Breast Neoplasms: Bloom KJ

Vance GH, Barry TS, Bloom KJ, Fitzgibbons PL, Hicks DG, Jenkins RB, Persons DL, Tubbs RR, Hammond ME, Anonymous00034. · Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA. · Arch Pathol Lab Med. · Pubmed #19391661 No free full text.

Abstract: CONTEXT: Intratumoral heterogeneity of HER2 gene amplification has been well documented and represents subclonal diversity within the tumor. The reported incidence of intratumor HER2 amplification genetic heterogeneity ranges in the literature from approximately 5% to 30%. The presence of HER2 genetic heterogeneity may increase subjectivity in HER2 interpretation by the pathologist. OBJECTIVES: To define HER2 genetic heterogeneity and to provide practice guidelines for examining and reporting breast tumors with genetic heterogeneity for improvement of HER2 testing in breast cancer. DESIGN: We convened an expert panel to discuss HER2 gene amplification testing by fluorescence in situ hybridization. Components addressed included a definition of HER2 amplification heterogeneity, practice guidelines for examination of the tissue, and reporting criteria for this analysis. RESULTS: Genetic heterogeneity for amplification of HER2 gene status in invasive breast cancer is defined and guidelines established for assessing and reporting HER2 results in these cases. These guidelines are additive to and expand those published in 2007 by the American Society of Clinical Oncology and the College of American Pathologists. CONCLUSION: Standardized methods for analysis will improve the accuracy and consistency of interpretation of HER2 gene amplification status in breast cancer.

Ross JS, Fletcher JA, Bloom KJ, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L, Hortobagyi GN. · Department of Pathology and Laboratory Medicine, Albany Medical College, NY 12208, USA. · Am J Clin Pathol. · Pubmed #15298144 No free full text.

Abstract: The testing of newly diagnosed breast cancer specimens for HER-2/neu status has achieved "standard of practice" status for the management of breast cancer in the United States. The discussion as to the best method to determine HER-2/neu status in these samples continues, with the fluorescence in situ hybridization method gaining popularity owing to the recent evidence that it, in comparison with immunohistochemical analysis, might more accurately predict clinical responses to trastuzumab-based therapies. With trastuzumab achieving excellent results in the treatment of HER-2/neu-positive advanced disease and under extensive evaluation in major clinical trials for its potential efficacy when used at earlier clinical stages, the potential role(s) for HER-2/neu testing as a predictor of response to other therapies being resolved by large prospective clinical outcome studies, and the more convenient gene-based chromogenic in situ hybridization technique "waiting in the wings," the saga of HER-2/neu testing in breast cancer will continue to unfold over the next several years.

Ross JS, Fletcher JA, Bloom KJ, Linette GP, Stec J, Symmans WF, Pusztai L, Hortobagyi GN. · Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY 12208, USA. · Mol Cell Proteomics. · Pubmed #14762215 links to  free full text

Abstract: The HER-2/neu oncogene, a member of the epidermal growth factor receptor or erb gene family, encodes a transmembrane tyrosine kinase receptor that has been linked to prognosis and response to therapy with the anti-HER-2-humanized monoclonal antibody, trastuzumab (Herceptin, Genentech, South San Francisco, CA) in patients with advanced metastatic breast cancer. HER-2/neu status has also been tested for its ability to predict the response of breast cancer to other therapies including hormonal therapies, topoisomerase inhibitors, and anthracyclines. This review includes an analysis of 80 published studies encompassing more than 25,000 patients designed to consider the relative advantages and disadvantages of the various methods of measuring HER-2/neu in clinical breast cancer specimens. Southern blotting, PCR amplification detection, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is reviewed along with the current studies designed to test whether HER-2/neu status can predict the response to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review will also evaluate the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.

