ANNUAL MEETING ABSTRACTS 25A expression had shorter overall survival and disease-free survival at 3 or 5 years than those 2064 Beta-Human Chorionic Gonadotropin Expression in Giant Cell with low HDGF expression, respectively. In addition to distant metastasis, multivariate Tumors analysis demonstrated that HDGF expression was an independent strong predictor for ME Lawless, MH Rendi. University of Washington, Seattle, WA. OS and DFS in Ewing sarcoma/PNET patients (p=0.002, p<0.001, respectively). HDGF Background: Giant cell tumors are benign yet locally aggressive neoplasms of bone knockdown dramatically inhibited proliferation, migration, invasion, colony formation and tendons with a high incidence of recurrence after surgical resection. Additionally, and G1/S transition of cell cycle, but induced apoptosis in Ewing sarcoma cells. they have the potential to metastasize to distant sites such as the lung and lymph nodes, Conclusions: In conclusion, our results firstly suggest that nuclear HDGF expression and the diagnosis can be difficult to establish histologically in this setting. The beta is a significant adverse prognostic factor for overall survival and disease-free survival subunit of human chorionic gonadotropin (hCG) is expressed in benign and malignant in patients with Ewing sarcoma/PNET. Targeting HDGF may be a novel strategy for syncytiotrophoblasts, several non-gynecological neoplasms, and is commonly used as Ewing sarcoma/PNET treatment. a serum marker in diagnosis and therapeutic monitoring of the associated gynecological tumors. Furthermore, due to the young age of patients with giant cell tumor of bone, 93 Fibromatosis of the Breast: Complication of Previous Radiation urine beta-hCG is often performed to evaluate for pregnancy during the work-up of a and Surgery lesion. At our institution, we have observed multiple cases of elevated urine beta-hCG in Y Zhang, S Libson, E Avisar, M Jorda, C Gomez, AE Rosenberg. University of Miami/ patients with giant cell tumors, which has led to diagnostic difficulty and in one instance, Jackson Memorial Hospital, Miami, FL. concern for metastatic choriocarcinoma. The aim of this study is to determine if giant Background: Fibromatosis of the breast is uncommon, and comprises less than 0.2% cell tumors of bone express beta-hCG, and if so, at what frequency. of all breast tumors. It is a locally aggressive neoplasm and often recurs. Fibromatosis Design: We evaluated 15 cases of giant cell tumors consisting of 11 recurrent neoplasms harbors mutations in the beta catenin gene and predisposing factors include Gardner and 4 metastatic lesions. The neoplasms were analyzed for expression of beta-hCG syndrome and a history of previous surgery. Herein, we report five cases of fibromatosis by immunohistochemistry using the Dako polyclonal rabbit beta-hCG antibody and of the breast in patients with a history of prior surgery and radiation. staining was reviewed independently by 2 pathologists. Design: Departmental and consultation files were searched from 2003 -2012 for Results: 40% of cases (n=6) exhibited staining for beta-hCG. 13% had weak (1+) cases diagnosed as “desmoid” or “fibromatosis” arising in the breast. Eleven patients staining, while 20% demonstrated moderate staining (2+), and 7% had strong staining with pathologically confirmed breast desmoid tumor were identified. Records were (3+). retrospectively reviewed. Conclusions: Beta-hCG is frequently expressed by recurrent/metastatic giant cell Results: Among those eleven patients, one patient had Gardner syndrome, five patients tumors of bone and may be of diagnostic utility when establishing this diagnosis. In had a history of surgery for breast cancer, two patients had a history of surgery for addition, it is also important for clinicians and pathologists to be aware of beta-hCG bilateral breast augmentation, and the remaining three had no documented surgery expression by the neoplasm and the potential for elevated urine and serum beta-hCG prior to the development of fibromatosis. Among the seven patients who had surgery, levels in patients with giant cell tumor of bone so as to avoid misdiagnosis of pregnancy five of them, including one of the patients with breast augmentation, received radiation or gestational trophoblastic disease. Currently, the clinical and prognostic significance therapy before developing fibromatosis. The five patients were female and ranged in of beta-hCG expression by giant cell tumors as well as expression by primary tumors age from 36-57 (mean of 48.8) years. The fibromatosis tumors presented as palpable is unclear and further studies will address these questions. masses, clinically and radiographically suspicious for carcinoma. Four patients had a history of breast cancer treated with surgery and radiation (6 weeks of 6040 CGY). One patient had multiple bilateral breast augmentations and had superficial electron bean Breast radiation for cosmetic reasons to prevent scar formation. Fibromatosis was diagnosed at a median of 28 months post-radiation therapy. The tumors ranged from 2.7 cm to 5 95 The Expression of Cytokeratin 19 in Breast Carcinoma and Its cm. All of the patients had their tumor resected and 1 patient had a local recurrence 8 Association with Hormone Receptor Status months after the initial excision with positive margins. The remaining patients were NA Aardsma, JV Groth, SV Prabhu, EL Wiley. University of Illinois Hospital & Health disease free at last follow up (0 to 9 years). Microscopically, the tumors showed the Sciences System, Chicago, IL. classic features of fibromatosis. There was no significant cytologic atypia, pleomorphism Background: Cytokeratin 19 (CK19) is the lowest molecular weight cytokeratin and or necrosis. Immunohistochemically, the tumor cells expressed beta-catenin in all cases. is commonly used to mark pancreaticobilliary carcinoma in the liver and papillary Conclusions: The development of fibromatosis is a well recognized complication of carcinoma of the thyroid. CK19 has been shown to be expressed in normal breast tissue different types of therapeutic interventions. Fibromatosis of the breast is uncommon and breast carcinoma. CK19 mRNA is also used to detect circulating tumor cells and and should be considered when a new mass develops in the breast following surgery micro-metastases of breast carcinoma. CK19 expression and its relationship to breast and/or radiation. Excision of the mass with or without adjuvant medical therapy should predictive markers (BPM) has yet to be elucidated. The purpose of this study is to be considered as possible forms of therapy. identify a relationship, if any, to BPM and other prognostic factors. Design: Tissue microarrays of 125 cases of varying grades of breast carcinoma had 94 Primary Malignant Myoepithelial Tumor of Bone been developed for quality assurance purposes and stained for Estrogen Receptor (ER), Y Zhang, GP Nielsen, K Raskin, N Cipriani, AE Rosenberg. University of Miami, Progesterone Receptor (PR), and HER2/neu and scored using techniques prescribed Miller School of Medicine-Jackson Memorial Hospital, Miami, FL; Massachusetts by CAP and ASCO. CK 19 staining was performed and considered positive if any General Hospital, Boston, MA. cytoplasmic or membranous stainging was present. Hormone expression was used to Background: Primary malignant myoepithelial tumor of bone is a rare malignancy classify each tumor into the following subtypes: ER positive/HER2-neu negative (ER+), which can resemble morphologically a variety of different tumors including chordoma, HER2-neu positive (HER2+) and triple negative (TN). Each subtype was compared myxoid chondrosarcoma, and carcinoma. To increase our understanding of the to tumor expression of CK19 and reported as a percentage. Prognostic factors such as clinicopathologic features of this unusual neoplasm we reviewed our experience with tumor size, tumor grade, and lymph node status were also compared to CK19 staining. 6 cases. Chi square analysis was performed to identify any significant findings. Design: The study cohort was identified from the authors consult files and from the Results: Overall expression of CK19 in breast carcinoma was 87.2%. A significant surgical pathology files of Massachusetts General Hospital and University of Miami difference was seen when comparing TN to the other subtypes: 98% of (ER+), 88% Hospital. The clinical and pathological features of the tumors including their histologic, of (HER2+), and 73% of (TN) expressed CK19 (χ 2 =14.34, p<0.0007). With regard immunohistochemical, and EWSR1 status were analyzed. to tumor grade, 100% of Grade 1, 94% of Grade 2, and 84% of Grade 3 carcinomas Results: The patients included 3 females and 3 males who ranged in age from 18-57 express CK19; however, this difference was not significant. An association between (mean of 41.3) years. The tumor location included: 1 in the maxilla, 1 in the distal tumor size and lymph node status when compared to CK19 expression was not identified. tibia, 1 in the calcaneus, 2 in the mandible, and 1 in the distal femur. Imaging studies Conclusions: Cytokeratin 19 is expressed diffusely in breast carcinoma. Our data showed that the tumors were lytic with cortical destruction and extra-osseous extension. suggests CK19 expression does not correlate with tumor grade, tumor size, and lymph Microscopically, four cases were composed of clusters of large epithelioid and spindle node status. However, we found a significant number of triple negative breast carcinoma cells enmeshed in a prominent basophilic myxoid stroma. The other two cases were lack CK19 expression. Thus independent use of CK19 may lack sensitivity in detecting hypercellular and composed of clusters of epithelioid cells and spindle cells with metastatic breast carcinoma, specifically in those tumors that lack ER and HER2-neu solid and reticular pattern. The tumor cells demonstrated pleomorphism, and high receptor expression. mitotic activity and variable amounts of necrosis. The tumors also infiltrated into the surrounding soft tissue. Immunohistochemically, the tumor cells were focally positive 96 HER2 Testing: Central Laboratory Assessment of HER2- for either S100/ or SMA and EMA/or Keratin. Four cases were analyzed by FISH for Equivocal (2+ Staining) Samples Using IHC and Dual SISH detection of EWSR1 gene rearrangement and one case had a rearrangement, one tumor GI Acosta Haab, IL Frahm, SI Sarancone, MC Horsburgh. Hospital de Oncologia demonstrated monosomy of chromosome 22, one case was inconclusive, and one case Marie Curie, Caba, Buenos Aires, Argentina; Sanatorio Mater Dei, Caba, Buenos Aires, was negative. All patients underwent wide resection. Argentina; Laboratorio Quantum Clinica de Diagnostico Medico, Rosario, Santa Fe, Conclusions: Primary malignant myoepithelial tumor is a rare bone tumor, is Argentina; Productos Roche SAQeI, Tigre, Buenos Aires, Argentina. heterogenous morphologically, and can be confused with a variety of different Background: In breast cancer overexpression or amplification of HER2 is associated neoplasms. Immunohistochemically these tumors usually express S100 and EMA and with higher metastatic risk and shorter overall survival. In these patients, however, have genetic aberrations involving chromosome 22. As very few of these tumors have therapies targeting HER2 prolong overall, progression-free and disease-free survival. been described their optimal treatment and natural history needs to be better defined. Immunohistochemistry (IHC) is the method most frequently used to assess HER2 overexpression, but equivocal results (2+) have to be further studied in order to classify them as positive or negative. Design: In August 2003 a network of Pathology Laboratories trained in HER2 assessment by IHC was set up in Argentina, supervised in turn by an Advisory Board 26A ANNUAL MEETING ABSTRACTS (AB) made up of a Coordinator and two consultants. Regular training and internal count scores for all observations. Deming regression and descriptive statistics were and external quality control (performed by Nordicq) ensure standardization and used to analyze correlation between observers and reported estimates. reproducibility of results. External Laboratories submit samples with scores of 2+ to one Results: Good correlation between two observers was found when 1000 cells were of three laboratories of the AB for re-testing with IHC and/or study of gene amplification counted to ascertain Ki67 index (y = 1.08x + 0.08, R2= 0.82). The standard deviation by in situ hybridization (ISH) techniques. We report here the results of 74 equivocal (SD) per read ranged from 0-9.9%, with the highest coefficent of variation (CV) at samples that we received and re-tested with IHC and the novel Dual SISH Ventana Kit. low Ki67 index values. The mean 1000 cell count scores had good correlation with the Results: Of the 74 initially equivocal samples, upon re-testing by IHC 40 (54%) were clinically reported estimates (y = 0.89x - 2.97, R2= 0.86), with a slight negative bias scored as 3+, and 39 showed HER2 gene amplification by Dual SISH (in 29 cases to the reported estimate. Using a 14% threshold for low vs high Ki67, 100% of cases amplification was high). The remaining sample produced an error. Twenty one samples classified as low by 1000-cell count were also classified as low by routine estimation. (28%) were either negative or 1+, and none of these showed gene amplification. The However, only 75% of cases classified as high by 1000-cell count were high by routine rest of the initially equivocal samples (i.e. 13- 18%) were classified as 2+. Of these, estimation. When counts were analyzed in 100-count increments, 200-cell counts only 6 had gene amplification (high in 2 cases). were found to be within 5% of the 1000-cell count method, but 800-cell counts were Conclusions: Accurately identifying HER2 positive tumours allows for correct decision required to be within 2.5% of the 1000-cell method, with 95% frequency in each case. making regarding therapy of breast cancer. Equivocal cases by IHC must be further Conclusions: Routine Ki67 estimation underestimates the Ki67 index when compared studied in order to clarify HER2 status. In our experience, even with properly trained to 1,000-cell count methods. There is good correlation between pathologists when and quality-controlled Pahtology laboratories, repeating IHC centrally can positively 1000-cell counting methods are used, however, there is inter-observer variation, which categorize up to 82% of initially equivocal samples, leaving just 18% for ISH techniques. is increased at low Ki67 index values. Counting fewer than 1000 cells can produce Optimizing IHC results is important because ISH techniques are more costly, and are results similar to 1000-cell counts and may be a more practical in clinical practice. less widely available. There is a high degree of overall concordance between central ICH and central SISH. 99 Host Inflammation and Breast Cancer Molecular Subtypes: Results from a TCGA Sub-Analysis 97 The Presence of Extensive Retraction Clefts in Invasive Breast KH Allison, Y-Y Chen, SC Lester, NB Johnson, RE Factor, GMK Tse, SJ Shin, DA Carcinomas Correlates with Lymphatic Invasion and Nodal Metastasis Eberhard, PH Tan, SJ Schnitt, LC Collins, KC Jensen, K Korski, FM Waldman, AH and Predicts Poor Outcome – A Prospective Validation Study of 2702 Beck. Cancer Genome Atlas (TCGA) Breast Cancer Expert Pathology Committee, Consecutive Cases Bethesda, MD. G Acs, N Khakpour, J Kiluk, MC Lee, C Laronga. Moffitt Cancer Center, Tampa, FL; Background: Tumor-associated inflammation is highly variable between patients. Women’s Pathology Consultants, Ruffolo Hooper & Associates, Tampa, FL. The association of tumor molecular and pathological characteristics with host immune Background: Retraction clefts (RC) around tumor cell nests are frequently seen in response is not well understood. Utilizing inflammation scores on the histology of formalin-fixed paraffin embedded histologic material. We have previously reported that breast cancer samples submitted for comprehensive molecular analyses for The Cancer extensive RC in early stage breast cancers correlates with lymphatic vessel density and Genome Atlas (TCGA), we examined associations between host inflammation and lymphatic tumor spread, and proposed that this feature may represent an early stage breast cancer molecular and pathologic features. of lymphatic invasion (LVI). In the present study we have prospectively validated our Design: Experts in breast pathology reviewed the digital slides of breast cancer samples prior results in a large independent series of breast cancers. submitted for comprehensive molecular profiling to the TCGA and scored each case for Design: We prospectively selected 2702 consecutive pT1-3 stage invasive breast the level of inflammation present (high/moderate vs mild/minimal). We tested pairwise carcinomas for the study. Cold ischemic time and duration of fixation was documented associations between host inflammation and molecular subtypes (DNA copy-number, for all cases. All H&E stained slides were prospectively reviewed to determine tumor RNA expression, RPPA defined subtypes, miRNA subtypes, methylation subtypes) and type and grade. The presence and extent of RC around tumor cell nests and the presence pathological features by performing Chi-Square analyses. Multiple hypothesis testing of LVI was also determined. LVI and micropapillary features in carcinomas were correction was performed using the Bonferroni method. confirmed with immunohistochemical stains for podoplanin and epithelial membrane Results: Morphological features (including inflammation) were scored by pathological antigen, respectively. Micropapillary and mucinous carcinomas were excluded. review of 309 breast cancer cases that underwent molecular profiling as part of TCGA. Results: Axillary lymph node assessment was performed in all cases. The median The strongest associations were seen between inflammation and the methylation number of nodes removed was 3 (range 1-66). Nodal metastasis was present in 1069 subtypes (p = 4E-10), with 66% of methylation cluster 5 cases showing at least moderate cases; the median number of positive nodes was 2 (range 1-44). The correlation of inflammation, compared with only 16% of non-cluster 5 cases. Inflammation also was the extent of RC with various clinicopathologic tumor features is summarized in the strongly associated with the PAM-50 molecular subtypes (p = 6E-9), with 67% of Basal table. The presence of extensive RC was associated with LVI and nodal metastasis and molecular subtype cases showing at least moderate inflammation, compared with only predicted poor recurrence-free (p=0.0024) and overall (p=0.0075) survival. 9% of Luminal A, 25% of Luminal B, and 42% of HER2-enriched cases. Inflammation Correlation of extent of RC with patholgic features was strongly associated with breast cancer Nottingham grade, with 51%, 18%, and Percent RC 15% of cases showing inflammation among grade 3, 2, and 1 cases respectively (p = N Median Mean±SEM p* 1.9e-7). Mitotic activity and nuclear pleomorphism were both strongly associated with Histologic type Ductal (NST) 2257 15 24.2±0.6 <0.0001 inflammation(p = 3.3E-8 and 2.0E-5, respectively); however, there was no significant Lobular 320 0 3.3±0.5 Other special 125 0 2.8±0.9 association of inflammation with pathological assessment of tubule formation (p=0.40). Histologic grade Low 606 0 9.5±0.7 <0.0001 Conclusions: In this integrative analysis of breast cancer molecular profiling data Intermediate 1286 10 20.0±0.7 with histopathological analysis of cancer-associated inflammation, we identify strong High 810 25 30.2±1.0 associations between breast cancer molecular and pathological features and the host pT stage 1A 135 0 17.0±2.7 <0.0001 inflammatory response. These associations provide new insights into breast cancer 1B 476 0 12.8±1.1 1C 1137 10 19.6±0.7 molecular and morphologic factors driving tumor-associated inflammation. 2/3 954 20 26.5±0.9 Lymphatic invasion Absent 2078 7.5 14.8±0.5 <0.0001 100 Prognostic Significance of Fibroblast Growth Factor Receptor Present 624 30 40.2±1.1 Nodal metastasis Absent 1633 5 15.7±0.6 <0.0001 3 (FGFR-3) Overexpression in Invasive Mammary Carcinoma (BRCA) Present 1069 20 28.6±0.9 RN Al-Rohil, MJ Presta, BVS Kallakury, GM Sheehan, CE Sheehan, AB Boguniewicz, *Kruskal-Wallis test JS Ross. Albany Medical College, Albany, NY; Georgetown University Hospital, Conclusions: Our results confirm in a large prospective study that the presence of Washington, DC. extensive RC in breast carcinomas highly significantly correlates with lymphatic tumor Background: The fibroblast growth factor receptor (FGFR) family includes at least 4 spread and predicts poor outcome. Our results further support the hypothesis that RC tyrosine kinase receptors which have been linked to the development and progression are the morphologic reflection of biologic changes in the tumor cells likely playing a of a variety of human cancers including head and neck squamous cell carcinoma, role in lymphatic tumor spread. hepatocellular carcinoma, urothelial carcinoma and multiple myeloma. FGFR-3 expression has not been widely studied as a prognostic factor for BRCA. Design: Formalin-fixed, paraffin-embedded tissue sections from 126 cases of BRCA 98 To Count or Not To Count? Scoring Ki67 in Breast Cancers [101 ductal carcinomas (IDC) and 25 lobular carcinomas (ILC)] were immunostained According to International Working Group Recommendations by a manual method using rabbit polyclonal FGFR-3 (sc 123; Santa Cruz Biotech, KH Adamson, EQ Konnick, SM Dintzis, MH Rendi, HM Linden, KH Allison. University Santa Cruz, CA). Nuclear and cytoplasmic immunoreactivity was semiquantitatively of Washington, Seattle, WA. scored based on staining intensity and distribution and the results were correlated with Background: Proliferation related markers can be used to both identify breast cancers morphologic and prognostic variables. that may respond to a therapy and provide a measurement of therapy response. An Results: Intense diffuse cytoplasmic FGFR-3 overexpression was observed in 26/126 International Ki67 in Breast Cancer Working (IKBCWG) published Ki67 assessment (21%) tumors and correlated overall and within the ER- subgroup, with PR+ status recommendations, which proposed 1000 cell counts per case. We tested the practical (p=0.013, p=0.026) with a trend toward PR+ status within both the IDC (p=0.084) application of their recommendations and compared results between observers as well and ILC (p=0.067) subgroups; and overall with disease recurrence (p=0.05), while as with routinely reported results. showing a trend toward disease recurrence within the ER+ subgroup (p=0.06). Nuclear Design: 43 cases of ER positive breast cancer were counted independently by two FGFR-3 overexpression was present in 30/126 (24%) tumors and correlated overall pathologists using the IKBCWG recommendations with 1,000 cells counted per case. and within the IDC subgroup, with ER+ status (p=0.041, p=0.05) and non-recurrent Percent Ki67 positive cells (Ki67 index) were compared both between observers and disease (p=0.004, p=0.003). Within the ER+ subgroup, nuclear FGFR-3 overexpression routinely reported Ki67 results (did not require counting cells). A Ki67 index threshold correlated with non-recurrent disease (p=0.017). On multivariate analysis, early age at of 14% was used to classify cases into low verses high. Lastly, scoring was analyzed by diagnosis (p<0.0001), advanced stage (p=0.004), lack of nuclear FGFR-3 overexpression 100-cell count increments to determine accuracy per 100-cell count vs final 1,000-cell (p=0.015), and intense diffuse cytoplasmic FGFR-3 overexpression (p=0.026) ANNUAL MEETING ABSTRACTS 27A independently predicted disease recurrence; while metastatic disease (p<0.0001) and We recommend breast cancer screening starting before age of 40 and a larger cases positive node status (p=0.001) independently predicted overall survival. sample to be studied in the future to draw a better conclusion. Conclusions: In summary, loss of nuclear expression and intense cytoplasmic FGFR- 3 expression correlate with hormone receptor status and are independent prognostic 103 Multifocal Flat Epithelial Atypia: Possible Precursor of Breast indicators in BRCA. Further study of FGFR-3 as a prognostic factor and potential target Carcinoma of therapy in BRCA appears warranted. MS Amin, M Hassan, SJ Robertson, S Islam. University of Ottawa, Ottawa, ON, Canada. Background: The biological behaviour and management of flat epithelial atypia in 101 Metaplastic Breast Carcinoma: A Clinico-Pathological and breast is not yet established. Whether or not extensive or multifocal FEA is associated Immunohistochemical Study with genetic changes that predispose to more advanced breast lesions has not been FJ Altaf, EAS Emam, GA Mokhtar, RY Bokhary, SM Al Amoudi, LA Baharith, HH Ali. extensively studied. The aims of this study were: 1. To characterize the radiological King Abdulaziz University, Jeddah, Western, Saudi Arabia. presentation, distribution and clinical outcome of FEA; and 2. Evaluate association of Background: Metaplastic breast carcinoma (MBC) is characterized by the expression FEA distribution with atypical hyperplasia and in situ or invasive carcinoma. of epithelial and or mesenchymal tissue morphologically or genotypically within Design: We retrospectively studied 58 breast biopsies and resections at our institution the same tumor. There is little information in the literature about clinically relevant between 2008 and 2010 that contained FEA. Among these 42 were core needle pathologic and immunohistochemical features that can affect tumor prognosis and biopsies (CNB), a subsequent resection was done in 25 of these. 16 cases were treatment modalities. The aim of the present study is to evaluate the clinicopathological resection specimens with secondary FEA diagnosis. FEA distribution was assessed features of metaplastic breast carcinoma seen at King Abdulaziz University Hospital semi-quantitatively as: focal (1-2 biopsy fragments or areas per section), multifocal and to investigate the value of immunohistochemical study in diagnosis, classification (3 and more fragments or sections but < 50% of total number) and extensive (>50% and treatment of such cases. of fragments or sections). Design: Seven cases of MBC were evaluated for Clinical, radiological and Results: Radiologic microcalcifications were present in 43 cases. FEA was the sole pathological features as well as treatment and follow up data were tabulated. Routine& diagnosis in 15 CNB cases: 2 of these had multifocal FEA, and one case subsequently immunohistochemistry for ER, PR, and HER-2. CK Pan, HCG, PLAP,Vimentin and presented with atypical hyperplasia. FEA was associated with atypical hyperplasia in 15 other stains were evaluated whenever needed and their interpretation were discussed. cases: 8 of these (53%) had mutifocal or extensive FEA, and 3 subsequently presented Results: The mean age of the patients was 36 years with range of (23-69 years). All with DCIS within 1 year. FEA was associated with an in situ or invasive carcinoma in cases presented with breast mass and large tumor size range (5-13cm). Histological 9 CNB cases: 5 of these (55%) had multifocal FEA. Multifocal FEA was also present examination revealed that three out of seven cases (43%) were composed of squamous in 2 of the 16 resections (12%) for carcinoma. cell carcinoma, four cases showed mixture of malignant epithelial and mesenchymal Conclusions: We are the first to report that multifocal or extensive FEA is more components: three cases (43%) showed infiltration by giant syncitiotrophoblastic frequently associated with atypical hyperplasia, in situ or invasive carcinoma. Therefore cells proved to be positive to HCG and PLAP antibodies. Four patients (50%) had focality of FEA must be mentioned in the diagnosis. Further molecular and genetic axillary lymph node metastasis. Local recurrence was seen in 50 % of the patients, studies are required to elucidate whether or not multifocal FEA is a premalignant and distant metastasis was present in 25%. Immunohistochemical staining revealed condition. negative imumnoreactivity of the cases to estrogen receptors, progesterone receptors and HER-2 neu. 104 Histology of Breast Cancer in Patients with Lynch Syndrome Conclusions: MBC in our population is an aggressive disease. Our patients are younger B Aqil, B Arun, N Sneige, AM Gutierrez-Barrera, D Rosen, S Abrahams, E Resetkova, than other studies. They are presented with large tumor size and more advanced stage. Y Wu, CT Albarracin. Baylor College of Medicine, Houston, TX; MD Anderson Cancer All cases are shown immunohistochemically to be contained in the triple negative Center, Houston, TX. category of breast cancer and so may follow similar treatment modalities. Majority Background: Germline mutations for Lynch syndrome (LS) are associated with colon, of them developed local recurrence and distant metastasis in a relatively short period small intestine, stomach, endometrium, upper urinary tract and sebaceous tumors of the of time.The relationship between the presence of syncitial cells histologically and skin. While breast cancer is not considered to be a LS-associated tumor, more recent pregnancy clinically, that is associated with advance stage at presentation, and very studies have described early onset of breast tumors in patients with LS. Our goal is to aggressive behavior require further investigation. evaluate the histopathological features of breast cancers associated with LS. Design: Women with primary breast cancer who had biopsy or /and surgery at our 102 Invasive Carcinoma of Breast Morphology and institution were included in this study. A search recovered 24 cases of LS-associated Immunohistochemical Classification – King Abdulaziz University Hospital breast cancers from 1987-2012. We were able to retrieve the slides for 13 patients. Experience Results: The mean age of diagnosis of invasive breast cancer in Lynch syndrome cases FJ Altaf, EAS Emam, GA Mokhtar, RY Bokhary, LA Baharith, HH Ali, ZK Al- Gaithy. was 51.5 years. The most common accompanying tumors included colon cancer in 39% King Abdulaziz University, Jeddah, Western, Saudi Arabia. (13 of 33) and endometroid carcinoma grade I in 15% (5 of 33). Breast tumor types Background: Management and prognosis of Invasive breast carcinoma (IBC) does not included invasive ductal carcinoma (21 of 25), invasive lobular carcinoma (1 of 25) and depend any more on pure morphology only, but it also includes the Immunohistochemical metaplastic carcinoma (1 of 25). Only 2 cases of DCIS were identified not associated features of the tumor based on its content of Estrogen Receptors(ER),Progesterone with invasive tumor. Infiltrating ductal carcinomas had low grade in 12% (3 of 23), Receptors(PR),and HER 2 Receptors (HER2). Large numbers of studies publish the intermediate grade in 26% (6 of 23) and high grade in 30% (7 of 23). The biologic new classification based on Immunostains and molecular findings. markers were tested in 15 invasive ductal, 1 invasive lobular and 1 metaplastic breast Design: A retrospective study is designed to reclassify cases of invasive breast tumors that showed, 18% (3 of 17) were ER-/HER2+, 18% (3 of 17) were ER+/HER2 +, carcinoma received and diagnosed at Anatomical pathology of king Abdulaziz 53% (9 of 17) were ER+ /HER2 – and 12% (2 of 17) were ER-/HER2- . DCIS was low University hospital.We identified 155 cases that fit our inclusion criteria.Patient’s age grade in 6.6% (1 of 15), intermediate grade in 53% (8 of 15)and high grade in 40% (6 were recorded. We classify them histologically into,invasive ductal carcinoma not of 15). 27% of DCIS in Lynch patients also showed periductal fibrosis. Out the 2 DCIS otherwise specified(IDCNOS), pure special type (PST) and mixed type (MT).Tumor cases not associated with invasive tumor only 1 case was tested for biologic markers size was identified, grade was applied to all of the three groups. In addition certain and was ER+/HER2-. Immunohistochemical stains and mutational analysis for DNA immunohistochemical markers including ER,PR,HER2, P63, Ki67 and other when mismatch repair (MMR) gene products showed loss of MLH1 alone in 14% (3/21), necessary were applied. PMS2 alone in 4.8% (1/21), MSH2 alone in 9.5% (2/21), MSH 6 alone in 4.8% (1/21), Results: We identify 7 groups (G), Luminal A(ER+,PR+ &HER2-). Lumina B MLH1/PMS2 in 57% (12/21), MSH2/MSH6 in 9.5% (2/21). Lynch patients showed (ER+,PR+&HER2+). While HER2+(ER-,PR-&HER2+) & triple negative (ER-,PR- fibrocystic changes(46%) and with 17% of cases having intraductal papillomatosis &HER2-). We also have apparent groups(AG 5&6) of Luminal A&B that they lack PR Conclusions: Invasive breast tumors and DCIS in Lynch syndrome tend to have an positivity. Apparent HER2+(G7) that lack ER+. Majority of cases 59% are presented intermediate to high nuclear grade, be ER+/HER2-, and show loss of MLH1/PMS2 between 41-60 years of age, while 13% were below the age of 40yrs. All of our cases markers. Approximately half of the patients with Lynch syndrome showed fibrocystic were presented with large tumor sizeT2&T3.(Table 1) changes with one third having intraductal papillomatosis. Groups, AverageTumor sizeTS,Frequancy %,Grad & Invasive Types Groups ATScm Frequancy((N) %n155 Grade(g) IDC PPST MTT 105 Invasive Ductal Carcinoma with Lobular Features Can Be Group 1 LuminalA 3.25 58 37,4% g1=19,g2=32,g3=7 42 10 6 Group 2 LuminalB 3.62 24 15.5% g1=6,g2=16,g3=2 18 3 3 Diagnosed on Breast Core Biopsies and May Be Important To Distinguish Group 3 HER+ 3.88 17 11% g1=1,g2=10,g3=6 15 1 1 from Pure Invasive Ductal Carcinomas Group 4 Triple 3.86 26 16,8% g1=3,g2=6,g3=17 22 3 1 D Arps, C Kleer, J Pang. University of Michigan, Ann Arbor, MI. Negative Group5 App. Background: Invasive ductal carcinoma with lobular features (IDC-L) is not recognized 2.86 18 11.6% g1=5,g2=12,g3=1 14 2 2 Luminal A as a distinct subtype of breast cancer by pathologists. It is a category used when there Group6 App. 2.5 10 6.5% g1=8,g2=2,g3=0 10 0 0 is the presence of both ductal and lobular histology in the same lesion. In routine Luminal B practice, many of these tumors are diagnosed as mammary carcinoma with ductal and Group7 App.HER+ 3.25 2 1.3% g1=0,g2=2,g3=2 2 0 0 Total 155 100.00% 123 19 13 lobular features, IDC-L, or classified as ductal or lobular type arbitrarily. A study at our institution on resection specimens suggested that IDC-L may be a distinct variant n= number. cm= centimeter of invasive ductal carcinoma (IDC), with more than 90% of cases being E-cadherin Conclusions: Lack of breast cancer screening program in the Kingdom resulted in positive, but has clinicopathological characteristics more similar to invasive lobular advanced presentation of our cases.Majority of the cases 53% are of luminal group of carcinoma (ILC). In addition, re-excision rates for obtaining negative margins were intermediate grade that will benefit from hormonal treatment. While 11% only exhibit higher for patients with IDC-L (49%) in our series than the overall rate of re-excisions high grad tumors that will benefit from Herceptin treatment. Almost 17% will have for our institution (25%). We sought to determine the accuracy of diagnosing IDC-L more aggressive tumors that does not respond to hormonal or Herceptin treatment, with on core biopsies, as patients with IDC-L may need further margin evaluations, and high tumor grade. There is no morphological difference in the seven groups identified. 28A ANNUAL MEETING ABSTRACTS their eligibility for specific treatments may depend on the diagnosis (e.g. cryotherapy Results: and partial breast radiation such as mammosite used for IDC are not recommended Comparison of GH in primary breast carcinoma and its axillary metastasis for patients with IDC-L). HER2 in primary carcinoma No. of cases HER2 in axillary lymph node metastases Design: 183 cases of IDC-L from 1996-2011 were identified from the pathology HER2- GH- HER2- GH+ HER2+ database. 149 cases had core biopsies. Cases initially diagnosed as IDC on core biopsy HER2- GH- 9 8 1 0 HER2- GH+ 28 1 25 2 were re-reviewed. Available slides from resections were previously reviewed (n=150). HER2+ 5 0 0 5 Lobular features were defined as small cells individually dispersed, arranged in linear + positive; - negative cords, or in loose aggregates without the formation of tubules or cohesive nests. GH was present in the LN metastasis of 89% (25 cases) of the breast carcinomas with Results: IDC-L was diagnosed on core biopsies in 92 cases (62%), while 44 (29%) GH. Of these 25 cases, 3 showed a >2 fold increase in GH whereas 3 demonstrated were diagnosed as IDC, ten (7%) as ILC, one as poorly differentiated carcinoma, and a >2 fold decrease in GH in the metastasis as compared to the primary. HER2 was two as ductal carcinoma in-situ (DCIS). Of the 44 core biopsies diagnosed as IDC, 30 amplified in the lymph node metastasis in 2 of the 3 cases demonstrating regional HER2 cases with available slides were re-reviewed and of those, 24 (80%) were re-classified amplification comprising 5 to 15% of the primary carcinoma. as IDC-L. Of the remaining six cases, four had slides available for review from their Conclusions: Concurrent axillary metastases from HER2 genetically heterogeneous resection. Three cases had < 30% lobular morphology on the resection. One case had breast carcinomas: 90% lobular morphology on resection; however the biopsy target was for calcifications 1) demonstrate variable genetic heterogeneity in the majority of cases. on mammogram and the core showed predominantly high grade DCIS with only a small 2) are HER2 amplified in a minority, particularly in those with small areas of HER2 focus of invasive carcinoma. amplification in the primary carcinoma. Such patients may benefit from the evaluation Conclusions: IDC-L can be diagnosed on core biopsies with good correlation on of lymph node metastases prior to the instigation of therapy. resection and may be important to distinguish from IDC as there are treatment and prognostic implications. 108 Dual-Color In Situ Hybridization (Dual ISH) for Evaluation of HER2/Neu (HER2) Amplification in Breast Cancer 106 High Mobility Group A1 (HMGA1) Proteins Associate with ER- K Astvatsaturyan, S Bose. Cedars-Sinai Medical Center, Los Angeles, CA. Negativity and High Nuclear Grade in Breast Cancer in a Cohort of Asian Background: HER2 amplification is a well recognized prognostic and predictive Women marker for breast cancer. Amplification is traditionally analyzed by flourescent in RJ Asch, A Cimino-Mathews, L Cope, A Meeker, YK Bae, LMS Resar. Johns Hopkins situ hybridization (FISH) which is an expensive and laborious procedure that limits University School of Medicine, Baltimore, MD; Johns Hopkins University School of widespread use. Dual ISH, a promising alternative to FISH, has recently been FDA Medicine, Baltimore, Korea; Yeungnam University College of Medicine, Daegu, Korea. approved for HER2 testing in breast cancer. This study is designed to compare Dual Background: Breast cancer remains a leading cause of death in women worldwide. ISH to FISH for determining HER2 amplification in breast cancer. Here, we investigate HMGA1 immunoreactivity as a biomarker for more advanced Design: Dual ISH was performed on 51 cases of infiltrating breast carcinomas retrieved disease in a cohort of Asian women with breast cancer. HMGA1 encodes the from our files using Ventana Medical System’s INFORM Her2 Dual ISH DNA Probe HMGA1a/1b chromatin remodeling proteins, which function in regulating gene Cocktail which detects HER2 copies via silver ISH and CEP17 copies via chromogenic expression.HMGA1 is highly enriched in embryonic stem cells and some high-grade Red ISH in formalin-fixed, paraffin-embedded sections as per the manufacturer’s tumors with poor outcomes. We focused on Asian women because they present at a guidelines. All of these cases had been subjected to prior HER2 analysis by FISH using younger age and with a more advanced stage than other women with breast cancer, the PathVysion HER2/neu kit (Abbott-Vysis, USA). All protocols were in keeping with suggesting that HMGA1 could be important in tumor progression in these women. the CAP guidelines. Of the 51 cases, 14 were Her2 amplified, 33 were Her2 negative Design: Tissue microarrays (TMAs) were generated from paraffin-embedded tissue and 4 were Her2 equivocal. Dual ISH was successfully performed in 50 cases which sections. HMGA1 protein levels were assessed by immunohistochemistry. TMAs formed the basis of this study. HER2 to CEP 17 ratio was calculated manually for each contained cores from 594 primary tumors from a Korean cohort with detailed case. Results were compared. histopathologic and clinical data (tumor grade, stage, metastases, survival, ER/PR/ Results: HER-2, P-cadherin status). Normal tissues were included as controls. Positive staining Table 1: Comparison of the overall HER2 and CEP17 signal counts obtained by FISH and Dual was identified as nuclear immunoreactivity and was scored using a 3 point scale based ISH on the product of staining intensity (weak =1, moderate =2, strong =3) and staining Signal Counts FISH Dual ISH extent (% of positive cells; <30% =1, 30-60% =2, >60% =3). The highest score per HER2 mean count 8.8 5.8 case was used for subsequent analyses. HER2 median count 4.2 2.7 CEP17 mean count 2.9 2.0 Results: HMGA1 immunoreactivity was observed in 515 of 594 cases of primary breast CEP17 median count 2.8 1.9 tumors, with intensity scores ranging from 0-3. Of positive cases, the immunoreactivity score was <3 in 64 (12%), 3-6 in 215 (42%) and >6 in 236 (46%). There was a significant, positive correlation between HMGA1 staining and ER-negativity. In addition, HMGA1 Table 2: Comparison of HER2 results obtained by FISH and Dual ISH FISH Dual ISH levels correlated positively with advanced nuclear grade. HER2 negative HER2 amplified Total Conclusions: We demonstrate for the first time that HMGA1 correlates with both ER- HER2 negative 30 2 32 negativity and advanced nuclear grade in primary tumors from Korean women with HER2 equivocal 4 0 4 breast cancer. Although further studies are needed, our results suggest that HMGA1 HER2 amplified 0 14 14 contributes to tumor progression and could represent a useful biomarker and effective Total 35 15 50 therapeutic target. Although we noticed lower signal counts for both HER2 and CEP17 (Table1) by Dual ISH, HER2 negative and HER2 amplified cases showed 94% and 100% concordance respectively (Table 2). The two HER2 negative cases that were amplified by Dual 107 Her2/Neu (HER2) Intratumoral Genetic Heterogeneity in Breast ISH showed ratios of 2 and 2.1. All four of the HER2 equivocal cases were negative Cancer and Its Concurrent Axillary Metastases by Dual ISH. K Astvatsaturyan, S Bose. Cedars-Sinai Medical Center, Los Angeles, CA. Conclusions: Dual ISH: Background: Per the recent guidelines of the College of American Pathologists breast is a viable alternative to FISH for evaluation of HER2 status and offers many advantages carcinomas are defined as being genetically heterogeneous for HER2 when 5-50% of (light microscopic evaluation, preservation of tissue morphology, capabilities for neoplastic cells demonstrate HER2 amplification. We have previously observed HER2 archiving, re-evaluation and use in retrospective studies). intratumoral GH in 26% of breast carcinomas. The significance of this finding is unclear has a high concordance rate with FISH. at present although it is postulated that this may result in refractoriness to therapy in offers lower rates of equivocal results as compared to FISH. HER2 negative cancers. Knowledge of the HER2 status in concurrent axillary metastasis shows lower HER2 and CEP17 signal counts as compared to FISH. from breast cancers with HER2 GH may thus be beneficial in determining therapy. This study is therefore designed to evaluate HER2 GH in paired samples of primary breast carcinoma and its concurrent axillary lymph node (LN) metastases. 