Dr. Gregory Bean

All Publications

• Primary mammary angiosarcomas harbor frequent mutations in KDR and PIK3CA and show evidence of distinct pathogenesis. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Beca, F., Krings, G., Chen, Y., Hosfield, E. M., Vohra, P., Sibley, R. K., Troxell, M. L., West, R. B., Allison, K. H., Bean, G. R. 2020

  Abstract

  Angiosarcoma (AS) is the most frequent primary sarcoma of the breast but nevertheless remains uncommon, accounting for <0.05% of breast malignancies. Secondary mammary AS arise following radiation therapy for breast cancer, in contrast to primary AS which occur sporadically. Essentially all show aggressive clinical behavior independent of histologic grade and most are treated by mastectomy. MYC amplification is frequently identified in radiation-induced AS but only rarely in primary mammary AS (PMAS). As a heterogeneous group, AS from various anatomic sites have been shown to harbor recurrent alterations in TP53, MAP kinase pathway genes, and genes involved in angiogenic signaling including KDR (VEGFR2) and PTPRB. In part due to its rarity, the pathogenesis of PMAS has not been fully characterized. In this study, we examined the clinical, pathologic, and genomic features of ten cases of PMAS, including one patient with bilateral disease. Recurrent genomic alterations were identified in KDR (70%), PIK3CA/PIK3R1 (70%), and PTPRB (30%), each at higher frequencies than reported in AS across all sites. Six tumors harbored a KDR p.T771R hotspot mutation, and all seven KDR-mutant cases showed evidence suggestive of biallelism (four with loss of heterozygosity and three with two aberrations). Of the seven tumors with PI3K alterations, six harbored pathogenic mutations other than in the canonical PIK3CA residues which aremost frequent in breast cancer. Three AS were hypermutated (?10 mutations/megabase (Mb)); hypermutation was seen concurrent with KDR or PIK3CA mutations. The patient with bilateral disease demonstrated shared alterations, indicative of contralateral metastasis. No MYC or TP53 aberrations were detected in this series. Immunohistochemistry for VEGFR2 was unable to discriminate between KDR-mutant tumors and benign vascular lesions of the breast. These findings highlight the underrecognized frequency of KDR and PIK3CA mutation in PMAS, and a significant subset with hypermutation, suggesting a pathogenesis distinct from other AS.

  View details for DOI 10.1038/s41379-020-0511-6

  View details for PubMedID 32123305

• NTRK Fusion Cervical Sarcoma: A Report of 3 Cases, Emphasizing Morphological and Immunohistochemical Distinction from Other Uterine Sarcomas, including Adenosarcoma. Histopathology Rabban, J. T., Devine, P., Sangoi, A. R., Poder, L., Alvarez, E., Davis, J. L., Rudzinski, E., Garg, K., Bean, G. R. 2020

  Abstract

  A unique fibrosarcoma-like tumor of the uterine cervix harboring a rearrangement of a neurotrophic tyrosine kinase receptor gene (NTRK1 or NTRK3) has recently been described in 11 young women, some with recurrence and/or metastasis. To expand the morphological spectrum of this tumor we report three additional cases which exhibited adenosarcoma-like features not previously described, one of which is the first reported to respond to targeted therapy. We also evaluated 19 conventional uterine adenosarcomas for evidence of NTRK rearrangement.Three patients presented with a polyp or mass confined to the cervix. The constellation of polypoid growth, spindle cell morphology, entrapped endocervical glands and intraglandular stromal projections raised diagnostic consideration for adenosarcoma with stromal overgrowth. Deep cervical wall invasion was present in two cases at hysterectomy while the third was removed by polypectomy. All three stained for S100 and pan-Trk but were negative for a spectrum of other diagnostic markers. All three harbored NTRK rearrangement (TPM3-NTRK1, TPR-NTRK1, and SPECC1L-NTRK3). One patient developed pleural metastases at 16 months, received the neurotrophic tyrosine kinase inhibitor larotrectinib, and is free of disease 15 months later. Two others are alive without disease. None of the uterine adenosarcomas showed any S100 or pan-Trk staining or rearrangement of NTRK1, NTRK2 or NTRK3 by next generation sequencing.Unusual adenosarcoma-like spindle cell neoplasms of the cervix may represent an NTRK fusion sarcoma, which can be detected by S100 and pan-Trk staining and confirmed by NTRK molecular testing. Conventional uterine adenosarcomas do not harbor NTRK rearrangements.

  View details for DOI 10.1111/his.14069

  View details for PubMedID 31971278

• CRTC1-MAML2 fusion in mucoepidermoid carcinoma of the breast HISTOPATHOLOGY Bean, G. R., Krings, G., Otis, C. N., Solomon, D. A., Garcia, J. J., van Zante, A., Camelo-Piragua, S., van Ziffle, J., Chen, Y. 2019; 74 (3): 463?73

  View details for DOI 10.1111/his.13779

  View details for Web of Science ID 000459597000010

• DICER1 mutations are frequent in müllerian adenosarcomas and are independent of rhabdomyosarcomatous differentiation. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc Bean, G. R., Anderson, J., Sangoi, A. R., Krings, G., Garg, K. 2018

  Abstract

  Müllerian adenosarcomas are biphasic epithelial-mesenchymal tumors with benign epithelial and malignant mesenchymal components. The sarcoma component may be low or high grade; the latter is often seen in the presence of stromal overgrowth, which correlates with worse clinical outcome. Heterologous differentiation may also occur, usually in association with stromal overgrowth. DICER1 mutations have been reported primarily in a small subset of adenosarcomas with rhabdomyosarcomatous elements, but whether these are specific to the rhabdomyosarcomatous phenotype is unclear. In this study, we examined the clinical, pathologic, and genomic features of 19 müllerian adenosarcomas enriched for tumors with rhabdomyosarcomatous differentiation, as well as eight uterine carcinosarcomas with a rhabdomyosarcoma component. Somatic hotspot mutations in the RNase IIIb domain of DICER1 were identified in 8/19 (42%) adenosarcomas, of which four showed rhabdomyosarcomatous differentiation. DICER1 mutations were detected in 4/6 (67%) cases with a rhabdomyosarcoma component and in 4/11 (36%) cases without rhabdomyosarcoma. At least two DICER1 mutations were identified in 7/8 (88%) tumors, of which four had a truncating mutation. The hotspot DICER1 mutation in the remaining tumor was hemizygous and associated with loss of heterozygosity. Other less frequent recurrent somatic pathogenic alterations included Ras or PI3K/PTEN pathway aberrations (5/19 each, 26%), CDK4/MDM2 amplifications (3/19, 16%), and mutations in TP53 (3/19) and ARID1A (3/19). Two tumors demonstrated homozygous BAP1 deletion. One tumor harbored an ESR1-NCOA3 fusion gene. Carcinosarcomas with rhabdomyosarcomatous differentiation showed frequent mutations in TP53 (7/8, 88%) and the PI3K/PTEN pathway (6/8, 75%) but lacked DICER1 mutations. The findings highlight the importance of DICER1 mutations in müllerian adenosarcoma tumorigenesis and show that these alterations are not exclusive to heterologous rhabdomyosarcomatous differentiation.