Ross JS, Fletcher JA, Linette GP, Stec J, Clark E, Ayers M, Symmans WF, Pusztai L, Bloom KJ. · Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York 12208, USA. · Oncologist. · Pubmed #12897328 links to  free full text

Abstract: The HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor. In this review, the association of HER-2/neu gene and protein abnormalities with prognosis and response to therapy with trastuzumab and to other therapies in breast cancer is presented. By considering a series of 80 published studies encompassing more than 25,000 patients, the relative advantages and disadvantages of Southern blotting, polymerase chain reaction amplification, and fluorescence in situ hybridization assays designed to detect HER-2/neu gene amplification are compared with HER-2/neu protein overexpression assays performed by immunohistochemical techniques applied to frozen and paraffin-embedded tissues and enzyme immunoassays performed on tumor cytosols. The significance of HER-2/neu overexpression in ductal carcinoma in situ and the HER-2/neu status in uncommon female breast conditions and male breast cancer are also considered. The role of HER-2/neu testing for the prediction of response to trastuzumab therapy in breast cancer is presented as well as its potential impact on responses to standard and newer hormonal therapies, cytotoxic chemotherapy, and radiation. The review also evaluates the status of serum-based testing for circulating HER-2/neu receptor protein and its ability to predict disease outcome and therapy response.

Dowlatshahi K, Francescatti DS, Bloom KJ, Jewell WR, Schwartzberg BS, Singletary SE, Robinson D. · Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, 1725 W. Harrison St., Suite 848, Chicago, IL 60612, USA. · Am J Surg. · Pubmed #11720684 No free full text.

Abstract: BACKGROUND: Widespread screening mammography has resulted in detection of many breast cancers smaller than one cm. Image-guided percutaneous needle sampling provides accurate diagnostic and prognostic information for adjuvant therapy. Less invasive methods based on imaging techniques are emerging as an alternative to wire localization and lumpectomy. DATA SOURCES: Information presented in this overview was provided by seven investigators from five medical centers in the United States. These researchers are currently developing various techniques of image-guided percutaneous therapy of small (Tis, 1) breast cancers. CONCLUSIONS: Several percutaneous treatment modalities for treatment of early breast cancer, either excisional or in-situ ablative, are described in this overview and their potential applications are discussed.

Dowlatshahi K, Fan M, Gould VE, Bloom KJ, Ali A. · Department of Surgery, Rush-Presbyterian St Luke's Medical Center, 1653 W Congress Pkwy, Suite 791 Jelke Southcenter, Chicago, IL 60612, USA. · Arch Surg. · Pubmed #11074894 links to  free full text

Abstract: HYPOTHESIS: Mammographically detected breast tumors can be completely ablated with laser energy. DESIGN: Nonrandomized control trial. SETTING: A university hospital ambulatory care center. PATIENTS: Thirty-six patients with mammographically detected well-defined breast tumors were selected. INTERVENTIONS: The diagnosis of malignant neoplasms and determination of prognostic factors were established by image-guided needle-core biopsy. Patients were treated on a stereotactic table, using a 16- to 18-gauge laser probe, with an optic fiber transmitting a predetermined amount of laser energy. A multisensor thermal probe was inserted into the breast adjacent to the laser probe to monitor treatment. In the last 10 patients, the tumor blood flow was evaluated before and after laser therapy with contrast-enhanced color Doppler ultrasound. One to 8 weeks after laser therapy, the tumors were surgically removed and serially sectioned. MAIN OUTCOME MEASURE: Complete necrosis in 66% of tumors. RESULTS: Total tumor ablation with negative margins was observed whenever 2500 J/mL of tumor was given or the thermal sensors recorded 60 degrees C. Microscopic examination at 1 week showed disintegration of malignant cells, with peripheral acute inflammatory response and at 4 to 8 weeks extensive fibrosis. Contrast-enhanced color Doppler ultrasound revealed loss of tumor circulation after therapy, and positron emission tomography scan correlated well with histologic findings. There were no systemic adverse effects. Two patients sustained 3 x 4-mm skin burns around the laser needle. CONCLUSION: A stereotactically guided minimally invasive technique may be effective for the treatment of mammographically detected breast cancer.

Dowlatshahi K, Dieschbourg JJ, Bloom KJ. · Department of General Surgery, Rush University Medical Center, Chicago, Illinois 60612, USA. · Breast J. · Pubmed #15125752 No free full text.

