bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genomic characterization of rectal cancer and molecular determinants of response to neoadjuvant chemoradiotherapy.
Authors: Chatila Walid Khaled , Marco Michael , Qu Xuan , Walch Henry S. , Luthra Anisha , Chen Chin-Tung , Kundra Ritika , Schultz Nikolaus , Yaeger Rona , Smith Jesse Joshua , Sanchez Vega Francisco , Garcia-Aguilar Julio 
Published: February 2020

192 Background: Rectal cancers are clinically different from colon cancers and have not yet been molecularly characterized due to the scarcity of pretreatment specimens. Discovery of molecular determinants of response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II and III) are needed to select those who can avoid surgery and benefit from watch-and-wait strategies. Methods: We profiled 371 pre-treatment specimens using targeted-exome sequencing of 468 cancer genes (n = 325), whole-exome sequencing (n = 100), and RNA-sequencing (n = 113). The targeted-sequencing cohort included patients with stage I (n = 44), II (n = 41), III (n = 176), and IV (n = 64) disease. Primary tumors were divided into lower (LR: 0-4 cm to anal verge, n = 62), middle (MR: 4-8 cm, n = 115), and upper rectum (UR: 8-12 cm, n = 107). We examined molecular determinants of complete response (CR) and relapse free survival (RFS) in LARC patients treated with chemoradiotherapy only (CRT: n = 39), induction chemotherapy + CRT (INCT: n = 87) and consolidation chemotherapy after CRT (CCNT: n = 63). Results: Among MSS cases, oncogenic gene and signaling pathway alterations did not vary by clinical stage. WNT pathway alterations, driven by APC mutations, were more frequent in the UR (89% UR v 86% MR v 60% LR, p < 0.001) while RTK/RAS alterations were more frequent in the LR (54% UR v 69% MR v 72% LR, p < 0.03). A set of genes enriched in mTOR signaling, G2M checkpoint, EMT transition, and DNA repair were overexpressed in the UR (FDR < 0.1). The 5-yr RFS rate for LARC was 75% (CI: 68%-82%) and 24% of the cases had a CR (n = 45). MSI cases had a higher rate of CR compared to MSS cases (50% v 23%, p = 0.07) and none relapsed (n = 8). KRAS-altered MSS tumors exhibited worse RFS in cases treated with CNCT (5-yr: 74% v 97%, p = 0.01), but not in cases treated with INCT (5-yr: 67% v 72%, p = 0.7). Conclusions: WNT alterations are more frequent in the UR while RTK/RAS alterations are more frequent in the LR, suggesting differences in tumor biology between proximal and distal rectal cancer. Further, we report correlations between distinct molecular profiles and response to treatment paradigms that could guide the design of future clinical trials.
Results of a fifty-gene breast cancer RNA subtype classifier applied to 167 colorectal cancer (CRC) patients.
Authors: Reddy Sandeep K. , Seery Tara Elisabeth , Szeto Christopher 
Published: February 2020

20 Background: ERBB2 (HER2) is thought to be a target in <10% of CRC patients versus 20% of breast cancers, 15% of gastroesophageal cancers, and 10% of biliary cancers, based on FISH or IHC. Intrinsic molecular subtype is used to classify cancers into distinct biologic subtypes (eg. CMS 1-4 in CRC). A 50-gene qPCR assay (PAM50) identifies 5 intrinsic biological subtypes: luminal A, luminal B, HER2-enriched, basal-like, and normal-like in breast cancer. The HERACLES trial (trastuzumab plus lapatinib) resulted a 32% ORR and median TTP of 5.5 months in heavily pre-pretreated HER2+ CRC patients. We determined molecular subtypes using the 50-gene breast cancer classifier to identify an expanded CRC patient population eligible for HER2 therapy. Methods: Retrospective analysis on Whole exome (WES) DNA tumor and paired germline and matched deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) data from NantHealth was performed. Breast Cancer Intrinsic Subtypes based on RNAseq was used to classify CRC into 5 BC subtypes. Results: 167 CRC patients were classified using the Nant50 Breast Cancer classifier: 15.0% as Luminal B, 13.1% as Luminal A, and 1.8% as Basal-like. Surprisingly, 117/167 (70%) classified as HER2-enriched (HER-E). 15/167 (9.0%) had over-expression of ERBB2 by RNAseq or CN gain, which is consistent with published data of HER2+ CRC. ERBB2 is very significantly differentially expressed in HER2-E subtyped CRC (p=<0.001), more than ERBB2 CN gain, suggesting that HER2-E may be more HER2 driven. Across subtypes APC and TP53 were the most commonly mutated genes at 65.3% and 52.6% respectively, however both were more enriched in HER2-E CRC (APC OR=3.3, p=0.001, TP53 OR=2.6, p=0.007). Other known drivers of CRC such as PIK3CA, KRAS, and BRAF, were not differentially mutated in HER2-E CRC, however NRAS mutants were significantly more enriched in non-HER2-E CRC (OR=4.6, p=0.02). Conclusions: Even after excluding known HER2 over-expression and CN gain, PAM50-like gene classifier identifies a far higher than expected percentage of HER-E subtype CRC (99/167 = 59%) which may represent an under appreciated population for HER2 directed therapy and clinical trials.