  View details for DOI 10.1038/s41379-018-0132-5

  View details for PubMedID 30266945

• Recurrent GNAQ mutations in anastomosing hemangiomas MODERN PATHOLOGY Bean, G. R., Joseph, N. M., Gill, R. M., Folpe, A. L., Horvai, A. E., Umetsu, S. E. 2017; 30 (5): 722-727

  Abstract

  Anastomosing hemangiomas are recently described benign vascular lesions that occur chiefly in the genitourinary tract and paravertebral soft tissues. Owing to their rarity and unusual cytoarchitectural features, anastomosing hemangiomas are frequently confused with low-grade angiosarcomas. The specific genetic alterations underlying these lesions are currently unknown. We performed capture-based next-generation DNA sequencing analysis on 13 anastomosing hemangiomas and identified frequent somatic mutations in the heterotrimeric G-protein alpha-subunit, GNAQ. Nine of 13 cases (69%) harbored a somatic mutation at GNAQ codon 209, a known hotspot that is commonly mutated in uveal melanoma and blue nevi, as well as various congenital vascular proliferations. No other pathogenic or likely pathogenic mutations were identified in these genetically simple lesions. The finding of a recurrent driver mutation in the G-protein signal transduction pathway provides strong evidence that anastomosing hemangiomas are indeed clonal vascular neoplasms.

  View details for DOI 10.1038/modpathol.2016.234

  View details for Web of Science ID 000400480100010

  View details for PubMedID 28084343

• Molecular Characterization of Metanephric Adenomas Beyond BRAF: Genetic Evidence for Potential Malignant Evolution. Histopathology Chan, E., Stohr, B. A., Croom, N. A., Cho, S., Garg, K., Troxell, M. L., Higgins, J. P., Bean, G. R. 2020

  Abstract

  AIMS: Metanephric adenomas (MA) are conventionally regarded as rare renal tumors with indolent behavior; limited case reports describe MA with aggressive features. Conventional MA harbor hotspot BRAF V600E mutations. A BRAF V600E senescence pathway, mediated by CDKN2A/p16, has been proposed to confer MA benignity. Aside from BRAF, the molecular landscape in both conventional MA and those with aggressive features has not been fully characterized. In this study we molecularly profiled a series of MA to investigate the correlation between genomic findings and clinical outcome.METHODS AND RESULTS: We retrospectively examined the histomorphology and patient outcomes of 11 conventional MA and one MA with aggressive features. Each was subjected to capture-based next-generation DNA sequencing of 479 cancer-related genes and immunohistochemical profiling. All tumors were positive for WT1 immunostaining and BRAF V600E mutation. One conventional MA contained an additional somatic BRCA2 pathogenic mutation. The MA with aggressive features showed a biphasic appearance: one component was epithelial with areas morphologically consistent with conventional MA; the second component was sarcomatous with areas of solid and angiosarcomatous growth. Differential profiling of the two populations revealed identical BRAF, EIF1AX, and TERT promoter hotspot mutations in the epithelial and sarcomatous components. Deep deletion of CDKN2A and MYC amplification were identified only in the sarcomatous component.CONCLUSIONS: While the vast majority of MA show indolent behavior, rare pathogenic alterations can occur in conventional MA in addition to BRAF. Molecular profiling of a case with aggressive clinical and pathologic features shows genetic evidence for malignant evolution in MA.

  View details for DOI 10.1111/his.14094

  View details for PubMedID 32064677

• Pathologic and molecular responses to neoadjuvant trastuzumab and/or lapatinib from a phase II randomized trial in HER2-positive breast cancer (TRIO-US B07). Nature communications Hurvitz, S. A., Caswell-Jin, J. L., McNamara, K. L., Zoeller, J. J., Bean, G. R., Dichmann, R., Perez, A., Patel, R., Zehngebot, L., Allen, H., Bosserman, L., DiCarlo, B., Kennedy, A., Giuliano, A., Calfa, C., Molthrop, D., Mani, A., Chen, H., Dering, J., Adams, B., Kotler, E., Press, M. F., Brugge, J. S., Curtis, C., Slamon, D. J. 2020; 11 (1): 5824

  Abstract

  In this multicenter, open-label, randomized phase II investigator-sponsored neoadjuvant trial with funding provided by Sanofi and GlaxoSmithKline (TRIO-US B07, Clinical Trials NCT00769470), participants with early-stage HER2-positive breast cancer (N=128) were recruited from 13 United States oncology centers throughout the Translational Research in Oncology network. Participants were randomized to receive trastuzumab (T; N=34), lapatinib (L; N=36), or both (TL; N=58) as HER2-targeted therapy, with each participant given one cycle of this designated anti-HER2 therapy alone followed by six cycles of standard combination chemotherapy with the same anti-HER2 therapy. The primary objective was to estimate the rate of pathologic complete response (pCR) at the time of surgery in each of the three arms. In the intent-to-treat population, we observed similar pCR rates between T (47%, 95% confidence interval [CI] 30-65%) and TL (52%, 95% CI 38-65%), and a lower pCR rate with L (25%, 95% CI 13-43%). In the T arm, 100% of participants completed all protocol-specified treatment prior to surgery, as compared to 69% in the L arm and 74% in the TL arm. Tumor or tumor bed tissue was collected whenever possible pre-treatment (N=110), after one cycle of HER2-targeted therapy alone (N=89), and at time of surgery (N=59). Higher-level amplification of HER2 and hormone receptor (HR)-negative status were associated with a higher pCR rate. Large shifts in the tumor, immune, and stromal gene expression occurred after one cycle of HER2-targeted therapy. In contrast to pCR rates, the L-containing arms exhibited greater proliferation reduction than T at this timepoint. Immune expression signatures increased in all arms after one cycle of HER2-targeted therapy, decreasing again by the time of surgery. Our results inform approaches to early assessment of sensitivity to anti-HER2 therapy and shed light on the role of the immune microenvironment in response to HER2-targeted agents.

  View details for DOI 10.1038/s41467-020-19494-2

  View details for PubMedID 33203854

• HER2 Dual In Situ Hybridization: Correlations and Cautions. Archives of pathology & laboratory medicine Troxell, M., Sibley, R. K., West, R. B., Bean, G. R., Allison, K. H. 2020

  Abstract

  Accurate HER2 testing in breast cancer is crucial for appropriate precision therapy. HER2 testing is most commonly accomplished by a combination of immunohistochemistry and in situ hybridization techniques, as gene amplification is closely tied to protein overexpression. During the last 5+ years, brightfield dual in situ hybridization (DISH) has replaced fluorescence methods (fluorescence in situ hybridization [FISH]) in some laboratories.To analyze routine HER2 DISH performance in the field.We reviewed our experience with HER2 DISH performed at outside laboratories and referred for patient care.Of 273 identified retrospective DISH results, 55 had repeated FISH testing at our institution; 7 (13%) were discordant. Additional cases had technical flaws hampering appropriate scoring. In 23 cases (42%), HER2 DISH was performed without immunohistochemistry. Slide review of a prospective cohort of 42 consecutive DISH cases revealed 14 (33%) with technical or interpretative limitations potentially jeopardizing results. Commonly identified problems include lack of or weak signals in most tumor cells, and silver precipitate or red signals outside of nuclei, resulting in false-negative or false-positive interpretations, respectively. Further, 44% (24 of 55) of DISH reports lacked complete data, specifically average HER2 signals/cell.While HER2 DISH can be an efficient and effective alternative to FISH, we illustrate pitfalls and reinforce that careful attention to slide quality and technical parameters are critically important. HER2 DISH cotesting with immunohistochemistry could help minimize false-negative or false-positive HER2 results.