109 Mutational and Clonal Intratumoral Heterogeneity of Breast Design: 42 cases of infiltrating breast carcinoma with axillary LN metastases (28 cases Cancer with GH, 9 cases without GH and 5 cases with HER2 amplification) were retrieved S Avril, LN Harris, H Hofler, HL Gilmore. Technische Universität München, Munich, from our files. HER2 GH was evaluated using Ventana Medical Systems’ two color Germany; Case Western Reserve University and University Hospitals Seidman Cancer chromogenic in situ hybridization in formalin-fixed, paraffin-embedded sections per Center, Cleveland, OH; Case Western Reserve University and University Hospitals the manufacturer’s guidelines. The HER2 gene was visualized as discrete black signals Case Medical Center, Cleveland, OH. and Chromosome17 as red signals by light microscopy. The ratio of HER2 gene to Background: Intratumoral heterogeneity of solid tumors has long been known. Chromosome 17 was calculated for each neoplastic cell in each primary carcinoma However, the full spectrum of mutational heterogeneity within different areas of a single and its LN metastases to determine the presence of GH. Breast carcinomas with GH breast carcinoma has not been assessed. The aim of this study is to assess the intratumoral demonstrated HER2 amplification in 5-44% of neoplastic cells present as single scattered heterogeneity of somatic mutations at the sequence level within primary breast cancers cells in 25 cases and as small clusters of amplified cells in 3 cases. and between axillary lymph node metastases from the same patient in order to define the extent of sampling needed to reliably represent the entire breast carcinoma. Design: In a pilot study, 25 prospectively collected formalin-fixed and paraffin embedded samples from 6 untreated primary breast cancers (2 Luminal, 2 HER2, and 2 Basal subtypes) were analyzed. These included samples from the peripheral, intermediate and central zones of each primary tumor and several axillary lymph node metastases per patient. All samples were analyzed by targeted deep-sequencing of 40 known candidate driver mutations using miSeq Custom Amplicon technology (Illumina). ANNUAL MEETING ABSTRACTS 29A The presence and frequency of mutations was compared between samples from different Conclusions: According to our results, with the biomarkers that we used it was not tumor zones and between different lymph node metastases from the same patient. possible to identify specific subgroups of TN tumors with specific marker expression Results: We found a large variation both in the presence and frequency of 40 candidate profiles. mutations in different regions of the same primary breast carcinoma, and between different lymph node metastases from the same patient. The majority of somatic 112 Novel Mutations in Neuroendocrine Carcinoma of the Breast: mutations were only present in some areas and not throughout the whole tumor. Possible Therapeutic Targets Conclusions: The considerable mutational heterogeneity demonstrated in this study may M Ballard, D Ang, C Beadling, A Warrick, A Schilling, R O’Gara, M Pukay, R West, lead to sampling bias when single tumor samples are used for assessing the mutational C Corless, M Troxell. Oregon Health & Science University, Portland, OR; Stanford, spectrum of a breast carcinoma. In addition, mutational heterogeneity may contribute to Stanford, CA. the development of resistant tumor cell clones, in particular when targeted therapies are Background: Primary neuroendocrine carcinoma of the breast is a rare variant, directed against mutated oncogenes. Multiple biopsies are required to fully characterize accounting for only 2-5% of diagnosed breast cancers. Literature on pathogenesis, a primary or metastatic breast cancer for the selection of targeted therapies or for prognosis and treatment options is extremely limited. Recent data suggest that prediction of treatment response. Future studies should address the question whether neuroendocrine breast cancer may have a relatively poor prognosis. Mutational profiling sequencing in greater depth of a single index lesion may in some instances replace the of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, need for multiple tumor samples. yet most studies have not included neuroendocrine carcinomas. In this study we sought to evaluate the mutational profile of this potentially aggressive breast cancer variant. 110 Harmonization of Immunohistochemical Stains for Breast Design: Twelve cases of neuroendocrine breast carcinoma were identified from Cancer Biomarkers – An Athena Pathology Collaboration pathology archives, and also by screening a tissue microarray with synaptophysin and R Balassanian, JA Engelberg, JW Bishop, AD Borowsky, RD Cardiff, PM Carpenter, chromogranin. Stains were then confirmed on full tissue sections, and cases with > Y-Y Chen, B Datnow, S Elson, F Hasteh, F Lin, NA Moatamed, B Perkovich, Y Zhang, 50% neuroendocrine differentiation, as defined by WHO criteria, were included in the SK Apple. University of California San Francisco, San Francisco, CA; University of study; tumors with a mucinous component were excluded. In four cases metastases were California Davis, Davis, CA; University of California Irvine, Irvine, CA; University tested. Using H&E stained slides as a guide, lesional tissue was punched from paraffin of California San Diego, San Diego, CA; University of California Los Angeles, Los embedded tissue blocks; genomic DNA was extracted and screened for a panel of known Angeles, CA. hotspot mutations using PCR and mass spectroscopy analysis (SequenomMassARRAY). Background: Four clinically important immunohistochemical stains (IHC4) for breast This mutation panel screens 643 point mutations in 53 genes, including PIK3CA, AKT1, cancer, including estrogen and progesterone receptors (ER/PR), Her-2 and Ki-67, can FGFR1/2/3/4 and KDR. Mutations were confirmed by Sanger sequencing. vary between different laboratories. Accuracy of IHC4 is important for breast cancer Results: Of the 12 cases, mutations were identified in 3 tumors, including 2 with treatment, and standardization of clinical protocols across different centers. There is PIK3CA exon 9 E542K mutations (both metastatic), and one with a KDR exon 24 limited standardization of technical methods or cross-center validation for IHC4. This A1065T mutation. One of the tumors with a PIK3CA mutation also harbored a FGFR4 study evaluated IHC4 scoring at 5 University of California (UC) laboratories using exon 13 V550M mutation. traditional scoring techniques and novel quantitative image analysis (QIA) with whole Conclusions: In this small study, 17% of neuroendocrine breast carcinomas had PIK3CA slide imaging (WSI). mutations, a mutation not uncommon in other breast carcinomas. Interestingly, both Design: Unstained slides from five phenotypically different breast cancer cases were FGFR4 and KDR mutations were also identified. Mutations in fibroblast growth factor sent to the 5 UC laboratories to perform IHC4 staining and to be scored by pathologists. receptors such as FGFR4 are exceedingly rare in the breast cancer literature, but have Digital whole slide images (WSI) were captured using an Aperio ScanScope XT and been reported in a subset of pediatric rhabdomyosarcomas. KDR (VEGFR2) encodes analyzed using quantitative image analysis (QIA) methods. The raw data was converted a receptor tyrosine kinase involved in angiogenesis. Activating KDR mutations have into graphs plotting the density of positive staining cells per mm2, at a given intensity. been reported in angiosarcomas and non-small cell lung cancers; the A1065T mutation The results were compared between the 5 UC laboratories. identified in this study has previously been shown to be sensitive to VEGFR kinase Results: No two UC laboratories used the same antibody clone (with the exception of inhibitors. Our findings demonstrate the utility of broad-based genotyping in the study Ki-67), detection system, and vendors for ER, PR and Her-2. Variance of pathologists’ of rare tumors such as neuroendocrine breast cancer. Further studies are needed to scores was observed, but was not sufficient to affect clinical care. QIA revealed patterns investigate potential roles for PIK3CA, FGFR4 and KDR in driving growth of these not observable from pathologist scores: 1) some laboratories produced similar IHC slides cancers. for histological results, 2) some slides that were scored unanimously by pathologists still demonstrated variance by QIA, 3) for Ki-67, technical staining variance by different 113 Clinico-Pathological Features of Fibroadenoma Occurring in a laboratories was observed significantly more than other IHC. Antibody vendor and Cohort of Young African-American Patients: Single Institution Experience different clone did not explain the variance. S Bandyopadhyay, B Alosh, M Bowles, M Cote, D Shi, N Salem, MH Kim, D Visscher, Conclusions: QIA data suggest that technical differences are the main source of variance R Ali-Fehmi. Wayne State University, Detroit, MI; Karmanos Cancer Institute, Detroit, between UC laboratories. WSI and QIA are novel tools for assessment of IHC4 variances MI; Mayo Clinic, Rochester, MN. between different laboratories, in both quantitative and qualitative analysis. WSI with Background: Fibroadenoma (FA) commonly seen in women in the 2nd and 3rd QIA is a robust method for harmonization of IHC4. decades of life, may be multiple and bilateral in approximately 15-20% of cases and are reportedly twice as common in young African American women compared to their 111 Can Triple Negative Breast Tumors Be Further Subdivided by Caucasian counterparts. Although surgical excision is the definitive treatment, it may Immunohistochemical Analysis of Expression of Protein Panels? be undesirable due to cosmetic/technical reasons. On the other hand, conservative S Balci, C Irkkan, CL Shapiro, K Huebner, G Guler. Yildirim Beyazit University, management is not acceptable to all patients. In our study, we analyzed clinical and Ankara, Turkey; James Cancer Hospital and Ohio State University Comprehensive histopathological features of FA occurring in an African American cohort of women Cancer Center, Columbus, OH; Ohio State University Comprehensive Cancer Center, less than 25 years of age at our institution. Columbus, OH. Design: We identified African American patients≤ 25 years of age who were diagnosed Background: Triple negative breast tumors (TNBC) are a morphologically and with FA from years 2003 to 2007. The clinical parameters recorded were age, number, molecularly diverse group of neoplasms. Recently there have been efforts in the laterality, size and number (single/multiple) of tumors. Recurrence, if any, was also literature to define subgroups according to protein expression profiles. Herein, we report documented from the medical charts over a 5 year follow up period. H and E slides the morphological and immunohistochemical profile of our TNBC cohort in terms were reviewed and the following pathological parameters were recorded: stromal expression of androgen receptor, claudin, p63 and stem cell, DNA damage response, cellularity (low, intermediate, high), epithelial hyperplasia (mild, moderate and florid), epithelial mesenchymal transition and basal markers. stromal mitoses (per 10 HPF) and stromal overgrowth (lack of epithelial units in 1 Design: 162 triple negative breast tumors were reviewed morphologically and TMA low power field). sections containing 2 cores from each tumor were used for immunohistochemistry. Results: A total of 95 women ≤ 25 years were diagnosed with FA ranged in size from Expression of individual proteins was not necessarily available for every case so the 0.5 cm to 12 cm with a median of 2.3 cm. Of these, 80 (84.2%) patients did not recur; valid percentages are given for each protein. in this group, multiple FA were identified in 4 (5%) of the patients and bilaterality was Results: Morphologically 123 (75.9%) cases were diagnosed as infiltrative ductal NOS. present in 2 (2.5%) patients. The tumors ranged in size from 0.5 to 2.8 cm with a median Of the 162 tumors; 91 (56.2%) were positive for basal markers: (77 (47.5%) for EGFR, of 2 cm. In the second group of 15 (15.8%) patients with multiple recurrences over the 28 (17.3%) for CK5/6); 33 (21.2%) were positive for AR. For DNA damage response follow up period, 6 patients (40%) had multiple tumors and bilaterality was seen in 4 proteins 148 tumors (91.4%) exhibited loss of Fhit, 152 (93.8%) loss of Wwox and 85 (26.7) patients. In this group, the tumors ranged in size from 2.8 cm to 12.1 cm with a (52.5%) loss of BRCA1 expression; 88 (55.7%) were p53, 124 (89.2%) γH2AX and median size of 3.8 cm. All recurrent FA were also diagnosed as FA. No differences in 82 (62.1%) pChk2 positive. 72 (48.6%) tumors were claudin 3 and 4 low. For stem histopathology were noted between these 2 groups. cell markers: 60 (42.0%) had at least one CD44P24N (P: positive; N: negative) cell, 90 Conclusions: In our cohort of patients, we identified a subset of patients who were (62.9%) had at least one CD24P44N cell and 23 (16.1%) had at least one CD44P24P prone to recurrence of FA. This group had a greater proportion of bilateral and multiple cell. Totally 62 (43.4%) tumors were CD44 positive. ALDH1 expression is observed tumors. Also, these FA tended to be larger than those which did not recur. However, none in 15 (17.9%) cases. For epithelial-mesenchymal transition markers: 84 (55.6%) were of these patients developed malignancy over a five year follow up period. Therefore, it AREB, 21 (13.7%) were ZEB2, 121 (79.1%) were SLUG and 125 (81.2%) were TGF appears that conservative management of FA occurring in young women with appropriate positive. p63 was positive in 29 (18.6%) of cases. On determining if subgroups with clinical and radiological follow up might be a viable treatment option. related biomarker expression can be identified among TN tumors by clustering analysis; we noted that although there are many one to one correlations between proteins we could not identify specific subgroups with similar protein expression profiles. 30A ANNUAL MEETING ABSTRACTS 114 Intratumoral Heterogeneity of HER2 in Invasive Breast associated inflammation (TAI), and perilobular inflammation (PLI) away from the mass, Carcinoma: How Much Testing Is Enough? to determine if these features were associated with worse prognosis. GC Bethune, MC Chang. Mount Sinai Hospital, Toronto, ON, Canada; University of Design: Our patient population consisted of 38 patients diagnosed with PABC within 2 Toronto, Toronto, ON, Canada. years of pregnancy (1998-2011, mean age:35.5, range 25-48). Age-matched nulliparous Background: The prognostic and predictive value of HER2 (human epidermal growth women with a diagnosis of breast cancer served as controls (mean age:37.5, range factor receptor 2/ERBB2) warrants routine testing in invasive breast carcinoma. Current 29-48). Pathologic tumor characteristics evaluated included histologic type, tumor testing guidelines recommend the reporting of intratumoral heterogeneity; however, grade, tumor size, presence of lymphatic vascular invasion (LVI), lymph node status, the clinical significance of heterogeneity remains unclear. There is some evidence breast tumor markers (ER, PR, HER2 and p53) and the presence of TAI and PLI. The that HER2-heterogeneous tumours fall between “positive” and “negative” cases with degree of TAI and PLI were graded as 0-absent, 1-mild (<10%), 2-moderate (10-50%) respect to prognosis and treatment response. The aim of this study is to evaluate the or 3-severe (>50%). value of testing multiple blocks within a single tumour, with an emphasis on detecting Results: Overall, women with PABC were more likely than controls to have grade 3 intratumoural heterogeneity. infiltrating ductal tumors (76% vs. 26%,p <0.0001) and triple negative phenotype (34% Design: As approved by the institutional research board, we searched (2006-2012) for vs. 3%, p <0.001). Women with PABC had more positive lymph nodes (61% vs. 45%) breast excisions in which a single focus of invasive breast carcinoma was tested for and higher rates of p53 expression (31% vs. 16%). 96% of PABC cases had TAI, 12 HER2 on more than one block. We identified 151 consecutive cases (144 with 2 blocks; mild, 8 moderate and 5 severe. Of interest, all 5 severe TAI were grade 3 ductal tumors 7 with 3 blocks); an audit of HER2 results was performed on each case. Cases with with positive lymph nodes. 73% of controls showed TAI, 13 mild and 3 moderate. PABC IHC having regional heterogeneity (clusters of HER2 positive cells) were tested by cases were more likely than controls to have moderate to severe TAI (50% vs. 14%, FISH. ‘Genetic heterogeneity’ was defined according to CAP 2009 consensus (at least p<0.04) and PLI (85% vs. 55%, p<0.05). There was no difference between PABC and 5% but fewer than 50% of nuclei with HER2/CEP17 ratio of >2.2). controls in regards to tumor size, LVI or HER2 status. Results: Multiple blocks were tested in tumors ranging from 0.5 to 10.8 cm in size (mean Conclusions: 1. PABCs diagnosed within 2 years of pregnancy are more likely to 2.3 cm). Reasons for additional testing included: large tumour size, equivocal result on be grade 3 triple negative ductal carcinomas often with positive lymph nodes 2. The a single block, discordance with a prior biopsy, phenotypic or genetic heterogeneity, and majority of PABC have TAI (50% moderate or severe) 3. Severe TAI is more likely clinician request. Testing of multiple blocks yielded no additional HER2 information in in PABC (1 in 5 cases) 4. Severe TAI is associated with lymph node involvement in 146/151 (97%) of cases. Of these, 122/146 (84%) were HER2-negative by IHC, 20/146 PABC and 5. PABC is more likely to have PLI. Our findings suggest that TAI and PLI (14%) were HER2-positive by IHC, and 4/146 (3%) were HER2-equivocal by IHC may play an important role in tumor metastasis in PABC and contribute to the poor and FISH. Although regional and/or genetic heterogeneity were seen in 2/146 (1%) prognosis in these patients. and 4/146 (3%) of these cases, the heterogeneous pattern could be seen on both blocks tested. Only 5/151 (3%) cases differed between blocks of the same tumor--all were tested 117 Multinucleation in Lobular Carcinoma In Situ: A Histologic by both IHC and FISH. These cases usually had high grade (4/5), heterogeneous IHC Feature Specific for the Pleomorphic Variant staining (4/5), and genetic heterogeneity if evaluated by FISH (4/4). By contrast, only LZ Blanco, Jr., A Mahajan, KP Siziopikou, B Susnik, ME Sullivan. Northwestern a minority (2/5) showed morphologic heterogeneity. The tumors in these discordant University Feinberg School of Medicine, Chicago, IL; Abbott-Northwestern Hospital, cases were not significantly larger (0.9 to 4.6 cm, mean 3.0 cm). Minneapolis, MN. Conclusions: In the vast majority of cases, unifocal invasive breast carcinomas do not Background: Classically, lobular carcinoma in situ (LCIS) is composed of dyshesive differ in HER2 status when multiple blocks are tested. Cases with all of the following: cells with intracytoplasmic vacuoles that expand the lobules, with some cells that are equivocal HER2 status, high grade, and heterogeneity in IHC/FISH, are more likely small, round and monotonous (type A) and others that are moderately enlarged with to yield a different result on testing of a second block. more abundant eosinophilc cytoplasm (type B). In contrast, pleomorphic LCIS (PLCIS) cells have marked nuclear enlargement similar to high grade ductal carcinoma in situ. 115 HER2 Testing of Multifocal Invasive Breast Carcinoma: How However, determining moderate vs. marked nuclear enlargement is subjective and can Many Blocks Are Enough? make distinguishing true PLCIS from CLCIS with abundant type B cells a challenge. GC Bethune, MC Chang. Mount Sinai Hospital, Toronto, ON, Canada; University of As the frequency of bi- and multinucleated (B/M) cells is generally greater in more Toronto, Toronto, ON, Canada. proliferative lesions, we evaluated cases of PLCIS and CLCIS for the presence and Background: The human epidermal growth factor receptor 2 (ERBB2) oncogene/ number of B/M to determine if this more objective morphologic feature helps in the oncoprotein, commonly known as HER2, is routinely evaluated in invasive breast differential diagnosis. carcinoma because of its predictive and prognostic value. When multiple ipsilateral Design: All cases diagnosed as PLCIS from 2001 to 2012 were identified in the invasive foci are present, the current CAP breast cancer protocol recommends evaluating pathology database, both with and without associated invasion (IC). The hematoxylin HER2 in 1 block of the largest tumor focus, and testing additional smaller foci if a higher and eosin (H&E) stained slides and the E-cadherin stains were reviewed to confirm grade or different histologic type is seen. The evidence supporting this recommendation the diagnosis (N=20). The number of B/M cells vs. the total cell count per high power is unclear. The objective of this study is to determine the diagnostic yield of testing field (HPF) was recorded. A group of consecutive CLCIS cases was subjected to the multiple blocks in cases of multifocal breast carcinoma. same analysis (N=26). Design: In accordance the protocol approved by the institutional research ethics board, Results: The number of LCIS cells counted per case ranged from 372 to 3530 we identified 246 consecutive cases of primary multifocal (ipsilateral) invasive breast (average=1455) with 1-3 HPF examined per case. The average age was 57.8 (range: carcinoma in which more than one tumor block were tested for HER2 between 2006 43-86 years) for the CLCIS group, and was 58.2 (range: 43-75 years) for the PLCIS and 2011. We performed an audit of HER2 results in all cases. For foci considered group. Overall, binucleated cells were more frequent in PLCIS compared to CLCIS equivocal by both immunohistochemistry (2+) and FISH (ratio between 1.8 and 2.2), (94% vs. 29%, p=0.0001). Multinucleated cells were present in 18% of PLCIS and the HER2 status was considered “negative” for a ratio of 2.0 or less, and “positive” if were absent in CLCIS (p=0.006). The frequency and quantity of B/M per HPF was the ratio was > 2.0. For all cases in which ipsilateral tumor foci showed differences in considerably less in CLCIS compared with PLCIS (Table 1). 15% of PLCIS cases had HER2 status, the tumor size, grade, and histologic features of each focus were recorded. >5 B/M in a HPF; this was never seen in CLCIS (p=0.016). There was no significant Results: Of 246 cases, 198 (80%) had 2 foci and 48 (20%) had 3 to 6 foci. In cases difference in B/M between PLCIS and PLCIS with IC (p=0.96). with 2 foci, both foci were tested. In cases with >2 foci, selected foci were tested (up Table 1: Bi and Multinucleated Cells per HPF to 5 blocks). Only 16/246 (6.5%) demonstrated a difference in HER2 status between 0 1-5 6-10 >10 Range Mean tumor foci. The largest tumor focus was HER2-positive in 12/16 (75%) of these cases; PLCIS 2 26 3 2 0-12 3.5 7/12 (58%) of these largest tumor foci were also the highest-grade foci. Overall, testing CLCIS 37 15 0 0 0-4 0.5 of the largest focus resulted in the most-positive result in 242/246 (98%) of cases. Of Conclusions: Binucleation was identified 3.25x as frequently in PLCIS compared to the remaining 4 discordant cases, 1/4 (25%) had 2 foci of the same size and 3/4 (75%) CLCIS. >5 B/M per HPF and multinucleation was seen exclusively in PLCIS. There were in smaller foci that also had higher grade or different histology. Not a single focus was no statistical difference in the frequency of B/M between patients with an IC vs. (0%) that was smaller and similar histologically yielded a more-positive HER2 result. those with in situ disease only. If PLCIS is being considered, frequent binucleated or any Conclusions: The large majority of multifocal breast carcinomas are not discordant multinucleated cells are an objective H&E feature that can assist in making the diagnosis. with respect to HER2 status. Testing of a single largest focus yielded the most-positive result in >98% of cases. Including equal-sized or smaller foci with a higher grade or 118 Comparison of Oncotype DX™ Recurrence Score by Histologic different histology would yield the most-positive result in 100% of cases. Tumour size, Subtype of Breast Carcinoma grade, and histologic type should therefore be examined if HER2 testing of more than PE Bomeisl, CL Thompson, LN Harris, HL Gilmore. University Hospitals Case Medical 1 focus is under consideration. Center, Cleveland, OH; Seidman Cancer Center, Cleveland, OH. Background: Oncotype DXTM is a commonly used 21 gene assay that predicts the 116 Tumor Associated Inflammation Correlates with Poor Prognosis benefit of chemotherapy for node-negative patients with all types and grades of estrogen in Pregnancy Associated Breast Cancer receptor-positive breast cancer. Results are used to determine which patients may benefit LZ Blanco, Jr., A Mahajan, SA Khan, ME Sullivan, SM Rohan, KP Siziopikou. from adjuvant chemotherapy. Though some data exists demonstrating that traditional Northwestern University Feinberg School of Medicine, Chicago, IL. pathologic features can predict score, there is little information published correlating Background: Pregnancy associated breast cancer (PABC) is diagnosed during or after specific histologic subtype of cancer and recurrence score (RS). TM recent pregnancy and is associated with a poor prognosis. It has been proposed that, Design: The pathology database was searched for all Oncotype DX reports from in some women, the mammary microenvironment might become tumor promoting 1/1/2008 to 10/1/2012. The corresponding original pathology reports were reviewed to after pregnancy as the mammary gland is remodeled to its pre-pregnant state. This determine histologic subtype, grade, and ER/PR/HER2 status. The pathologic features TM pro-inflammatory state is thought to support tumor cell dissemination. In this study were compared with the Oncotype DX report data. we evaluated pathologic characteristics of PABC and the presence and degree of tumor ANNUAL MEETING ABSTRACTS 31A Results: A total of 146 cases were identified. The histologic subtypes seen were invasive pathologic response (pCR, no residual invasive tumor identified), partial response ductal carcinoma (IDC, 71.2%), invasive lobular carcinoma (ILC, 15.8%), mixed (PR, residual invasive cancer identified), and no response (NR, no change in tumor invasive ductal and lobular carcinoma (ID+LC, 8.9%), invasive mucinous carcinoma size/cellularity). (IMC, 2%), mixed invasive ducal and mucinous carcinoma (ID+MC, 1.4%) and Results: Among these 84 cases, 21 had NR, 46 had PR and 17 had pCR. While the tubular carcinoma (TC, 0.7%). These categories were stratified by grade and RS. Not majority of tumors with NR (67%) and PR (52%) were ER+/PgR±/Her2-, the majority unexpectedly, amongst the IDCs, higher grades were associtaed with a much greater of tumors with pCR (70%) were ER-/PgR-/Her2-. Ki-67 was not associated with tumor likelihood of having a high RS (p<0.001). Most tumors with high RSs were IDCs. 15.4% response. With follow-up time of 6-60 months (average 31 months), the NR group had of all IDC vs. 2.1% of the remainder of the tumor histologic types fell into this category. 5 cases of recurrence (average time of 12.6 months before recurrence) and 3 cases of Recurrence scores (RS) by histologic subtype and grade death or hospice enrollment (average time of 19.3 before death/hospice); the PR group Low (0-17) Intermediate (18-30) High (31-100) had 10 cases of recurrence (average time of 20.7 months before recurrence) and 7 cases IDC (total) 52 36 16 of death or hospice enrollment (average time of 32.7 months before death/hospice); Grade 1 20 10 0 and the pCR had no cases of recurrence or death. Cases of recurrence and death did Grade 2 30 20 5 Grade 3 2 6 11 not show a correlation with a specific biomarker profile. ILC (total) 10 13 0 Conclusions: Tumor biomarker profile is a large predictor of response to neoadjuvant Grade 1 2 3 0 chemotherapy. pCR is associated with a better prognosis as compared to PR and NR Grade 2 8 10 0 groups in terms of tumor recurrence and survival. In the PR and NR groups, no specific ID+LC (total) 10 2 1 biomarker profile is identified in association with tumor recurrence or death. Further Grade 1 3 0 0 Grade 2 7 2 0 molecular studies in these groups of patients are necessary to identify additional Grade 3 0 0 1 predictive/prognostic factors. IMC (total) 0 3 0 Grade 1 0 3 0 ID+MC (total) 2 0 0 121 Diagnostic Utility of a 92-Gene Classifier in Triple-Negative Grade 2 2 0 0 Breast Carcinomas TC (total) 1 0 0 EF Brachtel, SE Kerr, J Anderson, Y Zhang, V Singh, MG Erlander, B Carey, WE Grade 1 1 0 0 Highsmith, CA Schnabel, SM Dry, PS Sullivan. Massachusetts General Hospital and Table 1 Harvard Medical School, Boston, MA; Mayo Clinic, Rochester, MN; bioTheranostics, Conclusions: IDCs have the highest likelihood of all subtypes to have high RSs. From Inc., San Diego, CA; University of California Medical Center, Los Angeles, CA. this data set, ILCs, IMCs (pure or mixed) and TCs were unlikely to have high RSs. Background: Triple-negative breast carcinomas (TNBCs) are often poorly differentiated Pure IMCs consistently had intermediate RSs despite the fact that they have favorable and can present as metastases of unknown origin since they are commonly negative for prognoses as compared with other subtypes. Clinicans may want to use Oncotype most immunohistochemical breast markers. Accurate and timely diagnosis of TNBC, TM DX more sparingly in certain histologic subtypes of carcinoma and should interpret which accounts for 10% to 20% of all breast cancers, is critical for appropriate treatment the results from special histologic subtypes with caution. Additional cases from special and participation in clinical trials. A recent multi-institutional validation study of a subtypes of breast carcinoma should be studied to confirm these results. 92-gene molecular cancer classifier showed 87% accuracy (n=790) to predict tumor origin of 28 tumor types. In this subgroup analysis, performance of the 92-gene assay 119 Microvessel Density and Distribution in MRI-Detected Benign in the classification of TNBCs was further examined. Breast Lesions Design: Formalin fixed paraffin embedded tumor samples were selected after a JS Bond, RM diFlorio-Alexander, VA Memoli, WA Wells, JD Marotti. Dartmouth- 3-pathologist-adjudicated review using whole slide imaging technology and ancillary Hitchcock Medical Center, Lebanon, NH. clinical information. Samples were tested in a blinded fashion using the CancerTypeID® Background: The majority of MRI-detected breast lesions are benign on biopsy, and 92-gene classifier (bioTheranostics, Inc), which makes tumor type predictions based therefore, many biopsies prove to be unnecessary. The aim of this study was to evaluate upon gene expression profiling of 87 gene targets and 5 reference genes by quantitative microvessel density (MVD) and distribution in benign breast lesions to better understand reverse transcriptase PCR; molecular results reported as rank probabilities were why they show increased MRI enhancement. compared to the pathological diagnosis. Design: The radiology database was retrospectively searched from 2005-2012 to Results: The current analysis included 34 breast cancers (65% metastatic) from female identify all MRI-guided breast biopsies. 10 clustered apocrine cysts, 10 benign patients. 77% (23/30) were high grade, 20% (6/30) grade 2/3, and 3% (1/30) grade papillary lesions, 10 invasive ductal carcinomas (IDC) and 4 pseudoangiomatous 1/3. 4 cases could not be graded. 21% (7/33) were ER-HER2- or TNBCs, 40% (13/33) stromal hyperplasia (PASH) cases were selected for immunohistochemical analysis ER+HER2-, 21% (7/33) ER-HER2+ and 18% (6/33) ER+HER2+. 1 case was without with CD31 and CD105. MVD was quantified independently by two pathologists (JM receptor/HER2 status. Overall, 74% (25/34 cases) were correctly classified as breast and JB) evaluating identical fields on a multi-headed microscope at 200X magnification. origin by the 92-gene classifier in this study. 86% of TNBCs (6/7) and 79% of ER+ breast Using the “hot-spot” technique, three areas of highest MVD in the center of each lesion, cancers (15/19) were correctly classified as breast origin. 7 incorrectly classified cases, and in stroma away from each lesion excluding lobular epithelium, were chosen for mostly HER2+, were identified as salivary gland origin. One case was unclassifiable. vessel count. Interobserver correlation was calculated using linear regression analysis TNBC (%) ER+HER2- (%) ER-HER2+ (%) ER+HER2+ (%) and comparison between lesional areas and stroma was analyzed by t-test. ANOVA Breast Cancer Cases N=33 7 (21) 13 (40) 7 (21) 6 (18) 92-Gene Classifier Prediction was used to compare MVD among the lesions. 6 (86) 11 (85) 3 (43) 4 (67) Breast Results: Between 2005 and 2012, 235 MRI-guided biopsies were performed, of Conclusions: Results from this analysis provide preliminary evidence of the diagnostic which 161 (69%) were benign. Overall correlation between pathologists was excellent utility of the 92-gene cancer classifier to identify TNBCs in both the primary and (R2=0.91). Compared to normal stroma, mean CD31 MVD was increased in clustered metastatic settings. In this limited data set, the diagnostic utility was equally high for apocrine cysts (33 vs. 14 p<0.0001), benign papillary lesions (63 vs. 19, p<0.001), and TNBCs as for ER+HER2- breast carcinomas. Validation on a larger cohort of TNBCs invasive ductal carcinoma (67 vs. 19, p<0.0001). Dense periductal vascular cuffing was is intended to corroborate these initial findings. seen in 90% of clustered apocrine cyst cases. Although not statistically significant, a trend towards higher MVD in PASH was seen (43 vs. 23). Mean CD105 MVD was higher in IDC (27.4) compared to benign papillary lesions (16.5) and clustered apocrine 122 Predicting OncoDx Recurrence Score with Immunohistochemical cysts (6.7), however, normalized lesion to stroma MVD ratio was not significantly Markers Including Stromelysin different among benign lesions and IDC for CD31 (p=0.138) and CD105 (p=0.221). SH Bradshaw, D Pidutti, DH Gravel, EC Marginean, X Song, SJ Robertson. Ottawa 90% of clustered apocrine cysts were enhancing foci (<5mm) or small masses < 9 mm. Hospital, Ottawa, ON, Canada; Carleton University, Ottawa, ON, Canada. Conclusions: MVD in MRI-detected benign breast lesions is increased compared Background: Recent reports suggest that immunohistochemistry (IHC) markers can be to normal stroma, with most clustered apocrine cysts having a prominent periductal used to give prognostic information in breast cancer that is similar to that contained in distribution similar to that previously reported in DCIS. Most clustered apocrine cysts the Genomic Health Inc. OncoDxR recurrence score (Onco-RS). Our own previous work were small enhancing foci or masses. Further studies are warranted to validate these suggests that the main drivers in predicting Onco-RS are ER PR and Ki67 proliferation findings, examine additional benign proliferative lesions, and to correlate with imaging index. One biological factor not included in our previous work is a measure of invasive to potentially identify predictive MRI features of benign lesions. potential. The goal of this study is to examine the potential predictive value of an IHC based measure of stromelysin-3 (ST-3/MMP-11) expression. 120 Outcomes of Invasive Breast Cancer after Neoadjuvant Therapy Design: A total ST-3 measure (both tumour and stromal) was selected as the first level and Its Association with Molecular Profiles of investigation. The patient population consists of 91 women with ER positive, HER2/ neu negative breast cancer who completed OncoDxR testing. Total ST-3 expression was J Boone, OW Tawfik, F Fan. University of Kansas Medical Center, Kansas City, KS. quantified on IHC stained slides using colour assisted image analysis and expressed Background: The clinical implications of the molecular profile and response to as a ratio of area showing positive staining to total area Also identifiable larger zones neoadjuvant chemotherapy (NACT) of invasive breast carcinoma and its association with of extracellular ST3 were removed from the primary analysis; however extracellular outcome are still being defined. This study is designed to collect outcome data on breast ST-3 was also assessed using a semiquanitative scoring scheme. cancer patients with invasive breast carcinoma treated with neoadjuvant chemotherapy. Results: There was no correlation between extracellular ST-3 and Onco-RS and no Design: We retrospectively identified 84 cases of biopsy proven invasive breast difference in the extracellular ST-3 score in the groups of patients with low (<18), carcinoma treated with NACT from 2007 to 2011 at our medical center. The tumor intermediate or high (> 31) Onco-RS. In contrast total ST-3 did show a significant biomarker profile [including estrogen receptor (ER), progesterone receptor (PgR), difference between these groups (ANOVA, p= .05) with post hoc analysis showing human epidermal growth factor receptor 2 (Her2) and Ki-67], pathological response, the difference is accounted for by a significantly higher score in the high risk group and patient follow-up data were recorded. Tumor response was divided into complete as defined by Onco-RS (figure 1). Stepwise multiple regression incorporating Ki67 32A ANNUAL MEETING ABSTRACTS percentage, total ST-3score, and semi-quantitative ER and PR scores showed no Table 1. Discordant ER/PR/HER2 cases between breast primary and recurrent cancers ER pos ER neg PR pos PR neg HER2 pos HER2 neg additional increase in ability to predict Onco-RS with inclusion of ST-3. Total to neg to pos to neg to pos to neg to pos LR 1 1 1 1 1 0 5/9 (55%) VM 4 2 9 1 5 3 24/54 (44%) BM 2 0 4 0 1 2 9/24 (38%) Total 7 (8%) 3 (3%) 14 (16%) 2 (2%) 7 (8%) 5 (6%) pos=positive, neg=negative Conclusions: Receptor status may change with clinically significant frequency between breast primary cancers and recurrences. The highest rates of change occur with loss of ER/PR/HER2 in the recurrences, with loss of PR being the most frequent change. Recent reports have correlated conversion to negative receptor status with decreased post-recurrence and overall survival. Thus, regardless of the receptor status of the primary breast cancer, all new breast cancer recurrences should be retested for ER/PR/ HER2 status to better direct future patient therapy.

