Lenvatinib plus Pembrolizumab Combination Therapy for Advanced or Recurrent Endometrial Cancer: A Single-Center, Retrospective Analysis.

A multi-kinase inhibitor, lenvatinib, plus an immune checkpoint inhibitor, pembrolizumab, became a viable therapeutic option for advanced or recurrent endometrial cancer in Japan by the end of 2021. The Japanese population has a relatively unique genetic background. Hence, the safety profile and effectiveness of lenvatinib plus pembrolizumab may differ between the Japanese and other populations. This single-center, retrospective study aimed to evaluate the treatment efficacy of lenvatinib plus pembrolizumab and the safety profile of the associated adverse events. The clinical records of 15 patients, who received lenvatinib plus pembrolizumab for advanced or recurrent endometrial cancer at the Tohoku University Hospital, were reviewed. Best overall response and disease control rates were 40.0% and 73.3%, respectively. Treatment was discontinued owing to disease progression and adverse events in six patients, respectively. As of the end of July 2023, treatment was ongoing in the remaining three patients. The median treatment and progression-free survival durations were 118 and 258 days, respectively. Relative dose intensity of lenvatinib was not positively associated with progression-free survival, neither during the first 4 weeks after treatment initiation nor during the entire treatment period. All patients experienced one or more adverse events, the most common of which were hypothyroidism (90%) and hypertension (83.3%). Among the 15 patients, 13 required lenvatinib dose reduction owing to adverse events. One patient developed grade 4 interstitial pneumonia requiring intensive care. Our results validate the short-term efficacy of lenvatinib plus pembrolizumab, and indicate that dose optimization of lenvatinib could be individualized without impairing efficacy.

A qualitative systematic review of the significance of adjuvant therapy in patients with low-risk endometrial cancer presenting positive peritoneal cytology: a relevant study to the guideline update for endometrial cancer by the Japan society of gynecologic oncology guideline committee.

In association with an update of the Japan Society of Gynecologic Oncology clinical practice guidelines for endometrial cancer in 2023, a systematic review was conducted about the therapeutic benefit of adjuvant therapy on patients with early-stage endometrial carcinoma, who presented positive peritoneal cytology (PPC) without the risk factors for recurrence. The systematic review only included two eligible retrospective studies. Both studies included patients with risk factors for recurrence. A nationwide study in the United States reported that adjuvant chemotherapy was associated with the reduced risk of death among patients with stages I-II endometrial cancer with PPC by multivariate, propensity score-adjusted analysis. Another single-center study in Japan reported no association between adjuvant chemotherapy and relapse-free survival among patients with stage IA endometrial cancer by univariate analysis. This systematic review identified that evidence was limited with conflicting results. Continuous evaluation is warranted to address this clinical question.

ExcLAMation marks in a pelvic lymph node.

Extrapulmonary lymphangioleiomyomatosis (LAM) can present as incidental nodal LAM in gynecological surgery specimens, that warrants systemic investigation and follow-up of concurrent and subsequent development of pulmonary and extrapulmonary LAM.

An Attempt to Develop a New Treatment Strategy for Rare Refractory Gynecological Malignancies: The Japanese Gynecologic Oncology Group.

Platinum-based combination chemotherapy has been a frontline therapeutic strategy for advanced ovarian cancer. Although patients with ovarian high-grade serous carcinoma (HGSC) respond well to the combination therapy, those with relatively rare histologic subtypes, such as mucinous or clear cell carcinoma of the ovary (OCCC), show resistance to platinum-based chemotherapy. Even with the recently developed maintenance therapies using molecular targeted inhibitors for ovarian cancers, such as bevacizumab or poly (ADP-ribose) polymerase (PARP) inhibitors, the prognosis of non-HGSC ovarian cancers is unsatisfactory. To overcome the limitations in the treatment of rare ovarian cancers, the Japanese Gynecologic Oncology Group (JGOG) has launched a comprehensive project utilizing publicly available genomic databases, including a national clinico-genomic database maintained by the Center for Cancer Genomics and Advanced Therapeutics (C-CAT). JGOG, a leading group in Japan that conducts clinical trials for the treatment of gynecological malignancies, also established a nationwide network through the long-standing efforts of all participants. Currently, JGOG is engaged in a phase II international clinical trial (CYH33-G201: jRCT2031210216), targeting OCCC with PIK3CA hotspot mutations. The CYH33-G201 trial is sponsor-initiated, and JGOG, in collaboration with pharmaceutical companies, is actively recruiting participants. To expand the functions of the nationwide network that JGOG had already established, we held explanatory meetings for this clinical trial in nine different areas throughout Japan to promote the penetration of the CYH33-G201 trial. Through C-CAT database analysis, we estimated that approximately 40% of the patients with OCCC harbored at least 1 of the 17 PIK3CA hotspot mutations designated in the CYH33-G201 trial. JGOG will continue the challenge of establishing novel treatment strategies for rare refractory cancers that will benefit patients suffering from gynecological malignancies, especially those who do not receive satisfactory standard treatment and care.