  View details for DOI 10.5858/arpa.2019-0510-OA

  View details for PubMedID 32101450

• Efficacy of affibody-based ultrasound molecular imaging of vascular B7-H3 for breast cancer detection. Clinical cancer research : an official journal of the American Association for Cancer Research Bam, R., Lown, P. S., Stern, L. A., Sharma, K., Wilson, K. E., Bean, G. R., Lutz, A. M., Paulmurugan, R., Hackel, B. J., Dahl, J., Abou-Elkacem, L. 2020

  Abstract

  Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for pre-clinical ultrasound (US) imaging with anti-B7-H3-antibody functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted-MB.Binding of ABYB7-H3 was confirmed with soluble and cell-surface B7-H3 by flow-cytometry. MB were functionalized with ABYB7-H3 or anti-B7-H3-antibody (AbB7-H3). Control and targeted-MB were tested for binding to hB7-H3-expressing cells (MS1hB7-H3) under shear stress conditions. US imaging was performed with MBABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 co-implanted with MS1hB7-H3 or control MS1WT cells and a transgenic mouse model of breast cancer development.ABYB7-H3 specifically binds to MS1hB7-H3 and murine-B7-H3-expressing monocytes. MBABY-B7-H3 (8.5 ± 1.4 MB/cell) and MBAb-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (p<0.0001) binding to the MS1hB7-H3 cells compared to control MBNon-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MBABY-B7-H3 produced significantly higher (p<0.04) imaging signal in orthotopic tumors co-engrafted with MS1hB7-H3 (8.4 ± 3.3 a.u.) compared to tumors with MS1WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MBABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (p<0.0002) imaging signal compared to MBNon-targeted (1.3 ± 0.3 a.u.), while MBABY-B7-H3 signal in normal mammary glands and tumors with B7-H3-blocking significantly reduced (p<0.02) imaging signal.MBABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.

  View details for DOI 10.1158/1078-0432.CCR-19-1655

  View details for PubMedID 31924738

• Evaluation of ductal carcinoma in situ grade via triple-modal molecular imaging of B7-H3 expression. NPJ breast cancer Bachawal, S., Bean, G. R., Krings, G., Wilson, K. E. 2020; 6: 14

  Abstract

  Ductal carcinoma in situ (DCIS) will account for 62,930 cases of breast cancer in 2019. DCIS is a pre-invasive lesion which may not progress to invasive carcinoma, yet surgery remains the mainstay treatment. Molecular imaging of a specific marker for DCIS grade for detection and active surveillance are critically needed to reduce potential overtreatment. First, breast cancer marker B7-H3 (CD276) expression was evaluated by immunohistochemical staining in 123 human specimens including benign epithelium (H-score 10.0±8.2) and low (20.8±17.7), intermediate (87.1±69.5), and high (159.1±87.6) grade DCIS, showing a positive association with DCIS nuclear grade (P<0.001, AUC 0.96). Next, a murine DCIS model was combined with ultrasound molecular imaging of B7-H3 targeted microbubbles to differentiate normal glands from those harboring DCIS (n=100, FVB/N-Tg(MMTVPyMT)634Mul, AUC 0.89). Finally, photoacoustic and fluorescence molecular imaging with an anti-B7-H3 antibody-indocyanine green conjugate were utilized for DCIS detection (n=53). Molecular imaging of B7-H3 expression may allow for active surveillance of DCIS.

  View details for DOI 10.1038/s41523-020-0158-y

  View details for PubMedID 32377564

• Pan-TRK Immunohistochemistry: A Useful Diagnostic Adjunct For Secretory Carcinoma of the Breast. The American journal of surgical pathology Harrison, B. T., Fowler, E., Krings, G., Chen, Y., Bean, G. R., Vincent-Salomon, A., Fuhrmann, L., Barnick, S. E., Chen, B., Hosfield, E. M., Hornick, J. L., Schnitt, S. J. 2019

  Abstract

  Secretory carcinoma is a special-type breast carcinoma underpinned by a recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. Immunohistochemistry (IHC) using a pan-TRK antibody has been recently shown to help identify NTRK rearrangements in other tumor types. The purpose of this study was to assess the diagnostic utility of pan-TRK IHC in secretory carcinoma of the breast. Pan-TRK IHC was performed using a rabbit monoclonal antibody on whole sections of 24 breast secretory carcinomas and tissue microarray sections of other breast carcinoma types (n=203) and histologic mimics (n=15). Cases were assessed for staining intensity and localization. The 24 patients with secretory carcinoma had a median age of 44 years and a median tumor size of 1.0cm. ETV6 fluorescence in situ hybridization was positive in all cases tested (n=20). Twenty-three cases (95.8%) showed staining with pan-TRK, which was exclusively nuclear in 19, primarily nuclear with weak cytoplasmic staining in 3, and primarily cytoplasmic with focal nuclear staining in 1. The nuclear staining was diffuse in 17 and at least focally strong in 17. The only pan-TRK negative case was a core biopsy with limited tumor. Among the 203 nonsecretory carcinomas, 21 (10.3%) showed focal, weak nuclear staining in <5% of tumor cells and 1 (0.5%) showed focal membranous staining. All histologic mimics were negative. In conclusion, diffuse and/or at least focally strong nuclear pan-TRK staining is a sensitive and specific marker for secretory carcinoma of the breast.

  View details for DOI 10.1097/PAS.0000000000001366

  View details for PubMedID 31498178

• Synchronous Breast Implant-associated Anaplastic Large Cell Lymphoma and Invasive Carcinoma: Genomic Profiling and Management Implications. Plastic and reconstructive surgery. Global open Mukhtar, R. A., Holland, M., Sieber, D. A., Wen, K. W., Rugo, H. S., Kadin, M. E., Bean, G. R. 2019; 7 (4): e2188

  Abstract

  A 59-year-old woman with a history of cosmetic implants developed ipsilateral synchronous breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) and invasive ductal carcinoma in the left breast. Each tumor was subjected to next-generation sequencing, and separate analyses revealed mutually exclusive aberrations: an activating STAT3 mutation in the lymphoma and a PIK3CA in-frame deletion in the carcinoma. The patient was treated with removal of implants, capsulectomy, partial mastectomy, sentinel node biopsy, radiotherapy, and endocrine therapy with no evidence of recurrence for 1 year. This case illustrates the importance of obtaining thorough evaluation for concomitant malignancies in the breast at the time of diagnosis of BIA-ALCL. Herein, we review the current recommendations for evaluation and management of BIA-ALCL.