Abstract: Breast cancer is the commonest female malignancy in the United States and is increasingly being detected at a non-palpable stage by annual mammography. Minimally invasive methods of its local treatment have been recently introduced as alternative to its surgical removal. The case reported in this article illustrates the advantages of in-situ ablation with minimal discomfort to the patient and deformity of the breast. More importantly, it demonstrates the absence of any adverse effect on health and survival of the patient during this intermediate period of follow-up.

Coogan CL, Estrada CR, Kapur S, Bloom KJ. · Department of Urology, Rush Medical College, Chicago, Illinois, USA. · Urology. · Pubmed #15072912 No free full text.

Abstract: OBJECTIVES: To assess HER-2/neu gene status by fluorescence in situ hybridization and protein expression by immunohistochemistry in human bladder transitional cell carcinoma (TCC). In breast carcinoma, HER-2/neu gene amplification and receptor protein overexpression are tightly correlated and have prognostic and therapeutic implications. METHODS: We used 54 randomly selected TCC specimens obtained from 1998 to 2000. Each specimen was fixated in 10% neutral-buffered formalin and embedded in paraffin. Of the 54 specimens, 7 were grade 1 (13%), 26 were grade 2 (48%), and 21 were grade 3 (39%); 36 (67%) were superficial (Stage Ta or T1) and 18 (33%) were invasive (Stage T2 or T3). The specimens were analyzed for HER-2/neu protein overexpression by immunohistochemistry and for gene amplification using fluorescence in situ hybridization. RESULTS: Of the 54 specimens, 14 (26%) were positive for protein overexpression. One (14%) of the 7 grade 1 tumors was positive for protein overexpression, 3 (12%) of 26 grade 2 tumors were positive, and 10 (48%) of 21 grade 3 tumors were positive (P = 0.0195). Six (17%) of 36 Stage Ta or T1 specimens and 8 (44%) of 18 Stage T2 or T3 specimens were positive for protein overexpression (P = 0.01). None of the 54 TCC specimens showed amplification of the HER-2/neu gene using fluorescence in situ hybridization. CONCLUSIONS: HER-2/neu protein overexpression is present in human bladder TCC, with a statistically significant increase in overexpression in grade 3, and invasive specimens. Gene amplification does not appear to be the mechanism of protein overexpression. The prognostic significance of these findings and the application of HER-2/neu in treatment needs additional investigation.

Burak WE, Agnese DM, Povoski SP, Yanssens TL, Bloom KJ, Wakely PE, Spigos DG. · Department of Surgical Oncology, Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Medical Center, Columbus, Ohio 43210, USA. · Cancer. · Pubmed #14508822 links to  free full text

Abstract: BACKGROUND: Radiofrequency ablation (RFA) is gaining acceptance as a treatment modality for several tumor types. However, its use in patients with breast carcinoma remains investigational. The current study was undertaken to determine the feasibility of treating small breast malignancies with RFA and to evaluate the postablation magnetic resonance imaging scans (MRI) and histologic findings. METHODS: Patients with core-needle biopsy-proven invasive carcinoma (< 2 cm in greatest dimension) underwent ultrasound-guided RFA under local anesthesia. Surgical excision was undertaken 1-3 weeks later. All patients had breast MRI scans performed before ablation and repeated within 24 hours of surgery. RESULTS: Ten patients completed the treatment and experienced minimal or no discomfort. The mean tumor size was 1.2 cm (range, 0.8-1.6 cm). The mean time required for ablation was 13.8 minutes (range, 7-21 minutes). There were no treatment-related complications other than minimal breast ecchymosis. A pre-RFA MRI scan showed enhancing tumors in 9 of 10 (90%) patients. A post-RFA MRI scan revealed no residual lesion enhancement in 8 of these 9 patients (89%), and the zone of ablation was demonstrated in all patients. One patient had residual enhancement anteriorly consistent with residual tumor, which was confirmed histologically. Evaluation of the remaining ablated lesions revealed a spectrum of changes ranging from no residual tumor to coagulation necrosis with recognizable malignant cells. Immunostains for cytokeratin 8/18 were negative in these recognizable malignant cells. CONCLUSIONS: RFA of small breast malignancies can be performed under local anesthesia in an office-based setting. A postablation MRI scan appears to predict histologic findings, although tumor viability needs to be assessed in a long-term study.