Identification of predictive biomarkers for endometrial cancer diagnosis and treatment response monitoring using plasma metabolome profiling.

BACKGROUND: Endometrial cancer (EMC) is the most common female genital tract malignancy with an increasing prevalence in many countries including Japan, a fact that renders early detection and treatment necessary to protect health and fertility. Although early detection and treatment are necessary to further improve the prognosis of women with endometrial cancer, biomarkers that accurately reflect the pathophysiology of EMC patients are still unclear. Therefore, it is clinically critical to identify biomarkers to assess diagnosis and treatment efficacy to facilitate appropriate treatment and development of new therapies for EMC. METHODS: In this study, wide-targeted plasma metabolome analysis was performed to identify biomarkers for EMC diagnosis and the prediction of treatment responses. The absolute quantification of 628 metabolites in plasma samples from 142 patients with EMC was performed using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The concentrations of 111 metabolites increased significantly, while the concentrations of 148 metabolites decreased significantly in patients with EMC compared to healthy controls. Specifically, LysoPC and TGs, including unsaturated fatty acids, were reduced in patients with stage IA EMC compared to healthy controls, indicating that these metabolic profiles could be used as early diagnostic markers of EMC. In contrast, blood levels of amino acids such as histidine and tryptophan decreased as the risk of recurrence increased and the stages of EMC advanced. Furthermore, a marked increase in total TG and a decrease in specific TGs and free fatty acids including polyunsaturated fatty acids levels were observed in patients with EMC. These results suggest that the polyunsaturated fatty acids in patients with EMC are crucial for disease progression. CONCLUSIONS: Our data identified specific metabolite profiles that reflect the pathogenesis of EMC and showed that these metabolites correlate with the risk of recurrence and disease stage. Analysis of changes in plasma metabolite profiles could be applied for the early diagnosis and monitoring of the course of treatment of EMC patients.

Diagnostic Performance of Preoperative Imaging in Endometrial Cancer.

BACKGROUND: Endometrial cancer is one of the most common gynecological malignancies. Because the findings mentioned in radiogram interpretation reports issued by diagnostic radiologists influence treatment strategies, we aimed to evaluate the diagnostic accuracy of preoperative computed tomography (CT) and magnetic resonance imaging (MRI) interpretation results in clinically relevant settings. METHODS: The clinical records of patients diagnosed with endometrial cancer treated at Tohoku University Hospital from January 2012 to December 2021 were reviewed. The preoperative and pathologically estimated cancer stages were compared based on the results mentioned in the radiogram interpretation report. RESULTS: The preoperative and postoperative cancer stages were concordant in 70.0% of the patients. By contrast, the cancer stage was underdiagnosed and overdiagnosed in 21.7% and 8.2% of the patients, respectively. The sensitivities of MRI for deep myometrial invasion, cervical stromal invasion, vaginal invasion, and adnexal metastasis were 65.1%, 58.2%, 33.3%, and 18.4%, respectively. The sensitivity and specificity for pelvic lymph node metastasis using a combination of CT and MRI were 40.9% and 98.4%, respectively. Those for para-aortic lymph node metastases using CT were 37.0% and 99.5%, respectively. CONCLUSIONS: The low sensitivity observed in this study clarified the limitations of preoperative diagnostic performance in current clinical practice.

Human papillomavirus vaccine impact on invasive cervical cancer in Japan: Preliminary results from cancer statistics and the MINT study.