  View details for DOI 10.1097/GOX.0000000000002188

  View details for PubMedID 31321184

  View details for PubMedCentralID PMC6554181

• Adipocyte size variability in benign and malignant lipomatous tumors and morphologic mimics: a quantitative definition using digital pathology. Human pathology Bean, G. R., Wen, K. W., Horvai, A. E. 2018; 72: 52?58

  Abstract

  Among well-differentiated lipomatous lesions, variability in adipocyte size has been proposed as a morphologic feature of malignancy. Specifically, normal adipose tissue and benign lipomas tend to contain adipocytes of uniform size, whereas atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) is described as containing adipocytes with a conspicuous variation in cell size. However, this proposed variance has never been objectively, quantitatively correlated with diagnosis. Using whole-slide scanning combined with semiautomated digital image analysis, we aimed to quantitatively test the hypothesis that variance in adipocyte size is a feature of malignancy in well-differentiated lipomatous tumors. Whole-slide scanning was performed on representative hematoxylin and eosin-stained slides selected from 130 cases representing benign (lipoma, spindle cell lipoma) and malignant lipomatous neoplasms (ALT/WDL) and morphologic mimics (normal adipose tissue, fat necrosis, fat atrophy). A previously validated, open source software package (Fiji-based Adiposoft) was used in semiautomated analysis of adipocyte size from a representative 1-cm2 portion of each slide. Median, range, and variance of cell sizes were compared across all diagnoses. Fat atrophy demonstrated smaller adipocyte cell size compared with other diagnoses. Among the remaining diagnostic groups, no significant differences were identified in adipocyte size or variance by objective quantitative morphologic analysis. However, the maximum range of adipocyte size was significantly higher in ALT/WDL than conventional lipoma and spindle cell lipoma. These data quantitate the morphology of ALT/WDL and its mimics and more specifically define the somewhat subjective "variability" of cell size as maximum range, rather than variance, of adipocyte size.

  View details for DOI 10.1016/j.humpath.2017.10.030

  View details for PubMedID 29128479

• Recurrent GNA14 mutations in anastomosing haemangiomas. Histopathology Bean, G. R., Joseph, N. M., Folpe, A. L., Horvai, A. E., Umetsu, S. E. 2018; 73 (2): 354?57

  View details for DOI 10.1111/his.13519

  View details for PubMedID 29574926

• Genomic profiling of breast secretory carcinomas reveals distinct genetics from other breast cancers and similarity to mammary analog secretory carcinomas. Modern pathology Krings, G., Joseph, N. M., Bean, G. R., Solomon, D., Onodera, C., Talevich, E., Yeh, I., Grenert, J. P., Hosfield, E., Crawford, E. D., Jordan, R. C., van Zante, A., Zaloudek, C., Shin, S. J., Chen, Y. 2017

  Abstract

  Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.

  View details for DOI 10.1038/modpathol.2017.32

  View details for PubMedID 28548128

• Arginine Deprivation Inhibits the Warburg Effect and Upregulates Glutamine Anaplerosis and Serine Biosynthesis in ASS1-Deficient Cancers CELL REPORTS Kremer, J. C., Prudner, B. C., Lange, S. E., Bean, G. R., Schultze, M. B., Brashears, C. B., Radyk, M. D., Redlich, N., Tzeng, S., Kami, K., Shelton, L., Li, A., Morgan, Z., Bomalaski, J. S., Tsukamoto, T., McConathy, J., Michel, L. S., Held, J. M., Van Tine, B. A. 2017; 18 (4): 991-1004

  Abstract

  Targeting defects in metabolism is an underutilized strategy for the treatment of cancer. Arginine auxotrophy resulting from the silencing of argininosuccinate synthetase 1 (ASS1) is a common metabolic alteration reported in a broad range of aggressive cancers. To assess the metabolic effects that arise from acute and chronic arginine starvation in ASS1-deficient cell lines, we performed metabolite profiling. We found that pharmacologically induced arginine depletion causes increased serine biosynthesis, glutamine anaplerosis, oxidative phosphorylation, and decreased aerobic glycolysis, effectively inhibiting the Warburg effect. The reduction of glycolysis in cells otherwise dependent on aerobic glycolysis is correlated with reduced PKM2 expression and phosphorylation and upregulation of PHGDH. Concurrent arginine deprivation and glutaminase inhibition was found to be synthetic lethal across a spectrum of ASS1-deficient tumor cell lines and is sufficient to cause in vivo tumor regression in mice. These results identify two synthetic lethal therapeutic strategies exploiting metabolic vulnerabilities of ASS1-negative cancers.

  View details for DOI 10.1016/j.celrep.2016.12.077

  View details for Web of Science ID 000396474300014

  View details for PubMedID 28122247

  View details for PubMedCentralID PMC5840045

• Fibroepithelial lesions; The WHO spectrum. Seminars in diagnostic pathology Krings, G., Bean, G. R., Chen, Y. Y. 2017; 34 (5): 438?52

  Abstract

  Fibroepithelial lesions of the breast comprise a morphologically and biologically heterogeneous group of biphasic tumors with epithelial and stromal components that demonstrate widely variable clinical behavior. Fibroadenomas are common benign tumors with a number of histologic variants, most of which pose no diagnostic challenge. Cellular and juvenile fibroadenomas can have overlapping features with phyllodes tumors and should be recognized. Phyllodes tumors constitute a spectrum of lesions with varying clinical behavior and are graded as benign, borderline or malignant based on a set of histologic features according to recommendations by the World Health Organization (WHO). Recent developments have significantly expanded our understanding of the pathogenesis of fibroepithelial lesions, highlighting fibroadenomas as true neoplasms and underscoring a commonality with phyllodes tumors in the form of recurrent MED12 exon 2 mutations. In addition, sequencing studies have elucidated pathways associated with phyllodes tumor progression. Accurate diagnosis and grading of phyllodes tumors are important for patient management and prognosis, as grade broadly correlates with increasing local recurrence risk, and essentially only malignant tumors metastasize. However, classification of fibroepithelial lesions in many cases remains challenging on both core biopsy and excision specimens. A commonly encountered problem at the benign end of the spectrum is the distinction of benign phyllodes tumor from cellular fibroadenoma, which is largely due to the subjective nature of histologic features used in diagnosis and histologic overlap between lesions. Grading is further complicated by the requirement to integrate multiple subjective and ill-defined parameters. On the opposite end of the histologic spectrum, malignant phyllodes tumors must be distinguished from more common metaplastic carcinomas and from primary or metastatic sarcomas, which can be especially difficult in core biopsies. Immunohistochemistry can be useful in the differential diagnosis but should be interpreted with attention to caveats. This review provides an overview and update on the spectrum of fibroepithelial lesions, with special emphasis on common problems and practical issues in diagnosis.

