Immunohistochemical assessment of HER2 low breast cancer: interobserver reproducibility and correlation with digital image analysis.

PURPOSE: The recent findings from the DESTINY-Breast04 trial highlighted the clinical importance of distinguishing between HER2 immunohistochemistry (IHC) scores 0 and 1 + in metastatic breast cancer (BC). However, pathologist interpretation of HER2 IHC scoring is subjective, and standardized methodology is needed. We evaluated the consistency of HER2 IHC scoring among pathologists and the accuracy of digital image analysis (DIA) in interpreting HER2 IHC staining in cases of HER2-low BC. METHODS: Fifty whole-slide biopsies of BC with HER2 IHC staining were evaluated, comprising 25 cases originally reported as IHC score 0 and 25 as 1 +. These slides were digitally scanned. Six pathologists with breast expertise independently reviewed and scored the scanned images, and DIA was applied. Agreement among pathologists and concordance between pathologist scores and DIA results were statistically analyzed using Kendall coefficient of concordance (W) tests. RESULTS: Substantial agreement among at least five of the six pathologists was found for 18 of the score 0 cases (72%) and 15 of the score 1 + cases (60%), indicating excellent interobserver agreement (W = 0.828). DIA scores were highly concordant with pathologist scores in 96% of cases (47/49), indicating excellent concordance (W = 0.959). CONCLUSION: Although breast subspecialty pathologists were relatively consistent in evaluating BC with HER2 IHC scores of 0 and 1 +, DIA may be a reliable supplementary tool to enhance the standardization and quantification of HER2 IHC assessment, especially in challenging cases where results may be ambiguous (i.e., scores 0-1 +). These findings hold promise for improving the accuracy and consistency of HER2 testing.

A Comparative Evaluation of TRPS1 and GATA3 in adenoid cystic, secretory, and acinic cell carcinomas of the breast and salivary gland.

GATA3 is the most used marker to determine tumors' breast origin, but its diagnostic value in triple-negative breast cancer (TNBC) is limited. The newly identified TRPS1 is highly sensitive and specific for breast carcinoma, especially TNBC. Here, we compared the utility of TRPS1 and GATA3 expression in a subset of salivary gland-type breast tumors (including adenoid cystic, acinic cell, and secretory carcinomas [AdCC, ACC, and SC, respectively]), and we compared TRPS1 and GATA3 expression of such tumors with head and neck (H&N) and AdCC of upper respiratory tumors. TRPS1 was strongly expressed in basaloid TNBC and AdCCs with solid components, including 100 % of mixed and solid breast AdCCs. However, TRPS1 was positive in only 50 % cribriform AdCCs. Expression patterns of TRPS1 in H&N and upper respiratory AdCC were similar. TRPS1 was positive in 30 % of H&N cribriform AdCCs but was strongly expressed in mixed AdCC (67 %) and solid AdCC (100 %). In the upper respiratory AdCCs, TRPS1 was positive in 58.4 % of cribriform AdCCs and positive in 100 % of AdCCs with solid components. On the contrary, GATA3 was negative in predominant AdCCs of the breast, H&N, and upper respiratory tract. These data show that GATA3 and TRPS1 expression varies AdCCs. In addition, TRPS1 and GATA3 expression patterns were similar SC and ACC of breast and H&N. Both markers were positive in SC and negative in ACC. Therefore, TRPS1 and GATA3 cannot be used to differentiate salivary gland-type carcinomas of breast origin from those of upper respiratory or H&N origin.

Nuclear staining for pan-Trk by immunohistochemistry is highly specific for secretory carcinoma of breast: pan-Trk in various subtypes of breast carcinoma.