Orucevic A, Reddy VB, Bloom KJ, Bitterman P, Magi-Galluzzi C, Oleske DM, Phillips M, Gould VE, Cobleigh M, Wick MR, Gattuso P. · Department of Pathology, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA. · Breast J. · Pubmed #12390357 No free full text.

Abstract: It is important to identify T1-substage breast carcinomas (BCs) which are inherently aggressive, so that these can be managed more assertively. The purpose of this study was to distinguish those T1 BCs with the potential to metastasize to axillary lymph nodes from those lacking that ability by multiparametric analysis of several clinicopathologic features. The authors studied 197 patients with invasive BC who had undergone modified radical mastectomy; 161 tumors were ductal and 26 were lobular BCs. The study group was stratified by age into two groups: </=34 years (n = 34) and 35-84 years (n = 153). Pathologic lymph node status was correlated with estrogen receptor (ER) and progesterone receptor (PR) tumor positivity, MIB-1 proliferation index, and immunoreactivity for mutant p53 protein. These factors were studied immunohistologically using standard methodology and microwave-mediated epitope retrieval. Statistical analyses employed accepted techniques. Women in this study ranged from 22 to 84 years of age; 39 (21%) had positive lymph nodes. ER-positive tumors comprised 73% of the total; similarly, 65% were PR positive. The MIB-1 index was greater than 10% in 44% of lesions, and 14% demonstrated labeling for mutant p53 protein. Using crude odds ratio data, the MIB-1 index was the only indicator found to predict lymph node metastasis significantly (p < 0.001). Moreover, even when adjustments were made for patient age, logistic regression analysis confirmed the utility of MIB-1-values of greater than 10% in this context, with a 4.4 greater likelihood of metastasis (p < 0.001). MIB-1 indices of greater than 10% are associated with a risk of lymph node metastasis from T1 BCs, independent of patient age. Hormone receptor status and immunohistologic p53 status are not predictors of nodal involvement in this specific setting.

Dowlatshahi K, Francescatti DS, Bloom KJ. · Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA. · Am J Surg. · Pubmed #12383903 No free full text.

Abstract: BACKGROUND: Widespread mammography has resulted in the increased detection of breast cancer <1.5 cm. It may be possible to treat these small tumors with in-situ laser ablation. Prior to ablation tumor size is determined by ultrasound and mammogram. Histologic diagnosis and determination of prognostic factors are obtained from image-guided needle core samples. Invasive and in-situ tumors may be percutaneously ablated by a stereotactically guided laser needle and subsequently evaluated by imaging methods and needle biopsy. METHODS: Fifty-four patients (50 invasive, 4 in-situ); 51 mass, 3 microcalcification; mean diameter 12 (5 to 23) mm were treated by a stereotactically guided 805 nm laser beam via a fiber in a 16G needle delivered to the cancer. One to 8 weeks later the coagulated lesions were surgically removed for pathologic evaluation. In 2 additional patients, the laser-treated tumors were not removed but were monitored by mammography, ultrasonography, and needle core biopsy. RESULTS: None of the patients sustained any adverse effect. The average treatment time was 30 minutes. Pathology analysis revealed a 2.5 to 3.5 hemorrhagic ring surrounding the necrotic tumor. Under steady conditions, in two groups of 14 patients, 93% and 100% of the tumors showed complete destruction, with no residual cancer report. In the 2 unresected cases kept under surveillance for 6 to 24 months, the laser-treated tumors first showed shrinkage, followed by a 2 to 3 cm oil cyst. Fibrosis was demonstrated on needle core biopsies. CONCLUSIONS: Laser energy delivered through a stereotactically guided needle appears to ablate mammographically detected breast cancer. A multicenter clinical trail is planned.