  View details for DOI 10.1053/j.semdp.2017.05.006

  View details for PubMedID 28688536

• A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas CELL DEATH & DISEASE Bean, G. R., Kremer, J. C., Prudner, B. C., Schenone, A. D., Yao, J., Schultze, M. B., Chen, D. Y., Tanas, M. R., Adkins, D. R., Bomalaski, J., Rubin, B. P., Michel, L. S., Van Tine, B. A. 2016; 7

  Abstract

  Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

  View details for DOI 10.1038/cddis.2016.232

  View details for Web of Science ID 000387358800001

  View details for PubMedID 27735949

  View details for PubMedCentralID PMC5133958

• PUMA and BIM Are Required for Oncogene Inactivation-Induced Apoptosis SCIENCE SIGNALING Bean, G. R., Ganesan, Y. T., Dong, Y., Takeda, S., Liu, H., Chan, P. M., Huang, Y., Chodosh, L. A., Zambetti, G. P., Hsieh, J. J., Cheng, E. H. 2013; 6 (268)

  Abstract

  The clinical efficacy of tyrosine kinase inhibitors supports the dependence of distinct subsets of cancers on specific driver mutations for survival, a phenomenon called "oncogene addiction." We demonstrate that PUMA and BIM are the key apoptotic effectors of tyrosine kinase inhibitors in breast cancers with amplification of the gene encoding human epidermal growth factor receptor 2 (HER2) and lung cancers with epidermal growth factor receptor (EGFR) mutants. The BH3 domain containing proteins BIM and PUMA can directly activate the proapoptotic proteins BAX and BAK to permeabilize mitochondria, leading to caspase activation and apoptosis. We delineated the signal transduction pathways leading to the induction of BIM and PUMA by tyrosine kinase inhibitors. Inhibition of the mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway caused increased abundance of BIM, whereas antagonizing the phosphoinositide 3-kinase (PI3K)-AKT pathway triggered nuclear translocation of the FOXO transcription factors, which directly activated the PUMA promoter. In a mouse breast tumor model, the abundance of PUMA and BIM was increased after inactivation of HER2. Moreover, deficiency of Bim or Puma impaired caspase activation and reduced tumor regression caused by inactivation of HER2. Similarly, deficiency of Puma impeded the regression of EGFR(L858R)-driven mouse lung tumors upon inactivation of the EGFR-activating mutant. Overall, our study identified PUMA and BIM as the sentinels that interconnect kinase signaling networks and the mitochondrion-dependent apoptotic program, which offers therapeutic insights for designing novel cell death mechanism-based anticancer strategies.

  View details for DOI 10.1126/scisignal.2003483

  View details for Web of Science ID 000316795100005

  View details for PubMedID 23532334

  View details for PubMedCentralID PMC3753585

• BID, BIM, and PUMA Are Essential for Activation of the BAX- and BAK-Dependent Cell Death Program SCIENCE Ren, D., Tu, H., Kim, H., Wang, G. X., Bean, G. R., Takeuchi, O., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., Cheng, E. H. 2010; 330 (6009): 1390-1393

  Abstract

  Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are associated with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.

  View details for DOI 10.1126/science.1190217

  View details for Web of Science ID 000284902100045

  View details for PubMedID 21127253

  View details for PubMedCentralID PMC3163443

• CpG Island Tumor Suppressor Promoter Methylation in Non-BRCA-Associated Early Mammary Carcinogenesis CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Vasilatos, S. N., Broadwater, G., Barry, W. T., Baker, J. C., Lem, S., dietze, E. C., Bean, G. R., Bryson, A. D., Pilie, P. G., Goldenberg, V., Skaar, D., Paisie, C., Torres-Hemandez, A., Grant, T. L., Wilke, L. G., Ibarra-Drendall, C., Ostrander, J. H., D'Amato, N. C., Zalles, C., Jirtle, R., Weaver, V. M., Seewaldt, V. L. 2009; 18 (3): 901-914

  Abstract

  Only 5% of all breast cancers are the result of BRCA1/2 mutations. Methylation silencing of tumor suppressor genes is well described in sporadic breast cancer; however, its role in familial breast cancer is not known.CpG island promoter methylation was tested in the initial random periareolar fine-needle aspiration sample from 109 asymptomatic women at high risk for breast cancer. Promoter methylation targets included RARB (M3 and M4), ESR1, INK4a/ARF, BRCA1, PRA, PRB, RASSF1A, HIN-1, and CRBP1.Although the overall frequency of CpG island promoter methylation events increased with age (P<0.0001), no specific methylation event was associated with age. In contrast, CpG island methylation of RARB M4 (P=0.051), INK4a/ARF (P=0.042), HIN-1 (P=0.044), and PRA (P=0.032), as well as the overall frequency of methylation events (P=0.004), was associated with abnormal Masood cytology. The association between promoter methylation and familial breast cancer was tested in 40 unaffected premenopausal women in our cohort who underwent BRCA1/2 mutation testing. Women with BRCA1/2 mutations had a low frequency of CpG island promoter methylation (15 of 15 women had

  View details for DOI 10.1158/1055-9965.EPI-08-0875

  View details for Web of Science ID 000264226100030

  View details for PubMedID 19258476

  View details for PubMedCentralID PMC2667866

• ESR1 promoter hypermethylation does not predict atypia in RPFNA nor persistent atypia after 12 months tamoxifen chemoprevention CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Baker, J. C., Ostrander, J. H., Lem, S., Broadwater, G., Bean, G. R., D'Amato, N. C., Goldenberg, V. K., Rowell, C., Ibarra-Drendall, C., Grant, T., Pilie, P. G., Vasilatos, S. N., Troch, M. M., Scott, V., Wilke, L. G., Paisie, C., Rabiner, S. M., Torres-Hernandez, A., Zalles, C. M., Seewaldt, V. L. 2008; 17 (8): 1884-1890

  Abstract

  Currently, we lack biomarkers to predict whether high-risk women with mammary atypia will respond to tamoxifen chemoprevention.Thirty-four women with cytologic mammary atypia from the Duke University High-Risk clinic were offered tamoxifen chemoprevention. We tested whether ESR1 promoter hypermethylation and/or estrogen receptor (ER) protein expression by immunohistochemistry predicted persistent atypia in 18 women who were treated with tamoxifen for 12 months and in 16 untreated controls.We observed a statistically significant decrease in the Masood score of women on tamoxifen chemoprevention for 12 months compared with control women. This was a significant interaction effect of time (0, 6, and 12 months) and treatment group (tamoxifen versus control) P = 0.0007. However, neither ESR1 promoter hypermethylation nor low ER expression predicted persistent atypia in Random Periareolar Fine Needle Aspiration after 12 months tamoxifen prevention.Results from this single institution pilot study provide evidence that, unlike for invasive breast cancer, ESR1 promoter hypermethylation and/or low ER expression is not a reliable marker of tamoxifen-resistant atypia.