The first prophylactic vaccine against human papillomavirus (HPV) 16 and HPV18 was licensed in Japan in 2009. HPV vaccine effectiveness against high-grade cervical lesions has been demonstrated among young Japanese women, but evidence of its effects on invasive cervical cancer (ICC) is lacking. Using data from two different cancer registries, we compared recent trends of new ICC cases by age group using Poisson regression analysis. We also analyzed time trends in HPV16/18 prevalence among 1414 Japanese women aged <40 years newly diagnosed with ICC in the past decade. Based on the population-based cancer registry, the incidence of ICC among young women aged 20-29 years showed a significant decline from 3.6 to 2.8 per 100 000 women-years during 2016-2019, but no similar decline was observed for older age groups (p < 0.01). Similarly, using data from the gynecological cancer registry of the Japan Society of Obstetrics and Gynecology, the annual number of ICCs among women aged 20-29 years also decreased from 256 cases to 135 cases during 2011-2020 (p < 0.0001). Furthermore, a declining trend in HPV16/18 prevalence in ICC was observed only among women aged 20-29 years during 2017-2022 (90.5%-64.7%, p = 0.05; Cochran-Armitage trend test). This is the first report to suggest population-level effects of HPV vaccination on ICC in Japan. Although the declining trend in HPV16/18 prevalence among young women with ICC supports a causal linkage between vaccination and results from cancer registries, further studies are warranted to confirm that our findings are attributable to vaccination.

Assessing the Significance of Lymphadenectomy in Older Patients with Stage I Endometrial Cancer: A Single-Center, Retrospective Cohort Study.

Advantages of lymphadenectomy for early stage endometrial cancer remain controversial. Lymphadenectomy had been routinely omitted for patients aged ≥ 70 years at our institute if lymph node metastasis was unsuspected due to an increased risk of peri- and postsurgical complications. Since 2013, with the introduction of minimally invasive surgery and considering the heterogeneous medical conditions, we started performing lymphadenectomy in patients who were considered well-tolerated. We retrospectively investigated our clinical database to assess the effect of lymphadenectomy in older patients with early stage endometrial carcinoma. Patients aged ≥ 70 years, preoperatively diagnosed with stage I endometrial carcinoma, and who underwent lymphadenectomy between 2013 and 2021 at Tohoku University Hospital were included in the lymphadenectomy group (n = 33), whereas patients who underwent surgery without lymphadenectomy before the end of 2012 were included in the no-lymphadenectomy group (n = 49). Clinical parameters and patient outcomes, such as disease-free survival (DFS) and disease-specific survival (DSS), were compared. The median age was significantly higher and fewer patients received adjuvant chemotherapy in the no-lymphadenectomy group. Neither DSS nor DFS differed significantly between the two groups. Five-year-DFS rates were 77.2% and 82.5% and 5-year-DSS rates were 89.7% and 97.8% for the lymphadenectomy and no-lymphadenectomy groups, respectively. No significant differences were observed in the subsequent survival analysis by substage, histological subtype, or risk of recurrence. Our results suggest that the indications for lymphadenectomy in older patients should be individually optimized according to the risk of recurrence and postoperative complications.

Surgically treated cervical cancer in a high-risk group in the era of the 2018 FIGO staging schema: a nationwide study.

The 2018 International Federation of Gynecology and Obstetrics (FIGO) revision to the staging criteria for uterine cervical cancer adopted pathological staging for patients who underwent surgery. We investigated the correlation between clinicopathological factors and prognosis in patients with high-risk factors in accordance with the FIGO 2018 staging criteria by analyzing a real-world database of 6,192 patients who underwent radical hysterectomy at 116 institutions belonging to the Japan Gynecologic Oncology Group. A total of 1,392 patients were categorized into the high-risk group. Non-squamous cell carcinoma histology, regional lymph node metastasis, pT2 classification, and ovarian metastasis were identified as independent risk factors for mortality. Based on pathological findings, 313, 1003, and 76 patients were re-classified into FIGO 2018 stages IIB, IIIC1p, and IIIC2p, respectively. Patients with stage IIIC2p disease showed worse prognoses than those with stage IIB or IIIC1p disease. In patients with stage IIIC1p disease, overall survival was significantly better if their tumors were localized in the uterine cervix, except for single lymph node metastasis, with a 5-year overall survival rate of 91.8%. This study clarified the heterogeneity of the high-risk group and provided insights into the feasibility of upfront radical hysterectomy for a limited number of patients harboring high-risk factors.