125 GATA3 Expression and Relationship between Clinicopathological Parameters in Invasive Breast Carcinomas A Cakir, O Ekinci, I Isik Gonul, B Cetin, M Benekli, O Uluoglu. Corlu State Hospital, Tekirdag, Turkey; Gazi University School of Medicine, Ankara, Turkey. Background: GATA3 is a transcription factor which regulates luminal cell differentiation in breast tissue and its expression is associated with estrogen receptor (ER) gene. Conclusions: Although total ST-3 showed a statistically significant separation of total Design: We immunohistochemically stained GATA3, CK5/6, EGFR, CK14 and vimentin ST-3 values associated with high Onco-RS, it was not found to be an independent factor on tissue microarray blocks of 457 invasive breast carcinomas. Tumor are subclassified in multivariate analysis which included ER, PR and Ki67 scores. Future work is planned as Luminal A, Luminal B, HER2 expressing, basal-like and null type according to to correct for tumor cellularity and to address the question of the relative importance ER, PR, cerbB2, CK5/6 and EGFR expressions. 13-84 months follow-up data for 254 of tumour cell versus a tumour stromal/ inflammatory cell source of ST-3 staining. cases were obtained. Association between GATA3 expression with clinicopathological parameters, CK5/6, CK14, EGFR, vimentin expression, molecular subtypes, disease 123 Semi-Quantitative Immunohistochemical Scoring System free survival (DFS) and overall survival (OS) were evaluated. Correlates with Oncotype DX® qRT-PCR Assay for Estrogen and Results: 215/457 (47%) tumors were GATA3 positive. GATA3 expression was inversely Progesterone Receptors correlated with mitotic count (p<0.0001), nuclear grade (p=0.001), histological grade W Bshara, L Yan, S Liu, T Khoury. Roswell Park Cancer Institute, Buffalo, NY. (p=0.001), tumor necrosis (p=0.001), nipple invasion (p=0.01), metastasis (p=0.03), Background: Therapeutic decision-making for women diagnosed with breast cancer vimentin (p=0.0003), EGFR (p=0.015) and CK14 (p=0.001) expressions; and directly requires accurate determination of the estrogen receptor (ER) and progesterone receptor associated with ER (p<0.0001) and PR (p<0.0001) expressions. No relationship can (PR). The cutoff for positive ER/PR is somewhat arbitrary. The aim of the study is to be showed between patients age, breast skin and fasia invasion, tumor size, axillary determine the accuracy of Oncotype DX® qRT-PCR assay comparing with IHC and lymph node metastasis, cerbB2, CK5/6 expression and GATA3. The highest GATA3 to define IHC cutoff that better predict Oncotype outcome. expression was in luminal A type (140/245) and the lowest expression was in basal- Design: Consecutive breast carcinoma cases (n=114) that had Oncotype DX® scoring like (1/42), (p<0.0001). Only 1 patient (1/15, 6.6%) died of breast carcinoma had for ER and PR from 2009 to 2011 were included in the study. IHC for ER and PR GATA3 expression (p=0.001). GATA3 positivity was associated with good DFS and (DAKO) was performed on the same tissue block that Oncotype was performed on. Then, OS (p=0.001 and p=0.0009). ER and PR stains were scored using image analysis (Aperio, Vista, CA) by tagging the Conclusions: These results showed that (1) ER and GATA3 expression have strong tumor cells. The percentage and the intensity stain (0+ to 3+) were recorded. Then, three correlation, (2) GATA3 expression is lost in mitotically active, high grade breast scoring systems, H-score, Q-score, and Allred-score were calculated. Spearman’s rank carcinomas, (3) LuminalA breast carcinomas have the highest GATA3 expression correlation coefficient test and area under the curve were used for statistical analysis. while basal-like carcinomas have the lowest and (4) GATA3 positivity can be used as Results: ER and PR tested by qRT-PCR were positive in 109 of 114 (97.3%) samples a prognostic factor to predict good DFS and OS. and in 85 of 114 (74.6%) samples, respectively. For ER, there was complete concordance between IHC and qRT-PCR when a cutoff ≥1% was used. One case became discordant 126 Incidence of Carcinoma and Atypia in Breast Reduction when Allred scoring system was used. For PR, 12 samples had discordant results (11 Mammoplasty Specimens false positive and 1 false negative). Only H-score, similar to qRT-PCR, produced a R Canas Marques, MF Lerwill. Instituto Portugues de Oncologia de Lisboa Francisco normal distribution, while Allred and Q-scoring systems produced skewed distribution Gentil, EPE, Lisboa, Portugal; Massachusetts General Hospital, Boston, MA. to the right for both markers. A cutoff for H-score >1.32 for ER and >13 for PR has Background: Reduction mammoplasty is a common cosmetic procedure. Occult an accuracy of 100% and 93.9% when compared with qRT-PCR assay, respectively. cancers and atypical epithelial proliferations are occasionally found in these specimens, Although more cases were discordant for PR than ER, the Spearman’s rank correlation although the incidence of such changes is not well established. Our aim was to define the coefficient was higher for PR than ER (0.88 and 0.63, respectively). incidence of carcinomas and atypical proliferations in a large series of mammoplasties, Conclusions: While there is no difference in ER testing by IHC and qRT-PCR, PR has with the goal of using this information to develop recommendations for pathology considerable discordance. When referring a breast cancer sample for an Oncotype test, an sampling protocols. ≥ H-score >1.32 or a percentage 1% for ER and H-score for PR>13 can be used as a guide. Design: 911 consecutive mammoplasty cases were retrieved from the Massachusetts General Hospital pathology database from 2005-2010. All specimens were processed 124 Discordance in Estrogen Receptor, Progesterone Receptor according to a standard protocol, requiring a minimum of 2 blocks per breast for patients and HER2 Status between Breast Primary Cancers and Respective <50 y of age with no history of breast cancer (HBC) and a minimum of 10 blocks per Recurrences breast for patients ≥50 y of age or with HBC. Clinical and pathological data were DA Budwit, SM O’Connor, CK Anders. University of North Carolina, Chapel Hill, NC. recorded. Findings were categorized as invasive carcinoma (IC), DCIS without IC, Background: Estrogen receptor (ER), progesterone receptor (PR) and (HER2) status atypical proliferations (LCIS, ADH, ALH, and flat epithelial atypia (FEA)), and benign. is of prognostic and targeted therapeutic importance for patients with breast cancer. Results: Patients ranged in age from 14-78 y (median, 43 y). 161/911 (18%) patients Receptor status may change between primary breast cancer and respective recurrences. had HBC. The median specimen weight was 520 g. The number of blocks ranged from Limited data are available regarding the discordance rates for receptor status overall for 2-37. 2 (0.2%) patients had occult IC, 1 case of grade 1 ILC and 1 case of grade 2 IDC. relapses, and less for the discordance rates for the subcategories of local recurrences 8 (0.9%) patients had DCIS without IC, which was grade 1 in 5 cases, grade 2 in 1 case, (LR), distant visceral metastases (VM) and bone metastases (BM). and grade 3 in 2 cases. Both patients with IC had HBC, whereas only 1 of 8 patients Design: Pathology computer files searched from 1/1/2006 to 7/1/2012 revealed with DCIS had HBC. Of the remaining 901 patients without IC or DCIS, 167 (19%) 82 patients with 87 breast cancer recurrences (9 LR, 54 VM, 24 BM) for which had at least one form of atypical proliferation. LCIS was found in 50 (6%) patients, ER/PR/HER2 receptor status had been determined. ER and PR were evaluated by ADH in 75 (8%), ALH in 47 (5%), FEA in 44 (5%), and other epithelial atypia in 5 immunohistochemistry (IHC), and were considered positive if 1% or more of cells (0.5%). The median age of patients with atypia/carcinoma was 52 y, while the median had positive nuclear staining. HER2 was evaluated by IHC for overexpression and/or age of those with benign findings was 40 y. 113/303 (37%) patients≥ 50 y had atypia/ fluorescence in situ hybridization (FISH) for gene amplification. HER2 was considered carcinoma, while only 64/608 (11%) patients <50 y did so. When stratified according positive if the IHC score was 3+ for membranous overexpression and/or positive for to HBC, 59/161 (37%) HBC+ and 118/750 (16%) HBC- patients had atypia/carcinoma. gene amplification by FISH. The receptor status of the recurrences was compared to Conclusions: Occult IC or DCIS was found in 1% of patients. Atypical proliferations that of the respective primary breast cancers. Discordance rates were determined for were found in 19% of the remaining patients. In total, 177/911 (19%) patients had overall recurrences and for the LR, VM and BM subcategories. significant pathologic findings, either atypia or carcinoma. More than 1/3 of patients≥ 50 Results: Fifty-four (66%) of patients were concordant for all three ER/PR/HER2 results y of age had significant findings, as did >1/3 of patients with HBC. Increased sampling between breast primary cancers and recurrences. Overall discordance rates were ER in patients ≥50 y and those with HBC is recommended for the detection of incidental 11%, PR 18% and HER2 14%. Of 14 triple negative primary breast cancers, 2 (14%) carcinoma and atypia, which can have important clinical implications. were ER positive in the recurrences. The results for discordant cases are summarized in Table 1. ANNUAL MEETING ABSTRACTS 33A 127 The Prognostic Value of STAT1 Overexpression and Design: Whole-genome methylation array analysis was carried out using the Illumina Clinicopathologic Features in Breast Cancer Patients Receiving Adjuvant Infinium HumanMethylation27 Bead- Chip on a 22 fresh frozen DNA samples: 14 Radiotherapy primary BC: 5 ER+, 4 triple negative (TNBC), 5 ER-HER2+, and 8 matched normal, W-C Chen, C-L Wu, Y-H Lai, H-Y Chen, HHW Chen. National Cheng Kung University to quantify the proportion of methylated cytosines (5mC) to total cytosines at 27,578 Medical College and Hospital, Tainan, Taiwan. different CpG dinucleotides. Data were analyzed by GenomeStudio software (Illumina). Background: Adjuvant radiotherapy is a key component of breast cancer management, Locus methylation was calculated as a β-value (low to high ranging from 0 to 1). With significantly reducing the risk of locoregional relapse (LRR) and improving overall 8 sets of paired tumor/normal tissue, GEE was performed to account for the correlation survival. The aim of this study was to evaluate our institutional experience of among such pairs which permitted the 6 unpaired tumor samples to be included in the radiotherapy in patients with operable primary breast cancer and study the prognostic same analysis. To identify methylated genes associated with ER- subtypes (TNBC value of signal transducer and activator of transcription 1 (STAT1) and clinicopathologic and ER-HER2+) and distinct from ER+, a 3-tiered approach to call out genes in which features on LRR. methylation changed dramatically between ER+ and ER- subtypes was used. Tier 1: Design: We retrospectively reviewed 1623 consecutive patients with operable computed adjusted FDR values for all CpGs/ (or their averages for each gene) to be breast cancers which received complete course of adjuvant radiotherapy at our 0.05 or lower. Tier 2: the CpGs/genes to include genes with a 2-fold change (ratio >= institution between 1988 and 2009. Clinicopathologic and outcome parameters were 2.0 or ratio <= 0.5). Tier 3: CpGs/genes to include genes with an absolute difference analyzed. Kaplan-Meier product limit method was used to calculate loco-regional between the mean β of =>0.2. recurrence-free survival (LRFS). Cox proportional hazards models were performed Results: When compared to normal, 2454 were differentially methylated genes (DMG) to assess the prognostic significance of these clinicopathologic parameters on LRFS. in BC at Tier 1, 342 at Tier 2 (238 hyper & 104 hypomethylated), and 77 genes at Tier Immunohistochemical study of STAT1 on 61 formalin-fixed, paraffin-embedded 3 (75 hyper & 2 hypomethylated). For ER+ vs TNBC, of the 187 DMG at Tier 2 (172 primary tumors from patients with LRR and a cohort of 145 consecutive patients who hypermethylated, 15 hypomethylated), Tier 3 restriction included 91 such genes, 87 had no evidence of disease for at least 10 years were performed. The prognostic value hyper- and 4 hypomethylated. For ER+ vs ER-HER+, of 101 significantly DMG (70 of STAT1 on LRR was also evaluated. hyper-, 31 hypomethylated) at Tier 2, Tier 3 restriction held on to 52 such differentially Results: With a median follow-up of 6.1 years, a total of 83 (5.1%) patients developed methylated genes (48 hyper-, 4 hypomethylated). For TNBC vs HER2+, there were no LRR in 4.8 months to 12.5 years (median, 2.0 years). There was a higher rate of DMG at Tiers 2 and 3. However, at Tier 1, only 2 FDR adjusted genes were noted: HAL LRR for patients with triple-negative (TN) or HER2-positive tumors than those with (hypermethylated) and HGDF (hypomethylated). hormone receptor (Rec)+/HER2- tumors. The 10-year LRR for TN, Rec-/HER2+, and Conclusions: ER+ BC are typified by many genes that are hypermethylated relative Rec+/HER2+ were 8.5%, 14.2% and 7.6%, respectively, while the 10-year LRR for to normal tissue or to the TNBC and HER2+subtypes. The study shows feasibility of Rec+/HER2- was 3.9% (P < 0.01). Multivariate analyses showed that patients with distinguishing ER- subtypes of basal-like and HER2+ breast cancers, and this suggests advanced T stage, nodes positive, HER2 positive or TN tumors had shorter LRFS (all the potential to predict outcome based on CpG DNA methylation. P < 0.05). In 206 patients with immunohistochemical studies, STAT1 overexpression was correlated with shorter LRFS (p = 0.02) and was an independent predictor of LRR 130 GATA-3 Is Helpful in Differentiating Primary Mammary after radiotherapy (p < 0.001). Neuroendocrine Carcinomas from Metastatic Neuroendocrine Tumors Conclusions: Patients with TN or HER2-positive breast cancer had a greater risk of S Chopra, S Kim, S Bose, R Mertens, D Dhall. Cedars-Sinai Medical Center, Los LRR after adjuvant radiotherapy. STAT1 overexpression identified a subset of patients Angeles, CA. with a significantly higher risk of LRR. Background: Metastatic neuroendocrine tumors in the breast (mNETs) can show histologic as well as immunohistochemical overlap with primary mammary carcinomas 128 Diagnostic Uncertainty of Fibroepithelial Lesions on Core showing neuroendocrine differentiation (MC-NEs). Since the management of these Biopsy Results in Increased Likelihood of Excision and Final Diagnosis tumors is quite different, it is of utmost importance to classify them accurately. GATA-3 of Phyllodes Tumor is a transcription factor expressed by the luminal epithelial cells in the breast and is SJT Chen, JC Pang, L Zhao, JM Jorns. University of Michigan, Ann Arbor, MI. closely associated with estrogen receptor α in breast carcinoma. This study investigates Background: Fibroepithelial lesion (FEL) is a broad term encompassing fibroadenoma the utility of GATA-3 in differentiating mNETs from MC-NEs. (FA) and phyllodes tumor (PT). FA and PT are treated differently but can have Design: The anatomic pathology files of our institution were searched for MC-NEs overlapping histologic features, making diagnosis challenging on core biopsy. The and mNETs. Tissue blocks were available for 14 MC-NEs and 5 mNETs. In addition, designation of FEL may be used when there is diagnostic uncertainty. Study aims were 11 pancreatic, 8 ileal, and 12 pulmonary primary NETs were selected. GATA-3 to examine the frequency and significance of core biopsy diagnosis (CBD) of FELs and immunohistochemistry was performed on all cases. The H&E slides, previously investigate which features may better predict PT vs. FA. performed immunohistochemical studies, and medical records were extensively Design: An electronic database query (1/07-1/12) identified patients with CBD of reviewed to confirm the primary site. FEL (N=603). Cases were separated into 4 groups: 1) FA (N=573); 2) FEL, favor FA Results: The mean age of patients with MC-NEs and mNETs was 63 and 55, (N=19); 3) FEL, favor PT (N=10) and 4) PT (N=1). In excised cases, biopsy and excision respectively. Four of 5 mNETs were from ileum and 1 was from ovary. The histologic diagnoses were compared and clinicopathologic features assessed. Core biopsies from appearance of MC-NEs and mNETs were quite similar, both showing neuroendocrine cases with follow-up excision were blindly reviewed by 2 breast pathologists for overall features. All mNETs were strongly and diffusely positive for synatophysin and favored diagnosis and histologic features (based on a scoring system for architecture, chromogranin, whereas variable positivity for synaptophysin and chromogranin was cellularity, stromal atypia and stromal mitoses). noted in MC-NEs. All 14 (100%) MC-NEs stained strongly and diffusely for GATA-3, Results: Of cases with known follow-up 45/573 (7.9%) FA and 29/29 (100%) group whereas all 5 mNETs and all 31 primary NETs of other sites were negative. All GATA-3 2-4 cases were later excised. Biopsy-excision correlation is shown in Table 1. Of note, positive cases were moderately to strongly positive for ER. One of the mNETs that 1 group 3 patient had excision elsewhere with unknown results. displayed weak ER positivity was negative for GATA-3. The immunohistochemical Table 1. Biopsy-Excision Correlation results are tabulated in Table 1. Excision Immunohistochemical staining pattern of MC-NEs and mNETs CBD FA PT Diffuse and strong positivity 1) FA 44 (97.8%) 1 (2.2%) for synaptophysin and/or Positivity for ER Positivity for GATA-3 2) FEL, favor FA 13 (72.2%) 5 (27.8%) chromogranin 3) FEL, favor PT 4 (45.4%) 5 (55.6%) Primary mammary 4) PT 0 1 (100%) carcinomas with NE 10/14 (71%) 14/14 (100%) 14/14 (100%) differentiation Among excised patients time from biopsy to excision was significantly decreased if Metastatic 5/5 (100%) 1/5 (20%) 0/5 (0%) CBD was not FA (groups 2-4) vs. FA, with mean times to excision of 4.0 and 13.9 neuroendocrine tumors months, respectively (p=0.002). Final diagnosis of PT was associated with a 1.8 fold Conclusions: Metastatic NETs in the breast closely mimic primary breast carcinomas increase in tumor size (p=0.003). Age at diagnosis and laterality were not associated with neuroendocrine differentiation. GATA-3 and ER are helpful in distinguishing with biopsy or final diagnosis. Blinded review of core biopsies resulted in accurate the two entities. prediction of 6/12 (50%) excision-confirmed PT cases. Histologic features analysis is currently pending statistical analysis. Conclusions: FELs are histologically heterogeneous and thus are subject to sampling 131 Classification of Recurrent MAST and NOTCH Mutations in error, resulting in possible diagnostic uncertainty on core biopsy. Intermediate CBD Breast Carcinoma, a Study of Pre-Malignant Lesions Indicates Early categories of FEL (groups 2-3) are useful in identifying patients that have increased Involvement risk of PT and require excision; however, these diagnoses should be used prudently as MR Clay, V Sushama, WW Robert. Stanford Hospital and Clinics, Stanford, CA. they may result in unnecessary surgical intervention. Background: Recurrent gene fusions and translocations can act as driver mutations affecting tumorigenesis. The Microtubule-Associated Serine/Threonine Kinase family (MAST1-4) is associated with gene fusions that have substantial phenotypic effects in 129 Distinct ER Negative Breast Cancer Subtype-Specific breast epithelial cells. Notch signaling is an evolutionarily conserved pathway that is Methylation Profiles associated with several inherited developmental diseases, and various types of cancer. DA Chitale, GW Divine, KM Chen, MJ Worsham. Henry Ford Hospital, Detroit, MI. Previous studies have identified gene rearrangements in MAST1, MAST2, NOTCH1, Background: Currently available markers routinely used in clinical practice are of and NOTCH2 in 5-7% of invasive breast cancers. - limited value to patients with estrogen receptor-negative (ER ) breast cancer (basal- Design: Using tumor microarrays (TMAs), we sought to identify these gene + like and HER2neu {HER2} positive {HER }), an aggressive subtype. Our aim was rearrangements in a large series of breast cancers. When rearrangements were identified - to identify differentially methylated genes informative of the biology of ER breast in carcinoma, we looked to identify earlier stages of involvement. We used probes against - cancer (BC) to aid in a refined classification of ER subtypes of basal-like and HER2+. the NOTCH1, NOTCH2, MAST1, and MAST2 genes in TMAs containing over 500 cases of breast neoplasia (289 carcinoma in situ, 288 invasive carcinoma, 52 normal). 34A ANNUAL MEETING ABSTRACTS Locus-specific fluorescent in situ hybridization (FISH) was performed using breakapart assays with BACs surrounding target genes. When positive cases were identified on the array, they were analyzed by full section FISH to identify involvement in earlier lesions. Results: For MAST2 and NOTCH1, FISH was evaluated on 1656 cores from 526 patients, of which 1398 hybridized. Of these, translocations were identified in 12 cases for MAST2 (1.8%) and 9 cases for NOTCH1 (1.3%). Despite multiple attempts, we were unable to generate a successful breakapart probe set for both the MAST1 and NOTCH2 genes. A subset of cases also had amplification, with and without associated translocations. During whole section FISH analysis, when invasive carcinoma was translocated, so were earlier lesions including ductal carcinoma in situ (4/5) and flat epithelial atypia (1/1). No translocations were identified in normal breast tissue, fibrocystic change, or columnar cell change.

Conclusions: We have shown a significant difference between the three testing modalities, with a relatively high percentage of discordant results but with few “complete discordances”. With a number needed to screen of 9, we feel the potential information gained by reporting all 3 results is significant enough to warrant this practice. Heterogeneity by IHC is associated with discrepant results, and may be utilized as an indicator for triple testing.