Establishing novel mutation subtypes in peritoneal carcinomatosis of appendiceal origin.
Authors: Garland-Kledzik Mary , Scholer Anthony Joseph , Khader Adam , Santamaria-Barria Juan , Orozco Javier , Pigazzi Alessio , Marzese Diego M. 
Published: February 2020

200 Background: Appendiceal cancer (AC) is a rare disease process with complex treatment strategies. The objective of this study was to identify mutation-based genetic subtypes that may differ from the current histological classification, compare genetic make-up of primaries and metastases, and to find novel targetable alterations. Methods: The analysis involved the curation and normalization of mutation panels from adenocarcinoma and mucinous adenocarcinoma (n = 196) stored in the AACR GENIE Database v6.0. Genes mutated in less than two patients and tumors profiled with incomplete mutation panels were excluded from the study. The optimal number of AC subtypes was established using the Non-negative Matrix Factorization algorithm. Statistical comparisons of mutation frequencies were performed by using the Pearson’s χ2 test. Results: AC patients were stratified into five mutation subtypes. AC0 had no mutations in the 41 genes in the study. The most frequently mutated genes varied between the subtypes. AC1: KRAS (91.9%) and GNAS (77.4%); AC2: KRAS (52.5%), APC (32.5%), and GNAS (30%); AC3: KMT2D (38.7%), TP53 (38.7%), KRAS (35.5%), EP300 (22.6%); and AC4: TP53 (97.2%), KRAS (77.8%), and SMAD4 (36.1%). Additionally, AC3 was less likely to be mucinous (22.6% v 50.0-74.2%, p < 0.001) and had a higher mutation frequency (3.6 v 0-3.1, p < 0.001). There were no differences between primaries and metastases (p = 0.35). Conclusions: Characterization of these subtypes suggest a need for molecular rather than anatomic staging for AC. Histone regulation by KMT2D and EP300 may be considered for targeted therapy for patients with AC3. A prospective comparison of subtype prognosis and response to surgery and adjuvant treatment is needed.
Prognostic and predictive value of DNA mismatch repair status in patients with locally advanced rectal cancer following neoadjuvant therapy.