AIMS: Secretory carcinoma of breast (SCB) typically harbours ETV6-NTRK3 gene fusion. Pan-Trk immunohistochemistry analysis (IHC) has been shown to be sensitive for SCB diagnosis. However, weak focal pan-Trk nuclear staining was previously found in 10% of non-secretory breast carcinomas. To further examine pan-Trk IHC specificity, we evaluated pan-Trk staining in various breast carcinoma subtypes. METHODS: The study cohort consisted of 346 invasive breast carcinomas (IBCs), including 8 SCBs and 48 triple-negative histological mimickers (36 metaplastic carcinomas, including 12 matrix-producing carcinomas; 5 adenoid cystic carcinomas; 5 apocrine carcinomas; 2 acinic cell carcinomas), 101 triple-negative IBCs of no special type, 101 estrogen receptor (ER)-positive/HER2-negative IBCs and 88 HER2-positive IBCs. Six salivary gland secretory carcinomas were also included. Pan-Trk IHC was performed on tumours using a rabbit monoclonal pan-Trk antibody. Any nuclear staining in the invasive carcinoma cells was considered positive. RESULTS: All 14 secretory carcinomas from breast and salivary gland exhibited moderate to strong pan-Trk nuclear staining. In contrast, no pan-Trk nuclear staining was identified in any of the 338 non-secretory IBCs. Focal cytoplasmic pan-Trk staining was observed in nine non-secretory IBCs (2.7%), and was considered nonspecific and negative. CONCLUSIONS: Our results indicate that pan-Trk nuclear staining is highly specific for SCB. In low-grade to intermediate-grade IBCs that share histological features with SCB, adding pan-Trk to a routing panel of estrogen receptor/progesterone receptor/HER2 is highly diagnostic. Our results also support using pan-Trk IHC to differentiate SCB from its triple-negative histological mimickers, such as adenoid cystic carcinoma, matrix-producing carcinoma, apocrine carcinoma and acinic cell carcinoma.

Breast Cancer With a HER2 FISH Group 2 Result: Should HER2 Tests be Repeated?

BACKGROUND: Breast cancer with fluorescence in situ hybridization (FISH) group 2 pattern (HER2 <4 and HER2/CEP17 ratio ≥2, a subset of monosomy CEP17) was historically considered HER2-positive, but mostly HER2-negative according to updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines unless 3+ by immunohistochemistry (IHC). Therapeutic relevance of this group remained elusive, therefore we assessed if repeat IHC and FISH can assist final HER2 classification. PATIENT AND METHODS: We retrospectively reviewed HER2 FISH performed at our institution from 2014 to 2018 and identified 23 of 3554 (0.6%) breast cancer cases with at least one-time measurement of HER2 FISH categorized as group 2. Repeat HER2 tests were performed for cases with available alternative tumor samples and compared with initial testing following 2018 ASCO/CAP guidelines. RESULTS: Only 1 of 23 group 2 cases was HER2-positive, 0/18 in primary and 1/5 in metastatic/recurrent tumors. Of 13 primary tumors with repeat HER2 results; 10 (77%) remained HER2-negative; 3 (23%) changed from HER2-negative (group 2 and IHC 2+) to HER2-positive (group 1 and IHC 2+). Among 8 of these 13 patients receiving neoadjuvant systemic therapy containing anti-HER2 agent, 3 (38%) achieved pathologic complete response (pCR). Two of 3 pCR cases were HER2-positive converters on repeat testing. Three pCR cases were ER-negative or -low positive and Ki67 ≥40%, while 5 partial responders were ER-positive and Ki67 <40% (P < .05). CONCLUSION: Breast cancer with HER2 FISH group 2 result may represent heterogeneous populations of tumor cells being originated de novo or preferentially selected secondary to therapy. Repeat HER2 tests on alternative samples may be considered to guide anti-HER2 therapy.

Immunohistochemical expression of TRPS1 in mammary Paget disease, extramammary Paget disease, and their close histopathologic mimics.