133 Does the Extent of Atypical Hyperplasia in a Benign Breast Biopsy Influence the Magnitude of Breast Cancer Risk? An Update from the Nurses’ Health Studies LC Collins, SA Aroner, RM Tamimi, JL Connolly, GA Colditz, SJ Schnitt. Beth Israel Deaconess Medical Center, Boston, MA; Channing Laboratory, Brigham and Women’s Hospital, Boston, MA; Washington University, St. Louis, MO. Background: Women who have had a benign breast biopsy (BBB) showing atypical hyperplasia (AH) are at increased risk for the development of breast cancer. However, the relationship between histologic type of AH (i.e., atypical ductal hyperplasia [ADH] vs. atypical lobular hyperplasia [ALH]), the extent of atypia and the magnitude of breast Conclusions: We have confirmed the presence of recurrent Mast and Notch family cancer risk are not well-defined. gene rearrangements in breast carcinoma. We have also shown these translocations Design: We conducted a case-control study of benign breast disease and breast cancer can be found in precancerous lesions. Knowing at which developmental stage these risk nested within the Nurses Health Studies. Cases were women with breast cancer translocations occur gives further understanding into the complex process of breast who had a prior BBB (n=470). Controls were women who also had a prior BBB but tumorigenesis. who were free from breast cancer at the time the corresponding case was diagnosed (n=1845). BBB slides were reviewed by pathologists blinded to the case/control status and categorized as non-proliferative, proliferative without atypia, or AH. Those showing 132 HER2 Status in Breast Cancer as Determined by Three ASCO/ AH were further categorized as ADH or ALH using the criteria of Page, et al. and the CAP Recognized Reporting Techniques Reveals Testing Discordance, and number of foci of AH were recorded. Emphasizes the Utility of Co-Testing with FISH and IHC Results: The adjusted OR for breast cancer among all women with AH was 3.3 (95%CI, MR Clay, EA Gilbert, CD Bangs, A Cherry, KC Jensen. Stanford Hospital and Clinics, 2.5 - 4.3). However, the magnitude of risk differed according to histologic type of AH. Stanford, CA; Veterans Hospital, Palo Alto, CA. The OR for the development of breast cancer among women with ALH (54 cases; 55 Background: HER2 positivity is found in 12-15% of newly diagnosed breast controls) was 6.7 (95%CI, 4.3-10.3) whereas the OR for those with ADH (52 cases; carcinomas. Standard practice includes immunohistochemistry (IHC) or fluorescent 118 controls) was 3.0 (95%CI, 2.0-4.4); this difference was statistically significant in situ hybridization (FISH) for primary HER2 status determination, and stipulates (p=0.002). Among women with ADH, there was no association between number of that regardless of whether FISH is reported as a ratio, or a raw count, all three of foci and breast cancer risk. The risk among women with ALH was minimally higher these reporting techniques are acceptable. We wanted to determine how often these for those with >5 than for those with <5 involved TDLUs. three reporting techniques yielded discrepant results in a large number of cases, and determine what if any information was gained by utilizing all three. We also evaluated Odds Ratios for Breast Cancer Risk According to Extent of Atypia Cases Controls OR (95%CI) HER2 heterogeneity by IHC. ADH, 1-2 foci 36 73 3.3 (2.1-5.2) Design: We selected the most recent 300 cases of breast carcinoma in our institution ADH, >3 foci 15 41 2.5 (1.3-4.7) to have had primary HER2 determination by FISH. All of these cases were also tested ALH, 1-4 TDLU 37 39 6.6 (4.0-11.0) by IHC, and FISH results were re-evaluated to include both the ratio and raw scores. ALH, >5 TDLU 13 12 7.0 (3.1-15.9) Heterogeneity on IHC staining was determined. The characteristics of discrepant cases Conclusions: This analysis continues to demonstrate that women with AH in a BBB were evaluated based on a number of clinicopathologic features. are at a substantially increased risk for the development of breast cancer. The risk is Results: We evaluated 91 core needle biopsies, 103 lumpectomies, 98 mastectomies, greater among those with ALH than those with ADH. The extent of AH (either ADH or and 8 metastasis resections. Overall, discordant findings were observed in at least one ALH) does not significantly contribute to breast cancer risk. The lack of a dose effect testing modality (IHC vs. FISHratio vs Fishraw) in 37 cases (12.33%). Discordance was between AH and breast cancer risk indicates that the extent of AH in a BBB should not associated with the presence of heterogeneity, some cancer subtypes, and ER negativity. influence management decisions. In particular, women with more limited foci of AH Discordance was not more likely with biopsies. It takes 9 patients to be screened by should not be managed differently from those with greater amounts of AH. all 3 ASCO/CAP recommended modalities in order to find one patient with discrepant testing (6.2-11.6 CI). Two cases were what we have termed completely discordant, in 134 Decreased Expression Levels of Phosphorylated Ser239- that they had different results for each of the 3 modalities (figure 1). Vasodilator-Stimulated Phosphoprotein Indicate Progression from In-Situ to Invasive Breast Carcinomas P Cotzia, A Bombonati, M Ali, JP Palazzo, R Birbe, G Pitari. Thomas Jefferson University Hospital, Philadelphia, PA. Background: Vasodilator-stimulated phosphoprotein (VASP) is an actin-binding protein promoting actin polymerization, dynamic membrane regions and epithelial cell barrier function. VASP activity is precisely regulated through Ser phosphorylation. Recent studies have shown that changes in VASP expression modulate adhesion and proliferation of breast cancer cells. Design: To examine the relationship between VASP Ser phosphorylation and the carcinogenic process, VASP and its Ser phosphorylated forms (pSer157-VASP and pSer239-VASP) were examined by immunohistochemistry in paraffin-embedded specimens, assembled into 2 tissue micro-arrays (TMA) with each patient represented by duplicate samples. Human tissues were obtained from the tumor bank of the Department of Pathology, Anatomy and Cell Biology at Thomas Jefferson University Hospital, under a protocol approved by the Institutional Review Board. One TMA contained 24 ANNUAL MEETING ABSTRACTS 35A ductal carcinoma in-situ (DCIS): 9 grade I-II and 15 grade III. The second TMA had Conclusions: While H&E assessment remains the most important tool in assessing MI 69 invasive ductal carcinomas (IDC): 14 grade I and 55 grade II. Specific membranous in BC grading, PHH3 mitotic labeling can serve as a useful adjunct in the proper grading and/or cytoplasmic staining was scored semiquantitatively (0-to-3 scale: 0, absent; 1+, of equivocal cases, especially in small biopsies. Given that PHH3 may potentially label weak in less than 10% of cancer cells; 2+, moderate in 10-30%; 3+, moderate and/or more mitotically active cells than by H&E staining (and thus substantially upgrade a strong in more than 30%). significant proportion of BCs), integration of this marker into BC grading needs large- Results: Expression of all 3 VASP biomarkers was significantly decreased in IDC, scaled, multicenter studies. compared to DCIS. Of note, pSer239-VASP was the most compromised biomarker and represented an independent diagnostic discriminator of non-invasive versus invasive 137 Immunohistochemical Characterization of Cystic disease. In DCIS, pSer239-VASP expression was significantly decreased in grade III Hypersecretory Carcinoma compared to grades I-II. Further, in IDC grade II tumors exhibited significantly lower TM D’Alfonso, Y-F Liu, SJ Shin. Weill Cornell Medical College, New York, NY. levels of pSer239-VASP than those of grade I. Background: Cystic Hypersecretory Carcinoma (CHC) is a variant of DCIS uniquely Conclusions: - Decreased expression of VASP biomarkers is associated with the containing luminal secretion resembling thyroidal colloid. CHC is thought to behave progression from in-situ to invasive ductal carcinoma of the breast. in an indolent manner, but has the potential to give rise to invasive carcinoma, which is - pSer239-VASP is a novel predictor of breast stromal invasion, which could be used typically poorly-differentiated. Despite its characterization as a clinicopathologic entity as an immunohistochemical tool to identify those DCIS with high potential to progress over two decades ago, the immunohistochemical (IHC) profile of CHC remains unclear. to invasive disease. We set out to better define the IHC features of this uncommon lesion. Design: 10 cases of CHC were identified from our files. In each case, IHC stains were 135 Hormone Receptor and HER2/Neu Immunohistochemistry in performed using the Bond Max Autostainer (Leica) with appropriate controls and Multifocal/Multicentric Breast Carcinoma resulting slides were reviewed by two breast pathologists. S Covey, DD Baker, SM O’Connor. UNC School of Medicine, Chapel Hill, NC; Duke Results: All 10 cases were biopsies (9 excisional; 1 needle core) of women averaging University, Durham, NC. 63 years in age (range: 47-79). The clinical/radiographic presentation was a mass (5/10), Background: The prognostic and predictive value of ER/PR/HER2 status in breast calcifications (3/10), bloody nipple discharge (1/10), and unknown (1/10). No cases cancer is well established. In addition, response to hormone-directed therapy correlates showed concurrent invasive carcinoma. The microscopic size of CHC ranged from 0.2 with degree of ER reactivity. A moderate number of breast cancers are multifocal/ to 2.7 cm (mean, 0.9). The architecture was predominantly micropapillary (9/10; 90%) multicentric (MF/MC) and there is limited data to direct whether more than one focus whereas the nuclear grade was either intermediate (5/10; 50%) or high (5/10; 50%). should be immunostained. The largest and most recent study (65 ductal carcinoma Calcifications were present in CHC in 7/10 (70%) cases. One case was associated cases) suggests that receptor status may be adequately assessed by analysis of one with pregnancy-like hyperplasia. CHC arose in a background of cystic hypersecretory focus, particularly if foci are histologically similar and low grade. Our goal was to change/hyperplasia (CHChange/CHH) and atypical cystic hypersecretory hyperplasia evaluate a larger number of MF/MC cases and to determine if specific histologic (ACHH) in most cases (7/10; 70%) while the remaining 3 cases had only co-existing features correlate with IHC discrepancies and may be used to guide the decision to CHChange. Luminal secretions were present in all cases and characteristically exhibited stain more than one focus. retraction, “pock marks,” and “Venetian blinds.” CHC was ER+/PR+ in 4/10; ER+/ Design: MF/MC invasive breast cancer cases from 1/2000 to 5/2011 were identified PR- in 4/10 and ER-/PR- in the remaining 2/10. All were HER-2 negative. Androgen in CoPath. Reports and archived slides were reviewed to verify number of foci, MF vs receptor was positive in 3/10 (30%) cases. Myoepithelial stains p63, smooth muscle MC, grade, and morphology. ER/PR IHC was scored by intensity and percent of cells myosin (SMM), and CK5 showed circumferential staining in 9/10 (90%) cases while 1 staining at each intensity. Modified H score was calculated by multiplying intensity case was negative for p63, SMM, and CK5 in both CHC and adjacent CHChange. CK5 x percent tumor cells labeled, range 0 to 300. Qualitatively, tumors were considered showed variable staining in 3/10 (30%) cases. Basal markers EGFR, CK14, and CK5 ER/PR+ if ≥1% nuclei stained. HER2 IHC was scored using ASCO/CAP guidelines. were negative in all cases with the exception of 2 cases, each positive for EGFR or CK5. If IHC was not performed at UNC, different clones were used, or staining not well Conclusions: CHC is a distinct variant of DCIS that typically arises in a background of preserved, tissue blocks were retrieved to perform IHC. Sections were immunostained CHChange/CHH/ACHH and exhibits micropapillary architecture, intermediate to high- using BenchMark XT IHC/ISH staining modules with ER (Ventana, SP1), PR (Ventana, grade nuclei, and luminal colloid-like secretion. CHC is typically ER/PR positive and 1E2), and HER2 (Ventana, 4B5) antibodies. HER-2 negative. Basal markers are consistently non-reactive. Negative or discontinuous Results: A total of 104 cases (225 tumors) were evaluated. ER results were discordant reactivity with myoepithelial markers may be seen, despite its in-situ nature. in 4 cases. One of these 4 was multicentric, 3 multifocal. In two cases, foci were morphologically different - in one, foci were the same grade and in the other, different 138 BreastPRS Effectively Separates OncotypeDX Intermediate grades. In the remaining two cases, foci were morphologically similar and all high Risk Group to Low and High Risk Groups grade (9/9); foci were triple negative except for the weak ER positivity seen in one TM D’Alfonso, RK van Laar, R Flinchum, N Brown, L Saint John, SJ Shin. Weill Cornell focus from each case. No positive/negative HER2 discordancies were identified. PR Medical College, New York, NY; Signal Genetics, LLC, New York, NY. results were more variable. Background: Multi-gene prognostic tools resulting in patients classified as ‘intermediate Conclusions: In the majority of MF/MC breast cancer cases, it is sufficient to risk’ are of limited clinical utility, as the benefit of chemotherapy in patients in this immunostain a single focus. In order to assure that a patient is offered appropriate risk group is unclear. BreastPRS (Signal Genetics) is a new molecular characterization therapeutic options, it may be advisable to stain more than one focus for ER if 1) the assay developed from a meta-analysis of publically available genomic datasets. The index focus is high grade and triple negative or 2) it is ER negative and other foci are assay utilizes three unique yet complementary gene signatures to determine molecular morphologically different. MC vs MF and differences in grade (low or intermediate) grade (103 genes), molecular subtype (280 genes) and risk of recurrence (200 genes). among foci did not predict discordant IHC. BreastPRS is a binary assay that could potentially re-classify intermediate risk patients into more meaningful prognostic groups. We sought to re-classify Oncotype Dx (ODx) 136 The Utility of Phosphohistone H3 (PHH3) in Breast Cancer (Genomic Health) intermediate risk cases using BreastPRS. Grading Design: 307 whole genome expression profiles were generated from formalin-fixed X Cui, GP Siegal, S Wei. University of Alabama at Birmingham, Birmingham, AL. paraffin embedded tissue of patients’ tumors with known ODx recurrence scores. A set Background: Histologic grading has been recognized as a powerful prognostic indicator of 120 genes was found to hierarchically cluster patients according to ODx risk group. in breast cancer (BC). The widely accepted Bloom-Richardson grading system employs This gene set was used to interrogate data from 522 untreated ER+ breast cancer patients the combination of tubule formation, nuclear pleomorphism and mitotic indices (MI). obtained from public genomic data repositories, with associated outcome data. Those In practice, accurate assessment of MI on H&E stained sections is an issue that requires predicted to be intermediate risk according to the ODx-estimation (ODx-est) algorithm experience and is subject to interobserver variability. Commonly used Ki-67 staining were further analyzed using BreastPRS. The recurrence free survival differences between is a proliferation marker but is not specific for mitoses. Histone H3 Phosphorylation resulting high and low risk predictions were compared using Kaplan Meier analysis occurs only when chromosomes are fully condensed which is a strict mitotic event and and multivariate cox proportional hazards regression. thus may provide a more promising marker for MI. In this study, we sought to evaluate Results: The risk groups by ODx-est and ODx were concordant in 72% of cases (Chi the utility of PHH3 labeling in grading BC and its prognostic significance. Square P<0.001). Using ODx-est and BreastPRS, 522 ER+ untreated patients were Design: The Surgical Pathology database at the authors’ institution was searched classified into high/intermediate/low and high/low risk groups, respectively, with a to identify breast biopsy cases with a diagnosis of BC between 2004 and 2006. A high degree of statistical significance in outcome (15-year recurrence free survival, total of 61 consecutive cases with sufficient tissue were selected and subjected to P<0.0001). The 124 patients predicted to be intermediate risk by ODx-est were then immunohistochemical evaluation. Ki-67 was scored as the average fraction and PHH3 re-classified as high or low risk using BreastPRS. The reclassified risk groups exhibited was scored as the total number of labeled tumor cells, in 10 high power fields where differences in recurrence free survival that were statistically significant (P=0.0021, the immunolabeling was prevalent (hot spots). Chi-Square or student t-test was utilized Hazard ratio: 4.38, 95% CI: 2.24 to 8.57). The significance of this stratification was for statistical analysis, when appropriate. independent when adjusted for age at diagnosis and tumor grade (P=0.025). Results: Cells with mitotic activity could be easily identified by unequivocal nuclear Conclusions: The data strongly suggest that BreastPRS can reclassify intermediate risk PHH3 labeling. While there was a strong linear correlation and regression for MI patients by ODx into two groups with significant differences in recurrence-free survival. between H&E and PHH3 stains (r=0.71; p<0.0001), 28 cases (46%) were upgraded by PHH3 (Chi-Square=25.5; p<0.00001), exclusively in those labeled grade II BCs by H&E staining. In addition, a strong correlation between PHH3 MI and the Ki-67 proliferation index was also seen (r=0.63; p<0.0001). There was no significant association between PHH3 MI and ER, PR or HER2 status. Further, no significant differences in PHH3 MI were found in BCs with and without lymph node involvement, or those with and without associated distant organ metastasis either at the time of diagnosis or subsequently. 36A ANNUAL MEETING ABSTRACTS 139 Microvessel Density as Determined by Computerized Image score generated by the VIAS algorithm ranges from 2.5 to 3.5. However, adjusting the Analysis of CD105 Expression Correlates with Clinicopathological manufacturer’s positive cut-off values may allow for increased specificity for HER2 Features and Outcome in Triple-Negative Breast Cancer amplification by FISH. M De Brot, RM Rocha, FA Soares, H Gobbi. Federal University of Minas Gerais, Belo Design: 533 cases with IHC staining and FISH analysis for HER2 were retrospectively Horizonte, Minas Gerais, Brazil; AC Camargo Hospital, Sao Paulo, Brazil. analyzed. Tumor sections were stained with Ventana PATHWAY© anti-HER-2/neu Background: Triple-negative breast cancers (TNBCs) tend to relapse and metastasize (4B5) antibody using automated method. Image analysis was performed using the VIAS early with a distinct pattern of dissemination. Although the occurrence of metastasis platform according to the manufacturer’s specifications. The manufacturer’s cutoff is related to angiogenesis, literature on angiogenic characteristics of these tumors is scores for 0, 1+, 2+, and 3+ are 0-0.5, 0.51-1.5, 1.51-2.5, and 2.51-3.5 respectively. limited. We assessed topography and characteristics of blood vessels in TNBC and FISH analysis was performed using the PathVysion Kit. HER2 amplification was defined examined their association(s) with clinicopathological criteria, basal-like phenotype as a HER2/CEP17 ratio of >2.2, borderline amplification was 1.8 to 2.2 and negative and prognosis in a series of TNBCs. was <1.8. In our laboratory, the positive threshold for the 3+ IHC was increased from Design: Clinicopathological data were obtained from 133 TNBCs. For identification 2.51 to 3.5 and those below 3.5 were interpreted as 2+. Sensitivity and specificity for of blood vessels, FFPE whole tissue sections were stained with CD105; slides were HER2 positivity at the two levels were compared with FISH results. scanned using Aperio Digital Pathology System. Each digital slide was examined by Results: Raising the IHC cut-off score for the 3+ category from 2.51 to 3.5 moved 52 a pathologist and divided into three regions: intratumoral (IT), peripheral (PP) and out of 533 cases from the positive to intermediate (2+) IHC category. Of these, 28 were peritumoral (PT) areas. Three hot spots were selected in each region (IT, PP, PT - area of HER2 negative by FISH, 23 were HER2 positive, and 1 had borderline amplification. 6.0 mm2 each). Microvessel density (MVD) was measured by applying the Microvessel This increased the specificity from 91.3% to 99.2% and lowered the false positive rate Density v1 algorithm. MVD was categorized into high MVD and low MVD according from 5.8% to 0.6%. to its median value. Associations between MVD in each region and clinicopathological Table 1 criteria were evaluated in univariate and multivariate analysis. VIAS Score NON AMP Amp Borderline Mean Score Std Dev Results: Median time of follow-up was 40 months (range, 1-161 months). In all, 53 0 (0-0.5) n = 92 89 (96.7%) 1 (1.1%) 2 (2.2%) 0.216 0.146 (39.1%) patients had developed a recurrence by the time of last follow-up; distant 1+ (0.51-1.5) n = 81 75 (92.6%) 4 (4.9%) 2 (2.5%) 1.003 0.289 2+ (1.51-3.49) n = 231 188 (81.4%) 39 (16.9%) 4 (1.7%) 2.150 0.513 metastasis was detected in 49 (36.8%) patients, lungs, brain and bones representing the 3+ (3.5+) n = 129 3 (2.3%) 124 (96.1%) 2 (1.6%) 3.500 0 main sites. Overall, 101 (75.9%) TNBCs showed a basal-like phenotype. Blood vessels Conclusions: The VIAS is a valuable tool for assessing HER2 overexpression in breast were located largely in the peripheral area; TNBCs with a basal phenotype showed carcinoma. However, the manufacturer’s IHC cutoff for HER2 positivity resulted in a higher MVDs (p=0.028). A significant association was observed between high peripheral false positive rate of 5.8%. Raising the 3+ cutoff value from 2.51 to 3.5 decreased the MVD, recurrence (p=0.038) and development of distant metastasis (p=0.031). High false positive rate to 0.6% and increased specificity to 99.2%. FISH testing should be peritumoral MVD was associated with vascular invasion (p=0.015). Survival analysis performed for VIAS IHC scores of 1.51 to 3.49. showed that high peripheral MVD was strongly associated with poorer overall survival (OS, p=0.001) and disease-free interval (DFI, p=0.001). In multivariate analysis for OS and DFI, peripheral MVD retained its prognostic significance (p= 0.003) after 142 Incidental High Risk Lesions in Reduction Mammaplasty adjustment for significant variables (stage and lymph node status). Specimens: Analysis of 2351 Cases from Large Women’s Health Centers Conclusions: We demonstrate associations between MVD in different areas and various MM Desouki, O Fadare, C Zhao. Vanderbilt University School of Medicine, Nashville, parameters in TNBCs, indicating that tumors with aggressive features have higher TN; Magee-Womens Hospital, Pittsburgh, PA. MVDs. Neovessels are mainly located in the peripheral area of TNBCs and may have Background: Atypical proliferative lesions (APL) are occasionally found in breast a critical role in disease dissemination. reductions. The aim of the study is to investigate the prevalence of APL in reduction mammoplasty from patients that were treated primarily for macromastia. 140 GATA3 Expression in Different Subtypes of Invasive Breast Design: A retrospective chart review of pathology records on patients that underwent Carcinoma and Comparison with GCDFP15 and Mammaglobin reduction mammoplasty for macromastia from 2006-11 retrieved 2351 cases. Exclusion criteria were a history of invasive or in-situ carcinoma on prior excision. Laterality, G Deftereos, U Krishnamurti, JF Silverman. Allegheny General Hospital, Pittsburgh, PA. weight, and presence of APL were recorded and analyzed. APL included invasive Background: GATA3 (GATA binding protein 3) is a transcription factor that has a carcinoma, ductal or lobular carcinoma in-situ (DCIS or LCIS), atypical ductal or role in cell proliferation and differentiation in many tissues, including the breast and is lobular hyperplasia (ADH or ALH), and flat epithelial atypia (FEA). The presence of fairly specific for breast and urological cancers. It has been suggested that low GATA3 papillomas and radial scars were also noted. expression in breast carcinomas (BC) correlates with a worst prognosis. However, Results: Among 2351 cases of reduction mammoplasty, 108 (4.6%) cases were there is little data available on GATA3 expression in the various histologic subtypes diagnosed with an APL (table 1). Only one case of invasive duct carcinoma measured of invasive BC. 0.2 cm and was assigned a Nottingham grade 1 was fond. Cases of ADH were associated Design: 59 cases of invasive BC, including 14 ductal, 12 lobular, 12 micropapillary, with bilateral lobular neoplasia (n=4), ipsilateral ALH (n=6) and contralateral FEA (n=2). 10 pleomorphic lobular, 4 ductal with apocrine features, 4 mucinous and 3 Papillomas and radial scars identified in 78 cases (3.3%). The former were interpreted medullary carcinomas were selected and immunostained for GATA3, GCDFP15 and as unilateral intraductal papilloma (IDP), unilateral multiple IDPs/papillomatosis, mammaglobin. For GATA3, stain intensity was graded as 1+, 2+ and 3+ and an H-score bilateral single IDP and bilateral multiple IDPs in 50, 14, 3 and 2 cases, respectively. was calculated by multiplying the stain intensity with the percentage of tumor cells Radial scars were identified in 9 cases. Out of the 108 cases with APL, 101 (93.5%) staining. GATA3 staining was examined in the various histologic subtypes and compared underwent bilateral and 7 cases (6.5%) underwent unilateral reduction. The average with GCDFP15 and mammaglobin reactivity. Correlations with nuclear grade, ER, PR weight of right and left reductions with APL was 712 and 732 grams, respectively. and HER2 status were also investigated. Results: Of the 59 cases, GATA3 was positive in all 59 (100%) BC, while GCDFP15 and Frequency of atypical proliferative lesions in breast reduction mammoplasty performed for macromastia (n=2351) mammaglobin were positive only in 23 (38.98%) and 31 (52.54%) cases, respectively. Atypical Proliferative lesion Bilateral (%) Right (%) Left (%) Total (%) GATA3 was positive in all 26 (100%) cases with grade 3 nuclei, while GCDFP15 and Invasive carcinoma 0 0 1 1 (0.04) mammaglobin were positive only in 5 (19.23%) and 11 (42.31%) cases, respectively. DCIS 0 4 0 4 (0.2) ER-positive carcinomas had significantly higher GATA3 expression when compared ADH 6 22 16 44 (1.9) LCIS 1 8 7 16 (0.7) with ER-negative carcinomas (mean H-score of 279 vs. 161; p<0.0001). 100% (n=8) ALH 6 18 16 40 (1.7) cases of triple-negative carcinomas also stained with GATA3, significantly more than FEA 0 2 1 3 (0.1) GCDFP15 (37.5%, p<0.0001) or mammaglobin (12.5%; p<0.0001). Staining for GATA3 Total 13 (0.6) 54 (2.3) 41 (1.7) 108 (4.6) was also qualitatively superior compared to GCDFP15 and mammaglobin, showing less Conclusions: This is the largest retrospective study that has assessed the prevalence of background staining, particularly in cases of mucinous BC, where the mucin showed APL in reduction mammoplasty. The frequency of detection of these lesions in patients no staining, in contrast to the other two immunomarkers. with no history of invasive or in situ carcinoma is low (4.6%). Detection of invasive Conclusions: GATA3 shows a higher sensitivity, compared to GCDFP15 and and DCIS lesions is extraordinarily low at 0.2%. The most common APL is lobular mammaglobin in BC with uniform expression in all histologic subtypes and with lower neoplasia (56/2351; 2.4%). ADH and FEA represent 2% (47/2351). Our findings provide background staining. There is correlation between ER-positivity and higher GATA3 data on the distribution of these lesions in this setting, as well as some insight on the expression, regardless of the histologic subtype. GATA3 is significantly more sensitive prevalence in the general population. in both high nuclear grade and triple-negative BC than the other IHC breast markers. In addition, we believe that GATA3 is a useful antibody in the work-up of metastatic cancer where BC is considered in the differential diagnosis. 143 Nuclear Poly (Adenosine Diphosphate-Ribose) Polymerase (PARP) Expression Is Most Prevalent in BRCA Mutated and Basal-Like Breast Cancers 141 Evaluation of HER2 IHC Staining Using the Ventana Image PJ DiMaggio, TM D’Alfonso, Y-F Liu, Z Chen, RE Kaplan, SJ Shin. Weill Cornell Analysis System, Correlation with HER2 FISH Amplification Medical College, New York, NY. JA Dennis, R Parsa, D Chau, Y Peng, S Sahoo, YV Fang, VR Sarode. UT Southwestern, Background: Poly(adenosine diphosphate-ribose) polymerase (PARP) is a family of Dallas, TX. enzymes involved in DNA damage repair. When activated, PARP forms polymers which Background: Overexpression of HER2 is seen in 15 to 20% of invasive breast bind and modify functions of numerous cellular proteins. The role of PARP inhibitors carcinoma. HER2 positivity by IHC is graded as 0, 1+, 2+, and 3+ based on the extent as a new anticancer agent is particularly promising in BRCA mutated tumors failing to and intensity of cytoplasmic membrane positivity. HER2 FISH is considered the gold perform homologous recombination. PARP inhibition lacks homologous recombination standard for IHC positivity. Concordance between HER2 IHC positivity and FISH capacity and causes apoptosis. No studies have been performed evaluating PARP amplification is between 80 to 95%. The Ventana Image Analysis System (VIAS) is a expression by immunohistochemistry (IHC) in BRCA mutated breast cancers or computer-based method of IHC scoring that provides continuous values. The positive ANNUAL MEETING ABSTRACTS 37A molecular subtypes including the basal-like subtype comprised of ER/PR/HER2 (triple) Results: A total of 473 patients were identified with histology available for review. negative breast cancers. This knowledge is of future value if IHC could select patients Out of these patients 266, 143 and 63 patients had 1, 2 or 3 PLN respectively. ENTE whose tumors would be susceptible to PARP inhibitors. was present in 97 cases, and absent in the remaining 357 cases. Kaplan-Meier disease Design: Primary breast carcinomas of 637 patients were evaluated using tissue specific survival curves are seen below. microarrays (TMA). Four molecular subtypes of invasive breast carcinoma were represented: Luminal A-145; Luminal B/HER2 positive-104; Luminal B/HER2 negative and Ki-67>14%-98; Basal-like-132; HER2 enriched-94. Sixty-four cases of BRCA mutated breast cancer were also studied. TMAs were stained with anti-PARP (Abcam) using the Bond Max Autostainer (Leica) and appropriate controls. Nuclear immunoreactivity for PARP was scored via a modified H-system and intensity was scored on a 0 to 3 scale (0-negative, 1-weak, 2-moderate, 3-strong). Percentages of positive cells were categorized into 5 groups (0%, 1%-10%, 11%-50%, 51%-80%, >80%). Statistical analysis used the Chi-square test. Results: Nuclear immunoreactivity for PARP was significantly higher in the basal-like molecular subtype than Luminal A, Luminal B/HER2 positive, and Luminal B/HER2 negative and Ki-67>14% (p=0.004, 0.006, and 0.037). Nuclear immunoreactivity for PARP was significantly higher in BRCA mutated carcinomas than in the Luminal A, Luminal B/HER2 positive, and Luminal B/HER2 negative and Ki-67>14% subtypes(p=0.004, 0.004, and 0.029). No difference existed between basal-like molecular subtypes and BRCA mutated carcinomas (p=0.356). No significant differences were found between HER2 enriched subtypes and any other group. Conclusions: Our results confirm clinical observations that PARP expression is most prevalent in basal-like (triple negative) and BRCA mutated breast carcinomas. PARP expression by IHC may be of clinical utility for selecting breast cancer patients likely to benefit from therapy using PARP inhibitors.

