Granulomatous Slack Skin With Lung and Esophagus Involvement: A Case Report and Molecular Analysis.

ABSTRACT: Granulomatous slack skin (GSS) is a rare subtype of mycosis fungoides, and few cases have been known to spread to the blood, lymph nodes, or viscera. We present a case with early dissemination to the lung. A 27-year-old woman, previously healthy, presented with scattered disseminated scaly patches, associated with vulvar and intergluteal firm swelling and groin-skin induration for 1 year. She also reported mild fatigue and breathlessness on moderate exertion. The patient underwent blood tests, skin biopsies, and computed tomography scan. The skin biopsy showed a mildly atypical T-cell lymphoid infiltrate involving the dermis/hypodermis, with focal epidermotropism, associated with a granulomatous infiltrate and elastophagocytosis. The computed tomography scan revealed bilateral ground-glass lung nodular opacities. Positron emission tomography showed an increased signal in the skin and subcutis around the buttocks, inguinal and mediastinal lymph nodes, and lungs. The lung biopsy confirmed a dense T-cell infiltrate with numerous multinucleated giant cells. Subsequently, esophageal involvement was also observed following biopsy. Molecular analyses demonstrated identical T-cell clones in the skin and lung. After 6 cycles of chemotherapy/localized external radiotherapy, the patient had a partial skin response and stable lung disease. A preferred diagnosis of GSS with systemic spread was made based on clinical/histologic/molecular findings, after considering granulomatous mycosis fungoides and peripheral T-cell lymphoma, not otherwise specified. This case highlights the frequent diagnostic difficulty in distinguishing GSS from an inflammatory granulomatous dermatitis. Pulmonary and esophageal involvements are rare in GSS, and the simultaneous presentation of characteristic cutaneous GSS with systemic disease poses an additional classification challenge.

Homozygous Loss of CDKN2 in Primary Cutaneous CD8(+) Lymphoma NOS.

ABSTRACT: Primary cutaneous acral CD8(+) lymphoma (AL) has been accepted as primary cutaneous acral CD8-positive T-cell lymphoproliferative disorder in the revised WHO and updated WHO-EORTC lymphoma classifications. Commonly arising on the ears and comprising a clonal cytotoxic CD8 + T-cell infiltrate, almost all cases follow an indolent clinical course. A single aggressive case reported in the literature had a deletion at the CDKN2 locus at 9p21. We report an atypical CD8 + T-cell proliferation arising on the chest of an elderly man who had some similarities to AL but with a very high proliferation rate, absent p16 protein expression, and homozygous loss of the CDKN2 locus using FISH analysis. A diagnosis of peripheral T-cell lymphoma not otherwise specified (PTCL NOS) was preferred. Analyses of 4 cases of AL demonstrated often low p16 protein expression but intact CDKN2 loci. This case raises the problems of the boundaries between AL and PTCL NOS, and a possible role in the loss of p16 function in pathogenesis.

Massive parallel sequencing unveils homologous recombination deficiency in follicular dendritic cell sarcoma.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole genome sequencing (WGS) in one case and whole exome sequencing (WES) in additional twelve, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair related genes in 70% (9/13) of cases. This indicates that patients with high stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by FISH in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of haematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

UBE2C promotes leptomeningeal dissemination and is a therapeutic target in brain metastatic disease.

Background: Despite current improvements in systemic cancer treatment, brain metastases (BM) remain incurable, and there is an unmet clinical need for effective targeted therapies. Methods: Here, we sought common molecular events in brain metastatic disease. RNA sequencing of thirty human BM identified the upregulation of UBE2C, a gene that ensures the correct transition from metaphase to anaphase, across different primary tumor origins. Results: Tissue microarray analysis of an independent BM patient cohort revealed that high expression of UBE2C was associated with decreased survival. UBE2C-driven orthotopic mouse models developed extensive leptomeningeal dissemination, likely due to increased migration and invasion. Early cancer treatment with dactolisib (dual PI3K/mTOR inhibitor) prevented the development of UBE2C-induced leptomeningeal metastases. Conclusions: Our findings reveal UBE2C as a key player in the development of metastatic brain disease and highlight PI3K/mTOR inhibition as a promising anticancer therapy to prevent late-stage metastatic brain cancer.

Extramedullary T-lymphoblastic Crisis in a Myelodysplastic/Myeloproliferative Neoplasm with a t(12;22)/MN1::ETV6 Translocation.

Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are not a single disease, but rather a heterogenous group of entities which are increasingly subclassified according to recurrent genetic abnormalities. Chromosomal translocations involving meningioma 1 (MN1) and ETS variant 6 (ETV6) genes are extremely rare, but recurrent in myeloid neoplasms. We describe the case of a patient with a myelodysplastic/myeloproliferative neoplasm with neutrophilia, who developed an extramedullary T-lymphoblastic crisis with the t(12;22)(p13;q12) translocation as the only cytogenetic abnormality. This case shares several clinical and molecular features with myeloid/lymphoid neoplasms with eosinophilia. The treatment of this patient was challenging, as the disease proved to be highly refractory to chemotherapy, with allogenic stem cell transplantation as the only curative option. This clinical presentation has not been reported in association with these genetic alterations and supports the concept of a hematopoietic neoplasm originating in an early uncommitted precursor cell. Additionally, it stresses the importance of molecular characterization in the classification and prognostic stratification of these entities.

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.

Meta-Analysis of MS-Based Proteomics Studies Indicates Interferon Regulatory Factor 4 and Nucleobindin1 as Potential Prognostic and Drug Resistance Biomarkers in Diffuse Large B Cell Lymphoma.

The prognosis of diffuse large B cell lymphoma (DLBCL) is inaccurately predicted using clinical features and immunohistochemistry (IHC) algorithms. Nomination of a panel of molecules as the target for therapy and predicting prognosis in DLBCL is challenging because of the divergences in the results of molecular studies. Mass spectrometry (MS)-based proteomics in the clinic represents an analytical tool with the potential to improve DLBCL diagnosis and prognosis. Previous proteomics studies using MS-based proteomics identified a wide range of proteins. To achieve a consensus, we reviewed MS-based proteomics studies and extracted the most consistently significantly dysregulated proteins. These proteins were then further explored by analyzing data from other omics fields. Among all significantly regulated proteins, interferon regulatory factor 4 (IRF4) was identified as a potential target by proteomics, genomics, and IHC. Moreover, annexinA5 (ANXA5) and nucleobindin1 (NUCB1) were two of the most up-regulated proteins identified in MS studies. Functional enrichment analysis identified the light zone reactions of the germinal center (LZ-GC) together with cytoskeleton locomotion functions as enriched based on consistent, significantly dysregulated proteins. In this study, we suggest IRF4 and NUCB1 proteins as potential biomarkers that deserve further investigation in the field of DLBCL sub-classification and prognosis.

Extracellular Vesicles in Diffuse Large B Cell Lymphoma: Characterization and Diagnostic Potential.

Diffuse large B cell lymphoma (DLBCL) is an aggressive B cell lymphoma characterized by a heterogeneous behavior and in need of more accurate biological characterization monitoring and prognostic tools. Extracellular vesicles are secreted by all cell types and are currently established to some extent as representatives of the cell of origin. The present study characterized and evaluated the diagnostic and prognostic potential of plasma extracellular vesicles (EVs) proteome in DLBCL by using state-of-the-art mass spectrometry. The EV proteome is strongly affected by DLBCL status, with multiple proteins uniquely identified in the plasma of DLBCL. A proof-of-concept classifier resulted in highly accurate classification with a sensitivity and specificity of 1 when tested on the holdout test data set. On the other hand, no proteins were identified to correlate with non-germinal center B-cell like (non-GCB) or GCB subtypes to a significant degree after correction for multiple testing. However, functional analysis suggested that antigen binding is regulated when comparing non-GCB and GCB. Survival analysis based on protein quantitative values and clinical parameters identified multiple EV proteins as significantly correlated to survival. In conclusion, the plasma extracellular vesicle proteome identifies DLBCL cancer patients from healthy donors and contains potential EV protein markers for prediction of survival.

Hydropic leiomyoma: A radiologic pathologic correlation of a rare uterine tumor.