BACKGROUND: Trichorhinophalangeal syndrome type 1 (TPRS1) expression has been found to be highly sensitive and specific for breast carcinomas. The frequency of TRPS1 expression in cutaneous neoplasms such as mammary Paget disease (MPD) and extramammary PD (EMPD) is currently unknown. We assessed the utility of TRPS1 immunohistochemistry (IHC) in the evaluation of MPD, EMPD, and their histopathologic mimics, squamous cell carcinoma in situ (SCCIS) and melanoma in situ (MIS). METHODS: Twenty-four MPDs, 19 EMPDs, 13 SCCISs, and 9 MISs were subjected to immunohistochemical analysis using anti-TRPS1 antibody. The intensity (none, 0; weak, 1+ ; moderate, 2+ ; strong, 3+ ) and proportion (<1%, absent; 1%-25%, focal; 26%-75%, patchy; >75%, diffuse) of TRPS1 expression were recorded. Relevant clinical data were documented. RESULTS: TPRS1 expression was present in 100% (24/24) of MPDs, with 88% (21/24) of MPDs exhibiting strong, diffuse immunoreactivity. Sixty-eight percent (13/19) of EMPDs showed TRPS1 expression. Intriguingly, EMPDs lacking TRPS1 expression were consistently of perianal origin. TRPS1 expression was seen in 92% (12/13) of SCCISs but was absent in all MISs. CONCLUSIONS: TRPS1 may be useful to distinguish MPDs/EMPDs from MISs, but its utility is limited in distinguishing them from other pagetoid intraepidermal neoplasms such as SCCISs.

Metastatic nonhematopoietic neoplasms to the breast: a study of 238 cases.

The aim of this study was to review the clinicopathologic characteristics of metastatic nonhematopoietic malignancies to the breast, in order to identify salient features for practicing pathologists that are useful in distinguishing metastatic lesions from primary breast neoplasms. A total of 238 cases were identified during the period from January 2005 to January 2015. Clinicopathologic features of these cases were retrospectively reviewed. Primary tumors included melanoma (99, 42%), serous carcinoma (35, 15%), neuroendocrine neoplasm (32, 13%), sarcoma (23, 10%), and adenocarcinoma from various organs (47, 20%), and 2 others. Most metastases were unilateral (223, 94%) and unifocal (206, 87%) and were detected radiographically (167, 70%). Concurrent ipsilateral axillary metastasis occurred in 33 (14%) patients. Among 238 cases, 41 had metastatic disease to the breast concurrently or preceding the primary cancer diagnosis. Notably, in 39 (16%) cases, breast metastasis was the first clinical presentation of disease, and 16 (41%) of these cases were initially misdiagnosed as breast primaries. In contrast, with a known history of nonmammary primary tumors, only 4 of 197 (2%) cases were misdiagnosed (p < 0.0001). Metastatic tumors share many overlapping features with breast primary carcinomas. However, cases with a well-circumscribed tumor, lack of in situ component, estrogen receptor/progesterone receptor negativity, and unusual morphologic features should raise the consideration of metastatic disease. While clinical history is paramount for correct diagnosis, metastasis to the breast as the first clinical presentation is not uncommon.

TRPS1, GATA3, and SOX10 expression in triple-negative breast carcinoma.

A diagnostic dilemma can be encountered when primary triple-negative breast carcinoma (TNBC) without an in situ component or metastatic TNBCs lose the currently used organ-specific marker such as GATA3, raising concerns about metastatic carcinoma from other sites. In the current study, we compared the newly identified breast marker TRPS1 with currently used breast markers GATA3 and SOX10 in whole-tissue sections from 315 cases of various subtypes of TNBC. TRPS1 was highly expressed in 100% of triple-negative primary and metastatic invasive lobular carcinomas, 99% of triple-negative primary and metastatic invasive breast carcinoma of no special type (IBC-NST), and 95% of metaplastic breast carcinomas. In contrast, GATA3 and SOX10 were expressed in 94% and 0% of invasive lobular carcinomas, 63% and 74% of IBC-NST, and 50% and 49% of metaplastic breast carcinomas, respectively. For special-type TNBCs, both TRPS1 and GATA3 were negative in acinic cell carcinomas, most cribriform adenoid cystic carcinomas, and neuroendocrine carcinomas, but positive in secretory carcinomas. Triple-negative apocrine carcinoma was the only subtype of TNBC with positive GATA3 but negative TRPS1. These data indicate that TRPS1 is a highly sensitive marker for TNBCs with positivity not only in GATA3/SOX10-positive TNBCs but also in almost all GATA3/SOX10-negative TNBCs.