Authors: Hu Huabin , Wang Chao , Wu Zehua , Huang Yan , Zhang Jianwei , Cai Yue , Xie Xiaoyu , Ling Jiayu , Deng Yanhong 
Published: February 2020

210 Background: DNA mismatch repair deficient (dMMR) is a distinct molecular phenotype of colon cancer related to favorable outcome and resistant to 5-FU based adjuvant chemotherapy, however, little is known about dMMR rectal cancers. We investigated the value of dMMR on prognosis and predicted response to neoadjuvant therapy in rectal cancer patients. Methods: Between January, 2013 and December, 2018, a total of 855 consecutive patients with determined MMR status who underwent neoadjuvant therapy followed by curative surgery for locally advanced rectal cancer were included this retrospective study. Associations of MMR status with clinicopathologic variates and response to neoadjuvant therapy were determined using Chi-square or Fisher Exact tests. Local recurrence-free survival (LRFS) and disease-free survival (DFS) were analyzed using Cox proportional hazard models. Results: In this study population, dMMR was detected in 69 of 855 (8.1%) cases. Patients with dMMR showed similar clinicopathological characteristics including grade of differentiation, location from anal verge, clinical stage, neoadjuvant therapy regimen, pathological stage and postoperative chemotherapy to those who with pMMR, except younger age ( < 65 years, 89.1% vs. 77.7%, P = 0.034) and more mucinous adenocarcinoma in dMMR tumors. MMR status were not predictive for response to neoadjuvant therapy including pCR rate (18.8% vs. 12.9%, P = 0.185), downstaging rate (76.6% vs. 68.9%, P = 0.200) and tumor regression grade 0-1 (31.3% vs. 43.7%, P = 0.052), regardless of chemoradiation or chemotherapy. Multivariable analysis revealed that patients whose tumors had dMMR vs. pMMR had significantly longer DFS (HR = 0.39, 95%CI = 0.18 - 0.83, P = 0.014), but the correlation was not observed for LRFS (HR = 0.81, 95%CI = 0.25 - 2.60, P = 0.724). In the subgroup analysis, dMMR was a statistically significant prognostic factor for DFS only in patients with ypstage II-III (HR = 0.39, 95%CI = 0.17 - 0.88, P = 0.024). Conclusions: dMMR was significantly associated with longer DFS in patients with locally advanced rectal cancer, whereas was not a predictive marker for neoadjuvant therapy.
Wild-type APC and prognosis in metastatic colorectal cancer.
Authors: Wang Chongkai , Ouyang Ching , Sandhu Jaideep Singh , Kahn Michael , Fakih Marwan 
Published: February 2020

223 Background: Somatic mutations at adenomatous polyposis coli ( APC) gene, found in ~75% of colorectal cancers (CRC), are under-represented in microsatellite instable (MSI-H) tumors. While several studies have suggested worse outcomes for CRC patients (pts) with wild-type APC ( APC-WT), the prognostic implication of this genomic alteration in metastatic CRC (mCRC) is not well defined. Methods: APC prognostic value was evaluated in 331 stage IV microsatellite stable (MSS) CRC pts treated in our institution. Next-generation genomic analysis (FoundationOne) was used to characterize the molecular characteristics of APC-WT and mutant APC ( APC-MT) pts. Findings were validated on a public database of stage IV colon cancer from MSKCC. Results: APC-WT was present in 26% of mCRC patients. In comparison to APC-MT population (n = 244), APC-WT pts (n = 87) tended to be younger (median age: 49 vs. 58 years), right-sided (44% vs. 24%), BRAF-V600E mutated (25% vs. 5%), p53 WT (38% vs. 21%) and RAS WT (66% vs. 53%). APC-WT tumors were associated with other Wnt activating alterations ( CTNNB1, FBXW7, RNF43, ARID1A and SOX9). Among those, RNF43 and CTNNB1 were more significantly represented in the APC-WT vs APC-MT population (12% vs 1% and 11% vs 3%, respectively). APC-WT pts had a worse overall survival (OS) than APC-MT pts (30 vs 48 months, HR = 1.809, 95% CI 1.260-2.596, p < 0.0001). Using a multivariate model correcting for primary tumor location, RAS and BRAF status, APC-WT was predictive of poor survival (HR = 1.7, p = 0.001) in our data set. The prognostic implication of APC-WT on OS were confirmed further in a similar multivariate model of 433 stage IV pts from MSKCC public database (HR = 1.6, P = 0.01). Conclusions: APC-WT is associated with poor OS in MSS mCRC regardless of RAS, BRAF status. Compared with APC-MT mCRC tumors, APC-WT tumors were associated with other activating alterations of Wnt pathway, including RNF43 and CTNBB1.
Polymorphisms of genes encoding for regulatory proteins in the coagulation cascade to predict outcome for stage II and III colon cancer.