144 The Basal-Like Molecular Subtype of Breast Carcinoma Shows Reduced Expression for Androgen Receptor When Compared to Other Subtypes PJ DiMaggio, S Varma, TM D’Alfonso, Y-F Liu, Z Chen, RE Kaplan, SJ Shin. Weill Cornell Medical College, New York, NY. Background: Androgen receptor (AR) expressing breast cancers can potentially be targeted by anti-androgens such as bicalutamide. Patients with breast cancers belonging to a particular molecular subtype may be poised to benefit more from anti-androgen therapies than those of other subtypes. We set out to investigate the prevalence of AR expression by immunohistochemistry in molecular subtypes and additionally, identify any correlation with clinicopathologic features within any single molecular subtype. Design: Primary breast carcinomas of 573 patients were evaluated using a tissue microarray (TMA) platform. The four molecular subtypes of invasive breast carcinoma were represented as follows: Luminal A-145; Luminal B/ HER2 positive-104; Luminal B/HER2 negative and Ki-67>14%-98; Basal-like-132; HER2 enriched-94. TMA slides were immunostained with anti-AR (Novocastra) using the Bond Max Autostainer (Leica) with appropriate controls. Nuclear immunoreactivity for AR was scored using the H scoring system. Staining intensity was scored on a scale from 0 to 3 (0-negative, 1-weak, 2-moderate, 3-strong) and percentage of positive cells was recorded (0-100%). Statistical analysis (one-way ANOVA test and pairwise comparison) was performed. Within each molecular subtype, clinicopathologic variables (age, tumor type, tumor grade, tumor size, presence of lymphovascular invasion, nodal status, stage) were also correlated with AR expression. Results: The basal-like molecular subtype showed significantly less AR expression than all other subtypes. The mean difference with (95% confidence interval) of basal-like versus Luminal A was 38.4 (16.8,60.0); Luminal B/HER2 positive 74.2 (50.3,98.2); Luminal B/HER2negative Ki-67>14% 52.5 (28.5,76.5); and HER2 enriched 33.9 (9.9,57.9). Luminal B/HER2 positive subtypes also showed significantly less AR expression than Luminal A and HER2 enriched molecular subtypes (data not shown). Conclusions: The number of positive axillary lymph nodes is statistically significant in No clinicopathologic features were associated with AR expression in any of the regards to patient outcome. The presence of ENTE does not reach significance. 12 year molecular subtypes. disease specific survival for patients with stage II breast cancer approached 87%. When Conclusions: As a group, invasive breast carcinomas of the basal-like subtype appear looking to stratify patients with stage II breast cancer who may benefit from radiation least likely to benefit from anti-androgen targeted therapy than other molecular therapy, in our study the presence of ENTE did not show clear patient stratification. subtypes. Within any single molecular subtype, AR expression did not correlate with clinicopathologic features. 146 Clinicopathologic Features of Male Paget Disease of Breast: 11 Cases from a Single Institute 145 Metastatic Tumor Volume and Extranodal Tumor Extension: X Duan, A Gullett, N Sneige, D Rosen, E Resetkova, C Ersahin, Y Wu, I Bedrosian, CT Clinical Significance in Patients with Stage II Breast Cancer Albarracin. Loyola University Medical Center, Maywood, IL; MD Anderson Cancer E Drinka, A McBride, P Allen, T Buchholz, A Sahin. MD Anderson Cancer Center, Center, Houston, TX; University of Washington, Seattle, WA. Houston, TX. Background: Mammary Paget disease (MPD) is an uncommon tumor consisting of Background: Lymph node status and number of positive lymph nodes (PLN) are the 1-3% of total breast cancer. Male MPD is much rarer with only single case reports in the most important prognostic factors in breast cancer. Extranodal tumor extension (ENTE) English literature. As a result there is not much information regarding the histopathologic has been used as a key histopathologic feature classifying patients into high versus and clinical features of this entity. This retrospective study was designed to examine low risk for local recurrence. Radiation oncologists also use ENTE when identifying and characterize the clinicopathologic features of male MPD. patients who may benefit from post mastectomy radiation. However, in the current era Design: We searched our database from 1985 to 2010 at the Department of Pathology, of early detection and systemic therapy the prognostic significance of ENTE is not as M.D. Anderson Cancer Center and identified 11 cases of male MPD. We collected the well defined in patients with 1-3 PLN. We sought to determine if the amount of tumor following clinical information: presentation symptoms, methods of treatments, clinical burden in an axillary dissection, or the presence of ENTE provides any additional follow up; and evaluated histopathologic features including diagnosis of breast cancer, information regarding patient outcome in patents with 1-3 PLN. tumor markers and lymph node status. Design: Clinical and pathologic factors were identified for 473 patients at MDACC Results: Age of these patients at presentation ranged from 50 to 90 years (mean 62). with pT1 tumors and 1-3 PLN, treated by mastectomy with or without postmastectomy Seven patients presented with either skin changes, nipple inversion or nipple discharge radiotherapy between 1978 and 2007. The total number of PLN was recorded for each and three of them also had palpable masses. Remaining four patients presented with patient, as well as the presence or absence of ENTE. A Kaplan-Meier analysis was palpable masses only. All patients had underline breast cancers; two DCIS and nine generated to determine whether the number of PLN, and presence or absence of ENTE invasive ductal carcinoma (IDC, six T1, one T2 and two T4). IHC studies demonstrated was an independent predictor of patient outcome. that the underlying breast tumors were positive for ER in all nine patients tested while only two of seven patients tested were positive for Her-2. Nine patients underwent modified total radical mastectomy with axalliry lymph node dissections (five patients) or 38A ANNUAL MEETING ABSTRACTS sentinel node sampling (four patients). The other two patients were treated with single or Table 2 bilateral simple total mastectomy with axillary node dissections. Four patients with IDC P value Groups pCR No pCR for GRB7 had metastatic disease in lymph nodes (two N1, one N2 and one N3) while lymph nodes score in remainging patients were negative. Seven patients with IDC also underwent radiation, 9 of 27 (33%) cases with mean GRB7 18 of 27 (67%) cases with mean ER+/HER2+ 0.5181 chemo- or hormonal therapy; including one radiation and hormonal; four chemo- and score of 217 GRB7 score of 200 16 of 28 (57%) cases with mean 12 of 28 (43%) cases with mean hormonal; one chemotherapy only and one hormonal therapy only. Remaining four ER-/HER2+ 0.1547 GRB7 score of 272 GRB7 score of 242 patients (two IDC and two DCIS) had no further treatments. Follow-up time ranged pCR: Pathologic Complete Response from 1 month to 11 years. Six patients were alive without diseases and one was alive Conclusions: GRB7 protein expression level parallels HER2 protein expression level with disease. Four patients with invasive cancers died, including two died of diseases; and is also associated with hormone receptor negative and highly proliferative tumors. one died of other cause and one died of unknown cause. However, the predictive value of GRB7 to trastuzumab containing chemotherapy is Conclusions: In summary, male MPD, similar to female MPD, is associated with diminished once HER2+ tumors are classified according to hormone receptor status. underlyine breast cancer. Patients of male MPD often present at advanced stage and early diagnosis is important in survival. 149 Mucinous Carcinoma of the Breast: A Clinico-Pathological Assessment Based on the Percentage of Mucinous Component 147 Synchronous Breast Cancers: Comparison of Racial Z Eslami, G Omeroglu, A Omeroglu. McGill University Health Center, Montreal, Distribution in Pathologic Parameters of Aggressive Behavior QC, Canada. C Duckworth, L Xu, G Birdsong, I Trgovcic, M Mosunjac. Emory University, Atlanta, Background: Mucinous carcinoma (MC) of the breast is a rare disease and can be GA. divided into 3 categories based on the extent of mucinous component: 90% or more Background: Synchronous breast cancers (SBC) are defined as a second tumor mucin (Pure Mucinous Carcinoma / PMC), 50- to 90% mucin (Mixed Mucinous diagnosed within three months from the diagnosis of a first tumor. While a variety of Carcinoma /MMC) and less than 50% mucin (Ductal Carcinoma with Mucinous Features risk factors and prognostic parameters have been associated with the overall incidence / DCMF). In this study we have compared the three categories of MC analyzing clinical of breast cancer, little is known about cancers that are synchronous. We describe a and pathological parameters. relatively large retrospective clinicopathologic study investigating the racial distribution Design: Our database (2006-2011) was searched for breast cancers containing of SBC in patients from a tertiary care and an inner city hospital. a mucinous component. Tumors were compared based on ER/PR/ HER2 status, Design: A retrospective search of all bilateral breast biopsies performed at our tertiary tumour size, grade, patient age, Breast Imaging Reporting and Data System (BIRAD) care and inner city hospitals between the years 2005 and 2010 was performedAmong classification and lymph node status. In addition a Ki67 stain was performed on PMC these, 50 cases were identified as being temporally synchronous. Electronic medical cases to evaluate its correlation with HER status. The percentage of Ki-67 positive cells records were used for the extraction of demographic data, clinical notes, imaging was identified using a computerized system. studies and pathology reports. Results: A total of 97 of breast cancers with a mucinous component have been identified: Results: 30 patients were African American (AA), 17 were Caucasian (CAU); and 3 45 cases of PMCs (46%), 17 cases of MMCs (18%) and 35 cases of DCMF (36%). were Hispanic (H); all were female. The age range was 33-80 years, with median age Her-2 was positive in four cases of PMC (9%), two DCMF (6%) and one MMC (6%). of 55 years for H, 58 years for AA, and 62 years for CAU women, respectively. The Lymph node metastasis was higher in MMC (7/17, 41%) compared to PMC (1/45, 2%) most common histologic type of tumor was infiltrating ductal carcinoma, followed by and DCMF (4/35,11%). There was no significant difference in ER/PR status between ductal carcinoma in situ, infiltrating lobular carcinoma, and lobular carcinoma in situ. the three entities. The average age at diagnosis was 72 for DCMF, 70 for PMC and 67 AA patients comprised (3/4) 75% of those with T4 and (5/6) 83% of T3 pathologic for MMC. Ki67 proliferative index was slightly higher in Her2 negative PMC (5.02) stage, as well as (18/28) 64% of carcinomas with positive lymph node status. Among than Her2 positive PMC (4.36). BIRAD scores for MMC was: 4B:18%, 4C:18%, cancers with high grade histology, (22/26) 85% were seen in AA women. CAU 5:41%, N/A:23%, for PMC: 4A:11%, 4B:18%, 4C:18%, 5:27%, 6:7%, N/A:18%, and women comprised the remainder of patients with high stage cancers, (9/18) 32% of the DCMF: 4A: 9%, 4B:11%, 4C:11%, 5:35%, 6:14%, N/A:20%. The average tumor size carcinomas with positive lymph node status, and 7.6% those with high grade histology. was 1.9 cm for PMC and DCMF and 2.2 cm for MMC. Tumor grades were as follows: The cancers in H women were (1/28) 3.6% of carcinomas with positive lymph node PMC (52%:1, 43%:2, 7%:3), MMC (76%:2, 24%:1), DCMF (69%:2, 20%:1, 11%:3). status and (2/26) 7.7% of those with high grade histology. Assessment of hormones Conclusions: Classifying MCs based on percentage of mucinous component identifies receptor status revealed that 8 (7.8%) cases were found to be triple negative for ER, three categories of tumors with distinct pathological and clinical characteristics. DMCF PR, and HER2-neu, all of which were in AA patients. occurs in older patients and are higher grade tumors. Tumor size and incidence of Conclusions: Our study investigates the difference in racial distributions of SBC over a 5 lymph node metastasis are greatest in MMC group. MMC is also more likely to have a year time period. Overall, AA patients presented at a younger age, with worse histology, BIRAD 5 score. Unexpectedly, Her2 positivity is more common in PMC group. Ki67 and with a higher pathologic stage. Additionally, all reported triple negative carcinomas proliferative index does not correlate with Her2 positivity in PMC. were found exclusively in AA women. While it is known that AA women have an overall higher mortality rate from breast cancer, our study suggests that this may be due in part to more aggressive tumor biology. Synchronous cancers, like solitary cancers, 150 Insulin Growth Factor Receptor-1 (IGF-1R) Overexpression also appear to present at a younger age in AA women when compared to Caucasians. Is Associated with Hormone Receptor Positive Ductal Carcinomas in African-American Women AK Esnakula, LJ Ricks-Santi, WAI Frederick, Y Kanaan, TJ Naab. Howard University 148 Significance of GRB7 Protein Expression Level in Breast Hospital, Washington, DC. Cancer Background: Insulin-like growth factor receptor 1 (IGF-1R) is a transmembrane BD Ellsworth, D Dabbs, R Bhargava. University of Pittsburgh Medical Center, Magee- tyrosine kinase receptor protein, activated by insulin growth factors (IGF). IGF-1R Womens Hospital, Pittsburgh, PA. mediates the growth promoting actions of IGF-1 and IGF-2 by downstream activation Background: GRB7 is an adaptor protein that interacts with a number of signaling of multiple pathways leading to differentiation, proliferation and enhanced survival. The molecules. Although GRB7 is often co-amplified with HER2, GRB7 protein expression objective of our study is to correlate the immunohistochemical expression of IGF-1R by immunohistochemistry (IHC) has not been widely studied, and its significance with in the four major subtypes of breast carcinoma (luminal A, luminal B, HER2 positive, HER2 protein expression is unknown. and Triple Negative) and with clinicopathological factors in African American women. Design: GRB7 protein expression was semi-quantitatively analyzed via H-score in Design: Tissue microarrays (TMAs) were constructed from optimally-fixed formalin- 219 invasive breast cancers. Of the 219 cases, 195 were core biopsies (4 HER2 IHC fixed, paraffin-embedded tumor blocks from primary breast ductal carcinomas in 2+/FISH not amplified; 49 HER2 IHC 2+/FISH equivocal; 35 HER2 IHC 2+/FISH 203 African-American females. Two separate 1mm cores represented each case. amplified; 107 HER2 IHC 3+) and 24 cancers (HER2 IHC 0 and 1+) were represented Tumors were subtyped based on expression of ER, PR, HER2, vimentin and 34βE12. on a tissue microarray. Neo-adjuvant chemotherapy was administered in 64 cases. Immunohistochemical evaluation for IGF-1R was performed using rabbit monoclonal Results: GRB7 H-scores were significantly higher in unequivocally HER2 amplified antibody (G11, Ventana, AZ, USA). The sections were evaluated for the intensity of and IHC 3+ cases compared to HER2 equivocal and negative cases (see table 1). Higher reactivity (0-3) and the percentage of reactive cells; and H-score was calculated. Previous scores also correlated with ER neg, PR neg, and high Ki-67 labeling index (>25%). No studies have reported that normal breast tissue showed moderate expression of IGF1-R, difference was identified with respect to age, tumor size, grade, or lymph node status. hence cases with H-score <100 are considered reduced expression for IGF-1R and Of the 64 cases that received neo-adjuvant chemotherapy, 25 (39%) achieved pathologic cases with H-score >200 were considered as overexpression for IGF-1R expression. complete response (pCR). Of these 64 cases, 55 cases were unequivocally HER2 positive Bivariate analysis was done via χ2 analysis and multivariate analysis was done via (IHC 2+/FISH amplified or IHC 3+). The GRB7 score was significantly higher in tumors Cox’s proportional hazards model. Statistical significance was assumed if P < 0.05. that achieved pCR (mean 258 compared to 192 without pCR, p=0.00005), but when Results: Among our study population, 43.8% were Luminal A, 14.4% Luminal B, 8.5% these unequivocally HER2+ tumors were classified as ER-/HER2+ and ER+/HER2+, HER2 positive, and 33.3% triple negative of which 51% were basal-like phenotype. the GRB7 scores were not significantly different (see table 2). IGF-1R overexpression was significantly linked to ER+ (P<0.0001), PR+ (P=0.0186), Table 1 HER2- (P=0.0229), and Luminal A breast cancer subtype (P<0.0001). However, Groups based on HER2 result Mean Median Range Std Dev; SEM A: IHC 0 or 1+ (n=24) 85 100 0-120 36; 7 29% of triple negative cases showed high IGF-1R expression. IGF-1R expression B: IHC2+, FISH Namp (n=4) 87 100 50-100 25; 13 was not associated with age, grade, stage or survival. There was trend towards better C: IHC2+, FISH Eq (n=49) 111 100 0-260 55; 8 overall survival in low IGF-1R expressing ER+ tumors when compared to IGF-1R D: IHC2+, FISH Amp (n=35) 155 170 50-230 55; 9 overexpressed ER+ tumors. E: IHC3+ (n=107) 244 250 0-300 59; 6 p-values: A vs B 0.90; A vs C 0.04; A vs D <0.0001; A vs E <0.0001; B vs C 0.40; B vs D 0.02; B vs E <0.0001; C vs D 0.0005; C vs E <0.0001; D vs E <0.0001. Namp: Not Amplified; Eq: Equivocal; Amp: Amplified; SEM: Standard error of mean. ANNUAL MEETING ABSTRACTS 39A Conclusions: Our study found that IGF-1R overexpression was significantly associated 153 Fibroadenomatous Lesions in Pediatric Age Group with hormone positive breast cancer suggesting that IGF-1R signaling might play a S Faiz, V Tudor, G-P Yasim, S Badve. Indiana University School of Medicine, potential role in pathogenesis of ER+ tumors. Monoclonal antibodies targeting IGF- Indianapolis, IN. 1R are already under clinical investigation in treatment of various solid organ and soft Background: Fibroadenomatous lesions (FAs) are well described in adults but the tissue tumors. Targeting IGF-1R in chemotherapy resistant ER+ breast cancer could morphological features are poorly characterized in the pediatric age group (age<18). be beneficial. We investigated the morphological features of FAs in this age group. Design: From January 1999 to December 2011, 119 cases with the diagnosis of 151 Metabolomic Profiling Reveals Increased Arginine Metabolism fibroadenoma in females age 18 years or younger were identified and were reviewed by in ER- Breast Cancers from African-American Women two pathologists. FAs were divided into different groups based on their morphological AK Esnakula, TJ Naab, LJ Ricks-Santi, WAI Frederick, RL DeWitty, Jr., Y Kanaan. appearance into tubular (epithelial rich), classic, cellular (increased stromal cellularity), Howard University Hospital, Washington, DC; Howard University College of Medicine, juvenile (epithelial hyperplasia), and mixed types and phyllodes tumor (stromal Washington, DC. overgrowth) on the basic of predetermine criteria. Background: Estrogen receptor negative (ER-) breast cancer, which is associated Results: with chemotherapy resistance and poorer survival, is more common in younger Patient Demographic and Histopathology of Fibroadenoma Tub& Type of Tubular Juvenile Phyllodes African-American women. The various genetic and epigenetic differences between Cellular n=12 Classic n=62 Classic Fibroadenoma n= 18 n=18 n=4 ER - and ER+ breast cancers have been studied, but only rare studies have analyzed the n=5 dysregulated metabolic pathways between these two subtypes. Metabolomics, a study Mean Age ( yrs) 16 15 16 14 15 16 of cellular small-molecule metabolites, is used to analyze the metabolic differences Race (W/B/others) 4/14/0 3/9/0 34/22/6 8/7/3 2/3/0 1/3/0 Tubular between ER+ and ER- tumors. The goal of our study is to establish the metabolomic 2/16 0/12 0/62 0/18 0/5 0/4 adenosis(Y/N) profile of biochemicals in various amino acid pathways in ER- and ER+ breast cancers Sclerosing Adenosis 0/18 0/12 0/62 0/18 0/5 0/4 from African-American women. (Y/N) Design: Breast cancer tumor tissue methanol extracts from 15 ER- and 15 ER+ African- Cystic dilatation(Y/N) 2/16 3/9 5/57 7/11 3/2 1/3 Epithelial 0/18 0/12 0/62 18/0 0/5 0/4 American women were analyzed using liquid/gas-chromatography coupled to mass Hyperplasia(Y/N) spectrometry. Following log transformation and imputation with minimal observed ≤1=16, ≤1=11, values for each compound, Welch’s two-sample t-test was used to identify metabolites Mitosis/10hpf ≤3=1, ≤1=8, ≤2=4< ≤1=56, ≤3=6 ≤3=5, ≤1=5 ≤5=4 that differed significantly between experimental groups. Statistical significance was ≤5=1 ≤5=2 Cellularity(H/M/L) 12/6/0 12/0/0 0/55/7 13/5/0 2/3/0 4/0/0 assumed if P ≤ 0.05. Increase periductal 10/8 5/7 35/27 17/1 3/2 4/0 Results: Global biochemical reveal significant differences in amino acid metabolism cellularity (Y/N) <1=2, in ER+ and ER- tumors. The most significant difference was observed in arginine Ratio of stroma to <1=4, 1=6, <1=2, 1=57, <1=2, <1=18 1=10, >1=4 metabolism. ER- tumors showed significant increase in arginine when compared to epithelium >1=2 >1=3 1=3 >1=3 ER- tumors. Various metabolites of arginine metabolism, including citrulline, putrescine Intracanalicular (I), P=4, I=2, P=7, I=23, P=4, I=2, P=2, P13, I+P=5 I+P=4 and proline, were significantly increased in ER- tumors. Citrulline, an intermediate in pericanalicular (P) I+P=6 I+P=32 I+P=12 I+P=3 Periepithelial infitrate the production of nitric oxide from arginine, was increased by 16.2 fold in ER- tumors. 6/12 3/9 9/53 6/12 1/2 2/2 (Y/N) Nitric oxide mediates inflammatory signaling and vascular tone in tumors. ER- tumors Types of stroma[ also showed 15.1 fold increase in putrescine, indicating proliferative signaling via Fibrous=F, Periductal F=7, and fibrous F=12, F=4, M=16, F=6, M=1, F=3, this polyamine. Arginine metabolites associated with extracellular matrix remodeling PFIND=3, F=4 interductal=PFIND, PFIND=6 PFIND=42 PFIND=11 PFIND=2 were also increased in ER- samples, including proline, 4-hydroxyproline and proline- H=1, M=1 hydroxy-proline. Hyaline=H, Myxoid=M] Conclusions: Our study indicates robust utilization of arginine in ER- tumors which W=White, B= Black, H=High, M=Medium, L=low, Y=yes, N=No, hpf=high power field is correlated with increased metabolites linked to nitric oxide-mediated inflammatory Fibroadenoma is the frequent lesion in pediatric age group with classic FA being the most signaling, cell proliferation, energy metabolism and extracellular matrix remodeling. common type. Cases with overlapping features were present. Complex fibroadenomas This increase in arginine uptake and metabolism might be a potential influence on the were extremely unusual with only rare cases exhibiting fibrocystic changes. Mitoses aggressiveness of ER- tumors when compared to ER+ tumors. were observed in all subtypes. Stromal overgrowth was the only feature that reliably distinguished FA from phyllodes tumor. 152 Fumarate, an Oncometabolite Is Elevated in ER- Breast Cancers Conclusions: Epithelial hyperplasia is a cardinal feature of Juvenile FA. Stromal mitosis from African-American Women is a very common finding in pediatric FELs and does not necessitate a diagnosis of AK Esnakula, TJ Naab, LJ Ricks-Santi, WAI Frederick, RL DeWitty, Jr., Y Kanaan. phyllodes tumor. Howard University Hospital, Washington, DC; Howard University College of Medicine, Washington, DC. 154 Ki 67 in Breast Cancer: How Many Tumor Cells Do We Need To Background: Estrogen receptor negative (ER-) breast cancer, which is associated Count? with chemotherapy resistance and poorer survival, is more common in younger HF Faragalla, L Feeley, A Bane, AM Mulligan. University Health Network, Toronto, African-American women. The various genetic and epigenetic differences between ON, Canada; Cork University Hospital, Cork, Ireland; Juravinski Cancer Center, ER - and ER+ breast cancers have been studied, but only rare studies have analyzed the McMaster University, Hamilton, ON, Canada. dysregulated metabolic pathways between these two subtypes. Metabolomics, a study Background: Ki67 has been shown to be a prognostic marker in breast cancer with of cellular small-molecule metabolites, is used to analyze the metabolic differences high levels being shown to be associated with poor outcome. A major obstacle to its between ER+ and ER- tumors. The goal of our study is to establish the metabolomic routine use is the lack of a standardized assay approach which hinders its use as a tool profile of biochemicals in various energy pathways in ER- and ER+ breast cancers for selecting patient to chemotherapy or assigning them to a particular risk group. The from African-American women. aim of this study is to study how many tumor cells need to be counted on actual visual Design: Breast cancer tumor tissue methanol extracts from 15 ER- and 15 ER+ African- count for accurate assessment of Ki67 in invasive breast cancer. American women were analyzed using liquid/gas-chromatography coupled to mass Design: 112 invasive breast cancer tumor blocks were selected and stained with spectrometry. Following log transformation and imputation with minimal observed MIB1 antibody for assessment of Ki67 labeling index. The Ki67 labeling index was values for each compound, Welch’s two-sample t-test was used to identify metabolites estimated by visual counting of 1000 tumor cells from three different areas of each that differed significantly between experimental groups. Statistical significance was tumor block including hotspots and invasive tumor front. The count per each 100 is assumed if P ≤ 0.05. recorded. Statistical analysis was performed using paired student t-test with a P-value Results: ER- tumors when compared to ER + tumors showed significant increase in < 0.05 considered significant. various intermediates of Tricarboxylic acid cycle (TCA or Krebs cycle) which include Results: Our results show that on visual counting Ki 67 LI is higher in the initial 200- succinate, fumarate and malate. Fumarate levels were almost 8-fold higher in ER- 300 and 500 tumor cells, which tends to gradually decrease due to dilution effect. There tumors when compared to ER+ breast cancers. There was also significant increase in was a statistically significant difference on counting 200, 300 and 500 tumor cells at a P branched chain amino acids including aspartate, phenylalanine and methionine, all of value of 0.001, 0.02 and 0.03 respectively. However, there was no statistically significant which could contribute to substantial increase in fumarate. difference on counting 700 and 800 versus 1000 tumor cells. Dichotomization into Conclusions: Our study shows a substantial increase in fumarate in ER- tumors when categories of high and low using 20% as a cut off value resulted in 7 discrepant results compared to ER+ tumors. Fumarate has recently been identified as an oncometabolite. with Ki67 higher at 300 than 1000 tumor cells. The mean Ki67 proliferation value for Intracellular accumulation of fumarate is associated with diverse consequences. the initial 300 tumor cells are 21.9 and for final 1000 tumor cells are 20.9 respectively. Competitive inhibition of 2-oxygenase enzyme group leads to activation of oncogenic Conclusions: In this study there was a decrease in proliferation value with increasing factors like hypoxia-inducible factors (HIF). Enhanced HIF-1α signaling is associated number of evaluated tumor cells. This is due to dilution effect. This decrease is with tumor angiogenesis secondary to increased secretion of vascular endothelial growth statistically significant. The dilution effect is important in clinical practice if samples factor (VEGF) by the cancer cells. Fumarate also mediates non enzymatic succination of are dichotomized as only proliferation values near a chosen cut-off would be affected. various cellular proteins leading to dysregulation or loss of protein which also highlights the oncogenic potential of fumarate. Elevated levels of fumarate in ER- breast cancers could play an important role in the aggressiveness of ER- cases. 40A ANNUAL MEETING ABSTRACTS 155 Ki67 in Breast Cancer: The Effect of Different Length of Fixation frequency of LN involvement in cases of DIN/DCIS and the contribution of previous on Ki 67 Index instrumentation, we evaluated all pure DIN/DCIS lesions treated by total mastectomy HF Faragalla, L Feeley, A Bane, AM Mulligan. University Health Network, University and sentinel lymph node (SLN) evauation. of Toronto, Toronto, ON, Canada; Cork University Hospital, Cork, Ireland; Juravinski Design: At our institution, SLN assessment is routinely performed on all DIN/DCIS Cancer Center, McMaster University, Hamilton, ON, Canada. lesions treated by mastectomy. A total of 252 mastectomies with DIN/DCIS and a Background: Ki67 has been shown to be a prognostic marker in invasive breast SLN evaluation from 2002-2012 were retrieved. All clinical and pathological data cancer with high levels are associated with poorer outcome. Furthermore, patients with were reviewed. The clinicopathological features of the cases with any LN involvement HR positivity and a high Ki67 LI may benefit from adjuvant chemotherapy. A major were compared to those without LN involvement to determine if there was a correlation obstacle is lack of a standardized assay approach. The aim of this study was to address between any lesional or clinical characteristics (i.e. size or type of DIN/DCIS, number of specifically, the effect of counting methods and fixation length on the LI. procedures prior to mastectomy/sentinel lymph node biopsy, etc.) and the nodal status. Design: Paired tumour blocks were selected from 70 tumors for short (SF) and Results: Of the 252 cases evaluated, 9 (3.6%) had tumor cells in the SLN. All nine prolonged fixation periods (PF) and stained with MIB1. Two visual counting methods (100.0%) of these were isolated tumor cells (ITC) identified by immunohistochemistry were utilized: actual count (VC) of positive to negative cells in three different areas only. Overall, the 9 patients with nodal involvement had, on average, 3.7 samplings prior and a visual estimate (VE), similar to scoring HR. Statistical analysis was performed to the mastectomy compared to 2.1 among those patients with no nodal involvement. using t-test with a P-value < 0.05 considered significant. Conclusions: The frequency of LN involvement in DIN/DCIS has been reported to be Results: The mean fixation period for the SF group was 13hrs18 min (range 10hrs33 as high as 9%. The 3.6% in our study is substantially lower. This may reflect the more min–17hrs45min) and for the PF group, 79 hrs35min (range 73 hrs33min-102hrs30 stringent criteria used in our study with the exclusion of any case with micro-invasion min). There was no statistically significant difference between the LI for the SF vs PF and the limitation of our cases to mastectomy specimens. Given this low frequency groups using either counting methods; however, 23% had >10% difference in the LI and the fact that only ITC were found in the involved LNs, it would be reasonable between the fixation groups. When 14% was used as the cut-off to assign cases to high to hypothesize that these patients are at no higher risk for subsequent metastasis or or low Ki67 LI, 34% of cases were assigned into different risk categories depending on recurrence than patients with DIN/DCIS and no nodal involvement. Also, the presence fixation length. In both analyses, there was no trend towards an increased or decreased of neoplastic cells in the LN may well be secondary to prior sampling manipulations. LI according to fixation length, i.e. these cases were evenly distributed between those that showed a higher or lower LI with greater fixation. A significant difference between 158 Collagen XIa1. A New Marker for Breast Carcinoma Malignancy VC and VE was not seen; in 80% of cases, an underestimation of LI was seen with VE in Core Needle Biopsies compared with VC. In 9% of cases the difference was >10%; however a difference J Freire, S Dominguez, S Pereda, A De Juan, A Vega, L Simon, J Gomez. Hospital in risk category (high vs low) was seen in 14% of cases (VE range for incorrectly Universitario Marqués de Valdecilla, Santander, Spain; Oncomatrix, Bilbao, Spain. assigned cases = 6-15%). Background: The accurate diagnosis between invasive breast carcinoma and not Conclusions: Fixation does not appear to affect Ki67 LI in BC. While a high number infiltrating lesions, may be, sometimes, very complex in the everyday-work of the of cases were reassigned to different risk categories based on fixation length, this pathologist. It has been shown that the extracellular matrix plays an essential role reassignment was in both directions. This is likely a result of intratumoral heterogeneity in breast tumor development and progression, being the protein collagen its main in Ki67 LI, further emphasizing the need to determine the most appropriate approach component. Several collagen organ and pathology specific types have been described, to selecting areas to be counted. In most cases VE accurately predicted LI; however, a and thus it can be used as a potential target in epithelial-mesenchymal transition visual count is recommended when the LI is estimated to be low (approximately < 20%). changes. Collagen type XIα1 (COLXIα1) has been studied in colorectal and pancreas adenocarcinomas acting as a marker of advanced lesions. Our work sought to revise 156 The Diagnosis of Phyllodes Tumour amongst Participants of COLXIα1 role in differential diagnosis between invasive and not infiltrative breast Mammographic Screening lesions. G Farshid. BreastScreen SA, Wayville, South Australia, Australia. Design: Immunohistochemistry of COLXIα1 was performed in formalin fixed, paraffin Background: Recent studies have sought discriminatory features to distinguish embedded Core Needle Biopsy (CNB) samples of 105 patients with radiological phyllodes tumours (PT) from fibroadenomas on needle biopsies. This distinction is evidence of breast tumor. We finally examined 12 Ductal Carcinoma In Situ (DCIS), imprecise but impacts patient care. Excision is advised for PTs but is not mandatory 46 Infiltrating Ductal Carcinomas (IDC), 7 Infiltrating Lobular Carcinoma (ILC) and for fibroadenomas, particularly in the setting of screening where there is an imperative 40 non-neoplasic lesions (fibroadenoma, hyperplasia, fibrosis ...). to limit surgery for benign processes, such as fibroadenomas. We wished to review Results: Our results show a significant difference (p <0.001) between infiltrating the diagnosis of PT made in the setting of population based breast cancer screening. tumors (48 out of 53 positive) and the rest of the lesions, both in situ carcinomas (1 Design: We searched our prospective database for cases with a diagnosis of PT during out of 12) and non-neoplastic (2 out of 40). Positive staining was clearly observed 01/01/1989 to 22/08/2012. We tabulated demographic, imaging, biopsy and outcome in fibroblast’s cytoplasm on carcinoma progression areas, (Figure 1) whereas signal data and followed the women through their subsequent course. was not detected in any other lesions but two cases of intraductal papilloma (without Results: During this 23 year period we performed a total of 1,185,686 mammographic posterior surgical confirmation). screening examinations. Of these 14298 women (1.2%) underwent biopsy. PT was diagnosed in 30 women, reflecting an overall rate of 2.53 per 100,000 women screened (0.0025%). Of these 13 were benign, 8 borderline and 9 malignant. Follow up of at least 12 mths is available in 20 cases. One woman died of metastatic PT 43 months later, 3 died of inter-current disease, 1 developed breast cancer and the remaining 15 are alive and well. PT was diagnosed during screening in 22 women (73.3%) and after symptoms arose in the ensuing 8-69 mths following a negative screen in the remaining 8 women. A circumscribed mass, mean size 34.7mm (std 17.8mm), was the dominant imaging finding. The imaging grade was 3 in 10 cases, 4 in 7 cases and 5 in 5 cases. Enlargement was documented in 21 cases (70%). A fibroepithelial lesion was specified on cytology in 5 of 23 cases. A biphasic malignancy, mucinous lesion, fat necrosis, hyperplasia and a papillary lesion were other cytologic considerations. Core biopsy favoured PT Conclusions: These data suggest that stromal fibroblast overexpression of COL11α1 is in 8 of 15 cases, fibroadenoma in 4, sarcoma in 2 and a fibroepithelial lesion in 1 case. an infiltrative growth robust marker, with very high levels of sensitivity and specificity Conclusions: Our findings document the extreme rarity of PT in the setting of population greater than 90%. based screening. This contrasts with the high prevalence of fibroadenomas. This finding is plausible, since their large size at diagnosis favours presentation as a palpable mass 159 HER2 Amplification Status in Breast Cancer: The Effect of and prior studies have established a mean age of 40 at diagnosis, younger than the Adjusting for “Polysomy 17” target age group for screening (50-69). While the limited precision of percutaneous I Gagne, KJ Craddock, AM Mulligan. University Health Network, University of needle biopsies in subclassifying fibroepithelial lesions remains an enduring challenge, Toronto, Toronto, ON, Canada. awareness of the extremely low prior probability of PT, and the even more remote odds Background: Correction of the HER2 gene copy number with the copy number of diagnosing malignant PTs among screen detected lesions may assist in avoiding of chromosome 17 centromere (CEP17) (believed to be a surrogate for Ch17 copy unnecessary surgery in equivocal cases. number) has long been believed to be necessary for determination of true HER2 gene amplification. Recent evidence suggests that polysomy 17 is a very rare event in breast 157 Sentinel Lymph Node Status among Patients with Pure Ductal cancer (BC): the centromere may be included in HER2 amplicons, which could lead to Intraepithelial Neoplasia/Ductal Carcinoma In Situ Treated by Mastectomy misleading HER2 amplification status and a misclassification of patients eligible for C Flynn, DR Lannin, FA Tavassoli. Yale University School of Medicine, New Haven, trastuzumab therapy, if the ratio is taken as the sole indicator. A recent policy change CT; Yale New Haven Hospital, New Haven, CT. in Cancer Care Ontario states that patients with tumors showing polysomy 17 may be Background: Ductal intraepithelial neoplasia (DIN)/Ductal Carcinoma in Situ (DCIS) considered eligible for trastuzumab if the mean number of HER2 copies per cell is is a non-invasive process limited to the ducts and lobules of the breast. Nonetheless, greater than 6, even though the ratio is less than 2. To assess the likely impact of this as many as 9% of DIN/DCIS cases have been reported to have lymph node (LN) in clinical practice, we performed an audit on a series of BC on which HER2 FISH involvement by “metastatic carcinoma.” Among the previously reported series, however, data was available. were cases suspicious for microinvasion. A large number of the reported cases were Design: An audit was performed of a consecutive series of 961 tumors at a single also treated by lumpectomy and consequently an occult infiltrating carcinoma may institution between December 2008 and September 2012. ASCO/CAP guidelines have gone undiagnosed. Confounding the issue, patients regularly have at least one to define amplification status based on ratio of HER2 to CEP17 copy number were if not multiple biopsies and/or aspirations before the final excision. To determine the followed. Polysomy and monosomy 17 were defined as CEP17 copy number > 3 and < 1.4 respectively. ANNUAL MEETING ABSTRACTS 41A Results: Of 961 cases, 808 had HER2 immunohistochemistry data available: 3.2% cm) for C-ILC, p=0.31. 