Bloom KJ, Govil H, Gattuso P, Reddy V, Francescatti D. · Department of Pathology, Rush Medical College, Chicago, IL 60612, USA. · Am J Surg. · Pubmed #11720677 No free full text.

Abstract: BACKGROUND: HER-2 overexpression is seen in 20% to 30% of invasive female breast carcinomas. Besides being prognostic, HER-2 may also be predictive of response to therapy. Similar studies in male breast carcinoma are lacking. We compared the overexpression and amplification of HER-2 in female and male breast carcinoma. DESIGN: Formalin-fixed, paraffin embedded archival material from 58 invasive male breast carcinomas and 202 invasive female breast carcinomas were immunostained for HER-2. Scoring was performed according to established guidelines. Each case was also assessed for HER-2 gene amplification by fluorescence in-situ hybridization (FISH) utilizing the PathVysion assay (Vysis corporation, Downers Grove, Illinois). RESULTS: There were 58 male patients who ranged in age from 38 to 92 years (mean 63). Thirty-five (60%) were T1 lesions and 23 (40%) were T2 lesions. Twenty-five patients (43%) had positive lymph nodes. One (1.7%) of the 58 cases showed 3+ staining of HER-2. The remaining 57 cases did not show overexpression. There was no amplification of the HER-2 gene in any of the cases. There were 202 female patients who ranged in age from 26 to 96 years (mean 52). In all, 129 (64%) were T1 lesions, 61 (30%) were T2 lesions, and 13 (6%) were T3 lesions. Fifty-two (26%) showed positive staining with HER-2 (44 cases 3+, 8 cases 2+). The remaining 150 (74%) did not show overexpression. There was amplification of HER-2 gene in 55 (27%) of the cases. Two of the cases negative by FISH were 3+ positive by IHC. CONCLUSIONS: HER-2 is overexpressed in approximately 27% of female breast carcinomas. A high level of correlation is demonstrated between IHC and FISH techniques. Gene amplification of HER-2 does not play a role in male breast carcinoma. The rate of single-copy overexpression of HER-2 appears identical in male and female breast carcinoma.

Bloom KJ, Dowlat K, Assad L. · Department of Pathology, Rush Medical College, Rush Presbyterian-St. Luke's Medical Center, 1653 W. Congress Pkwy., Chicago, IL 60612, USA. · Am J Surg. · Pubmed #11720676 No free full text.

Abstract: BACKGROUND: Interstitial laser therapy (ILT) is a technique of destroying tumor cells via thermal energy. Prior studies have shown that the procedure is safe and efficacious in laboratory animals and that complete cell death, as assessed by tetrazolium staining, is achieved in the laser treated area. DESIGN: Forty women with localized, mammographically detectable, T1 breast cancers consented to be treated with ILT and then have the treated area subsequently excised. The delay period ranged from 5 to 42 days. Prior to ILT, the diagnosis of breast carcinoma was established by stereotactic needle core biopsy. RESULTS: All 40 cases showed a characteristic gross appearance, which consisted of a series of concentric rings surrounding a cavity corresponding to the laser needle tip. The tissue immediately adjacent to the cavity appeared coagulated and showed the same "wind-swept" nuclei seen with cautery artifact. Surrounding this was a white-tan ring that histologically showed recognizable tumor. No necrosis, increased apoptosis or inflammatory infiltrate was noted in this area on hematoxylin-eosin sections. Immunostains for cytokeratin were negative in the recognizable tumor cells despite intense staining in the epithelial cells outside the laser treated area. A ring of red tan tissue which histologically consisted of necrotic tumor was seen next and this, in turn, was surrounded by a rim of vascular proliferation and fat necrosis. The breast tissue outside the zone of fat necrosis appeared to be unaffected by the laser therapy. CONCLUSIONS: ILT appears to be an effective way to treat small localized breast cancer. It is important for the pathologist to recognize the changes seen after ILT, especially the zone containing pseudoviable tumor. Cytokeratin may be a marker to identify these likely non-viable but recognizable tumor cells. The extent of the laser affected area is defined by vascular proliferation and fat necrosis.