Authors: Berger Martin D. , Zlobec Inti , Cao Shu , Miyamoto Yuji , Suenaga Mitsukuni , Soni Shivani , Puccini Alberto , Tokunaga Ryuma , Naseem Madiha , Battaglin Francesca , Zhang Wu , Lugli Alessandro , Lenz Heinz-Josef 
Published: February 2020

227 Background: In cancer patients, an activated coagulation cascade might promote tumor cell dissemination. There are preliminary data suggesting that fibrinogen in combination with platelets can build a meshwork that entraps cancer cells enabling them to escape from immunosurveillance. We therefore hypothesize that variations in genes encoding for regulatory proteins within the coagulation pathway may predict outcome in patients with stage II and III colon cancer. Methods: The impact of three functional single nucleotide polymorphisms (SNPs) within the FGB, SERPINC1 and ITGA2B genes on time to recurrence and overall survival was evaluated in 209 patients with stage II and III colon cancer. Genomic DNA was isolated from formalin-fixed paraffin embedded tissue and the SNPs were analyzed by PCR-based direct sequencing. Results: Baseline characteristics were as follows: median age = 70y (19-91); female/male ratio = 42.8% / 57.2%; 111 patients had stage II, and 98 stage III colon cancer. The FGB rs4220 SNP showed significant association with recurrence rate in the overall population. Patients harboring any A allele had a higher 3-years recurrence rate compared to those with a G/G genotype (28% vs 17%) in both univariate (HR 1.83, 95% confidence interval (CI) 0.99-3.36, p = 0.048) and multivariate analyses (HR 1.94, 95% CI 1.05-3.57, p = 0.034). This trend was most evident among pts with stage II and especially the subgroup of high-risk stage II colon cancer. Again, A allele carriers had a higher 3-years recurrence rate compared to those having a G/G genotype (24% vs 10% and 44% vs 15% respectively) in both univariate (HR 3.32, 95% CI 1.26-8.74, p = 0.010 and HR 5.34, 95% CI 1.38-20.68, p = 0.006) and multivariate analyses (HR 3.34, 95% CI 1.27-8.78, p = 0.015 and HR 5.44, 95% CI 1.40-21.15, p = 0.015). Conclusions: Here, we demonstrate that the FGB polymorphism rs4220 might serve as a prognostic biomarker in stage II colon cancer. Assessment of FGB rs4220 might help us to identify those stage II colon cancer patients who will derive the most benefit from adjuvant chemotherapy.
Molecular landscape and prognostic value of HER2 low-expression on metastatic colorectal cancer.
Authors: Yagisawa Masataka , Sawada Kentaro , Nakamura Yoshiaki , Ishii Takahiro , Kubota Yohei , Nakajima Hiromichi , Jogo Tomoko , Sasaki Akinori , Mishima Saori , Kotani Daisuke , Kawazoe Akihito , Kuboki Yasutoshi , Shitara Kohei , Kojima Takashi , Doi Toshihiko , Ohtsu Atsushi , Fujii Satoshi , Yoshino Takayuki , Taniguchi Hiroya 
Published: February 2020

229 Background: HER2 dual blockade showed the promise in clinical trials in patients (pts) with HER2-positive (HER2-Pos) metastatic colorectal cancer (mCRC). A phase I/II study of a novel anti-HER2 antibody-drug conjugate (HER2-ADC) showed promising activities across different cancer subtypes including HER2-Pos mCRC, and also showed a hint of activity for low-expressing (HER2-L) mCRC, regardless of RAS mutation. However, molecular landscape and prognostic value of HER2-L on mCRC are unclear. Methods: The eligibility included pts with mCRC who had undergone surgical resection of primary tumor. Using the specimen, HER2 expression was evaluated by immunohistochemistry (IHC) and FISH. Definition of HER2 positivity was as follows; HER2-Pos as IHC 3+ or IHC 2+/FISH positive: HER2-L as IHC 2+/FISH negative or IHC 1+: Negative (HER2-Neg) as IHC 0+. NGS tests were also done. Results: Between 2005 and 2015, a total of 370 pts were analyzed; 60% were male, with a median age of 64 y/o. Fifteen pts (4%) with HER2-Pos, 21 (6%) with HER2-L, and 334 (90%) with HER2-Neg were identified. Proportion of left-sided primary tumor was similar among groups ranging from 72% to 78%. No difference was observed in terms of major clinicopathological characteristics. The proportion of co-altered RAS mutations in HER2-L was significantly higher than HER2-Pos ( p = 0.037) and similar to HER2-Neg (27% in HER2-Pos vs. 62% in HER2-L vs. 56% in HER2-Neg). As of median follow-up of 24.8 months, median overall survival (mOS) in HER2-L was significantly better than that in HER2- Pos ( p = 0.029, 18.2 months in HER2-Pos vs. 33.3 in HER2-L vs. 27.9 in HER2-Neg). In 58 pts harboring RAS wild-type who received an anti-EGFR monoclonal antibody therapy, median progression-free survival (mPFS) in HER2-L tended to be better than that in HER2- Pos, with 2.2 months in HER2-Pos, 7.8 in HER2-L, and 5.5 in HER2-Neg ( p = 0.099). Conclusions: Since HER2-L mCRC had the high prevalence of co-altered RAS mutations but showed a better prognosis and might benefit more from an anti-EGFR therapy than HES2-Pos, the HER2-L mCRC seems to have a different biological behavior from HER2-Pos in terms of molecular landscape and prognostic value on mCRC.