16(89%) TN-ILC showed pleomorphic features with apocrine were negative (0/1+), 96% were equivocal (2+), and 0.6% were positive (3+). Of 961 morphology. In contrast, none of the C-ILC showed these features. Mean mitotic count tumors, 131 (13.6%) were amplified, 784 (81%) were non-amplified and 46 (4.8%) were in TN-ILC was 7.7 per 10 hpf (range 1-56) vs 43 (range 1–28) for C-ILC, p=0.23. equivocal. 11.6% cases showed polysomy and 8% showed monosomy. Of 784 non- Proliferation index measured by MIB-1 was 6%(range 1-48%) for TN-ILC and 8%(range amplified cases, 11 (1.4%) had more than 6 copies of HER2. All had polysomy (CEP17 1-43%) for C-ILC, p=0.57. 7(39%) TN-ILC showed lymph node metastasis vs. 7(23%) copy number range: 3.5-5.2). Of 131 amplified cases, 30 had a HER2 copy number ≤ of C-ILC, p=0.32. 17(95%) TN-ILC cases were AR+. 6 (23%), 9 of which showed monosomy (CEP17 copy number range:1.1-2.4). Of 77 Conclusions: TN-ILC is a distinct entity with a characteristic pleomorphic & apocrine monosomy 17 cases, 62 were non-amplified, 3 were equivocal and 12 were amplified. morphology. Compared to our control group, their age at presentation is older. An Amongst the 12 monosomy 17 amplified cases, only 3 had > 6 HER2 gene copies and overwhelming majority of TN-ILC are AR+. The latter finding suggests that like other 9 had ≤ 6 HER2 gene copies. AR+ TN cancer, TN-ILC may benefit from anti-androgen therapy. Conclusions: We found that just 1.4% of patients with tumors considered non-amplified according to HER2:CEP17 ratio had > 6 copies of HER2 and would now be considered 162 Secretory and Cystic Hypersecretory Changes at Breast Core eligible for trastuzumab therapy under new Ontario guidelines. 23% of patients with Needle Biopsy: Clinical, Radiologic and Pathologic Findings in 67 Cases tumors considered amplified based on ratio had< 6 copies of HER2. Routine reporting L Galman, J Catalano, A Cordero, M Akram, E Brogi. Memorial Sloan-Kettering of HER2 copy number is recommended and patients with non-amplified ratios but >6 Cancer Center, New York, NY. copy numbers should be included in trials testing anti-HER2 therapies to determine Background: Focal secretory changes (SC) of the mammary epithelium, including the clinical significance in terms of response to therapy. cystic hypersecretory changes (CH-SC), can occur independent of pregnancy and lactation. Information about the significance of these findings at breast core needle 160 Correlation of Src Activation with Trastuzumab Response in biopsy (CNB) of radiologically-detected lesions is limited. Patients with HER2-Positive Breast Carcinoma Design: We searched for CNBs with diagnosis including the term “secretory” or A Gallardo, E Lerma, F Ortiz-Martinez, A Perez-Balaguer, E Adrover, A Tibau, A “hypersecretory” and performed at our center between 1992 and 2012. Patient (pt) Barnadas, F Aranda, G Peiro. Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; data were extracted from the e-medical records. CNBs with malignant diagnosis, from University General Hospital, Alicante, Spain. lactating/pregnant pts, or with diffuse SC in all cores were excluded from the study. A Background: Src is a non-receptor tyrosine kinase, experimental data indicate that pathologist (LG) reviewed all available slides from the CNBs and selected those with activated Src inhibits the PI3K/Akt/mTOR pathway which is related to trastuzumab SC for review with another pathologist (EB). Subsequent ipsilateral breast specimens resistance. Little is known about the clinical relevance of Src activation in the response were also reviewed. to trastuzumab. We investigated proteins involved in the PI3K/Akt/mTOR pathway and Results: We found 67 CNBs with SC, including 2 CH-SC. Patients (pts) had mean Src in a cohort of HER2+ breast carcinomas (BC) patients treated with and without age 46 years (range 29-72), with 17 (25%) >50 years. Thirty-five (52%) pts had family trastuzumab protocols. history (hx) of breast carcinoma, 12 had ductal carcinoma (10 invasive, 2 in situ; 11 Design: We selected 278 HER2+ BC patients: 65 with trastuzumab in the initial contralateral, 1 ipsilateral), 1 had ipsilateral atypical ductal hyperplasia (ADH), 2 had treatment, 76 in the metastatic disease and 124 without trastuzumab. Patients with atypical lobular hyperplasia (ALH) and 1 had lobular carcinoma in situ (LCIS). Two neoadjuvant treatment or stage IV were excluded from the survival analysis. We pts had hx of mantle radiation. At the time of CNB, 18/67 (27%) pts took hormone examined immunohistochemically pSrc (Tyr416) and pMAPK, ER, PR, HER2, EGFR, replacement therapy or oral contraceptives. CNB sampled calcifications (Ca2+) (42/67, Ki67, p110a, pAkt and p-mTOR on paraffin-embedded tissue microarrays. Expression 63%), MRI abnormality (17/67, 25%), and mammographic/sonographic mass (M/S) was assessed combining intensity and percentage (score 0-300): in the membrane for (8/67, 12%). Only 3/67 (4%) SC were atypical, including an atypical CH-SC showing pSrc-416 (cut-off >12), pMAPK, p110a, pAkt (cut-offs >150), EGFR (cut-off >10), nuclear crowding, hyperchromasia and mitoses. SC involved 2.8 lobules (range 1, 13) p-mTOR (cut-off >30) or Ki67 (nuclei, cut-off >14). IHC, clinicopathological factors per case and had mean span of 1.5 mm (range 0.4, 3). Of note, the lesion span in the and prognosis were correlated. two cases of CH-SC was 1.7 mm and 2.3 mm. SC, including one CH-SC, contained Results: Active pSrc-416 was seen in 35% of the tumors, predominantly of grade 3 Ca2+ and were the primary finding in 33/42 (78%) CNBs targeting Ca2+. No other (67%;p=0.09), with vascular invasion (32%/p=0.02), metastasis to the central nervous lesion beside SC was consistently present in CNBs targeting MRI abnormalities. In system (28%/p=0.01), pMAPK activation (28%/p=0.04), loss of ER/PR (53%/p=0.04) addition to SC, CNBs for M/S yielded 4 fibroadenomas, and papilloma, adenosis, and and no EGFR expression (9%/p=0.01). Increased active Src implied poor overall survival stromal fibrosis in the rest. Additional tissue from 9/67 pts was benign, and contained only in the group of patients with first line trastuzumab treatment. ADH+LCIS, ALH, and atypical CH-SC in one case each. Table 1 Conclusions: Focal SC, including CH-SC, are a rare finding at CNB. Most CNBs Trastuzumab in metastatic Trastuzumab in first-line. No Trastuzumab yielding SC target mammographic Ca2+ and SC with associated Ca2+ account for disease.(n=53) (n=40) treatment.(n=116) the radiologic lesion in over 75% of case. All CNBs with SC in our series are benign, OS DFS OS DFS OS DFS SRC416 n.s. n.s. p=0.034 n.s. n.s. n.s. including those with CH-SC. Our results can be useful to pathologists and clinicians mTOR n.s. p=0.042 p=0.001 p=0.079 n.s. n.s. who encounter these rare lesions in their practice. pAKT p=0.010 p=0.006 n.s. n.s. n.s. n.s. p110a n.s. p=0.013 p=0.002 p=0.008 n.s. n.s. 163 Sclerosing Papillary Hyperplasia without Myoepithelium: A Table 1: Survival analysis (Kaplan Meier) for the different tumor markers. All were associated with Variant of Papillary Thyroid Carcinoma? poor outcome. OS: Overall survival. DFS: Disease free survival. Conclusions: Our results suggest that Src plays a role in promoting trastuzumab RG Gamez, S Narendra, C Reynolds, DW Visscher. Mayo Clinic, Rochester, MN. resistance in combination with other proteins of PI3K/Akt/mTOR pathway in a subset Background: To this date 12 cases of the breast tumor resembling the tall cell variant of HER2 positive BC. Therefore, blocking this axis may prevent the development of of papillary thyroid carcinoma (BTRPTC) have been reported, two reports raised trastuzumab resistance in those patients. These biomarkers seem to have less prognostic the possibility of malignant potential due to metastatic disease. A recent review of significance in patients without trastuzumab treatment. our experience with solid and cystic papillary carcinoma showed a wide variety of histologic features, some overlaping with BTROTC (presence of nuclear grooves, clearing and inclusions). We report the clinical characteristics and follow up of six 161 Clinicopathologic and Immunophenotypic Characteristics of cases with overlapping clinical and histological features between BTRPTC and solid Triple Negative Invasive Lobular Carcinoma of the Breast and cystic papillary carcinoma. L Galman, M Akram, J Catalano, A Cordero, D Giri. Memorial Sloan-Kettering Cancer Design: All of the 6 cases had been seen as part of our consultation service. The available Center, New York, NY. H&E slides were reviewd. The clinical and histologic characteristics and follow up Background: 15-20% of breast cancers are triple (ER, PR, HER2) negative (TN). These were analyzed and recorded. tumors are clinically aggressive and are difficult to treat due to absence of target for Results: The mean age of diagnosis was 57 years (range 38-79), the mean follow up therapy. Recently (Gucalp A et.al., Cancer Res 2011), it has been reported that about time was 32.5 months (range 5 – 117), 83% lacked a myoepithelial layer, 100% (2 of 12% of TN breast cancers express Androgen receptor (AR) and that these tumors show 2) had negative sentinel nodes and no metastatic disease has been reported in any case. response to treatment with Bicalutamide (an anti-androgen agent). Although not well- Our previous review of the solid and cystic variants of papillary carcinoma showed recognized, a small proportion of invasive lobular carcinoma (ILC), fall in the category that besides the common papillary architecture, around 30% shared at least one or of TN. However, to our knowledge, there are no published studies on the characteristics more of the same histologic characteristics of the BTRPTC. The cells were columnar of these tumors and it is not known if any of these are AR positive (+). In this study, we or cuboidal in 40% and 25% of the cases, 25% had nuclear clearing or inclusions, 7% examine the clinical, morphologic and immunophenotypic characteristics of TN-ILC. had nuclear grooves and all lakced a myoepithelial layer. All of our 6 new cases have Design: A total of 18 cases of TN-ILC from our database (1998-2008) were identified columnar cells, nuclear clearing or inclusions, nuclear grooves, lack myoepithelial cells after morphologic review and immunohistochemical (using E-Cadherin, Clone and show a papillary architecture with a surrounding sclerotic stroma. EP700Y 1:200 Epitomics, and p120, Clone MRQ-5 1:200 Cell Marque) confirmation. Table 1 Clinical characteristics. For comparison, a control ILC (C-ILC) group of 30 ER+ and HER2 negative was Case Dx Year Age ER Myoepithelial Axillary LN Follow up (months) AWD randomly selected from the Surgical Pathology files. Patient age and menopausal status 1 2003 38 NP (-) ND 117 + were recorded. Pathologic features such as cytomorphologic characteristics (classical 2 2010 47 (-) Focally (+) ND 16 + vs pleomorphic), mitoses, lymphovascular invasion; associated in-situ carcinoma as 3 2011 79 (-) (-) ND 13 + 4 2011 52 (-) (-) (-) 5 + well as tumor size and lymph node status were assessed. Additional immunostains 5 2012 63 Focally (+) (-) (-) 6 + for MIB-1 (Clone MIB 1:200, DAKO) and AR (Clone AR441 1:300, abCAM) were 6 2009 63 NP (-) ND 38 + also performed. Fisher exact test and t-test were used to obtain statistical significance. All were wide local excisions, Dx: Diagnosis, NP not performed, ND not determined, AWD alive Results: The mean age was 71 years (range 42-86) for TN-ILC vs. 60 (range 35-85) without disease for C-ILC, p=0.02. 17(94%) TN-ILC were menopausal vs. 24(80%) for C-ILC, p=0.23. Mean tumor size was 2.4 cm (range 0.8-9 cm) for TN-ILC vs. 1.8 cm (range 0.5–6.5 42A ANNUAL MEETING ABSTRACTS Conclusions: The histological and clinical features of our cases overlap those of BTROTC and show resemble the solid and cystic variants of papillary carcinoma. This raises the possibility that these cases and BTRPTC might be a variant of papillary carcinoma. In our experience none of the tumors showed malignant (metastatic) potential as reported in a previously published case report and have an excellent prognosis. Until more cases and clinical follow up is available for clarification of its malignant potential, we propose the name sclerosing papillary hyperplasia without myoepithelium.

164 Expression of Beclin-1 Protein in Breast Cancer AV Gandhi, PJ Holmes, JP Palazzo. Thomas Jefferson University Hospital, Philadelphia, PA. Background: Autophagy is the self-digestion of intracellular organelles in cells that undergo stress. Beclin-1, the mammalian orthologue of Atg6 (autophagy-related gene), is fundamental to the formation of the autophagosome involved in this process. The end result is either adaptation or cell death, which may explain how both increased and decreased beclin-1 levels are linked to different types of cancer. In regards to breast cancer, beclin-1 may be of prognostic value if its role is fully elucidated. In this study, the staining of beclin-1 in benign breast tissue, ductal carcinoma in situ (DCIS), low- grade carcinoma, and high-grade carcinoma was evaluated to determine the correlation between beclin-1 and cancer progression. Design: We studied 521 patients who underwent surgery from 1999-2008 at TJUH. There were 113 cases of benign breast tissue, 47 cases of DCIS, 170 cases of low- grade carcinoma, and 191 cases of high-grade carcinoma. All cases were stained for Beclin-1 (1:200, EPR1733Y, Epitomics, Burlingame, CA) with appropriate controls. Cytoplasmic staining was considered positive and cases were categorized by intensity (weak, moderate, strong). The study was approved by the IRB. Results: Benign breast, DCIS, and low-grade cases demonstrated moderate-strong Conclusions: WT1 IHC may be used as an alternative marker for breast myoepithelial cytoplasmic staining of beclin-1 in 95/113 (84%), 38/47 (81%), and 131/170 (77%) cells in benign and malignant breast lesions. The fact that WT1 protein is expressed in of cases, respectively. However, only 38/191 (20%) of the high-grade cases showed the cytoplasm of breast myoepithelial cells suggests the possibility of pairing WT1 with moderate-strong staining, the majority (70%) being negative. a nuclear myoepithelial marker such as p63 and/or another complementary cytoplasmic Conclusions: Beclin-1 staining was increased in cases of benign tissue, DCIS, and marker such as calponin to develop a more robust IHC panel. low-grade carcinoma in contrast to decreased or negative staining in cases of high-grade carcinoma. These findings indicate that beclin-1 inversely correlates with histological grade and progression to high-grade cancer. Stress-induced inhibition of autophagy may propagate breast malignancy by reducing cell death. Thus, our results suggest that beclin-1 could be used as a valuable marker in human breast cancer.

165 WT1 Immunohistochemistry as a Novel Marker for Breast Myoepithelial Cells M Ghofrani, O Saglam, FA Tavassoli. PeaceHealth Laboratories, Vancouver, WA; Yale University, New Haven, CT. Background: The Wilms tumor-1 (WT1) gene plays a critical role in urogenital development. Originally reported as the main gene in the development of nephroblastomas, WT1 was later seen in a wide variety of benign and neoplastic tissues by immunohistochemistry (IHC). In breast, WT1 expression has been reported in mucinous carcinomas, invasive micropapillary carcinomas, and carcinomas with basal-like and ERBB2 profiles, as well as a marker of endothelial and myoepithelial differentiation. Design: Thirty benign and malignant breast tissues were stained to assess WT1 expression in myoepithelial cells. Patients, including 3 males, ranged in age from 15 to 78 years. Breast ducts from normal reduction mammoplasty tissue as well as a variety of benign, precancerous and malignant lesions were evaluated, including adenosis, gynecomastia, fibroadenoma, intraductal papilloma, ductal hyperplasia, ductal intraepithelial neoplasia (atypical ductal hyperplasia and in situ carcinomas of all grades) and invasive carcinoma. Patterns of WT1 expression were evaluated in myoepithelial cells compared to conventional markers such as p63 and calponin. Results: Breast myoepithelial cells within lobules as well as ducts of all calibers expressed WT1 protein in a diffuse cytoplasmic pattern. Myoepithelial cell nuclei were clearly negative for WT1 protein by IHC. In several cases cytoplasmic staining intensity 166 MUC1 Is Expressed at High Frequency in Early-Stage Basal-Like varied across ducts in the same field, but this heterogeneity seemed to complement the Triple Negative Breast Cancer variation in staining for calponin, another cytoplasmic marker of myoepithelial cells. HL Gilmore, FW Abdul-Karim, A Siroy, J Miedler, P Fu, J Baar. University Hospitals Case Medical Center, Cleveland, OH; Cleveland Clinic Foundation, Cleveland, OH; University Hospitals Seidman Cancer Center, Cleveland, OH. Background: Triple-negative breast cancer (TNBC) comprises 10-15% of newly- diagnosed breast cancer (BC) and lacks expression of the estrogen receptor (ER), progesterone receptor (PR) and the HER-2/neu receptor. Many of these tumors are basal-like, a molecular intrinsic subtype of BC that has been associated with particularly poor clinical outcomes. Patients with early-stage basal-like TNBC are at a high risk of relapse after adjuvant chemotherapy and may therefore benefit from novel therapies, including immunotherapy. MUC1 is a tumor antigen expressed on adenocarcinomas and represents an ideal target for MUC1-based vaccination. Design: We evaluated 52 cases of early-stage basal-like TNBC for MUC1 expression by immunohistochemistry (IHC). The intensity of staining was graded according to the intensity (negative (0), positive (1) or strongly-positive (2)) and percent (0 to 100%) of tumor cells staining for MUC1. A specimen was considered positive if immunoreactive cells were found in at least 5% of tumor cells. An overall score of 0 to 2.0 was calculated for each case by multiplying the intensity of staining by the percent of tumor cells staining positively. Results: Four staining patterns for MUC1 were identified: apical, membranous, cytoplasmic and combinations of the three patterns. Forty nine of the 52 cases of basal- like TNBC (94%) were positive for MUC1 expression. The mean score was 0.90 (range 0 - 1.9). Cases were evenly distributed over this range, where most (67%) exhibited moderate to strong MUC1 expression (score 0.5 to 1.90), 27% demonstrated weak MUC1 expression, and 6% lacked MUC1 expression. There was a significant difference ANNUAL MEETING ABSTRACTS 43A in MUC1 score and percent of MUC1+ cells in favor of the combination pattern. type (ST) carcinomas (low grade and >90% purity of pattern), including pure lobular, Conclusions: This study indicates that a large proportion of early-stage basal-like tubular, cribriform, and mucinous. We examined whether the prevalence of PIK3CA TNBC expresses MUC1 and provides a rationale for MUC1-based immunotherapy in mutations among ST BC would differ from NST BC. this high-risk patient cohort. Design: Using H&E stained slides, we typed (NST vs. ST) and graded the first 98 stage I-II ER+ operable tumors from postmenopausal patients enrolled in a clinical trial of 167 Determining Tumor Heterogeneity and Response to Treatment presurgical letrozole. Slides were reviewed by three expert breast pathologists with Ex Vivo Using 3D Mammosphere Cultures Derived from Primary Breast concordance. We correlated the histologic type with the pretreatment Ki67 index and Cancers and Metastases PIK3CA mutation status as determined by RNA-Seq. Results: Mutation data were available on 8 of 18 (44%) ST and 35 of 80 NST (44%) HL Gilmore, K Miskimen, A Difeo, LN Harris. Seidman Cancer Center, Cleveland, OH. tumors in the study group, with an overall mutation rate of 42% (exon 20, n=11; exon Background: One of the major limitations to using commercially available breast 9, n=7), consistent with the enrichment of luminal tumors in this cohort. Mutations were cancer (BC) cell lines for research is that they are not accurate representations of the more frequent in ST versus NST carcinomas (63% versus 37%). Baseline proliferation tumor heterogeneity that exists in patients with newly diagnosed disease. In addition, rates as measured by percent of Ki67+ tumor cells were higher in wild-type versus the response of commercial cell lines to current BC therapies does not give an accurate PIK3CA mutants (30.5% versus 19.4%, p=0.03). Although the cohort contained few portrayal of patient response in vivo. The studies described here focus on growing fresh high grade tumors (9%), exon 20 mutations were found exclusively in low-intermediate BC tissue in 3D cultures called mammospheres. It is believed that 3D cultures better grade, low proliferative rate tumors. There were no other recurrent histologic features reflect the in vivo growth properties of BC and retain tumor heterogeneity. The goal is among NST carcinomas predictive of a PIK3CA mutation. to use this model to predict responses to chemotherapeutic agents ex vivo. Conclusions: PIK3CA mutations are enriched in ST and NST carcinomas with low Design: Discarded fresh tissue samples from 5 patients with primary or metastatic BC baseline proliferation rates. Pending analyses of RNA-Seq and whole exome DNA-Seq were obtained. This included ER+/HER2-, ER+/HER2+ and ER-/HER2- disease. After for the remaining tumors may increase power and provide further insight into these digestion, a homogenous single cell suspension was plated in Matrigel, a laminin-rich relationships and their role in resistance to antiestrogen therapy. basement membrane substance that recapitulates the complex extracellular environment, and on Ultra Low Attachment (ULA) plates which are designed to inhibit cell attachment and select for tumor initiating cells. Growth was assessed at varying timepoints by 169 FISH MYC Amplification and MYC Protein Expression in Atypical brightfield, immunofluorescence and confocal microscopy. Vascular Lesions, Mammary Angiosarcomas and Angiosarcomas of Other Results: Mammospheres grew from all tumors types on both the Matrigel and ULA Sites plates. Growth and expansion of the mammosphere structures were seen over time by PS Ginter, TY MacDonald, TM D’Alfonso, RA Kaplan, JM Mosquera, MA Rubin, SJ brightfield microscopy in culture. The presence of epithelial cells was confirmed using Shin. Weill Cornell Medical College, New York, NY. immunofluorescence and confocal microscopy. Figure 1 shows (A) representative Background: Breast cancer patients who receive radiation therapy or develop brightfield images of two mammospheres and (B) shows representative confocal images. chronic lymphedema following breast surgery are at risk for developing secondary (2o) mammary angiosarcomas (AS) and radiation-induced atypical vascular lesions (AVL). Recent studies have demonstrated MYC amplification (amp) by fluorescencein situ hybridization (FISH) in 2o mammary AS, but not in AVL, primary (1o) mammary AS or AS of other sites. Additionally, these studies have shown good concordance between MYC amp and MYC protein expression (PE) by immunohistochemistry (IHC). In this study, we aimed to identify MYC amp and PE in a sizeable cohort of mammary AS (1o and 2o), radiation-induced AVLs, and 1o AS of other sites to validate recently reported findings. Design: Cases of AVL (n=6), 2o mammary AS (n=10), 1o mammary AS (n=17) and AS of other sites (n=19) were identified in our files. FISH analysis and IHC for MYC amp and PE, respectively, were performed on tissue microarray (TMA) or whole tissue slides. MYC amp was defined as a MYC/CEP8 ratio of≥ 2.0. Strong nuclear immunoreactivity in >10% of lesional cells was deemed positive for MYC PE. Results: All 2o mammary AS (10/10) while none of the AVL (0/6) showed MYC amplification. One of seventeen (6%) 1o mammary AS showed MYC amp. One case of cardiac AS showed MYC amp while the remaining eighteen 1o AS of other sites were not amplified (5%). MYC PE was observed in all but one 2o mammary AS (9/10). In contrast, none of the AVL (0/6) showed MYC PE. All but one 1o mammary AS (1/17=6%) were MYC negative which was also amplified. Nearly half of 1o AS of other sites (8/19=42%) showed MYC PE including the single amplified case of cardiac origin. Concordance between MYC amp and PE was 100% in cases of AVL (6/6), 1o mammary AS (17/17), and almost all 2o mammary AS (9/10). Concordance between FISH and IHC was 63% in 1o AS of other sites. Conclusions: Our findings confirm previously published data thatMYC amp by FISH is consistently present in 2o mammary AS but not in AVL, 1o mammary AS and AS of other sites. However, we have also identified MYC amp in one 1o mammary AS and one cardiac AS. These new findings suggest that MYC amp may not be specific to o2 mammary AS as previously thought. Additionally, we found that while FISH and PE was concordant in all AVL, 1o mammary AS and nearly all 2o mammary AS, this was not true in AS of other sites suggesting that while PE in mammary vascular lesions correlate highly with MYC amp, this does not hold true in non-mammary AS. Conclusions: Mammospheres may be grown from fresh primary and metastatic BC samples. Future directions include treating the 3D mammosphere cultures with relevant 170 MRI-Guided Needle Core Biopsies of Breast: Diagnostic Yield chemotherapeutic agents such as trastuzumab, bevacizumab, or paclitaxel. Genomic in 193 Cases changes that occur with treatment will be assessed using next-generation sequencing, PS Ginter, AJ Winant, SA Hoda. Weill Cornell Medical College, New York, NY. and signatures will be developed to predict response to therapy. Ultimately, these Background: Magnetic resonance imaging (MRI) is an increasingly employed adjunct optimized conditions for growing BC tumor cells ex vivo will be used on limited tissue to standard breast screening, and is primarily used in high-risk patients. Cumulative data samples from patients as part of clinical trials to guide therapeutic decision making indicate that MRI-guided needle core biopsies of breast (MRI-NCB/B) have relatively and predict response to therapy. high sensitivity but low specificity; however, information regarding diagnostic yield of particular types of lesions detected via this modality remain scant. Design: MRI-NCB/B performed over a 3.5-year period (2009-2012) were retrieved. 168 PIK3CA Mutations Are Enriched in Special Type Invasive Mammary All archived slides, and relevant records, were reviewed. Non-mass-like enhancement Carcinomas in high-risk patients was the indication in almost all cases (Fig. A). J Giltnane, J Balko, MG Kuba, N Wagle, E Van Allen, I Mayer, V Abramson, L Garraway, Results: 193 MRI-NCB/B, performed in 162 patients, were re-evaluated. 31 patients I Meszoely, C Arteaga, M Sanders. Vanderbilt University, Nashville, TN; Broad Institute had multiple (19%) and 9 had bilateral (6%) biopsies. Benign breast findings were of Harvard and MIT, Cambridge, MA. present in 147/193 (76%) cases. Invasive mammary carcinoma (ca) was diagnosed in Background: Aberrant signaling through the phosphatidylinositol-3 kinase (PI3K) 16 (8%) (ductal: 11, lobular: 4, mucinous: 1), ductal ca in situ (DCIS) in 12 (6%), and pathway is a common alteration in breast cancer (BC), with frequent activating mutations lobular ca in situ (LCIS) in 9 (5%). Atypical duct hyperplasia (ADH) was diagnosed in the PIK3CA gene helical (exon 9) and catalytic (exon 20) domains of the PIK3CA in 8 (4%). Benign diagnoses included cystic papillary apocrine hyperplasia: 36 (19%), (p110a catalytic subunit of PI3K) gene. PI3K pathway alterations are present in 40% of fibroadenoma: 11 (6%), papilloma: 18 (9%), radial scar: 7 (4%), benign intramammary estrogen receptor positive (ER+) breast cancer (BC); however, the literature is divided as lymph node: 6 (3%), fat necrosis: 6 (3%), duct ectasia: 5 (3%), and hemangioma: 2 (1%). to whether these alterations are prognostic. Recent data from the Breast Cancer Genome Some degree of apocrine change was evident in 5 (3%) cases of invasive ca (including in Atlas Network (doi:1-.1038/nature11412) indicates that PIK3CA mutations are highly 2 ductal, 2 lobular, and 1 mucinous) and 7 (4%) of DCIS. Apocrine change was evident enriched in the luminal BC subtypes, especially luminal A (45%). Histologically, the in benign lesions, including: 14 cases of sclerosing adenosis (apocrine adenosis), 3 of luminal A subtype includes low grade no special type (NST) carcinomas and the special complex fibroadenoma, and 8 of papilloma. Thus, some element of apocrine change 44A ANNUAL MEETING ABSTRACTS was present in association with various benign and malignant lesions in 75 (39%) cases. assessed by Ki-67 labeling index (LI). Neoadjuvant therapy (NAT) was administered This relatively common finding of apocrine-related lesions in MRI-NCB/B validates a in 12 cases which made it possible to assess pathologic complete response rate in these previously reported (Int J Breast Cancer; 2011; ID 613285) observation. low ER+/HER2 negative tumors. Conclusions: In this series of 193 cases, the positive-predictive value of MRI-NCB/B Results: The average age of the patients was 57 years. The mean tumor size was 2.0 cm. was 19%. Invasive carcinoma was diagnosed in 8% and in situ carcinoma in 10%. Most (37 cases, 97%) tumors were of ductal no special type carcinoma, and one (3%) was Cystic papillary apocrine hyperplasia in 19% was the most common histological a pleomorphic lobular carcinoma. Twenty-eight (74%) carcinomas were grade 3 with finding (Fig. B). average mitotic count of 23 mitotic figures/10 hpf. The mean Ki-67 LI was 73%. The tumors showed circumscribed borders in 42%, sheet-like growth pattern in 71%, spindle cells in 29%, apocrine differentiation in 26%, prominent nucleoli in 47%, lymphocytic infiltrate in 58%, necrosis in 39% and apoptosis in 15% cases. Lymphovascular space invasion was noted in 29% and positive lymph nodes in 38% cases. Of the 38 cases 35 (92%) were negative for progesterone receptor. Pathologic complete response (pCR) was seen in 4 of 12 cases (33%) that received NAT. The average tumor volume reduction in cases that failed to achieve pCR was 71%. Conclusions: Low ER+/HER2 negative breast cancers have morphologic features and a response to chemotherapy more similar to triple negative tumors rather than the usual type of ER positive tumors. These tumors form a distinct group within ER positive breast cancers. The diagnostic yield of MRI-NCB/B in breast screening and the preferential detection of apocrine lesions require further study. 