Expression of TRPS1 in phyllodes tumor and sarcoma of the breast.

When a sarcomatous neoplasm is identified in the breast, distinguishing metaplastic carcinoma, malignant phyllodes tumor (MPT), and primary sarcoma is a diagnostic challenge, especially on small biopsies, as all these tumors may have overlapping morphological features, thoroughly grossing with histological examination and immunohistochemical staining being the standard approach to aid in classifying these lesions. Recently, we identified a highly sensitive and specific breast carcinoma marker TRPS1 with high expression in metaplastic breast carcinoma. In the current study, we tested TRPS1 in MPTs and primary sarcoma of the breast. We found TRPS1 was highly expressed (95%) within spindle cell, chondro-osseous, and/or liposarcomatous components of MPTs, in all breast primary chondrosarcomas and extraskeletal osteosarcomas, but not in other sarcomas of the breast. In extramammary sarcomas, TRPS1 was expressed in 28% of conventional chondrosarcomas and 56% of osteosarcomas of bone, but rarely in undifferentiated pleomorphic sarcomas (UPSs), liposarcomas, and angiosarcomas. In summary, MPTs may share similar genetic background with metaplastic carcinoma exhibiting TRPS1 expression, and TRPS1 may play a role in chondro-osseous differentiation because of its expression in chondro-osseous sarcomas from both breast and extramammary sites. Our findings suggest TRPS1 may be clinically useful in distinguishing MPT and metaplastic carcinoma from primary breast sarcoma except for tumors with chondro-osseous differentiation.

HER2 testing in breast cancers: comparison of assays and interpretation using ASCO/CAP 2013 and 2018 guidelines.

PURPOSE: HER2 overexpression and gene amplification are routinely tested by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), respectively. In addition, HER2 mRNA expression is also tested by the Oncotype DX assay. Discordance between laboratories among the different assays remains a problem. To improve the routine HER2 reporting, the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) updated their guidelines in 2018. Our study will compare concordance of HER2 status by IHC and FISH using ASCO/CAP 2013 and 2018 guidelines with Oncotype DX. METHODS: We retrospectively reviewed 657 estrogen receptor positive primary breast cancer cases with available Oncotype DX tests between 2011 and 2018. Medical records were reviewed for HER2 results by IHC, FISH, and Oncotype DX. The HER2 results by different assays and between 2013 and 2018 guidelines were compared. RESULTS: Of the 657 cases, 280 were tested by IHC, FISH, and Oncotype DX. HER2-equivocal cases by IHC 2013 guidelines were all negative (67/67, 100%) by FISH 2018 guidelines and by Oncotype DX. HER2-equivocal cases by FISH 2013 guidelines were all negative (16/16, 100%) by FISH 2018 guidelines, while 15/16 (93.8%) negative and 1/16 (6.2%) equivocal by Oncotype DX. The HER2-equivocal and HER2-negative groups were similar in age, gender, histology, grade, and Ki67 score. CONCLUSIONS: HER2 concordance was highest between Oncotype DX (99.6%) and FISH per 2018 guidelines. This suggests that the ASCO/CAP 2018 guidelines improved the accurate stratification of HER2-equivocal cases.

Imaging Features of Triple Negative Breast Cancer and the Effect of BRCA Mutations.

OBJECTIVE: The purpose of this study is to review the mammographic and the ultrasound features of triple negative breast cancer (TNBC) patients and to investigate the potential effect of BRCA mutations on the imaging features of these patients. METHODS: One hundred and seven patients with TNBC were enrolled in a retrospective study following IRB approval and approval of waiver of informed consent. BRCA mutations were assessed using genetic testing. Imaging features on mammography and ultrasound (US) as well as pathology and clinical information were retrospectively reviewed and characterized according to the BI-RADS lexicon (fifth edition). The relationships between BRCA mutations and the imaging findings were examined. RESULTS: TNBC commonly presented as an irregular mass with obscured margins on mammography and as an irregular hypoechoic mass with microlobulated or angular margins on US. Approximately two thirds of TNBC cases had a parallel orientation and approximately one third had posterior enhancement, features often associated with benign masses. There was no statistically significant difference in the mammographic and the US features of BRCA positive and BRCA negative triple negative tumors. CONCLUSION: TNBC may have a parallel orientation and posterior enhancement, which are features often seen with benign masses. BRCA mutations do not affect the imaging features of triple negative breast tumors.