  View details for DOI 10.1158/1055-9965.EPI-07-2696

  View details for Web of Science ID 000258800800008

  View details for PubMedID 18708376

  View details for PubMedCentralID PMC2717700

• Morphologically normal-appearing mammary epithelial cells obtained from high-risk women exhibit methylation silencing of INK4a/ARF CLINICAL CANCER RESEARCH Bean, G. R., Bryson, A. D., Pilie, P. G., Goldenberg, V., Baker, J. C., Ibarra, C., Brander, D. M., Paisie, C., Case, N. R., Gauthier, M., Reynolds, P. A., Dietze, E., Ostrander, J., Scott, V., Wilke, L. G., Yee, L., Kimler, B. F., Fabian, C. J., Zalles, C. M., Broadwater, G., Tisty, T. D., Seewaldt, V. L. 2007; 13 (22): 6834-6841

  Abstract

  p16(INK4a) has been appreciated as a key regulator of cell cycle progression and senescence. Cultured human mammary epithelial cells that lack p16(INK4a) activity have been shown to exhibit premalignant phenotypes, such as telomeric dysfunction, centrosomal dysfunction, a sustained stress response, and, most recently, a dysregulation of chromatin remodeling and DNA methylation. These data suggest that cells that lack p16(INK4a) activity would be at high risk for breast cancer development and may exhibit an increased frequency of DNA methylation events in early cancer.To test this hypothesis, the frequencies of INK4a/ARF promoter hypermethylation, as well as four additional selected loci, were tested in the initial random periareolar fine needle aspiration samples from 86 asymptomatic women at high risk for development of breast cancer, stratified using the Masood cytology index.INK4a/ARF promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia and, importantly, in morphologically normal-appearing mammary epithelial cells; 29 of 86 subjects showed INK4a/ARF promoter hypermethylation in at least one breast. Importantly, INK4a/ARF promoter hypermethylation was not associated with atypia, and the frequency of hypermethylation did not increase with increasing Masood cytology score. The frequency of INK4a/ARF promoter hypermethylation was associated with the combined frequency of promoter hypermethylation of retinoic acid receptor-beta2, estrogen receptor-alpha, and breast cancer-associated 1 genes (P = 0.001).Because INK4a/ARF promoter hypermethylation does not increase with age but increases with the frequency of other methylation events, we predict that INK4a/ARF promoter hypermethylation may serve as a marker of global methylation dysregulation.

  View details for DOI 10.1158/1078-0432.CCR-07-0407

  View details for Web of Science ID 000251207100037

  View details for PubMedID 18006786

• Atypia in random periareolar fine-needle aspiration affects the decision of women at high risk to take tamoxifen for breast cancer chemoprevention CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Goldenberg, V. K., Seewaldt, V. L., Scott, V., Bean, G. R., Broadwater, G., Fabian, C., Kimler, B., Zalles, C., Lipkus, I. M. 2007; 16 (5): 1032-1034

  Abstract

  Random periareolar fine-needle aspiration (RPFNA) is a research procedure designed to (a) evaluate short-term breast cancer risk in women at high risk for developing breast cancer, and (b) track response to chemoprevention. Of import, cellular atypia in breast RPFNA is prospectively associated with a 5.6-fold increase in breast cancer risk in women at high risk. Among 99 women attending a clinic for high-risk breast cancer, we explored the effects of RPFNA cytology results on decision making pertaining to the use of tamoxifen for breast cancer chemoprevention. No patient with nonproliferative or hyperplastic cytology subsequently elected to take tamoxifen. Only 7% of subjects with borderline atypia elected to take tamoxifen. In contrast, 50% with atypia elected to take tamoxifen. These results suggest that the provision of a biomarker of short-term risk can affect the motivation to take tamoxifen for chemoprevention. This conclusion is informative given that tamoxifen, due to its side effects, is often underused by women at high risk of developing breast cancer. Further research is needed to determine the mechanisms through which RPFNA results affect the decision to use tamoxifen, or any other breast cancer chemopreventive agent.

  View details for DOI 10.1158/1055-9965.EPI-06-0910

  View details for Web of Science ID 000246649200031

  View details for PubMedID 17507634

• Interferon regulatory factor-1 regulates reconstituted extracellular matrix (rECM)-mediated apoptosis in human mammary epithelial cells ONCOGENE Bowie, M. L., Troch, M. M., Delrow, J., Dietze, E. C., Bean, G. R., Ibarra, C., Pandiyan, G., Seewaldt, V. L. 2007; 26 (14): 2017-2026

  Abstract

  Interactions between extracellular matrix (ECM) and mammary epithelial cells are critical for mammary gland homeostasis and apoptotic signaling. Interferon regulatory factor-1 (IRF-1) is a transcriptional regulator that promotes apoptosis during mammary gland involution and p53-independent apoptosis. We have recently shown that rapid cell surface tamoxifen (Tam) signaling promotes apoptosis in normal human mammary epithelial cells that were acutely damaged by expression of human papillomavirus type-16 E6 protein (*HMEC-E6). Apoptosis was mediated by recruitment of CREB-binding protein (CBP) to the gamma-activating sequence (GAS) element of the IRF-1 promoter, induction of IRF-1 and caspase-1/-3 activation. Here, we show that growth factor-depleted, reconstituted ECM (rECM), similar to Tam, promotes apoptosis in *HMEC-E6 cells through induction of IRF-1. Apoptosis was temporally associated with recruitment of CBP to the GAS element of the IRF-1 promoter, induction of IRF-1 expression and caspase-1/-3 activation. Small interfering RNA-mediated suppression of IRF-1 protein expression in *HMEC-E6 cells blocked (1) induction of IRF-1, (2) caspase-1/-3 activation and (3) apoptosis. These observations demonstrate that IRF-1 promotes rECM-mediated apoptosis and provide evidence that both rECM and rapid Tam signaling transcriptionally activate IRF-1 through recruitment of CBP to the IRF-1 GAS promoter complex.

  View details for DOI 10.1038/sj.onc.1210013

  View details for Web of Science ID 000245313400004

  View details for PubMedID 17016442

• Overweight and obese perimenopausal and postmenopausal women exhibit increased abnormal mammary epithelial cytology CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Seewaldt, V. L., Goldenberg, V., Jones, L. W., Peace, C., Broadwater, G., Scott, V., Bean, G. R., Wilke, L. G., Zalles, C. M., Demark-Wahnefried, W. 2007; 16 (3): 613-616

  Abstract

  High body mass index (BMI >or= 25 kg/m2) is associated with increased postmenopausal breast cancer incidence and mortality. However, few studies have explored associations between BMI and direct measures on target tissue. Epithelial cytology was assessed in 62 high-risk perimenopausal and postmenopausal women using random periareolar fine needle aspiration. Masood cytology index scores were significantly higher among women with BMIs >or=25 kg/m2 than in women with BMIs <25 kg/m2 (13.9 +/- 0.42 versus 12.7 +/- 0.29 kg/m2; P = 0.017). Overweight or obese women also had significantly higher random periareolar fine needle aspiration epithelial cell counts compared with those who were normal weight (1,230 +/- 272 versus 521 +/- 185; P = 0.028). These data suggest that overweight in perimenopausal and postmenopausal women is associated with direct cytologic abnormalities within the breast. Further research is needed to confirm these findings and to determine if this potential biomarker is responsive to changes in body weight resulting from diet and/or exercise interventions.