Hydropic leiomyoma is a rare leiomyoma subtype composed of a conspicuous zonal watery edematous stroma that causes compartmentalization of the smooth muscle cells. It exhibits atypical imaging features which can mimic malignancy, so differential diagnosis with malignant uterine tumors such as leiomyosarcoma is crucial for treatment decisions and patient follow-up. We describe the case of a 54-year-old postmenopausal woman presenting with a fast-growing abdominopelvic tumor associated with abdominal bloating, urinary frequency, and metrorrhagia. Radiologic evaluation depicted a voluminous, well-circumscribed, slightly lobulated, heterogeneous mass with mixed solid and cystic components arising from the uterus. Given the postmenopausal patient status, size of the tumor, and uncertainty about a possible malignant origin, an uneventful total abdominal hysterectomy with bilateral adnexectomy was performed as definitive treatment. However, as it is common practice in our institution, a second opinion report of the previous MRI was done before surgery, with the proposed diagnosis being hydropic leiomyoma. Pathologic examination of the surgical specimen revealed a large subserosal tumor with nodules separated by empty spaces and cysts due to watery exudate. Histologically, it was a mesenchymal neoplasm with trabecular and nested architecture, with tumor cells separated by watery fluid without mitosis or necrosis, securing the diagnosis of a hydropic leiomyoma.

Extramedullary Acute Leukemia-Still an Unforeseen Presentation.

Myeloid sarcomas (MS) are rare extramedullary (EM) hematological tumors that generally arise during the natural course of acute myeloid leukemia (AML), occurring concomitantly with the onset of systemic leukemia; it can also occur following onset but rarely before. Common sites of EM involvement include the lymph nodes, skin, soft tissue, bone and peritoneum. Herein, we report the case of a 63-year-old man who presented EM AML upon initial diagnosis involving the bone marrow, lymph nodes and skin (leukemia cutis). A diagnosis was made based on immunohistochemistry (IHC). This case presents a diagnostic dilemma due to its atypical presentation and the sites involved. It also highlights the importance of IHC in the diagnosis of EM AML. The potential role of hypomethylating agents and Venetoclax in cases not eligible for hematopoietic stem cell transplant are also discussed.

Primary Cutaneous Anaplastic Large Cell Lymphoma With 6p25.3 Rearrangement and Epidermotropism.

ABSTRACT: Primary cutaneous anaplastic large cell lymphoma may harbor a 6p25.3 rearrangement, which has been associated with an epidermotropic small cell component. We report the case of a patient with said lymphoma harboring that rearrangement. It presented as a forehead nodule, histologically composed of an intermediate-to-large cell dermal component alongside a small-to-intermediate cell epidermotropic component. After multiple cutaneous and regional lymph node relapses, disease progression has been documented to a distant lymph node, despite local radiotherapy of the cutaneous lesions, chemotherapy, and anti-CD30 therapy, albeit with an indolent course over 6 years. Cases of pcALCL with nonregional lymph node involvement are unusual. Nevertheless, in this case, progression to a distant lymph node was not associated with an aggressive transformation of the disease.

How to Diagnose and Treat CD5-Positive Lymphomas Involving the Spleen.

Patients with CD5-expressing lymphomas presenting with splenomegaly are frequently diagnosed with chronic lymphocytic leukemia. The most important differential diagnosis is mantle cell lymphoma, both in its classical and leukemic, non-nodal forms, given its prognostic and therapeutic implications. Other small B-cell neoplasms that frequently involve the spleen and occasionally express CD5 include the splenic marginal zone lymphoma, hairy cell leukemia and, rarely, lymphoplasmacytic lymphoma. The frequency of CD5 positivity depends in part on the sensitivity of the detection methods employed. Usually, a combination of morphological, immunophenotypic and molecular findings allows for a precise sub-classification of CD5-positive, low-grade B-cell lymphomas of the spleen. Some of these tumors may display a mixture of small and larger B cells, raising the possibility of more aggressive lymphomas, such as diffuse large B-cell lymphomas (DLBCL). Approximately 5-10% of DLBCL are CD5-positive and some may manifest as primary splenic lesions. When available, the morphology of DLBCL in the splenic tissue is distinctive and a leukemic picture is very rare. In conclusion, the appropriate morphological and clinical context assisted by flow cytometry panels and/or immunohistochemistry allows the differential diagnosis of CD5-positive, non-Hodgkin, B-cell lymphomas involving the spleen.

Rare Association Between Idiopathic Multicentric Castleman Disease, Nephrotic Syndrome and Polyneuropathy in an Immunocompetent Patient.