Dowlatshahi K, Fan M, Anderson JM, Bloom KJ. · Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois, USA. · Ann Surg Oncol. · Pubmed #11569784 No free full text.

Abstract: BACKGROUND: Up to 30% of patients with operable breast cancer and negative regional lymph nodes experience disease recurrence within 10 years. Serial sectioning and immunohistochemical staining of these nodes have revealed 9% to 30% occult metastases. METHODS: Sentinel nodes from 200 patients with T1 and T2 invasive breast carcinoma were step-sectioned at 2- to 3-mm intervals, fixed in 10% formalin, and embedded in paraffin. Sections were taken from the face of the blocks and stained with hematoxylin and eosin (H&E). The blocks were then cut completely, and sections at .25-mm intervals were stained with cytokeratin and examined. RESULTS: Tumor metastases were found in 34 patients when the sentinel nodes were examined at 2- to 3-mm intervals and in an additional 51 patients when the nodes were sectioned in their entirety at .25-mm intervals and stained with cytokeratin, bringing the total number of patients with metastases to 85. Of the 51 patients whose metastases were detected by 2- to 3-mm sectioning and cytokeratin staining, 27 had isolated tumor cells and 24 had clusters of innumerable malignant cells, all of which were visualized and confirmed by H&E staining of the adjacent sections. CONCLUSIONS: Histologic examination of sentinel nodes of patients with invasive breast cancer sectioned at 2- to 3-mm intervals and stained with H&E significantly underestimates nodal metastases. Sectioning of the entire sentinel nodes at .25-mm intervals and staining with cytokeratin detects metastases as either isolated cells or as clusters.

Dowlatshahi K, Fan M, Bloom KJ, Spitz DJ, Patel S, Snider HC. · Department of General Surgery, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA. · Cancer. · Pubmed #10491525 links to  free full text

Abstract: BACKGROUND: Thirty percent of lymph node negative patients with operable breast carcinoma experience disease recurrence within 10 years. Retrospective serial sectioning of axillary lymph nodes has revealed undetected metastases in 9-30% of these patients. These occult metastases have been shown to have an adverse effect on survival. Serial sectioning (SS) is impractical for all axillary lymph nodes harvested from Levels I and II, but it is feasible if applied only to sentinel lymph nodes. METHODS: Sentinel lymph nodes from 52 patients with invasive breast carcinoma were cut at 2 mm intervals, fixed in 10% formalin, and embedded in paraffin. Sections were taken from the blocks, stained with hematoxylin and eosin (H & E), and compared with cytokeratin-stained sections taken at 0.25 mm intervals throughout the entire blocks. RESULTS: Tumor metastases were found in 6 patients (12%) when the sentinel lymph nodes were sectioned at 2 mm intervals and stained with H & E, compared with 30 patients (58%) when the same lymph nodes were serially sectioned at 0.25 mm intervals and stained with cytokeratin. Of 24 patients whose metastases were detected by SS and cytokeratin staining, 12 had isolated tumor cells and 12 had colonies of several thousand malignant cells. CONCLUSIONS: Routine histologic examination of axillary lymph nodes, including sentinel lymph nodes, in cases of breast carcinoma significantly underestimates lymph node metastases. This deficiency may be overcome by SS of the entire lymph nodes and staining with a specific monoclonal antibody. The percentage of patients found to have colonies of cells that were missed by routine sectioning corresponds closely to the percentage of "lymph node negative" patients who would be expected to relapse. The true clinical significance of these occult metastases will be determined by long term follow-up. [See editorial on pages 905-7, this issue.] Copyright 1999 American Cancer Society.

Hanna WM, Hammond E, Taylor CR, Dabbs DJ, Penault-Llorca F, Bloom KJ, Bilous M, Badve S. · No affiliation provided · Mod Pathol. · Pubmed #18813329 No free full text.

This publication has no abstract.