Molecular characterization of appendiceal goblet cell carcinoid.
Authors: Arai Hiroyuki , Baca Yasmine , Johnston Curtis , Goldberg Richard M. , Philip Philip Agop , Seeber Andreas , Battaglin Francesca , Xiu Joanne , Wang Jingyuan , Soni Shivani , Zhang Wu , Hwang Jimmy J. , Shields Anthony Frank , Marshall John , Korn W. Michael , Lenz Heinz-Josef 
Published: February 2020

231 Background: Goblet cell carcinoid (GCC) is a distinct subtype of appendiceal neoplasm that exhibits unique clinical and pathologic features. There are a few reports focusing on the molecular differences between GCC and other appendiceal tumors such as adenocarcinoma and neuroendocrine tumor (NET). Methods: A total of 495 appendiceal tumor samples (53 GCCs, 428 adenocarcinomas and 14 NETs) were tested with Next-Generation Sequencing (NGS) on a 592-gene panel and immunohistochemistry (IHC). Microsatellite instability (MSI) / mismatch repair (MMR) status was tested with a combination of NGS, IHC and fragment analysis. Tumor mutational burden (TMB) was evaluated by NGS, and PD-L1 were tested by IHC (SP142). Molecular characteristics of GCCs are compared with those of adenocarcinomas and NETs, using Chi-square test. Results: The top five genes with most frequent mutation rate in GCCs were TP53 (24.0%), ARID1A (15.4%), SMAD4 (9.4%), KRAS (7.5%) and CHEK2 (4.0%). Compared to adenocarcinomas, GCCs showed significantly lower mutation rates in KRAS (7.5% vs 60.4%), GNAS (3.8% vs 34.4%), APC (1.9% vs 11.7%), while significantly higher mutation rates in CDH1 (3.8% vs 0.7%), CHEK2 (4.0% vs 0.3%), CDC73 (2.0% vs 0.0%), ERCC2 (2.0% vs 0.0%) and FGFR2 (1.9% vs 0.0%). Compared to NETs, GCCs showed significantly lower mutation rate in KRAS (7.5% vs 28.6%), APC (1.9% vs 28.6%), BRCA2 (0.0% vs 7.1%) and FANCA (0.0% vs 7.1%), with all p < 0.05. In GCCs, MSI-H/dMMR, TML-high (> 17mut/Mb) and PD-L1 expression were seen in 0.0%, 0.0% and 2.0%, respectively. No significant difference was observed in these immune-related markers’ frequency, compared to adenocarcinomas and NETs. Conclusions: GCCs had considerably distinct mutational profile compared to appendiceal adenocarcinomas and NETs. Understanding these molecular characteristics may be critical for a development of effective treatment strategy in GCC.
Variation in genetic polymorphisms and gene expression of HLA-E to predict outcomes in metastatic colorectal cancer (mCRC) patients (pts) treated with first-line FOLFIRI/cetuximab: Data from the phase III FIRE-3 trial.
