD25(+)FoxP3(+)-Treg frequencies were significantly associated with better survival, and were considered in a combination model. Patients (43.5%) presenting with the best biomarker signature had a 30% response rate and median survival of 16 months. In contrast, patients with the worst biomarkers (27.5%) had only a 3% response rate and median survival of 4 months. The occurrence of adverse events correlated with neither baseline biomarker signatures nor the clinical benefit of ipilimumab. In another model, limited to the routine parameters LDH, AMC, AEC, and RLC, the number of favorable factors (4 vs. 3 vs. 2-0) was also associated with OS (P < 0.001 for all pairwise comparisons) in the main study and additionally in an independent validation cohort. CONCLUSIONS: A baseline signature of low LDH, AMC, and MDSCs as well as high AEC, Tregs, and RLC is associated with favorable outcome following ipilimumab. Prospective investigation of the predictive impact of these markers following ipilimumab and other treatments, e.g., PD-1 antibodies, is warranted. Clin Cancer Res; 22(12); 2908-18. ©2016 AACR.