  View details for DOI 10.1158/1055-9965.EPI-06-0878

  View details for Web of Science ID 000245093900042

  View details for PubMedID 17372261

• Hypermethylation of the breast cancer-associated gene 1 promoter does not predict cytologic atypia or correlate with surrogate end points of breast cancer risk CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Bean, G. R., Drendall, C. I., Goldenberg, V. K., Baker, J. C., Troch, M. M., Paisie, C., Wilke, L. G., Yee, L., Marcorn, P. K., Kimler, B. F., Fabian, C. J., Zalles, C. M., Broadwater, G., Scott, V., Seewaldt, V. L. 2007; 16 (1): 50-56

  Abstract

  Mutation of the breast cancer-associated gene 1 (BRCA1) plays an important role in familial breast cancer. Although hypermethylation of the BRCA1 promoter has been observed in sporadic breast cancer, its exact role in breast cancer initiation and association with breast cancer risk is unknown. The frequency of BRCA1 promoter hypermethylation was tested in (a) 14 primary breast cancer biopsies and (b) the initial random periareolar fine-needle aspiration (RPFNA) cytologic samples obtained from 61 asymptomatic women who were at increased risk for breast cancer. BRCA1 promoter hypermethylation was assessed from nucleotide -150 to nucleotide +32 relative to the transcription start site. RPFNA specimens were stratified for cytologic atypia using the Masood cytology index. BRCA1 promoter hypermethylation was observed at similar frequency in nonproliferative (normal; Masood

  View details for DOI 10.1158/1055-9965.EPI-06-0598

  View details for Web of Science ID 000243550100009

  View details for PubMedID 17220331

• Long-term raloxifene in a woman at high risk for breast cancer NEW ENGLAND JOURNAL OF MEDICINE Bean, G. R., Kimler, B. F., Seewaldt, V. L. 2006; 355 (15): 1620-1622

  View details for Web of Science ID 000241160400029

  View details for PubMedID 17035661

• CREB-binding protein regulates apoptosis and growth of HMECs grown in reconstituted ECM via laminin-5 JOURNAL OF CELL SCIENCE Dietze, E. C., Bowie, M. L., Mrozek, K., Caldwell, L. E., Neal, C., Marjoram, R. J., Troch, M. M., Bean, G. R., Yokoyama, K. K., Ibarra, C. A., Seewaldt, V. L. 2005; 118 (21): 5005-5022

  Abstract

  Interactions between normal mammary epithelial cells and extracellular matrix (ECM) are important for mammary gland homeostasis. Loss of interactions between ECM and normal mammary epithelial cells are thought to be an early event in mammary carcinogenesis. CREB-binding protein (CBP) is an important regulator of proliferation and apoptosis but the role of CBP in ECM signaling is poorly characterized. CBP was suppressed in basal-cytokeratin-positive HMECs (CK5/6+, CK14+, CK8-, CK18-, CK19-). Suppression of CBP resulted in loss of reconstituted ECM-mediated growth control and apoptosis and loss of laminin-5 alpha3-chain expression. Suppression of CBP in normal human mammary epithelial cells (HMECs) resulted in loss of CBP occupancy of the LAMA3A promoter and decreased LAMA3A promoter activity and laminin-5 alpha-3 chain expression. Exogenous expression of CBP in CBP-negative HMECs that have lost reconstituted ECM-mediated growth regulation and apoptosis resulted in (1) CBP occupancy of the LAMA3A promoter, (2) increased LAMA3A activity and laminin-5 alpha3-chain expression, and (3) enhancement of reconstituted ECM-mediated growth regulation and apoptosis. Similarly, suppression of laminin-5 alpha3-chain expression in HMECs resulted in loss of reconstituted ECM-mediated growth control and apoptosis. These observations suggest that loss of CBP in basal-cytokeratin-positive HMECs results in loss of reconstituted ECM-mediated growth control and apoptosis through loss of LAMA3A activity and laminin-5 alpha3-chain expression. Results in these studies may provide insight into early events in basal-type mammary carcinogenesis.

  View details for DOI 10.1242/jcs.02616

  View details for Web of Science ID 000233678700012

  View details for PubMedID 16219677

• Retinoic acid receptor-beta 2 promoter methylation in random periareolar fine needle aspiration CANCER EPIDEMIOLOGY BIOMARKERS & PREVENTION Bean, G. R., Scott, V., Yee, L., Ratliff-Daniel, B., Troch, M. M., Seo, P., Bowie, M. L., Marcom, P. K., Slade, J., Kimler, B. F., Fabian, C. J., Zalles, C. M., Broadwater, G., Baker, J. C., Wilke, L. G., Seewaldt, V. L. 2005; 14 (4): 790-798

  Abstract

  Methylation of the retinoic acid receptor-beta2 (RARbeta2) P2 promoter is hypothesized to be an important mechanism for loss of RARbeta2 function during early mammary carcinogenesis. The frequency of RARbeta2 P2 methylation was tested in (a) 16 early stage breast cancers and (b) 67 random periareolar fine needle aspiration (RPFNA) samples obtained from 38 asymptomatic women who were at increased risk for breast cancer. Risk was defined as either (a) 5-year Gail risk calculation > or = 1.7%; (b) prior biopsy exhibiting atypical hyperplasia, lobular carcinoma in situ, or ductal carcinoma in situ; or (c) known BRCA1/2 mutation carrier. RARbeta2 P2 promoter methylation was assessed at two regions, M3 (-51 to 162 bp) and M4 (104-251 bp). In early stage cancers, M4 methylation was observed in 11 of 16 (69%) cases; in RPFNA samples, methylation was present at M3 and M4 in 28 of 56 (50%) and 19 of 56 (38%) cases, respectively. RPFNAs were stratified for cytologic atypia using the Masood cytology index. The distribution of RARbeta2 P2 promoter methylation was reported as a function of increased cytologic abnormality. Methylation at both M3 and M4 was observed in (a) 0 of 10 (0%) of RPFNAs with Masood scores of < or = 10 (nonproliferative), (b) 3 of 20 (15%) with Masood scores of 11 to 12 (low-grade proliferative), (c) 3 of 10 (30%) with Masood scores of 13 (high-grade proliferative), and (d) 7 of 14 (50%) with Masood scores of 14 of 15 (atypia). Results from this study indicate that the RARbeta2 P2 promoter is frequently methylated (69%) in primary breast cancers and shows a positive association with increasing cytologic abnormality in RPFNA.