Male Mammary Paget Disease: A Tale of 2 Contrasting Cases.

Mammary Paget disease (MPD) comprises 1.45% all male breast cancers, compared with only 0.68% of all female breast cancers. Patients usually present in the fifth and sixth decades of life with ulceration, eczematous changes, discharge, bleeding, itching, and induration of the nipple and areola. Typically, there is a delay in definitive diagnosis and treatment from the onset of symptoms because most patients are initially treated for a rash. At the time of diagnosis, about half of the patients may have palpable breast mass, positive lymph nodes, or both. In this article, we present 2 cases of male MPD representing the extremes of clinical, radiologic, and histopathologic spectrum of the disease. One patient presented with a rash of the nipple of several months duration without an underlying lesion, whereas the other presented with sensitivity and pain of the nipple for 1 year and an underlying mass. Biopsies were diagnostic of MPD in both cases, and definitive surgery revealed an underlying ductal carcinoma in situ in the first case and an invasive ductal carcinoma in the second, highlighting the importance of early biopsy to initiate appropriate management.

Ductal Carcinoma In Situ and Margins <2 mm: Contemporary Outcomes With Breast Conservation.

OBJECTIVE: To determine the relationship between negative margin width and locoregional recurrence (LRR) in a contemporary cohort of ductal carcinoma in situ (DCIS) patients. BACKGROUND: Recent national consensus guidelines recommend an optimal margin width of 2 mm or greater for the management of DCIS; however, controversy regarding re-excision remains when managing negative margins <2 mm. METHODS: One thousand four hundred ninety-one patients with DCIS who underwent breast-conserving surgery from 1996 to 2010 were identified from a prospectively managed cancer center database and analyzed using univariate and multivariate Cox proportional hazard models to determine the relationship between negative margin width and LRR with or without adjuvant radiation therapy (RT). RESULTS: A univariate analysis revealed that age <40 years (n = 89; P = 0.02), no RT (n = 298; P = 0.01), and negative margin width <2 mm (n = 120; P = 0.005) were associated with LRR. The association between margin width and LRR differed by adjuvant RT status (interaction P = 0.02). There was no statistical significant difference in LRR between patients with <2 mm and ≥2 mm negative margins who underwent RT (10-yr LRR rate, 4.8% vs 3.3%, respectively; hazard ratio, 0.8; 95% CI, 0.2-3.2; P = 0.72). For patients who did not undergo RT, those with margins <2 mm were significantly more likely to develop a LRR than were those with margins ≥2 mm (10-yr LRR rate, 30.9% vs 5.4%, respectively; hazard ratio, 5.5; 95% CI, 1.8-16.8, P = 0.003). CONCLUSIONS: Routine additional surgery may not be justified for patients with negative margins <2 mm who undergo RT but should be performed in patients who forego RT.

The isomiR-140-3p-regulated mevalonic acid pathway as a potential target for prevention of triple negative breast cancer.

BACKGROUND: Prevention of triple-negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis. METHODS: To identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represent early stages of TNBC development. We have identified direct gene targets of isomiRNA-140-3p and by using cell-based and in vivo model systems we have demonstrated the utility of targeting downstream pathways for prevention of TNBC. RESULTS: These analyses showed that 5'isomiRNA of miR-140-3p (miR-140-3p-1) and its novel direct gene targets, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, were deregulated in the normal-to-preneoplastic transition. Upregulation in the cholesterol pathway creates metabolic vulnerability that can be targeted. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin to inhibit growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin-induced HMGCR transcript levels to be directly correlated with the degree of histological progression of lesions, indicating that the extent of cholesterol pathway suppression directly correlates with abrogation of the tumorigenic process. To block the HMGCR feedback response to statins, we treated resistant preneoplastic cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin and metformin effectively abrogated the statin-induced aberrant upregulation of HMGCR and sensitized these resistant cells to fluvastatin. CONCLUSIONS: These results suggest the potential use of combined treatment with statin and aspirin for prevention of TNBC.