173 Expression of Androgen Receptor in Triple-Negative Breast Carcinomas and Inflammatory Breast Carcinomas 171 Immunohistochemical Profile of Breast Cancer with Respect Y Gong, W Wei, Y Wu, L Huo. University of Texas MD Anderson Cancer Center, to ER and HER2 Status Houston, TX. NC Gloyeske, J Yu, E Elishaev, AH Woodard, DJ Dabbs, R Bhargava. Magee-Womens Background: Androgen receptor (AR) is commonly expressed in breast cancers and Hospital of University of Pittsburgh Medical Center, Pittsburgh, PA. has been implicated in breast cancer biology. Triple-negative breast carcinoma (TNBC) Background: Immunohistochemical (IHC) stains are performed to confirm the identity and inflammatory breast cancer (IBC) both have generally aggressive clinical course of breast cancer when it metastasizes or presents as a tumor of unknown primary. There and either do not benefit from or frequently develop resistance to conventional targeted are very few studies that have evaluated a panel of stains on a single large dataset, which therapies. We herein sought to examine the prevalence of AR expression and explore its is required for direct comparison between different antibodies. Moreover, almost no data value as a prognostic marker and a potential therapeutic target in these two subgroups exists on IHC profiles of breast cancer with respect to receptor expression. of breast carcinoma. Design: A panel of IHC stains was performed on tissue microarrays of 198 primary breast Design: Tissue microarrays of 91 TNBCs (between 6/2005 and 11/2011) and 88 carcinomas with 3 fold redundancy. The panel was chosen to include breast specific inflammatory breast carcinomas (between 9/1994 and 8/2004) were stained with markers and markers that are expressed in tumors resembling breast. It included CK7, monoclonal antibody AR441 (dilution 1:30, DAKO) by immunohistochemistry. Nuclear CK20, PAX8, vimentin, GCDFP-15, mammaglobin, NYBR1, and androgen receptor. staining in greater than 10% of invasive tumor cells was defined as positive. Correlations ER, PR and HER2 results were available from pathology reports. between AR expression and pathologic parameters, biomarkers (Fisher’s exact test) Results: The individual marker positivity was 92% (172/186) for CK7, 80% (151/189) and between AR expression and overall survival (OS) (log-rank test) were examined. for AR, 55% (105/190) for NYBR1, 52% (99/189) for mammaglobin, 31% (59/191) for Results: The median follow-up and 5-year OS rate were 5.7 years and 63%, respectively, vimentin, 12% (22/195) for GCDFP-15, 0.5% (1/186) for CK20, and 0% (0/188) for for TNBC and 10.8 years and 46%, respectively, for IBC. AR was positive in 17% of PAX8. Data for all markers was available on 178 carcinomas that resulted in a total of TNBCs and 39% of IBCs. Positive AR expression was significantly associated with 41 profiles. The most common tumor profiles are summarized for each IHC/molecular lymphovascular invasion in both tumor subgroups (p = 0.02 and p = 0.01, respectively). class of breast cancer below. There was no significant association between AR positivity and Ki67 expression, other Profiles of ER+/HER2- Tumors pathologic parameters or OS in either subgroup. There was no significant association CK7+/CK20-/PAX8-/Vim-/GCD-/MGB+/NYBR1+/AR+/PR+ (17/125) between AR positivity and ER, PR and HER2 status in IBCs. In the IBC subgroup, CK7+/CK20-/PAX8-/Vim-/GCD-/MGB-/NYBR1+/AR+/PR+ (15/125) patients with AR-/ER- tumor had a significantly worse OS than those with AR-/ER+, CK7+/CK20-/PAX8-/Vim-/GCD-/MGB+/NYBR1-/AR+/PR+ (11/125) AR+/ER- or AR+/ER+ tumors (p = 0.03). Profiles of ER+/HER2+ Tumors Conclusions: Positive AR expression was found in 17% TNBC and 39% of IBC. The CK7+/CK20-/PAX8-/Vim-/GCD-/MGB+/NYBR1-/AR+/PR+ (3/17) IBC patients with AR-/ER- tumor had a significant worse OS than IBC patients with CK7+/CK20-/PAX8-/Vim-/GCD+/MGB+/NYBR1+/AR+/PR+ (2/17) other combinations of AR/ER expression. Perplexingly, AR positivity was significantly CK7+/CK20-/PAX8-/Vim-/GCD-/MGB-/NYBR1+/AR+/PR+ (2/17) associated with lymphovascular invasion in both TNBC and IBC subgroups. Further Profiles of ER-/HER2+ Tumors study with larger series is required to delineate the biologic mechanism of AR in these No consistent profile in 7 tumors but all CK7+, CK20-, PAX8-, PR- tumors. Profiles of ER-/HER2- Tumors CK7+/CK20-/PAX8-/Vim+/GCD-/MGB-/NYBR1-/AR-/PR- (15/29) 174 Expression of Enhancer of Zeste Homolog 2 (EZH2) in Invasive CK7+/CK20-/PAX8-/Vim+/GCD-/MGB-/NYBR1+/AR-/PR- (2/29) Neuroendocrine Carcinoma of the Breast CK7+/CK20-/PAX8-/Vim+/GCD-/MGB+/NYBR1-/AR-/PR- (2/29) Y Gong, J Chen, J Wang, L Radvanyi, DG Rosen, Y Wu. University of Texas MD Conclusions: Although the majority of breast carcinomas are CK7+/CK20-, an Anderson Cancer Center, Houston, TX. unusual profile (i.e. CK7-/CK20-) seen in∼ [/underline]8% cases can create confusion Background: Enhancer of Zeste homolog 2 (EZH2), a member of Polycomb Group in investigation of a tumor of unknown primary. Fortunately, most of the CK7 negative (PcG) proteins, is involved in the regulation of cell cycle progression and has been tumors are ER positive. Vimentin expression in triple negative breast cancer can create implicated in various human malignancies, including beast cancer, and also has been confusion in distinction from high grade endometrioid endometrial carcinoma and associated with poor clinical outcome of the patients. Invasive neuroendocrine carcinoma therefore PAX8 should always be included in this differential diagnosis. The results of (NEC) of the breast is a rare type of tumor that has not been well-studied. Our recent this study define the sensitivity of antibodies against each other and strongly support the study (Cancer 2010;116:4463–73) indicated that mammary NEC is a distinct type of use of immunohistochemical panels when investigating tumors of unknown primary. breast carcinoma associated with aggressive biologic behavior. In this study, we sought to determine the correlation between the expression of EZH2 and clinicopathologic 172 Low Estrogen Receptor Positive Breast Cancer: Is This a variables as well as survival outcome of the patients with mammary NEC. Distinct Group? Design: Mammary NEC was defined according to 2003 WHO classification of the NC Gloyeske, DJ Dabbs, A Mallon, R Bhargava. Magee-Womens Hospital of University breast and female genital organs. A total of 64 primary NECs of the breast that were of Pittsburgh Medical Center, Pittsburgh, PA. surgically removed between February 1996 and March 2009 were analyzed in this Background: Invasive breast carcinoma expressing estrogen receptor (ER) at low study. Immunhistochemical staining for EZH2 was performed using monoclonal levels by immunohistochemistry (IHC) constitute a minority of ER positive tumors. antibody (clone 6A10, dilution 1:200, Novocastra). Nuclear staining in greater than Clinically these tumors are considered within in the umbrella group of ER positive 10% of invasive tumor cells was defined as positive. Patients’ information, tumor breast cancers. However, recent studies have shown the clinical significance of semi- characteristics and prognostic and predictive biomarkers were retrospectively reviewed. quantitative hormone receptor levels and it is possible that these “low ER positive” The relationships between EZH2 expression and overall survival (OS), recurrence-free tumors are different from the usual ER positive tumors. survival (RFS), and other pathologic parameters were examined. Design: Of the 731 total ER positive invasive breast cancers in the year 2010 at our Results: The age of the patients ranged from 34 to 82 years (median: 62 years). The institution, 53 (7.3%) cases had H-scores of 1-100, which was used as the definition median follow-up was 36 (7-215) months and the 5-year OS and RFS rates were 82.6 of “low ER positive” cases. HER2 positive cases were excluded from this analysis. (95% confidence interval = 63.0% - 92.4%) and 61.7% (95% confidence interval = 40.7% A total of 38 ER+/HER2 negative cases were analyzed for various morphologic - 77.2%), respectively. EZH2 expression was found in 26 (40.6%) of the 64 tumors, parameters including tumor border, overall grade and mitotic activity. The following and was significantly associated with high nuclear grade (p < 0.001), high histologic features were considered present if identified in≥ 10% of the tumor: sheet-like growth grade (p < 0.001) and high Ki-67 index (p = 0.0001). However, there as no significant pattern, spindle cells, apocrine differentiation, intra-tumoral lymphocytic infiltrate, and correlation between EZH2 expression and OS (p = 0.183) or RFS (p = 0.101) rates. necrosis. Nucleoli were considered prominent if visible at 10X objective and substantial apoptosis was considered present if easily visible at 10X. Cell proliferative activity was ANNUAL MEETING ABSTRACTS 45A Conclusions: Positive EZH2 expression was significantly associated with high nuclear 177 ROR2 Expression in Breast Cancer grade, high histologic grade and high Ki67 index. Targeting EZH2 may provide a novel X Guo, P Sweeny, S Varma, K Montgomery, RB West, M van de Rijn. Stanford University strategy to improve clinical outcome in patients with mammary NEC. Further study with School of Medicine, Stanford, CA. larger series is required to delineate the biologic mechanism of EZH2 in mammary NEC. Background: ROR2 is an orphan receptor tyrosine kinase that is expressed by several cancer types. ROR2 mediates the WNT signaling pathway and is involved in tumor 175 Clinicopathologic Evaluation of Apocrine Carcinoma of the invasion in sarcomas and renal cancer. ROR2 is a potential therapeutic gene target for Breast cancer given the success of other receptor tyrosine kinases. Y Gong, S Sioshanshi, S Lu, D Kandil, A Khan. University of Massachusetts Medical Design: In this study, breast cancer tissue microarrays containing 236 cases of School, Worcester, MA; Rhode Island Hospital, Providence, RI. ductal carcinoma in situ, 223 cases of invasive ductal carcinoma, and 106 metastatic Background: Apocrine carcinoma of the breast is a rare histologic subtype accounting breast cancers were stained with an anti-ROR2 antibody. Cores were evaluated for for 0.4 to 4% of all breast cancers. Limited data exists in literature regarding these circumferential membranous staining, consistent with the HER2/neu scoring criteria. tumors due to the rarity of the subtype. The goal of this study is to evaluate the Results: 10.17% (24/236) of ductal carcinoma in situ cases and 11% (25/223) of immunohistochemical (IHC) expression pattern of a series of apocrine carcinomas and invasive breast carcinoma cases are ROR2-positive. Notably, in the invasive cancers, to correlate the findings with the clinical outcome. ROR2 expression was not correlated with HER2 amplification. Among ROR2-positive Design: A search in the medical records at our institution from 1998-2012 yielded 77 metastases, 100% (3/3) of the corresponding primary carcinomas lesions are also malignant cases of breast surgical specimens with apocrine differentiation. The tumors positive. Overall positivity in metastases is 5.5% (6/106). included 24 cases of ductal carcinoma in-situ (DCIS) and 53 cases of invasive ductal Conclusions: Overall, 10% of ductal carcinoma in situ and invasive carcinoma cases carcinoma (IDC). IHC study was performed on paraffin sections of the cases, including express ROR2. ROR2 expression may represent a clinically useful therapeutic target. markers for estrogen (ER), progesterone (PR), HER2/neu, androgen receptor (AR), insulin-like growth factor II mRNA-binding protein 3 (IMP3), epidermal growth factor 178 Significance of Myoepithelial Cell Layer around Ducts with receptor (EGFR) and cytokeratin 5/6 (CK 5/6). Clinical follow-up (FU) was available Papillary Ductal Epithelial Neoplasia (DIN 1-3) (Papillary Ductal Carcinoma on 17/24 of DCIS and on 31/51 of IDC patients, with a median follow-up period of 64 In-Situ, DCIS 1-3) and 56 months, respectively. M Harigopal, C Flynn, P Karam, M Orsaria, FA Tavassoli. Yale University School of Results: Of the total of 5698 breast carcinomas diagnosed between 1998-2012, 24/1388 Medicine, New Haven, CT. (1.7%) DCIS and 53/4310 (1.2%) IDC were identified to have apocrine differentiation. Background: While persistence of myoepithelial cells around a duct with papillary DIN 3/18 (15%) of apocrine DCIS and 18/53 (34%) of apocrine IDC exhibited a triple 1-3 (PDIN = Papillary DCIS) is helpful in excluding an invasion, the significance of its negative (TN)* profile; and of the TN cases, 100% of DCIS and 93% of IDC were AR absence in this setting is controversial. Furthermore, when the PDIN 1-3 is associated positive. However, there was a poor correlation between TN profile and basal phenotype with adjacent conventional invasive carcinoma (IC), the size and stage of the carcinoma markers, with 0/3 DCIS positive for EGFR, CK 5/6 and IMP3 and with only 7/18, 4/18, could vary substantially depending on whether the papillary component is interpreted and 2/18 IDC cases positive for EGFR, CK5/6 and IMP3 respectively. Of the 17 DCIS as invasive or intraepithelial. patients with FU, 1 patient developed concurrent invasive metaplastic carcinoma and Design: In a retrospective search of the pathology database at our institution for all died from the disease. The remaining 16 patients have no evidence of local recurrence PDIN 1-3 (Papillary DCIS) with excisional biopsy between 2001 to 2011, a total of 50 or metastases. Of the 31 IDC patients, 3 developed metastatic diseases, 4 died from (PDIN, solid), 2 (PDIN, intracystic), 12 PDIN with associated IC were identified and unrelated causes and the remaining 24 are disease-free to date. reviewed. 27 low grade PDIN of solid and intracystic types with or without associated IC *Triple negative (TN) = ER, PR and HER2 negative were further evaluated with 3 ME (p63, calponin and CD10) and 2 basement membrane Summary of IHC Study markers (collagen IV & laminin). ER PR HER2 IMP3 EGFR CK5/6 AR Results: All low grade PDIN (22) showed absence of ME (>90%) within the intraluminal DCIS 58% (14/24) 42% (10/24) 67% (12/18) 5% (1/20) 10% (3/21) 0% (0/20) 91% (19/21) papillary fronds and focal or discontinuous ME cell around the duct; BM markers showed IDC 38% (20/53) 21% (11/53) 40% (21/52) 13% (6/47) 26% (12/47) 17% (8/47) 85% (39/46) retention of BM around distended ducts with PDIN. PDIN with associated IC (n = 5) % positive (# positive/total # tested) showed complete absence of ME cells around the distended ducts with PDIN. BM was Conclusions: Apocrine carcinoma of the breast is a distinct subset of ductal carcinoma present around PDIN, but absent around foci of IC. The size of the PDIN varied from with a largely indolent behavior. While >1/3 of IDC were TN, only a minority of these 3 mm to 2.7 cm, while the adjacent invasive carcinoma (IC) varied from <0.1 to 1.3 showed markers associated with basal-like breast cancer. No prognostic impact was cm. Only one of the 22 cases had nodal metastases, this case had IC. observed with TN profile or the basal phenotype markers. Conclusions: The majority of papillary DIN 1-3 is non-invasive with an expansile growth in distended ducts. The IC associated with PDIN (5) was a conventional IDC (n 176 Bcl-2 Downregulation Is Predictive of Complete Pathologic = 4) or mucinous (n=1). The absence of ME cells around ducts in the absence of typical Response Following Neoadjuvant Chemotherapy in Breast Cancer IC architecture is not an indication of an invasive carcinoma. Ultimately, the prudent S Goodman, T Stockl, S Lyle, D Kandil, K Edmiston, R Quinlan, A Khan. UMass use of both histologic criteria and IHC in papillary DIN 1-3 with or without associated Memorial Medical Center, Worcester, MA. IC is critical to avoid overstaging and overtreatment. Papillary DIN 1-3 lesions devoid Background: Neoadjuvant chemotherapy has become standard of care in a subset of any ME cells have excellent prognosis similar to DIN 1-3 (DCIS, grades 1-3). of breast cancer. Certain chemotherapeutic agents including docetaxel act by binding tubulin, inhibiting mitsosis, and by phosphorylating bcl-2 to inactivate its 179 HER2 Expression in DCIS Is Highly Associated with DCIS overexpression. Bcl-2 is an oncogene, which inhibits apoptosis and restrains cell Associated Lymphocytes cycle entry. Overexpression of Bcl-2 prevents apoptosis in damaged cells and can J Hatem, R Batiste, K Lee, B Czerniecki, P Zhang. University of Pennsylvania, eventually lead to cancer. The aim of this study was to evaluate the expression pattern Philadelphia, PA. and predictive value of Bcl-2 in the outcome of breast cancer by examining the effect Background: HER2 expression is known to be higher in DCIS than invasive ductal of neoadjuvant therapy on breast cancer. carcinoma. Though not considered as a major therapeutic target in DCIS yet, the role Design: A total of 27-paired cases (pre-treatment biopsy and post-treatment resection of immunotherapy targeting HER2 has currently been explored in DCIS in clinical specimens) over a six year period (2006-2011) were obtained from the surgical pathology trials. The histologic characteristics of HER2+ DCIS, particularly those related to the files. All the slides were reviewed to confirm the diagnosis and evaluate pathologic host immunoresponse such as DCIS associated lymphocytes (DAL) have not been response in post treatment specimens. These consisted of invasive carcinoma of both well analyzed. lobular (n=3) and ductal types (n=24). Immunohistochemistry for Bcl-2 was performed Design: Representative H&E sections of 80 consecutive DCIS from 2011-2012 at on pretreatment biopsies and in post treatment specimens in which residual tumor was our institution were reviewed for histologic type and nuclear grade. DAL was semi- present. Positive bcl-2 staining in tumor cells was graded into three groups by intensity quantitatively analyzed as peri-DCIS lymphoid aggregate (PDLA) with or without direct of cytoplasmic staining, 1 as weak and 3 as strong. The chemotherapy used varied, and contact of DCIS (LDCD). Peri-DCIS stromal change (PDSC) was also evaluated. These included paclitaxel, docetaxel, cycophosphamide, doxorubicin, and in Her-2 positive histologic features were correlated to the results of HercepTest (Dako) performed at cases, Transtuzumab (Herceptin). Complete pathologic response (CPR) was defined as the time of the diagnosis in each case. no residual tumor seen in post treatment surgical resection specimen. Results: HER2 was 3+ in 32 (40%) and 2+ in 5 (6%) of all cases. There were 41 (51%) Results: Of the 27-paired cases, complete pathologic response (CPR) to therapy cribriform (HER2 3+ in 9 [22%]), 11 (14%) solid (HER2 3+ in 5 [45%]), 21 (26%) occurred in 9. Of these nine cases, 8 of them were Bcl-2 negative, and 1 was Bcl-2 comedo (HER2 3+ in 16 [76%]), 5 (6%) papillary/micropapillary (no HER2 3+) and 2 positive. Of the remaining 18 cases, 7 were Bcl-2 negative, and 11 were Bcl-2 positive (2.5%) and Paget (HER2 3+ in 2 [100%]). Table 1 for HER2 results for nuclear grade, (P value = 0.019, Fisher’s exact test). Of the cases with CPR, all 9 were invasive ductal PDLA, LDCD, and PDSC. carcinoma (8 grade III, 2 grade II). Positive Her-2 receptor status did not correlate Table 1 with Bcl-2 expression. Of the 9 cases with complete response, five cases were Her-2 NG PDLA LDCD PDSC positive and received Transtuzumab. Bcl-2 expression did not change after treatment <5% / 5-20% / Low / Int / High Yes / Focal / No Yes / No with neoadjuvant chemotherapy. >20% Conclusions: In summary bcl-2 down regulation is significantly associated with CPR All cases n=80 10 / 38 / 32 12 / 21 / 34 13 / 7 / 60 56 / 24 0(0%) / 10(26%) / 2(8%) / 3(14%) / 12(92%) / 3(42%) / HER2 3+ n 28(50%) / 4(17%) following neoadjuvant chemotherapy in breast cancer suggesting that a pro-apoptotic 21(65%) 27(79%) 17(28%) molecular signature may play a role in response to chemotherapy and bcl-2 expression HG DCIS n=32 4 / 8 / 20 9 / 2 / 21 1(25%) / 2(25%) / 9(100%) / 1(50%) / may be used to predict response following neoadjuvant chemotherapy. HER2 3+ n 18(90%) 11(52%) Conclusions: In addition to high grade (65%) and comedo morphology (76%), HER2 is highly expressed in DCIS with prominent PDLA (>20%) (79% vs. 8%) and presence of LDCD (92% vs. 28%) and PDCS (50% vs. 17%). The impact of PDLA and LDCD 46A ANNUAL MEETING ABSTRACTS does not seem to depend upon nuclear grade as it remains strong when only high Conclusions: The high affinity rabbit antibody against E-cadherin was more sensitive grade DCIS were analyzed. Since HER2 protein is known to be able to stimulate and than clone HECD-1 for immunohistochemical evaluation of ductal carcinomas, and activate host immunocytes in vitro, the high incidence of prominent PDLA and LDCD was negative in all lobular carcinomas; thus making it a reliable alternative for the in HER2+ DCIS suggests that the abnormally high HER2 expression could naturally distinction of infiltrating ductal cell vs. lobular carcinomas. function as a tumor antigen to induce host immunoresponse in these patients. Although the significance is yet to be determined, the assessment of PDLA and LDCD might 182 Genomic Profiling of Histological Special Types of Breast provide insight to host anti-tumor immunoresponse for predicting and monitoring Cancer therapeutic response. HM Horlings, B Weigelt, MB Lambros, EM Anderson, A Mackay, CKY Ng, FC Geyer, MJ Van de Vijver, JS Reis-Filho. Netherlands Cancer Institute, Amsterdam, Netherlands; 180 BTG2 Loss Is Associated with Poor Prognosis in HER2+ Breast Academic Medical Center, Amsterdam, Netherlands; Memorial Sloan-Kettering Cancer Cancer Center, New York, NY; Institute of Cancer Research, London, United Kingdom. MM Hefti, N Knoblauch, AH Beck. Beth Israel Deaconess Medical Center, Boston, MA. Background: Whilst the majority of invasive breast cancers are classified as invasive Background: B-cell translocation gene 2 (BTG2) is a key regulator of cell proliferation carcinomas of no special type (IC-NST), up to 25% of cases are of a histological and a tumor suppressor in breast cancer1,2. BTG2 inhibition enhances growth and ‘special type’. The aims of this study were to determine i) the constellation of gene migration of breast cancer cells via stabilization of the HER ligands NRG1b and copy number aberrations in histological special types of breast cancer, ii) whether these epiregulin.3 We thus examined the effect of BTG2 on survival in breast cancer, special breast cancer types are distinct from ER- and grade-matched IC-NSTs at the particularly in HER2-type cancers. genomic level, and iii) the genes whose expression correlates with gene copy number Design: We used a previously described collection of publicly available gene expression in each histological special type. profiling data sets4. Gene expression was scaled across the entire data set for the overall Design: A series of 59 breast cancers of ten histological special types and a cohort of survival analysis and within each subtype for the molecular subtype analysis. grade- and ER-matched IC-NSTs were subjected to microarray-based comparative Results: A total of 1173 patients had available survival data. BTG2 was significantly genomic hybridisation (aCGH) and microarray-based gene expression profiling predictive of overall survival (p<10-3) on univariate Cox regression but not in a model (Operon). An integrative analysis of aCGH and microarray-based gene expression that included HER2, ER, age, grade, nodal status, and size (p=.144). We then examined profiles of the 59 special type breast cancers was performed. each molecular subtype individually. BTG2 was associated with survival in HER2+ Results: Hierarchical cluster analysis revealed that the patterns of gene copy number molecular subtype (p=.002) but not for Luminal A, Luminal B, or Basal (p=.901, .325, aberrations segregated with ER-status and histological grade, and that, in general, and .579 respectively; Table 2). The interaction term BTG2*HER2 was significantly samples from each of the special types of breast cancer were preferentially allocated associated with survival (p=.031) when included in a multivariate model as above, but to one cluster. We confirmed the patterns of gene copy number aberrations previously BTG2*ER was not (p=.480). reported for lobular, micropapillary, medullary, metaplastic and mucinous carcinomas. Conclusions: Our data show that BTG2 is associated with prognosis only in HER2+ Metaplastic and medullary carcinomas were shown to have genomic profiles similar breast cancer. These data suggest that BTG2 may play a key functional role in regulation to those of IC-NST matched for grade and ER-status. The constellation of gene copy of breast cancer growth and metastasis by HER2. BTG2 warrants further investigation number aberrations found in invasive carcinomas with osteoclast-like stromal giant as a possible therapeutic target and mechanism for HER2-antagonist resistance. cells support the classification of this histological type as variant of IC-NSTs. Finally, Cox Regression of BTG2 on Survival by Breast Cancer Subtype integrative analysis of aCGH and gene expression data revealed copy number regulated Subtype n p-value OR 95% CI genes and genes significantly overexpressed when amplified, including FOXM1 and HER2 161 0.002 0.665 0.551 - 0.864 FGF6 mapping to the 12p13.33-13.32 amplicon in metaplastic carcinomas. Luminal A 294 0.901 1.016 0.786 - 1.314 Conclusions: Our results illustrate that together with histological grade and ER-status, Luminal B 408 0.325 0.910 0.753 - 1.099 Basal 280 0.579 1.055 0.874 - 1.273 histological type is also associated with the patterns and complexity of gene copy number aberrations in breast cancer. 1. Kawakubo H, Brachtel E, Hayashida T, et al. Loss of B-cell translocation gene-2 in estrogen receptor-positive breast carcinoma is associated with tumor grade and overexpression of cyclin d1 protein. Cancer research 2006;66:7075-82. 183 The Unique Role of ER-a36 in Maintaining a Close Relationship 2. Mollerstrom E, Kovacs A, Lovgren K, et al. Up-regulation of cell cycle arrest between ER-a66 and HER2 in Breast Cancer protein BTG2 correlates with increased overall survival in breast cancer, as detected by J Huang, CR Chitambar, B Behmaram, YC Cheng, Z Basir, A Walker. Medical College immunohistochemistry using tissue microarray. BMC cancer 2010;10:296. of Wisconsin, Milwaukee, WI. 3. Takahashi F, Chiba N, Tajima K, et al. Breast tumor progression induced by loss of Background: Many lines of evidence have demonstrated that crosstalk between wild- BTG2 expression is inhibited by targeted therapy with the ErbB/HER inhibitor lapatinib. type estrogen receptor alpha (ER-α66) and human epidermal growth factor receptor 2 Oncogene 2011;30:3084-95. (HER2) plays a critical role in intrinsic and acquired resistance to both endocrine and 4. Haibe-Kains B, Desmedt C, Loi S, et al. A three-gene model to robustly identify breast HER2-directed neoadjuvant therapies. However, this paradigm has been challenged cancer molecular subtypes. Journal of the National Cancer Institute 2012;104:311-25. by clinical evidence that ER-α66 is co-expressed with HER2 in only 10% of ER-α66- positive breast cancer patients. Therefore, an intrinsic relationship between ER-α66 and HER2 needs to be identified. ER-α36, a novel splice variant of ER-α66, is expressed 181 A New Rabbit Monoclonal E-Cadherin Antibody Shows Higher in both ER-α66-positive and ER-α66-negative breast tumors. Whether ER-α36 plays Sensitivity Than Mouse Monoclonal E-Cadherin [HECD-1] Antibody in an unique role in maintaining a close relationship between ER-α66 and HER2 has Infiltrating Ductal Cell Carcinomas not been explored. L Hoang, R Bremer, P Tang, DG Hicks, T Haas, D Tacha. Biocare Medical, LLC, Design: The expression of ER- α36, ER-α66 and HER2 was examined using Concord, CA; University of Rochester Medical Center, Rochester, NY; Mercy Health immunohistochemistry (IHC) in two cohorts of 471 formalin-fixed, paraffin-embedded System, Janesville, WI. tissue microarray slides of breast tumors. We evaluated the correlations among ER- Background: The loss of E-cadherin expression has been observed in breast cancer α36, ER-α66 and HER2, and confirmed their relationships in breast cancer cell lines. and is associated with a poor prognosis. Immunohistochemical studies have shown Statistical significance was measured using Fisher’s exact test. E-cadherin to be expressed in breast ductal carcinoma with loss of expression in lobular Results: The results showed that 1) ER-α66 is inversely associated with HER2 carcinoma. As a result, mouse monoclonal anti-E-cadherin [HECD-1] has been used by (P< 0.05); there is no relationship between ER-α66 and HER2 in ER-α36-negative pathologists to differentiate between ductal and lobular carcinomas of the breast, with tumors(P=1); Strikingly, there is a strong positive correlation between ER-α66 and currently published sensitivity and specificity of approximately 90%. Rabbit monoclonal HER2 in ER-α36-positive tumors (P<0.001). Knocking down ER-α36 expression by antibodies may combine the best properties of both mouse monoclonal antibodies and siRNA abrogated the interaction between ER-α66 and HER2 in both MCF7/HER2 rabbit antisera. This study compared the staining sensitivity of a new rabbit monoclonal and BT474 breast cancer cell lines. 2) ER-α36 is positively associated with ER-α66 E-cadherin vs. mouse monoclonal clone HECD-1. The two E-cadherin antibodies were (P<0.001); ER-α36 is more strongly associated with ER-α66 in HER2-negative tumors further applied in the discrimination of ductal from lobular carcinomas, in combination (P<0.0001); there is no association between ER-α36 and ER-α66 in HER2-positive with a p120 catenin antibody. tumors (P=0.78). 3) ER-α36 is also strongly positively associated with HER2(P<0.001); Design: Rabbit monoclonal E-cadherin and HECD-1 were tittered and optimized for ER-α36 is more strongly associated with HER2 in ER-α66-negative tumors (P<0.0001); immunohistochemical staining in infiltrating ductal breast cancers using an HRP- there is no association between ER-α36 and HER2 in ER-α66-positive tumors (P=0.14). polymer detection system and visualization with DAB. 31 cases of infiltrating ductal Conclusions: We demonstrated that there are strong dynamic relationships among cell carcinoma and 29 cases of lobular carcinoma were evaluated. A semi-quantitative ER-α36, ER-α66 and HER2 in breast cancer. ER-α36 plays a key role in maintaining scoring system for intensity of membrane staining with E-cadherin was applied: such relationships. ER-α66 and HER2 are competitively associated with ER-α36. These 0=negative, 1=weak to medium, 2=medium to strong, 3=strong and intensely strong. results further suggest that ER-α36 may be a novel therapeutic target for overcoming Results: The rabbit monoclonal anti-E-cadherin antibody showed much sharper staining resistance to both endocrine and HER2-directed neoadjuvant therapies. and increased sensitivity (30/31, 97%) compared to mouse monoclonal HECD-1 (27/31, 87%) [Table 1]. No staining was observed in any of the lobular carcinomas evaluated. All lobular carcinomas were confirmed by the absence of E-cadherin and the diffuse 184 Wnt Signalling Pathway Activation Plays Important Role in the cytoplasmic expression of p120 catenin. Oncogenesis of TNBC and Is Associated with Poor Survival Outcome Table 1: Rabbit Monoclonal E-cadherin and Mouse Monoclonal Clone HECD-1 in breast ductal J Iqbal, AA Thike, SS Ahmed, PH Tan. Singapore General Hospital, Singapore, carcinoma Singapore. Average Positive Antibody Score 0 Score 1 Score 2 Score 3 % Positive Background: Triple negative breast cancers (TNBC) are defined by the absence of score (n=31) Rabbit E-cadherin 1 1 7 22 2.6 30 97% estrogen receptor (ER), progesterone receptor (PR) and C-erbB2 expression. Oncologic Mouse E-cadherin managements options are limited in this group of patients. The Wnt/beta-catenin 4 10 15 2 1.5 27 87% (HECD-1) pathway has been implicated in the oncogenesis of invasive breast carcinomas via ANNUAL MEETING ABSTRACTS 47A inappropriate activation of the Wnt/beta-catenin signalling. Secreted frizzled-related carcinoma in situ (DCIS), apocrine type = 9, residual AAA = 3, no residual AAA = protein 1 (SFRP1), a tumor suppressor which antagonizes this pathway is frequently 11. Five of the 9 cases upgraded to DCIS were performed stereotactically and showed lost in breast tumors. The goal of this study was to investigate the expression of sFRP1 residual calcifications in both AAA and DCIS. The remaining 4 cases were obtained by and related downstream proteins in TNBC. MRI (1) and US (3); the latter 2 also had residual AAA on excision in addition to DCIS. Design: Immunohistochemistry was performed on paraffin-embedded tumor tissue of a Conclusions: AAA represents a borderline lesion in the spectrum from apocrine consecutive cohort of 640 female patients with TNBC diagnosed between 2001 to 2008. metaplasia to apocrine DCIS. Given its rarity, our series was limited by number but Antibodies to sFRP1, beta-catenin, cyclin D1 and E-cadherin were applied to sections nevertheless showed an upgrade rate to DCIS of 39% (9/23). Thus, AAA diagnosed on cut from tissue microarray blocks, using the streptavidin-biotin method. Intensity and core biopsy should be excised due to associated sampling error and possible upgrade proportion of tumor cells stained were assessed. Follow-up information was obtained to DCIS. from casenotes. Disease free survival (DFS) and overall survival (OS) were defined as time from diagnosis to recurrence or death respectively. Associations between 187 Negative Estrogen Receptor and HER2 Assays at Core Biopsy sFRP1, beta-catenin, cyclin D1 and E-cadherin expression with clinicopathological of Invasive Cancers Should Be Confirmed in the Surgical Specimens parameters, DFS and OS were evaluated using H-score. A p value of <0.05 defined D Jaggessarsingh, J Catalano, M Akram, E Brogi, M Murray. Memorial Sloan-Kettering statistical significance. Cancer Center, New York, NY. Results: Loss of sFRP1 (89%) was seen in majority of cases with increased expression Background: Expression of ER and HER2 in invasive breast carcinoma (IC) guides of cyclin D1 (77%) associated with aberrant (nuclear/cytoplasmic) expression of beta- patient (pt) management. Accurate assessment is critical to avoid false-negative results catenin (48%). Loss of sFRP1, cyclin D1 over expression and aberrant beta-catenin and withholding of therapy. We evaluate ERH2 status on core biopsies(CB) and retest the staining was associated with basal-like breast cancer. Loss of sFRP1 signals poor surgical specimen(SS) for any marker negative on CB. Our study evaluates discrepancies overall survival and was associated with nuclear grade and nodal status. Loss of both in ER and HER2 results in CB and SS to determine if repeat testing is necessary. sFRP1 and E-cadherin expression was correlated with aberrant beta-catenin expression Design: Review of our database found 301 IC from 306 pts in which ERH2 was (p=0.001). Significantly, nuclear beta-catenin expression was associated with poor OS performed on the CB, and negative stains repeated on the SS. For our study, and reduced E-cadherin expression. immunostains(IHC) were repeated simultaneously on CB and SS for the marker with Conclusions: These results show that (1) sFRP1 loss is significant in TNBC (2) its discordant results, using the same antibody. HER2FISH was done on the SS for all suppression leads to oncogenic activation of WNT signalling pathway via aberrant cases with a discordant HER2 IHC result. expression of beta-catenin (3) sFRP1 and its downstream molecules beta-catenin and Results: Concordance between CB and SS results was 98%(300/306);discordant results cyclin D1 may be used as prognostic markers to predict poor survival in a subgroup were found in 6/306(2%) cases (Table 1). Three cases were discordant in ER(including 2 of TNBC. Due to ubiquitous involvement of beta-catenin in multiple cell signalling cases that were PR(-) on CB and SS) and 3 cases were discordant for HER2. Discrepancy pathways it can be a surrogate target for new chemotherapeutic molecules in TNBC. was due to intratumoral heterogeneity in 2 cases. CB sampled the HER2(-) area of the tumor in 1 case, but staining of a larger section unveiled the positive focus; the 185 Expression of Cancer Stem Cell Markers Aldh1 and Ezh2 Are positve result was confirmed by HER2 FISH. The second case was an invasive lobular Increased in Triple Negative Breast Cancers carcinoma(IL) with mixed classical and histiocytoid morphology; only the classical J Iqbal, KW Lau, AA Thike, SS Ahmed, PH Tan. Singapore General Hospital, Singapore, IL was ER(+), but the CB sampled the histiocytoid IL. In 2 cases discrepancy resulted Singapore. from technical error. Both cases were prospectively interpreted as ER(-) on CB and Background: Breast carcinomas lacking expression of ER, PR, and HER2 (triple- ER(+) on SS, but repeat IHC for ER showed positive staining in both the CB and SS. negative breast carcinomas or TNBC) are characterized by aggressive behavior, distinct In 2 cases HER2(0/1+) on CB but equivocal(2+) in the SS; the equivocal result led to pattern of metastasis, and lack of targeted therapy. In this study we sought to evaluate reflex HER2 FISH with detection of low level HER2 amplification. Relying solely on the presence of cancer stem cell (CSC)-related markers in this group of cancers. CSCs the CB would have resulted in the misclassification of 2 tumors as triple negative; repeat are pluripotent tumor-initiating cells believed to be responsible for treatment failures stains on the SS showed that one was HER2(+) and the other ER(+). and recurrences. We assessed the expression of CSC-related markers ALDH1 and EZH2 Discordant cases in triple negative breast cancers, investigating their association with clinicopathological Case ER(%) HER2 HER2 FISH features and outcome. CB SS CB SS Design: Clinicopathological data were obtained from 131 triple negative breast cancers 1 0 0 0 3+ 4.4 2 0 95 1+ 1+ N/P and 114 hormone-positive invasive breast cancers diagnosed between 2001 and 2008. 