Authors: Naseem Madiha , Cao Shu , Stintzing Sebastian , Battaglin Francesca , Tokunaga Ryuma , Puccini Alberto , Berger Martin D. , Soni Shivani , Zhang Wu , Barzi Afsaneh , Heinemann Volker , Lenz Heinz-Josef 
Published: February 2020

245 Background: HLA-E is an MHC Class I antigen which inhibits NK cell activity by binding to CD94 receptor. Overexpression of HLA-E on crc cells is associated with poor prognostic outcomes and has recently been associated with microsatellite instability. As cet enhances NK cell activity which is dependent on HLAE, this study aims to assess whether differences in HLAE gene expression and polymorphisms lead to different outcomes. Methods: Genomic DNA from blood samples of pts treated with first-line FOLFIRI-cetuximab (cet, n = 129) and FOLFIRI-bevacizumab (bev, n = 107) was genotyped through the OncoArray, a custom array manufactured by Illumina. Gene expression levels were measured from 102 tumor samples of pts in the cet arm by HTG EdgeSeq Oncology Biomarker Panel. PFS and OS outcomes were investigated for an HLAE genetic polymorphism, rs1264457, where G > A leads to glycine > arginine, where glycine has stronger affinity for NK cell receptor. Results: FOLFIRI/cet and FOLFIRI/bev cohort characteristics: median FU (29.1/26.7mo); PFS (12.8/11.5mo); OS (49.8/31.4mo); RAS WT (64%/62%) and RAS mut (15%/16%). Overexpression of HLAE was associated with poor OS (log2 > 13, 30 vs 34 mo) in cet arm (P < .05). No significant association was observed with PFS. Multivariable analysis showed that HLAErs1264457 carriers with any A allele (n = 65) had better PFS than pts with G/G genotype (n = 18) in RAS WT pts treated with FOLFIRI/cet (12.9 vs 12.3 mo; HR = 2.36; 95%CI = 1.14-4.88; p = 0.021). A non-significant trend of improved OS was seen among carriers of A allele than G/G genotype (56.2 vs 40 mo). These effects were not observed in Ras mut pts. No significance was observed in FOLFIRI/bev overall or FOLFIRI/bev RAS WT pts. Conclusions: Overexpression of HLAE leads to poor OS among pts treated with FOLFIRI/cet. Poor PFS was seen among pts treated with cet who are RAS wt carrying polymorphisms allowing for increased binding to NK cell receptor, which in turn enhances HLAE inhibitory function of NK cell lysis. Addition of mAb against HLAE, which have been shown to restore NK cell mediated cell lysis, needs investigation in this pt population.
The influence of adjuvant chemotherapy dose intensity on overall survival in resected colon cancer: A multicenter retrospective analysis.
Authors: Lakkunarajah Suganija , Breadner Daniel Adam , Zhang Hanbo , Spratlin Jennifer L. , Mulder Karen E. , Gill Sharlene , Sha Aaron , Abadi Shirin , Kong Shiying , Cheung Winson Y. , Loree Jonathan M. , Welch Stephen 
Published: February 2020

248 Background: Colorectal cancer remains the second leading cause of cancer death in developed countries. The benefit of using fluorouracil-based chemotherapy with oxaliplatin, such as FOLFOX (fluorouracil (5-FU), leucovorin, oxaliplatin) and CAPOX (capecitabine and oxaliplatin) is well established. The optimal dose intensity (DI) under which overall survival (OS) is inferior is not established. Methods: Patients (pts) treated with adjuvant chemotherapy between 2006 and 2011 for resected stage III colon cancer (CC) from four academic cancer centres in Canada were retrospectively analysed. Patients that received CAPOX and FOLFOX were examined for the relationship between DI and OS. Results: A total of 625 pts with resected high risk stage II or stage III CC that received adjuvant chemotherapy were analysed. The median age was 63. Pts with T4 and N2 disease comprised 35.4% and 29.9% of pts, respectively. Median follow-up was 3.2 years. There was available survival data for 319 pts. The median oxaliplatin DI was 70%. The frequency of pts reaching an oxaliplatin DI of > 80% was 43%, while 76.6% of pts had a dose intensity of > 80% for their FU component. An oxaliplatin DI of > 80% was associated with a significant improvement in survival, HR = 0.42 (95%CI 0.21 – 0.81, p < 0.01). Achieving a DI of > 80% for capecitabine or 5-FU did not improve OS. Other factors associated with inferior OS included T4 (HR = 3.5, p = 0.03) and N2 (HR = 5.27, p = 0.0005) subgroups. The improvement in OS was not significant when restricting the analysis to pts with non-T4 and non-N2 disease (n = 144), HR = 0.16 (0.02 – 1.26; p = 0.08). Conclusions: Oxaliplatin DI of > 80% is associated with improved OS in patients receiving chemotherapy for high risk stage II and stage III CC.