Identification of predictive biomarkers for diagnosis and radiation sensitivity of uterine cervical cancer using wide-targeted metabolomics.

AIM: Uterine cervical cancer (UCC) is the fourth most common cancer in women, responsible for more than 300 000 deaths worldwide. Its early detection, by cervical cytology, and prevention, by vaccinating against human papilloma virus, greatly contribute to reducing cervical cancer mortality in women. However, penetration of the effective prevention of UCC in Japan remains low. Plasma metabolome analysis is widely used for biomarker discovery and the identification of cancer-specific metabolic pathways. Here, we aimed to identify predictive biomarkers for the diagnosis and radiation sensitivity of UCC using wide-targeted plasma metabolomics. METHODS: We analyzed 628 metabolites in plasma samples obtained from 45 patients with UCC using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The levels of 47 metabolites were significantly increased and those of 75 metabolites were significantly decreased in patients with UCC relative to healthy controls. Increased levels of arginine and ceramides, and decreased levels of tryptophan, ornithine, glycosylceramides, lysophosphatidylcholine, and phosphatidylcholine were characteristic of patients with UCC. Comparison of metabolite profiles in groups susceptible and non-susceptible to radiation therapy, a treatment for UCC, revealed marked variations in polyunsaturated fatty acid, nucleic acid, and arginine metabolism in the group not susceptible to treatment. CONCLUSIONS: Our findings suggest that the metabolite profile of patients with UCC may be an important indicator for distinguishing these patients from healthy cohorts, and may also be useful for predicting sensitivity to radiotherapy.

Retrospective analysis of local recurrence pattern by computed tomography image-guided intracavitary and interstitial brachytherapy for locally advanced cervical cancer in a single Japanese institution.

PURPOSE: The purpose of this study was to investigate the treatment results with focus on local control (LC) by computed tomography (CT)-guided intracavity brachytherapy and interstitial brachytherapy (ICBT/ISBT) for locally advanced cervical cancer (LACC). METHODS AND MATERIALS: Patients with LACC undergoing ICBT/ISBT at least once in our institution between January 2017 and June 2019 were analyzed retrospectively. The primary endpoint was local control (LC), and the secondary endpoints were progression-free survival (PFS), overall survival (OS), and late toxicities. Differences between patient subgroups for prognostic factors in LC, PFS, and OS were analyzed using the log-rank test. The recurrence patterns of LC were also investigated. RESULTS: Forty-four patients were included in the present study. The median high-risk clinical target volume (HR-CTV) at the initial brachytherapy was 48.2 cc. The median total dose of HR-CTV D90 (EQD2) was 70.7 Gy. The median followup period was 39.4 months. The 3-year LC, PFS and OS rates in all patients were 88.2%, 56.6%, and 65.4% (95% CI 50.3-78.0%), respectively. Corpus invasion and large HR-CTV (70 cc or more) were significant prognostic factors in LC, PFS, and OS. Marginal recurrences at the fundus of the uterus were detected in 3 of 5 patients in whom local recurrence was observed. Late toxicities of Grade 3 or higher were detected in 3 patients (6.8%). CONCLUSIONS: Favorable LC was achieved by performing CT-guided ICBT/ISBT for LACC. The brachytherapy strategy for patients with corpus invasion or large HR-CTV may need to be reconsidered.

Precautions during Direct Oral Anticoagulant Introduction in Gynecologic Malignancies: A Single-Center Retrospective Cohort Study.

The risk factors for venous thromboembolism (VTE) recurrence/exacerbation or a change from a direct oral anticoagulant (DOAC) to another anticoagulant in patients with gynecologic cancer using DOACs have not been thoroughly elucidated. Here, we aimed to investigate the risk factors for a composite primary outcome, including VTE recurrence/exacerbation, or a change from a DOAC to another anticoagulant, in this population. A total of 63 patients were analyzed. Risk factors for a primary outcome within 2 years after DOAC initiation were investigated using multiple logistic regression analysis. Among the 63 patients, 10 developed a primary outcome. Clear cell carcinoma of the ovary (adjusted odds ratio (aOR), 18.9; 95% confidence interval (CI), 2.25-350.74), pulmonary embolism (PE) or proximal deep vein thrombosis without PE (aOR, 55.6; 95% CI, 3.29-11,774.66), and D-dimer levels in the third tertile (≥7.6 µg/dL) when VTE was first diagnosed (aOR, 6.37; 95% CI, 1.17-66.61) were associated with increased odds of a primary outcome in patients with gynecologic cancer using DOACs. Patients with one or more risk factors for a primary outcome require careful follow-up after DOAC initiation for the early recognition of treatment failure.