Multicentric Castleman disease (MCD) represents a group of poorly understood lymphoproliferative disorders related to proinflammatory hypercytokinaemia. In immunocompetent patients the aetiology is still unknown, hence the designation of idiopathic MCD (iMCD). To successfully diagnose iMCD, diagnostic criteria must be fulfilled and a large array of alternative diagnoses excluded. Peripheral neuropathy and nephropathy are relatively common findings in cases associated with POEMS syndrome, but very rarely reported in iMCD. We present the case of a 64-year-old man with iMCD (HIV- and HHV-8-negative) with nephrotic syndrome and severe motor polyneuropathy. Alternative diagnoses were excluded. The patient was treated with intravenous glucocorticoid followed by rituximab. Complete clinical and laboratory remission was achieved and maintained at the 2-year follow-up. LEARNING POINTS: iMCD is a lymphoproliferative disease in immunocompetent patients with no known cause.To diagnose iMCD major and minor criteria must be fulfilled, and alternative diagnoses must be excluded.Nephrotic syndrome and motor polyneuropathy are rare in iMCD and all alternative diagnoses must be excluded before relating all of these.

Proteomic Landscape of Extracellular Vesicles for Diffuse Large B-Cell Lymphoma Subtyping.

The role of extracellular vesicles (EVs) proteome in diffuse large B-cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state-of-the-art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole-cell and secreted EVs proteomes of the two cell-of-origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole-cell proteome and 228/608 proteins from EVs (adjust p-value < 0.05/p-value < 0.05). In our preclinical model system, we demonstrated that the EV proteome and the whole-cell proteome possess the capacity to separate cell lines into ABC and GCB subtypes. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B-cell receptor (BCR), Fc_gamma R-mediated phagocytosis, ErbB signaling, and endocytosis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow-up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.

Peripheral blood cell ratios as prognostic factors in canine diffuse large B-cell lymphoma treated with CHOP protocol.

Diffuse large B-cell lymphoma (DLBCL) is the most common haematopoietic tumour in dogs and recognized as clinical model for its human counterpart. Recently, neutrophil-to-lymphocyte (NLR) and lymphocyte-to-monocyte (LMR) ratios have been shown to predict time-to-progression (TTP) and lymphoma-specific survival (LSS) in dogs with DLBCL treated with CHOP-based chemotherapy. We retrospectively evaluated in 59 dogs diagnosed with DLBCL the prognostic value of haematological parameters and derived ratios: NLR, LMR, platelet-to-lymphocyte (PLR) and platelet-to-neutrophil (PNR) ratios for TTP, LSS and associated secondary end-points (time-to-progression-rate [TTPR] and lymphoma-specific survival-rate [LSSR]) as rates at 180 and 365 days. PNR is an independent prognostic marker (p ≤ .001) for TTPR/180 and 365 days, dogs with a PNR above 0.032 were more likely to progress before 180 days (sensitivity 46.5%, specificity 87.5%, p = .004). On univariate analysis, NLR showed a prognostic significance for LSSR/180 (p = .006) and LSSR/365 (p = .009). A baseline NLR value below 7.45 was positively associated with survival at 180 days (sensitivity 52%, specificity 85.3%, p = .025). The presence of substage b, was associated with early progression and decreased survival at 180 days (p = .031). Anaemia significantly reduced LSSR at 365 days (p = .028). This is the first study evaluating PLR and PNR in canine DLBCL and demonstrates that PNR could be a predictor of early lymphoma progression. Since peripheral blood cell composition can be affected by several non-oncological causes, the development of larger multicenter studies with homogeneous inclusion criteria could help to better determine the true predictive values of blood cell ratios in dogs' DLBCL treated with CHOP chemotherapy.

Label-free quantitative mass spectrometry from formalin-fixed paraffin-embedded samples of nasopharyngeal carcinoma: Preliminary results from a non-endemic European cohort of patients.