Pseudoangiomatous Stromal Hyperplasia of the Breast: Multimodality Review With Pathologic Correlation.

Pseudoangiomatous stromal hyperplasia (PASH) is a rare benign breast condition. PASH is thought to be hormonally responsive, and it is usually identified in premenopausal and perimenopausal women. PASH may also be seen in postmenopausal woman on hormone replacement therapy (HRT). Approximately 53% of patients with PASH present with abnormalities on screening mammography, and 44% of patients with PASH present with palpable abnormalities. On imaging studies, PASH appears similar to fibroadenomas. On mammography, PASH is usually seen as a noncalcified, circumscribed mass. On ultrasound, PASH often appears as an oval, circumscribed, hypoechoic mass. On magnetic resonance imaging, PASH usually has progressive (Type 1) enhancement, and high-signal slit-like spaces may be seen on T2-weighted and short tau inversion recovery (STIR) images. The slit-like spaces correspond to empty clefts within acellular hyalinized stroma on histopathology. PASH may be mistaken for a low-grade angiosarcoma on pathologic examination. While angiosarcoma has true vascular spaces, PASH has a network of pseudoangiomatous slit-like clefts. Women with biopsy-proven PASH usually undergo follow-up imaging. Surgical excision may be considered for larger lesions and in women at an increased risk for developing breast cancer. In the future, additional studies are needed to provide definitive data regarding appropriate management and long-term outcomes for women with PASH. PASH has become increasingly recognized, but the literature regarding the imaging features of PASH is scarce. This paper reviews the imaging and pathologic features of PASH and some processes that may simulate PASH are discussed. Features of PASH on mammography, ultrasound, MRI, and nuclear medicine studies are discussed with pathologic correlation.

High incidence of germline BRCA mutation in patients with ER low-positive/PR low-positive/HER-2 neu negative tumors.

BACKGROUND: The 2015 National Comprehensive Cancer Network guidelines recommend that genetic counseling and germline BRCA mutation testing be offered to women under age 60 with triple-negative breast cancer (TNBC). As a result of the 2010 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines for breast cancer, patients with breast cancers that are estrogen receptor (ER) or progesterone receptor (PR) low-positive (1%-9% on immunohistochemistry) are no longer strictly considered to have TNBC and may not be referred for genetic counseling. However, the incidence of BRCA mutation in patients with hormone receptor (HR) low-positive breast cancers remains unknown, and current ASCO/CAP guidelines may result in undertesting for BRCA mutations. METHODS: A prospectively maintained research database of breast cancer patients evaluated at The University of Texas MD Anderson Cancer Center between 2004 and 2014 was reviewed; 314 patients were identified with HER2/neu-negative breast cancers expressing ER and PR <10% with known BRCA mutation status. RESULTS: Three hundred fourteen patients had breast cancers expressing ER and PR <10%; 238 (75.8%) had HR-negative cancers (<1% ER and PR), and 76 (24.2%) had HR-low-positive cancers (1%-9% ER and/or PR). Among patients with HR-negative tumors, 86 of 238 (36.1%) had a BRCA1/2 mutation, whereas in the HR-low-positive group, 30 of 76 (39.5%) had a BRCA1/2 mutation. In multivariate analysis, HR status (<1% vs 1%-9%) was not significantly associated with BRCA1/2 mutations. CONCLUSIONS: The incidence of BRCA1/2 mutations is similar in patients with HR-low-positive breast cancer and patients with HR-negative breast cancer. Genetic counseling and BRCA testing should be offered to patients under age 60 who have HR-low-positive breast cancers. Cancer 2015;121:3435-43. © 2015 American Cancer Society.

Annexin A1 Preferentially Predicts Poor Prognosis of Basal-Like Breast Cancer Patients by Activating mTOR-S6 Signaling.