  View details for Web of Science ID 000228351300006

  View details for PubMedID 15824145

• Interferon-regulatory factor-1 is critical for tamoxifen-mediated apoptosis in human mammary epithelial cells ONCOGENE Bowie, M. L., Dietze, E. C., Delrow, J., Bean, G. R., Troch, M. M., Marjoram, R. J., Seewaldt, V. L. 2004; 23 (54): 8743-8755

  Abstract

  Unlike estrogen receptor-positive (ER(+)) breast cancers, normal human mammary epithelial cells (HMECs) typically express low nuclear levels of ER (ER poor). We previously demonstrated that 1.0 microM tamoxifen (Tam) promotes apoptosis in acutely damaged ER-poor HMECs through a rapid, 'nonclassic' signaling pathway. Interferon-regulatory factor-1 (IRF-1), a target of signal transducer and activator of transcription-1 transcriptional regulation, has been shown to promote apoptosis following DNA damage. Here we show that 1.0 microM Tam promotes apoptosis in acutely damaged ER-poor HMECs through IRF-1 induction and caspase-1/3 activation. Treatment of acutely damaged HMEC-E6 cells with 1.0 microM Tam resulted in recruitment of CBP to the gamma-IFN-activated sequence element of the IRF-1 promoter, induction of IRF-1, and sequential activation of caspase-1 and -3. The effects of Tam were blocked by expression of siRNA directed against IRF-1 and caspase-1 inhibitors. These data indicate that Tam induces apoptosis in HMEC-E6 cells through a novel IRF-1-mediated signaling pathway that results in activated caspase-1 and -3.

  View details for DOI 10.1038/sj.onc.1208120

  View details for Web of Science ID 000225165100004

  View details for PubMedID 15467738

• Re: Active tamoxifen metabolite plasma concentrations after coadministration of tamoxifen and the selective serotonin reuptake inhibitor paroxetine JOURNAL OF THE NATIONAL CANCER INSTITUTE Ratliff, B., Dietze, E. C., Bean, G. R., Moore, C., Wanko, S., Seewaldt, V. L. 2004; 96 (11): 883-883

  View details for DOI 10.1093/jnci/djh170

  View details for Web of Science ID 000221715500019

  View details for PubMedID 15173275

• Tamoxifen and tamoxifen ethyl bromide induce apoptosis in acutely damaged mammary epithelial cells through modulation of AKT activity ONCOGENE Dietze, E. C., Troch, M. M., Bean, G. R., Heffner, J. B., Bowie, M. L., Rosenberg, P., Ratliff, B., Seewaldt, V. L. 2004; 23 (21): 3851-3862

  Abstract

  Normal human mammary epithelial cells (HMECs), unlike estrogen receptor-positive (ER+) breast cancers, typically express low nuclear levels of ER (ER-'poor'). We previously demonstrated that 1.0 microM tamoxifen (Tam) induced apoptosis in ER-'poor' HMECs acutely transduced with human papillomavirus-16 E6 (HMEC-E6) through a rapid mitochondrial signaling pathway. Here, we show that plasma membrane-associated E2-binding sites initiate the rapid apoptotic effects of Tam in HMEC-E6 cells through modulation of AKT activity. At equimolar concentrations, Tam and tamoxifen ethyl bromide (QTam), a membrane impermeant analog of Tam, rapidly induced apoptosis in HMEC-E6 cells associated with an even more rapid decrease in phosphorylation of AKT at serine-473. Treatment of HMEC-E6 cells with 1.0 microM QTam resulted in a 50% decrease in mitochondrial transmembrane potential, sequential activation of caspase-9 and -3, and a 90% decrease in AKT Ser-473 phosphorylation. The effects of both Tam and QTam were blocked by expression of constitutively active AKT (myristoylated AKT or AKT-Thr308Asp/Ser473Asp). These data indicate that Tam and QTam induce apoptosis in HMEC-E6 cells through a plasma membrane-activated AKT-signaling pathway that results in (1) decreased AKT phosphorylation at Ser-473, (2) mitochondrial membrane depolarization, and (3) activated caspase-9 and -3.

  View details for DOI 10.1038/sj.onc.1207480

  View details for Web of Science ID 000221242500012

  View details for PubMedID 14990993

• CBP/p300 induction is required for retinoic acid sensitivity in human mammary cells BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Dietze, E. C., Troch, M. M., Bowie, M. L., Yee, L., Bean, G. R., Seewaldt, V. L. 2003; 302 (4): 841-848

  Abstract

  The coactivators CBP and p300 are recruited by retinoic acid receptors (RARs) during retinoid mediated transcriptional regulation. To assess the role of CBP/p300 in all-trans-retinoic acid (ATRA)-mediated growth arrest in mammary epithelial cells, two systems were tested: (1) ATRA resistant MCF-7 cells were transduced with a functional RAR-beta 2; (2) normal human mammary epithelial cells (HMECs) were transduced with a pan-RAR dominant negative, RAR-alpha 403. Expression of RAR-beta 2 in MCF-7 cells resulted in increased sensitivity to ATRA-induced growth arrest and correlated with induction of CBP/p300 mRNA and protein. Inhibition of RAR function in HMECs resulted in resistance to ATRA-induced growth arrest and loss of CBP/p300 induction. Antisense suppression of CBP/p300 in HMECs resulted in decreased retinoic acid response element reporter trans-activation and decreased ATRA-mediated growth arrest. Thus, in human mammary epithelial cells, CBP/p300 were both modulated by an ATRA signaling pathway and were required for a normal response to ATRA.

  View details for DOI 10.1016/S006-291X(03)00266-3

  View details for Web of Science ID 000181881300032

  View details for PubMedID 12646247

• Retinoids and retinoic acid receptors regulate growth arrest and apoptosis in human mammary epithelial cells and modulate expression of CBP/p300 MICROSCOPY RESEARCH AND TECHNIQUE Dietze, E. C., Caldwell, L. E., Marcom, K., Collins, S. J., Yee, L., Swisshelm, K., Hobbs, K. B., Bean, G. R., Seewaldt, V. L. 2002; 59 (1): 23-40

  Abstract

  Retinoids and retinoic acid receptors (RARs) are important mediators of normal epithelial cell homeostasis. To assess the role of retinoids and RARs in regulating growth arrest and apoptosis in benign and malignant mammary epithelial cells, two model systems were developed: 1) RAR function was suppressed in retinoid-sensitive normal human mammary epithelial cells (HMECs) by the dominant-negative retinoic acid receptor, RARalpha403 (DNRAR), and 2) retinoid-resistant MCF-7 breast cancer cells were transduced with a functional RARbeta2. Inhibition of RAR function by the DNRAR in HMECs resulted in retinoid-resistance, increased proliferation, and dysregulated growth when cells were cultured in reconstituted extracellular matrix (rECM). Expression of RARbeta2 in MCF-7 cells resulted in sensitivity to retinoid-induced growth arrest and apoptosis. The CREB-binding protein (CBP) and the homologous protein p300 are tightly regulated, rate-limiting integrators of diverse signaling pathways and are recruited during retinoid-mediated transcriptional activation. The relationship between retinoid receptor expression, growth regulation, and transcriptional regulation of CBP/p300 is poorly understood. Inhibition of RAR function in HMECs by DNRAR suppressed expression of CBP/p300 and expression of RARbeta2 in MCF-7 cells promoted induction of CBP/p300 when cells were treated with 1.0 microM all-trans-retinoic acid (ATRA). These results suggest that ATRA and RARs regulate growth arrest of HMECs and modulate CBP/p300 protein expression. Since CBP and p300 are normally present in limiting amounts, their regulation by ATRA and RARs may be an important element in the control of transcriptional activation of genes regulating growth arrest and apoptosis.

  View details for DOI 10.1002/jemt.10174

  View details for Web of Science ID 000178264400004

  View details for PubMedID 12242694