AIM: Report our results of biomarker discovery in formalin-fixed paraffin-embedded (FFPE) nasopharyngeal carcinoma (NPC) via proteomic analysis. BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare cancer in Western countries. Proteomic analysis have already been reported as a useful tool to provide biomarkers. Formalin-fixed paraffin-embedded (FFPE) samples, despite largely underused, can provide invaluable information for biomarker research via proteomic analysis. METHODS: FFPE samples of NPC were submitted to protein extraction followed by FASP-digestion and label-free quantitative mass spectrometry (MS). Patients' received concurrent chemoradiation with or without adjuvant chemotherapy as per Intergroup 0099 trial. IMRT was delivered following the RTOG0615 specifications. Toxicity was scored using the CTCAE 4.03 tables. Survival was estimated using Kaplan-Meier curves. Log-rank was used to detect differences. KEGG ontology graphics were generated. RESULTS: 28 FFPE samples from NPC patients were used. Patients were: 79% male, 97% Caucasians, 86% WHO type 3, 40% T1, 10% T2, 25% T3, and 25% T4. With a median follow up of 37 months, local control was 83 (T1, 100% T2, T3 and T4), overall survival was 84%, and six patients developed distant metastases. All five patients that died were due to metastatic disease. Tumor samples contained a median of 75% of tumor material. We found Epstein-Barr (EBV) and Herpes simplex (HSV) viruses' related proteins significantly present in early-stage primary NPC (T1 and T2, p < 0.01). A pool of 10 proteins was statistically up-regulated in the metastatic group of patients (p < 0.01). Median survival from this M1 group was <1 year (p < 0.001). CONCLUSIONS: FFPE samples yielded adequate material for MS analysis. We found EBV and HSV related proteins on early-stage NPC, and proteomic profiling associated with distant metastases, potential candidates of disease biomarkers. Validation is needed.

Improved long-term results of intensity-modulated radiotherapy for a non-endemic European nasopharyngeal carcinoma cohort: single-center retrospective study.

PURPOSE: Report our matured outcomes of European nasopharyngeal carcinoma (NPC) treatment from a non-endemic region in the IMRT era. METHODS: We reviewed 109 consecutive patients with biopsy proven NPC treated between 2009 and 2013. All received IMRT as per RTOG 0615. Toxicity was scored accordingly to CTCAE 4.03. Platinum-based chemotherapy was delivered following the Intergroup 0099. RESULTS: Median age of 53 years; 97% Caucasian; 74% male; 72% WHO grade III; 43% T1; 14% T2; 18% T3, 25% T4; 17% N0; 17% N1; 39% N2; 27% N3. Compliance to adjuvant chemotherapy was 88%. With a median follow up of 56 months, the 4-year local control was 90.2% (88.6% for T1; 100% for T2; 85% for T3; and 91.7% for T4), the 4-year distant metastases-free survival was 86% and an overall survival rate was 77%. Local control and survival were better in G3 (p < 0.001 and p = 0.032, respectively). Xerostomia was the most frequent late toxicity in 55% (n = 60). Hypothyroidism requiring hormonal reposition occurred in 15.5% (n = 17). From the 36 deaths, 20 were due to distant metastases, 3 grade 5 toxicity, 2 from local progression, 5 non-cancer deaths and unknown cause in the remaining 6. On multivariable analysis, age (p = 0.017), local recurrence and distant metastases were associated with death (p < 0.001, both). CONCLUSION: Our matured data from the IMRT era showed a major improvement from our 3D cohort series reaching excellent local and regional control, even in T4. Local recurrences, despite few, and distant metastases were correlated with the risk of death.

Reproducibility of histologic prognostic parameters for mantle cell lymphoma: cytology, Ki67, p53 and SOX11.

Mantle cell lymphoma (MCL) shows a clinical aggressiveness that varies from patient to patient. Despite major advances in outcomes with current immunochemotherapy, the future development of therapies requires risk stratification to tailor therapy intensity. Within the group of reference pathologists for the ongoing trials of the European MCL Network, we performed a round robin test on a tissue microarray to evaluate the reproducibility in assessing the biomarkers of outcome in MCL. Cytological subtype, Ki67-index and expression of p53 and SOX11 were evaluated on 20 diagnostic tumour samples by eight participating labs independently. We demonstrate that the assessment of the proliferation index by counting the Ki67 positive cells as well as assessment of SOX11 and p53 expression status is reproducible between labs. For the most established prognostic biomarker, Ki67, the intra-class correlation coefficient was very good when assessed as a continuous parameter (0.87). The agreement was lower when the values were analysed in a dichotomized way applying the commonly used cutoff of 30% (kappa = 0.65, complete concordance of all labs in 13/20 (65%)). Cases with discrepant results between labs in the dichotomized analysis showed mean values close to the cutoff of 30%. Centralised scoring and digital image analysis revealed results in line with the scores from individual labs. All cases in our cohort were additionally assessed for gene expression signatures and of TP53 gene alterations. Given the good reproducibility when guidelines of assessment are applied, the biomarker studied in this inter-laboratory test presents potential candidates to be enhanced for risk-stratification in the future clinical trials.