INTRODUCTION: Annexin A1 (ANXA1) is an anti-inflammatory protein reported to play a role in cell proliferation and apoptosis, and to be deregulated in breast cancer. The exact role of annexin A1 in the biology of breast cancer remains unclear. We hypothesized that the annexin A1 plays an oncogenic role in basal subtype of breast cancer by modulating key growth pathway(s). METHODS: By mining the Cancer Genome Atlas (TCGA)-Breast Cancer dataset and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. RESULTS: Our in-silico analysis of TCGA-breast cancer dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer overall survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer than luminal and Her2/neu-positive breast cancer. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway likely through activation of AMPK but had no impact on the MAPK, c-Met, and EGFR pathways. In a cell migration assay, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells, which could be responsible for poor patient prognosis in basal like breast cancers that are known to express higher annexin A1. CONCLUSIONS: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be in part due to the role of annexin A1 in activating mTOR-pS6 pathway.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer.

PURPOSE: Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. EXPERIMENTAL DESIGN: In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. RESULTS: We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. CONCLUSIONS: Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer.

Molecular cytogenetic characterization of mammary neuroendocrine carcinoma.

Primary mammary neuroendocrine carcinoma (NEC) is an uncommon entity that accounts for 2% to 5% of breast carcinomas. Recent reports have shown that NEC of the breast is an aggressive subtype of mammary carcinoma that is distinct from invasive ductal carcinoma, not otherwise specified, and have suggested that these tumors have a poorer prognosis than invasive ductal carcinoma, not otherwise specified. In this study, we provide the first cytogenetic characterization of mammary NEC using both conventional G-banding and spectral karyotype on a group of 7 tumors. We identified clonal chromosomal aberrations in 5 (71.4%) cases, with 4 of them showing complex karyotypes. Of these, recurrent numerical aberrations included gain of chromosome 7 (n = 2) and loss of chromosome 15 (n = 2). Recurrent clonal structural chromosomal aberrations involved chromosomes 1 (n = 3), 3 (n = 2), 6q (n = 3), and 17q (n = 3). Of the 4 (57.1%) cases with complex karyotypes, 2 showed evidence of chromothripsis, a phenomenon in which tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. One of these had evidence of chromothripsis involving chromosomes 1, 6, 8, and 15. The other also had evidence of chromosome 8 chromothripsis, making this a recurrent finding shared by both cases. We also found that mammary NEC shared some cytogenetic abnormalities--such as trisomy 7 and 12--with other neuroendocrine tumors in the lung and gastrointestinal tract, suggesting trisomy 7 and 12 as potential common molecular aberrations in neuroendocrine tumors. To our knowledge, this is the first report on molecular cytogenetic characterization of mammary NEC.

Suppression of Akt-mTOR pathway-a novel component of oncogene induced DNA damage response barrier in breast tumorigenesis.

DNA damage has been thought to be directly associated with the neoplastic progression by enabling mutations in tumor suppressor genes and activating/and amplifying oncogenes ultimately resulting in genomic instability. DNA damage causes activation of the DNA damage response (DDR) that is an important cellular mechanism for maintaining genomic integrity in the face of genotoxic stress. While the cellular response to genotoxic stress has been extensively studied in cancer models, less is known about the cellular response to oncogenic stress in the premalignant context. In the present study, by using breast tissues samples from women at different risk levels for invasive breast cancer (normal, proliferative breast disease and ductal carcinoma in situ) we found that DNA damage is inversely correlated with risk of invasive breast cancer. Similarly, in MCF10A based in vitro model system where we recapitulated high DNA damage conditions as seen in patient samples by stably cloning in cyclin E, we found that high levels of oncogene induced DNA damage, by triggering inhibition of a major proliferative pathway (AKT), inhibits cell growth and causes cells to die through autophagy. These data suggest that AKT-mTOR pathway is a novel component of oncogene induced DNA damage response in immortalized 'normal-like' breast cells and its suppression may contribute to growth arrest and arrest of the breast tumorigenesis.