Comparison of treatment outcomes between first-line chemotherapy with or without bevacizumab for advanced ovarian, fallopian tube, and primary peritoneal cancer (Tohoku gynecologic cancer unit: TGCU-RS001 study).

BACKGROUND: Outcomes with and without bevacizumab as first-line chemotherapy in Japanese-only ovarian cancer patients have not been reported. In this study, we report a retrospective study conducted at the Tohoku Gynecologic Cancer Unit. PATIENTS AND METHODS: The study included 453 patients with stage III/IV ovarian, fallopian tube, and primary peritoneal cancer who received first-line platinum-based chemotherapy. The patients were divided into two groups: bevacizumab (168 patients) and without bevacizumab (285 patients). The primary endpoint was the rate of platinum-resistant recurrence and the secondary endpoints were the antitumor response, progression-free survival, overall survival, and adverse events. RESULTS: The objective response rates for patients with measurable diseases treated with and without bevacizumab were 84.5% and 73.0%, respectively (P = 0.0066). Platinum-resistant recurrence in the groups treated with and without bevacizumab was noted in 31 (18.4%) and 111 (38.6%) patients, respectively (P < 0.0001). The median progression-free survival for the bevacizumab and without bevacizumab groups was 23 and 15 months, respectively (P = 0.0002), and the median overall survival was not reached and 49 months, respectively (P = 0.0005). Hypertension of grade 3 or higher was observed in 21 patients (12.5%) in the bevacizumab group (P < 0.001), and proteinuria was observed in 18 patients (10.7%) and 1 patient (0.3%) in the bevacizumab and without bevacizumab groups, respectively (P < 0.001). Intestinal perforation was observed in only one patient (0.6%) in the bevacizumab group. CONCLUSION: Combination and maintenance with bevacizumab in primary chemotherapy for advanced ovarian, fallopian tube, and primary peritoneal cancer was effective in reducing platinum-resistant recurrence rates and prolonging progression-free and overall survival.

Prognostic factors of 2018 FIGO stage IB-IIA cervical cancer with absence of high/ intermediate surgical-pathological risk factors.

OBJECTIVE: This retrospective analysis of a real-world database of open radical hysterectomy in Japan aimed to reveal the clinicopathological findings and clinical outcomes of low-risk patients with stage IB-IIA cervical cancer. METHODS: A total of 1143 stage IB1, IB2 and IIA1 (reclassified by FIGO 2018 staging system) patients with cervical cancer who underwent radical hysterectomy between January 2004 and December 2008 from the Japanese Gynecologic Oncology Group database were analyzed. Low-risk patients were defined as those without a tumor size exceeding 4 cm, parametrial tumor involvement, deep (outer half) stromal invasion, lymphovascular space invasion or lymph nodal metastasis. RESULTS: 61.2% (772/1262) patients with stage IB1, 32.1% (229/932) with stage IB2 and 16.9% (72/294) of stage IIA1 were classified into the low-risk group. The 5-year overall survival and disease-free survival rates were 98.4 and 93.7%, respectively. Histological classification did not affect the survival rates, but stage IIA cases had significantly lower overall survival and disease-free survival (83.5 and 93.8%, respectively) than stage IB cases. The independent prognostic factors for disease-free survival were older age (≧50), histology, clinical stage and clinical stage as independent prognostic factors for overall survival. Regarding recurrence, older age, non-SCC and stage IIA1 were independent risk factors for local recurrence, but stage IIA1 was the only independent risk factor for distant metastasis. CONCLUSION: We found that stage IIA1 was the strongest risk factor for survival and recurrence of low-risk uterine cervical cancer (FIGO, 2018). In low-risk cases, stage IIA1 should be considered separately from stage IB.

A Retrospective Analysis of Clinical Biomarkers for Olaparib Maintenance Therapy in Patients with Recurrent Ovarian Cancer.

Poly(ADP-ribose) polymerase (PARP) inhibitors theoretically promote synthetic lethality in cancer cells with homologous recombination deficiency (HRD). However, clinical evidence indicates that PARP inhibitors are also effective for treating HRD-negative ovarian cancer. The PARP inhibitor olaparib became available in Japan as a maintenance therapy for platinum-sensitive recurrent ovarian cancer regardless of homologous recombination status in April 2018. The purpose of this study was to identify potential clinical biomarkers for olaparib sensitivity in patients with recurrent ovarian cancer. Clinical information about the patients with recurrent ovarian cancer treated with olaparib maintenance therapy (OMT) was retrospectively collected. OMT duration was used as an indicator for olaparib sensitivity. The relationship between OMT duration and clinical parameters was statistically analyzed. We found a positive correlation between OMT duration and progression-free survival (PFS) or treatment free interval (TFI). In some cases, OMT duration exceeded PFS before olaparib introduction. We also found that more than half of the patients with measurable target lesions at the time of OMT introduction showed partial or complete response to OMT. These results validated the effectiveness of OMT and identified PFS and TFI as potential clinical markers for olaparib sensitivity in the patients with recurrent ovarian cancer.

Risk assessment in the patients with uterine cervical cancer harboring intermediate risk factors after radical hysterectomy: a multicenter, retrospective analysis by the Japanese Gynecologic Oncology Group.

BACKGROUND: Adjuvant therapy is usually considered for surgically treated patients with uterine cervical cancer harboring intermediate risk (IR) factors such as large tumor diameter, stromal invasion to the outer half, and lymphovascular space invasion (LVSI). However, the indications and types of adjuvant therapy for the IR group remain controversial. This study aimed to analyze the differences in patient outcomes in the IR group to provide novel insights for tailoring adjuvant therapy. METHODS: Data from 6192 patients with cervical cancer who underwent radical hysterectomy at 116 institutions belonging to the Japanese Gynecologic Oncology Group were reviewed. RESULTS: In total, 1688 patients were classified into the IR group, of whom 37.3% did not receive adjuvant therapy. Conversely, approximately equal proportions of the remaining patients received adjuvant radiotherapy, concurrent chemoradiotherapy, and chemotherapy. Patients with all three risk factors showed worse overall survival than those with one or two risk factors. In addition to LVSI, non-squamous cell carcinoma histology, and vaginal invasion were identified as independent risk factors for both recurrence and mortality in multivariate analyses. Tumor diameter greater than 40 mm and surgical center volume were identified as independent risk factors for recurrence. Stromal invasion to the outer half and ovarian metastasis were identified as independent risk factors for mortality. CONCLUSIONS: This study revealed the significant differences in prognosis in the IR group. The indications for adjuvant therapy should be further studied, focusing on conventional risk factors and other pathological findings.

MicroRNA-152 Regulates Endometrial Serous Carcinoma Cell Motility by Suppressing Matrix Metalloproteinase 10 Expression.

MicroRNA-152 (miR-152) expression has been reported to be associated with poor prognosis in patients with endometrial serous carcinoma (ESC). However, the function of miR-152 in ESCs is not fully understood. The present study aimed to investigate the involvement of miR-152 in ESC progression. The influence of miR-152 overexpression on cell proliferation and motility was assessed by transfecting two human ESC cell lines, USPC-1 and SPAC-1-L, with a miR-152 precursor. MiR-152 overexpression increased apoptosis and inhibited the proliferation of the two ESC cell lines. Cell motility was also suppressed in both cell lines following precursor transfection. Conversely, miR-152 inhibitor transfection led to an increase in cell migration ability, suggesting the involvement of miR-152 in ESC cell motility. Results of the analysis of publicly available messenger RNA dataset indicated that high expression of matrix metalloproteinase 10 (MMP10), one of the predicted targets of miR-152 by microRNA target prediction database, was a poor prognostic factor for ESC. In vitro examination results revealed that miR-152 overexpression reduced MMP10 expression, and knockdown of MMP10 significantly reduced cell motility. This study elucidates the function of miR-152 as a tumor suppressor in ESCs. We demonstrated that miR-152 plays an important role in ESC cell motility by regulating MMP10 expression.