3 0 100 1+ 1+ N/P Tissue microarray was constructed and immunohistochemistry for ALDH1 and EZH2 4 5 50 1+ 1+ N/P was performed using the streptavidin-biotin method. Intensity and proportion of tumor 5 90 N/P 0-1+ 1-2+ 2.6 cells stained were assessed. Molecular subtype was based on immunophenotype of ER/ 6 90 N/P 1+ 2+ 3.8 PR/Her2. Follow-up information was obtained from case-notes. Disease free survival N/P-not performed (DFS) and overall survival (OS) were defined as time from diagnosis to recurrence Conclusions: Concordance in the ER and HER2 results between CB and SS was or death respectively. Cytoplasmic expression of ALDH1 and nuclear expression of high(98%), but 2% of cases were discordant. Factors associated with discordance EZH2 were evaluated. P values less than 0.05 were considered statistically significant. included intratumoral heterogeneity, technical error, and equivocal findings. A triple Results: Tumour cells exhibited cytoplasmic expression of ALDH1 in 32% of TNBC negative profile on CB converted to either ER(+) or HER2(+) after staining on SS in 2 cases while increased EZH2 expression was noted in 79% of TNBCs. EZH2 expression cases (<1% of all cases), impacting pt management. Our findings suggest that any ER was highest in the basal-like breast cancer subgroup (83%). Luminal B subtype showed and/or HER2 negative result obtained at CB should be confirmed on the SS to ensure highest expression of ALDH1 (30%). Tumour cell expression did not correlate with any proper pt management. of the clinicopathologic parameters. Increased expression of either biomarker showed non-statistically significant trend of poor DFS in invasive ductal carcinoma but not in 188 Male Breast Carcinoma: Clinicopathologic and Molecular triple negative breast cancer patients. Analysis of 89 Cases Conclusions: In this study (1) EZH2 expression is increased in triple negative breast D Jaggessarsingh, J Catalano, M Akram, E Brogi, M Murray. Memorial Sloan-Kettering cancers/basal-like breast cancers indicating association with aggressive tumor behavior Cancer Center, New York, NY. (2) DFS is worse in patients with increased expression of either CSC marker ALDH1 Background: Male breast cancer(MBC) is a rare disease and remains under studied or EZH2. Further studies of CSC markers in the context of TNBC are warranted to despite evidence of rising incidence. We investigated the molecular subtype profiles identify new chemotherapeutic targets. of MBCs and subsequent clinical outcome. Design: We searched our institutional database over a 20 year period (1992- 186 Atypical Apocrine Adenosis of the Breast on Core Biopsy 2012) for MBC. Tissue microarrays were constructed from 89 invasive MBC and S Jaffer, S Frost, A Nayak, C Nagi, IJ Bleiweiss. Mount Sinai Medical Center, New immunoperoxidase stains (IHC) were performed. Patient(pt) clinical and follow-up(F/U) York, NY. data were extracted from the e-medical records. Background: Atypical apocrine adenosis (AAA) of the breast is defined by sclerosing Results: Median age is 64y(range 32-96y); 3 (3%)pts were <40y. Initial presentation was adenosis infused by atypical apocrine epithelium (characterized by at least a three-fold palpable mass 76, nipple discharge 3, nipple retraction 7, and 3 identified on imaging increase in nuclear size and nucleolar enlargement). It is an uncommon condition performed for other conditions. 27 pts had a personal history of carcinoma and 20 pts and therefore only a few small series have attempted to assess the associated risk of had a family history of BC in a 1stº relative. All pts were surgically treated; 82(92%) subsequent malignancy, particularly when diagnosed on core biopsy. mastectomies, 7(8%) excisions. Tumor types were 72(80.9%) ductal NOS, 7(7.9%) Design: Using our computerized database, we retrospectively identified, reviewed papillary, 6(6.7%) micropapillary, 3(1.1%) mucinous features, 1(1.1%) lobular. Lobular and confirmed the diagnosis of AAA in 32 cases (.02%) on core biopsy from 2000 to CA was confirmed by E-cadherin and p120 IHC on whole tissue sections. Mean tumor present. We excluded all cases with associated necrosis or conventional atypical duct size was 2.1 cm(range 0.8-4.7cm). 82 pts had lymph node(LN) biopsy(BX); 60(73%) hyperplasia (ADH). All clinical information including follow up information was sentinel LN BX. Most pts had positive LNs 53(65%). 87 MBCs were Luminal A gathered and radiologic-pathologic correlation was performed. Excision specimens phenotype and 2 were Luminal B. Clinical FU and IHC results are in Table 1. Most were also reviewed for pathology and previous biopsy site changes. MBCs were AR(72/80, 90%) and EZH2 63/84(75%) positive. Recurrence or distant Results: The patients ranged in age from 33 to 79 years (mean=56.5). The method of core metastasis occured in 9(10%). After a F/U ranging from 7 days to 17 yrs, 6.7% are alive biopsy was as follows: stereotactic for calcifications = 19, ultrasound (US) guided for with disease, 3.4% are dead of disease and 76.4% are alive with no disease. a nodular mass = 7 and MRI for enhancement = 6 (4 with a recent or remote history of breast carcinoma). The indications for the calcifications on stereotactic biopsies were as follows: clustered = 16, new = 1, granular = 1, and linear = 1. Follow up information was as follows: excision = 23, patient refused excision = 3 (with >2 years stable radiologic follow up), and lost to follow up = 6. The diagnoses on excision were as follows: ductal 48A ANNUAL MEETING ABSTRACTS

Luminal A N=87(%) Luminal B N=2(%) and within the ER negative subgroup, with advanced stage (p=0.027) and disease Age(mean, y) 65 60 recurrence (p=0.002), with a trend toward shortened survival (p=0.06). Nuclear FGFR-2 Size(median, cm) 2.1 2.1 overexpression was present in 55/126 (44%) and correlated overall and within the IDC NG 3 39 (45) 2(100) subgroup with ER+ status (p=0.05, p=0.05), HER2- status (p=0.042, p=0.05), with LVI 39(45) 2(100) LN BX 81(93) 1(50) a trend toward non-recurrent disease (p=0.069, p=0.067) and early stage (p=0.085, LN+ 53(65) 0 p=0.08); within the ILC subgroup, with tumor size >3.0cm (p=0.034) and PR- status Ki-67>15% 19 (24.4)(N=78) 1 (50) (p=0.035); within the ER+ subgroup, with HER2- status (p=0.021) and lengthened Ki-67≤15% 59 (75.6)(N=78) 1 (50) survival (p=0.043). On multivariate analysis, early age at diagnosis (p<0.0001), AR+ 70 (89.7)(N=78) 2 (100) metastatic disease (p<0.0001) advanced stage (p=0.004), and intense diffuse cytoplasmic Bp53 80 (100)(N=80) 2 (100) EZH2 61 (74.4)(N=82) 2 (100) FGFR-2 overexpression (p=0.013) independently predicted disease recurrence; while Id1 & Id4 0 0 advanced stage (p<0.0001) and metastatic disease (p<0.0001) independently predicted Median FU (y) 2.65 3.05 overall survival. Local recurrence only 2 0 Conclusions: Cytoplasmic overexpression of the FGFR-2 protein is an independent Bone(3)Lung(2)Brain+Bone(1)Lung+Bone(1) Distant metastases 0 prognostic factor in BRCA and correlates with both hormonal receptor and HER2 status. Mediastinum(1)Pleura(1) DOD(3) AWD(6) NED(67) AUK(2) DOC(8) Further study of the prognostic significance and potential role as a target of therapy for Patient status NED(2) DUC(1) FGFR-2 in BRCA appears warranted. NG-nuclear grade; NED-no evidence of disease; DOD-dead of disease; AWD-alive with disease; DOC-dead other causes; AUK-alive unknown; DUC-dead unknown cause 191 Non-Mass Like Enhancement (NMLE) in Breast Magnetic Conclusions: In our cohort Luminal A was the most common phenotype, while luminal Resonance Imaging: A Proposal of Pathological-Radiological Correlation B was extremely rare. No basal-like or Her2 overexpressing tumors were found. Distant metastases/recurrences and LN+ were only seen in the Luminal A subtype. EZH2, a S Kandel, P Kumar, Q Liu, S Liu, T Khoury. Roswell Park Cancer Institute, Buffalo, NY. marker of aggressive BC, is positive in 75% MBC; however majority of pts are NED. Background: Radiological correlation with the pathological findings in breast core With the incidence of MBC rising molecular subtypes, IHC and clinical outcomes needle biopsy (CNB) is essential in breast disease management. Unlike mammography should be further studied. and breast sonogram, there is no data to suggest correlation between NMLE seen by Magnetic resonance imaging (MRI) and the pathology results. The purpose of this study is to test this correlation. 189 Basal-Like Breast Cancer Is Associated with Both an Enhanced Design: The radiological and pathological findings of non-palpable mammographically Cytotoxic and Regulatory T-Cell Response occult 87 consecutive NMLE lesions targeted by CNB were reviewed. MRI imaging was E Kalife, S Lu, S Mangray, R Tavares, L Noble, M Resnick, E Yakirevich. Warren Alpert performed utilizing a 1.5Tesla magnet and appropriate breast coil. Pre and post contrast Medical School of Brown University, Providence, RI. dynamic fat subtracted images were obtained. The radiologic findings were classified Background: Tumor infiltrating lymphocytes (TILs), the primary immune component according to the established lexicon by American College of Radiology. The distribution infiltrating solid tumors, are considered a manifestation of the host antitumor reaction. was classified into linear/ductal, regional, or segmental and enhancement pattern was The majority of TILs are CD3+ T-cells which include CD8+ cytotoxic T-lymphocytes classified into focus, clumped, homogenous or heterogeneous. The histologic findings (CTLs) and FoxP3+ regulatory T-cells (Tregs). These subsets have a unique role in were recorded including carcinoma, fibroadenoma, radial scar, papilloma, stromal promotion or suppression of the immune response and have proven prognostically fibrosis, inflammation, pseudoangiomatous stromal hyperplasia (PASH), sclerosing significant in some tumors. Our goal was to investigate the type, distribution and density adenosis, UDH/ADH/DCIS, apocrine metaplasia, duct ectasia, columnar cell lesion of TILs in different molecular subtypes of breast carcinoma. and fat necrosis. One or more histologic entity could be present in one biopsy and one Design: Tissue microarrays were constructed from 189 consecutive cases of high grade or more radiologic feature could be present in the same MRI image. These lesions were invasive ductal carcinoma. On the basis of IHC expression, the tumors were stratified into histologically graded from 0 to 3, based on the degree of involvement. 2 X 2 tables 54 luminal (ER+), 59 HER2 positive (HER2+) and 76 basal-like (ER-, HER2-, CK5/6 Fisher’s exact test is used to test the radiological/pathological correlation. and/or EGFR+). TILs were assessed by IHC for CD3, CD8, granzyme B (activated Results: Invasive carcinoma was seen in 4 cases (4.6%). MRI was performed for CTLs) and FoxP3. The TIL subsets were quantitated separately in intratumoral and screening in 3 patients, for staging in 61, and for high risk in 23. All carcinoma cases stromal compartments. The mean number of positively stained TILs was normalized were seen in the staging group. The MRI features with correlated pathology features 2 per 0.785 mm of tumor. are presented in table 1. Results: The number of intratumoral CD3+ TILs was higher, albeit not significantly, Conclusions: Our results suggest it is possible to test the MRI finding of NMLE in the basal-like type, as opposed to luminal and HER2 types (44.9±6.3, 37.1±13.9, correlation with a specific pathologic finding. The study is slightly limited, especially 40.1±11.8). Significantly higher numbers of intratumoral CD8+ CTLs were found in the for invasive carcinoma; due to the limited number of cases in each category. basal-like type compared to luminal and HER2 types (26.7±4.0, 11.6 ±1.9, 19.6±4.0; MRI findings correlation with histological findings. p=0.02). Granzyme B+ activated CTLs were also significantly increased in the basal-like NMLE MRI feature Histologic correlation n (%) p value* type compared to the luminal and HER2 types (18.0±3.8, 3.3±1.2, 10.8±3.7; p=0.018). Linear Papilloma 5/9 (55.6%) 0.042 Paradoxically, basal-like tumors contained significantly higher numbers of FoxP3+ Clumped Papilloma 7/9 (77.8%) 0.03 Tregs than the luminal and HER2 types (15.4±2.5, 6.1±1.35, 8.9±1.7; p=0.02). Using Linear and Clumped Papilloma 4/9 (44.4%) 0.019 Segmental PASH 4/7 (57.1%) 0.025 the median number as cutoff, we found that the basal-like type was associated with Focus PASH 1/17 (5.9%) 0.002 significantly higher recruitment of both granzyme B+ high and FoxP3+ high TILs than Focus Fat necrosis 4/4 (100%) 0.021 the luminal type (60% vs 7.4%; p=0.0001). No significant differences were found among Clumped UDH or ADH or DCIS 18/30 (60%) 0.013 the number of stromal TILs in the different tumor subtypes. Survival analysis revealed Focus and Clumped Apocrine metaplasia 4/9 (44.4%) 0.044 that tumor stage was predictive of overall survival (p<0.0001). The TIL distribution *p value is calculated based on the odd ratio was not associated with patient survival. Conclusions: The distribution of activated granzyme B+ CTLs and FoxP3+ Tregs differs 192 Intraoperative Margin Assessment Significantly Improves among the molecular subtypes of breast cancer. The basal-like type is characterized Quality of Care in Patients Undergoing Breast Conserving Surgery by a unique immune response comprised of both increased cytotoxic and regulatory SG Karak, LP Middleton, M Yi, KK Hunt, AA Sahin. University of Texas MD Anderson TILs. This may be one of the reasons that despite having increased numbers of CTLs, Cancer Center, Houston, TX. the basal-like type confers a poorer prognosis. Background: The goal of treating breast cancer patients with breast conserving therapy (BCS) is to obtain negative margins, as positive margins are associated with local/ 190 Prognostic Significance of Fibroblast Growth Factor Receptor regional recurrence. BCS aims to achieve a balance between complete resection of the 2 (FGFR-2) Overexpression in Invasive Mammary Carcinoma (BRCA) tumor with negative margins and avoiding excessive resection of non-neoplastic breast BVS Kallakury, RN Al-Rohil, MJ Presta, GM Sheehan, CE Sheehan, AB Boguniewicz, tissue to provide favorable cosmesis for the patient. The reported rate of re-excision for JS Ross. Georgetown University Hospital, Washington, DC; Albany Medical College, margin control after BCS is 48-59%. We investigated the relevance of intra-operative Albany, NY. assessment of breast specimens in this era of improved mammographic techniques. Background: FGFRs are tyrosine kinase receptors implicated in carcinogenesis and Design: In the six month period of January to June 2010, 315 patients underwent BCS tumor progression. FGFR-2 expression has been associated with distant metastasis and under needle localization 164 (52%), ultrasound guidance (39%), or other 8 (9%) poor prognosis in colorectal cancer and shortened survival in pancreatic cancer. Although techniques with intraoperative margin analysis to include gross examination, sliced there is substantial evidence linking aberrant FGFR expression in breast cancer, this is specimen radiography, with or without frozen section. the first report to demonstrate prognostic significance of increased FGFR-2 expression Results: 211 (67%) patients with invasive ductal carcinoma (IDC), 81 (26%) patients in BRCA clinical specimens. with ductal carcinoma in situ (DCIS), 17 (5%) patients with invasive lobular carcinoma Design: Formalin-fixed, paraffin-embedded tissue sections from 126 cases of BRCA (ILC) and 6 (2 %) with other pathology were excised. Median age 57 (range 25 to 89) [102 ductal carcinomas (IDC) and 24 lobular carcinomas (ILC)] were immunostained 50 patients (15.87%) received pre-operative chemotherapy. 138 (43%) patients were by a manual method using rabbit polyclonal Bek (FGFR-2) (sc 122; Santa Cruz Biotech, evaluated and found to have negative margins during intraoperative examination. 112 Santa Cruz, CA). Nuclear and cytoplasmic immunoreactivity was semiquantitatively of these patients had true negative margins after final pathologic evaluation (81.15%). scored based on staining intensity and distribution and the results were correlated with Negative predictive value 81.5%, positive predictive value 75.14%. 13 patients had morphologic and prognostic variables. positive or close margins identified in frozen section and had true positive margins after Results: Intense diffuse cytoplasmic FGFR-2 overexpression was observed in final histologic evaluation (100%). 164 patients had positive or close margins determined 35/126 (28%) tumors and correlated overall with tumor type (46% ILC vs 24% IDC, intraoperatively. 26 cases (8.25%) showed discordance with intraoperative assessment p=0.028), disease recurrence (p=0.031) and shortened survival (p=0.048); within the IDC subgroup, with disease recurrence (p=0.001) and shortened survival (p=0.018); ANNUAL MEETING ABSTRACTS 49A of negative margins and positive final margins. Half of the discordant cases (13) were Acquired cell adhesion and cell motility genes in DCIS patients with DCIS, 30.8% (8) were patients with IDC and DCIS, and the remaining Gene Status Location Function 5 (19.2%) were cases of IDC. SPAG1 Gain 8q22.2 Pro- cell motility Conclusions: Intraoperative assessment of margins assisted in identifying positive/close RHPN1 Gain 8q24.3 Coordinated assembly of focal adhesions SCRIB Gain 8q24.3 Pro-cell migration and cell polarity margins and allowed greater than 90% of the patients to obtain negative margins with MIR661 Loss 11q.25 Anti- cell motility re-excision during the initial operation. Despite advances in radiologic techniques, the NTM Loss 11q.25 GPI-anchored cell adhesion molecule role for intraoperative assessment remains necessary. Intraoperative multidisciplinary OPCML Loss 11q.25 Cell adhesion molecule margin evaluation is a value added component in the management of patients with JAM3 Loss 11q.25 Junctional adhesion molecule breast cancer and can reduce the need for additional surgery by 40%. 195 Delay to Formalin Fixation Negatively Affects on HER2 Test by 193 Pleomorphic Lobular Carcinoma In Situ of the Breast: Dual-Color Silver-Enhanced In Situ Hybridization (SISH) Clinicopathological Review of 57 Cases and a Proposed New T Khoury, Q Liu, S Liu. Roswell Park Cancer Institute, Buffalo, NY. Histopathologic Classification Background: A woman is decided to receive an effective target therapy of anti-HER2 T Khoury, R Karabakhtsian, L Yan, S Syriac, S Liu, MM Desouki. Roswell Park Cancer antibody (trastuzumab) based on HER2 test. Therefore, accurate determination of HER2 Institute, Buffalo, NY; University of Kentucky, Lexington, KY; Vanderbilt University, is essential. One of the variables that negatively affects HER2 test is delay to formalin Nashville, TN. fixation (DFF). We and others have demonstrated the effect of DFF on HER2-FISH Background: Pleomorphic lobular carcinoma in situ (PLCIS) of the breast is a and HER2-IHC. The purpose of this study is to determine the effect of progressive distinctive entity, that its definition and management is unclear. The purpose of this study DFF on HER2-SISH. is to review a large number of cases and propose a new histopathologic classification Design: Ten palpable invasive breast cancers were resected and underwent immediate according to the clinical outcome. gross evaluation. For each case, the procured tumor was divided into 8 parts and Design: Cases of PLCIS (n=57) from three academic institutions along with cases of consecutively fixed after 0, 10, 30 minutes, 1, 2, 4, and 8 hours; one section was kept classic LCIS (CLCIS) (n=39) and DCIS (n=615) over 12 year-period were reviewed. The in saline and stored overnight at 4C°. Two tissue microarray blocks were constructed. clinical, radiological and pathological findings were recorded. Immunohistochemical Then, HER2 was tested SISH (Ventana). HER2 and cep17 signal and HER2/Cep17 ratio markers (ER, PR, HER2, Ki-67, p53, GCDFP-15, mammaglobin, CK7, CK17, CK5/6, was calculated in 10 cells for each core. The percentage of cells that had complete loss EGFR, and BCL-2) were performed on 20 PLCIS cases. PLCIS cases were classified of signal was calculated. The core was considered affected by artifact when nuclear based on the histologic pattern into conventional pleomorphic (>10% of cells have bubbling and/or haze or dust was present in more than 50% of the cells. Statistical pleomorphism), necrotic (comedo type), macroacinic, and mixed subtypes. PLCIS analyses Page’s L test, Fisher’s exact test and Chi square test were used. cases are also classified based on the cell type into apocrine, non-apocrine, signet ring, Results: All ten cases were invasive ductal carcinomas. One case had HER2 histiocytic and mixed subtypes. amplification and the rest were not amplified. DFF had no statistically significant Results: For PLCIS, 47 (82.5%) cases had mixed histologic pattern and 28 (49.1%) had effect on HER2/Cep17 ratio, absolute number of HER2, or Cep17 signals. The trend mixed cell type. Seven of 57 (12.3%) PLCIS cases had tumor recurrence. All recurred of the percentage of cells that lost signal with time of DFF was statistically significant cases had mixed histologic pattern. The cell type was apocrine (n=1), non-apocrine (n=2) (p<0.001), more notably after 1 hour of DFF (figure 1). Nuclear bubbling and/or haze and mixed (n=4) in cases with local recurrence. Local recurrence was more common or dust was seen in 4 cases (p=0.009, Chi square test). These changes started to appear in younger patients (mean age 53-years vs. 60-years) and in cases with positive margin at 30-minute to 1-hour DFF (p=0.025 and 0.003, Fisher’s exact test respectively). for CLCIS (p=0.02 and p=0.01, respectively). Both PLCIS and DCIS more commonly Conclusions: DFF has no effect on the HER2-SISH results. However, the interpretation involved postmenopausal patients when compared to CLCIS (p<0.0001). While PLCIS could be limited due to the artifact caused by DFF. Therefore, the tissue should be fixed was more likely to develop local recurrence compared to grade I- and grade II-DCIS within 1 hour of tissue harvesting and should not be stored overnight. (p=0.06 and 0.04, respectively), it had similar local recurrence rate to grade III-DCIS. All patients who developed local recurrence were not treated with radiation therapy. ER, PR, HER2, GCDFP-15, mammaglobin, BCL-2, CK7, were positive in 70%, 65%, 30%, 75%, 55%, 90%, and 100%, respectively. CK17, CK5/6, EGFR tested negative in all PLCIS cases. The median and range for Ki-67 and p53 was 5% (2%-40%) and 20 (0 to 240), respectively. Conclusions: The proposed classification of PLCIS is based on the histologic pattern and predominant cell type. This classification could be useful to better recognize PLCIS. This study is the first to provide an insight to the clinical behavior of PLCIS and therefore to provide the patient with the proper management.

194 Intracystic Papillary Carcinoma: A Closer Look into Its Relationship with In Situ and Invasive Carcinoma: Array Comparative Genomic Hybridization Study of 14 Cases T Khoury, Q Hu, S Liu, J Wang. Roswell Park Cancer Institute, Buffalo, NY. Background: Classifying ICPC under invasive (IDC) or in situ carcinoma (DCIS) is still a matter of debate. The purpose of this study is to explore the genomic relationship of this tumor to its concurrent IDC or DCIS using array comparative genomic hybridization (aCGH). Design: ICPC cases (n=14) were classified into 3 categories, pure ICPC (n=7), with concurrent DCIS (n=5) and with concurrent IDC (n=2). Each component was dissected using laser capture microdissection. DNA was extracted and aCGH was performed. The test of difference in copy number changes among ICPC, DCIS and IDC was carried out using CGHMultiArray. Results: ICPC had common copy number change including 16p gain, 16q loss, and 196 Phenotypes of Breast Cancers Heterogeneous for HER2 Gene 1q gain. In addition, 7q loss, 8p23.3-8p21.1 loss, 11q14.1 to 11q22.1 loss, 11q22.1 to Amplification: Looking for Biologically Relevant Thresholds 11q25 loss, 9p13.1-9q12 loss, and 1q21.3-1q23.1 gain were found in variable number of MR Kilgore, JS Kranseler, SM Dintzis, RA Schmidt, PE Swanson, MH Rendi, HM cases. Hierarchical agglomerative clustering of samples using probe log2 ratio revealed Linden, KH Allison. University of Washington, Seattle, WA. that the three components could not be separated into different cluster. Moreover, 4 Background: In 2009, a College of American Pathologists expert panel (CAP-EP) of 5 DCIS cases and both IDC cases clustered with the concurrent ICPC. The median published supplemental HER2 testing recommendations suggesting that cases with and range for the proportions of changed genome was 8% (3.7% to 19.1%) for ICPC, between 5% and 50% individual cells amplified by fluorescence in situ hybridization 6.2% (5.9% to 11.9%) for DCIS, and 2.2 (0.09% to 10.9%) for IDC (p=0.06, Wilcox (FISH) be reported as “heterogeneous for HER2 gene amplification.” rank sum test). The mean probability of copy number changes for DCIS is similar to Design: Data from counting sheets of 1329 consecutive breast cancer cases analyzed ICPC with the following additional focal changes: 8q22.2 to 8q22.3 gain, 8q24.3 gain, by FISH for HER2 at our institution were imported to an SQL Server database for and 11q25 loss in 3 of 6 cases, and 12q24.31-12q24.33 loss in 2 of 6 cases. A group analysis and statistics. Cases were categorized as heterogeneous if between 5-50% of of genes involved in cell adhesion and motility was found in DCIS, listed in table 1. cells counted had HER2:CEP17 ratios > 2.2. For cases meeting criteria for heterogeneity Conclusions: 1. ICPC shares all major copy number variation with concurrent DCIS and the following parameters were analyzed when available: HER2 IHC, estrogen receptor IDC, which suggest that all tumors arise from one stem cell. 2. The overall molecular (ER) and progesterone receptor (PR), Nottingham grade, age, and histologic type. The change in ICPC is closer to DCIS than to IDC, which may explain its indolent clinical percentage of HER2 amplified cells per case was divided into 5% incremental bins behavior. 3. DCIS acquires motility/adhesion molecular changes. So, we suggest that beginning with 5-9% and ending with 45-49%. Each bin was analyzed with respect these changes permits the neoplastic cells to migrate from ICPC and colonize the to the aforementioned parameters, as well as the cumulative averages across bins. surrounding ducts, but does not give it the required genetic changes for stromal invasion. Results: Out of 1329 cases, 283 cases met the CAP-EP criteria for HER2 heterogeneity by FISH. Of these, 84.1% were not amplified by mean HER2:CEP17 ratios, 13.4% were equivocal, and 2.5% were amplified. Most cancers that met the CAP-EP definition of HER2 heterogeneous were in women older than 50 (73.1% > 50, 20.5% 40-50, 6.4% <40) and 93.7% were invasive ductal carcinoma (6.3% lobular). Of the 250 cases with 50A ANNUAL MEETING ABSTRACTS ER/PR data available, 79.2% were ER positive and 43.2% were PR positive. Of 240 199 Correlation of Ki-67 between Biopsy and Resection Specimens: cases with Nottingham grade available, 44.6% were grade 3, 44.2% grade 2, and only Does “Hot-Spot” Counting Matter? 11.2% grade 1. Interestingly, all cases with between 35-49% HER2 amplified cells were Z Kos, DH Gravel, RJ Susan. Ottawa Hospital and University of Ottawa, Ottawa, equivocal or amplified by mean ratios. ER/PR status remains relatively consistent over ON, Canada. the range of percent HER2 amplified cells but all cases with 35-49% amplified cells Background: The neoadjuvant setting is being increasingly explored as a means for the were grade 2-3 carcinomas. 18.8% of cases in the 35-49% amplified range occurred in evaluation of therapeutic strategies in women with breast cancer. This setting permits women younger than 40 versus 5.6% of cases with < 35% of amplified cells. study of the biological characteristics of responsive versus resistant tumours in vivo, Conclusions: When looking at minority HER2 positive cell populations by FISH, using surrogate markers of response. The Ki-67 proliferation index is emerging as an more aggressive features are associated with cancers with 35-49% amplified cells important predictive and prognostic marker, and changes in Ki-67 labelling in response versus 5-34%. This higher threshold for the definition of HER2 heterogeneity should to short-term challenge with anti-estrogens and chemotherapeutic agents have been be examined for potential response to HER2 targeted therapies to determine if it is reported to be predictive of the long-term clinical effectiveness of the drug. However, clinically relevant. studies specifically addressing the correlation between Ki-67 scores on biopsies and resection specimens are lacking. In the past few years, there has been a shift towards 197 Prediction of a Pathological Complete Response Defined as hot-spot counting, suggesting a possibility of higher Ki-67 scores in resection specimens ypT0/is ypNx in the Setting of a Preoperative Systemic Therapy (EC-T+- versus biopsies as a direct result of selective counting and biopsy sampling discrepancies. Trastuzumab) of Invasive Breast Carcinoma by Evaluating bcl2 and TP53 Design: Biopsy and resection specimens from 20 patients with estrogen receptor-positive on the Pretherapeutic Core Needle Biopsy Specimen breast carcinomas were selected. Ki-67 scores were determined by image-assisted hot- D Klein, KC Siegmann-Luz, M Hahn, TN Fehm, UF Vogel. University Hospital, spot counting of 1000 cells in the resection specimens and biopsies when possible; in Eberhard-Karls-University, Tuebingen, Baden-Wuerttemberg, Germany. smaller biopsy samples, all neoplastic cells were counted. The samples were analysed Background: Preoperative systemic therapy may be the best tool to acquire information for correlation using a two-tailed paired t-test. on the response of a malignant tumor to chemotherapy. To avoid unnecessary toxicity Results: The mean Ki-67 index for the biopsies was 28.3 + 24.3%, with a range of 0.4 it would be helpful to predict the pathological complete response (pCR) for a certain to 99.1%. The mean labelling index for resection specimens was 32.2 + 26.0 %, with chemotherapy regimen on the pretherapeutic core needle biopsy specimen (pCNBS). a range of 4.8 to 91%. The resection specimens had a mean Ki-67 score that was 3.9% Design: 126 patients with invasive breast cancer were treated preoperatively higher than on biopsy, but this was not statistically significant (p = 0.2). Difference in (GeparQuattro study: Epirubicin, Cyclophosphamide, Docetaxel, +-Trastuzumab). individual counts, however, ranged from 13% lower to 43 % higher on the resection Besides ER, PGR and HER2 TP53 and bcl2 were determined immunohistochemically specimen as compared to the biopsy, including 3 cases in which the Ki-67 would on the pCNBSs. Additional molecular analysis of TP53 was done in a few selected cases. give a score that would be different with respect to the values suggested as surrogate Results: Using the TP53 and bcl-2 expression three groups of breast carcinomas could cut-offs (10 to 14 % range) for designating a tumour as lower risk luminal A versus be designed to predict the pCR defined as ypT0/is ypNx: higher risk Luminal B. 1. bcl2(% positive tumor cells) > TP53 (%): Prediction: No pCR. Conclusions: These data show that despite the practice of hot-spot counting there is n = 71; no pCR: 69/71 (97%). Especially invasive lobular carcinomas belong to this no significant difference between the Ki-67 labelling index determined on biopsies group. and subsequent resection specimens. This study also predicts that for interventions in Subgroup: HER2+: non-pCR: 7/8 (87%). The pCR case: unusual strong bcl2. trials dealing with groups, changes in Ki-67 scores may be predictive in most cases; 2. bcl2 < TP53 with TP53 not 0 or not 90-100%: Prediction: pCR. however, interpretation of results, on a case by case basis, does require some caution. n = 17; pCR: 15/17 (88%); pCR: 15/16 (94%) if an adenosquamous carcinoma is excluded. 200 Presence of Lobular Features Predicts Enrichment for PI3K Subgroup: HER2+: pCR: 10/10 (100%). Pathway Mutations among No Special Type Breast Cancers 3. bcl2 <= TP53 with TP53 = 0 or TP53 = 90-100%: Prediction: Not possible without MG Kuba, GM Jennifer, JM Balko, IA Mayer, V Abramson, I Meszoely, W Pao, Z an additional molecular analysis of TP53. Su, D Lai, DR LaFrance, CL Vnencak-Jones, CL Arteaga, ME Sanders. Vanderbilt n = 38; pCR: 14/38 (36%). Preliminary sequence analysis of a few cases of this group University, Nashville, TN. revealed TP53 mutations in the exons 5-8 in non-pCR cases, whereas a wild type TP53 Background: The PI3K pathway is the most frequently mutated pathway in breast could be found in the pCR cases. cancer (BC). Recent data from The Breast Cancer Genome Atlas Network (doi:1-.1038/ Subgroup: HER2+ TP53>=90%: n = 6; pCR: 5/6 (83%). The non-pCR case: no nature11412) indicates these mutations occur across all BC subtypes (36%), with the trastuzumab; point mutation exon 5. highest frequency (∼[/underline]45%) in luminal A/ER+ BC. Other studies found Subgroup: HER2+ TP53 = 0: n = 4; non-pCR: 3/4 (75%). One of these cases sequenced: increased mutation frequency among special type (ST) breast cancers and metaplastic point mutation exon 7. carcinomas. Histologic features among no special type (NST) BC correlating with Conclusions: The immunohistological determination of TP53 and bcl2 on the pCNBSs mutation status have not been described. allows the definition of three groups of breast carcinomas to predict the therapy response. Design: We characterized the histopathologic features of 169 primary or recurrent BC According to literature the evaluation of the expression signature for the TP53 status previously screened for 18 hotspot mutations in the PIK3CA (14), PTEN(3) and AKT(1) seems to be superior to the sole sequence analysis of TP53. The prediction of ypN0 genes utilizing a SNaPshot multiplex platform developed at our institution. Tumors cannot be achieved by this classification. were typed and graded according to WHO and AJCC criteria. ST carcinomas are low grade with >90% purity of pattern with invasive lobular carcinoma (ILC) variant tumors 198 Proliferation Markers PHH3, Ki67 and Mitotic Count All Show separately recognized based on intermediate combined histologic grade. Relationship of Significant Associations with Features of Aggressive Breast Carcinomas mutation status to the presence of ST features (<70% of tumor), growth pattern (pushing and Reduced Survival vs. infiltrative), nuclear pleomorphism, mitotic index, density of lymphocytic response G Knutsvik, IM Stefansson, K Collett, LA Akslen. University of Bergen, Bergen, Norway; and stromal type (sclerotic vs. desmoplastic) were examined. Slides were reviewed by Haukeland University Hospital, Bergen, Norway. three breast pathologists for concordance. Background: Determination of proliferation in breast carcinomas is highly prognostic Results: There were 15 ST/ILC variant tumors (1 tubular, 1 mucinous, 2 invasive and may also predict response to chemotherapy. However, there is no consensus on cribriform, 1 ILC, 4 ILC variants), 1 metaplastic and 154 NST carcinomas. 27% of issues such as counting area or cut-off values. Our aim was to evaluate Ki67 counts in tumors carried mutations: 88% PIK3CA, 8% AKT and 4% PTEN. Five (36%) ST/ILC different tumor areas as compared with the novel mitosis marker phosphohistone H3 variant (all ILC) and 42 (27%) NST carcinomas carried PI3K mutations. Among 137 (PHH3) and standard mitotic count. Furthermore, we aimed to assess the prognostic NST carcinomas, a higher mutation rate was observed in BC with lobular features (13/28 value of these proliferation markers. or 46%) versus BC without lobular features (25/109 or 23%; p=0.01). The frequency Design: We examined a retrospective, population-based series (n = 556) as part of of mutations was inversely correlated with histologic grade (52% low vs. 9% high; the Norwegian Breast Cancer Screening Program, including all women (50-69 years) p<0.0001) and proliferative rate (low 41% vs. high 12%; p=0.002). 94% of mutations diagnosed with breast cancer in one county of Norway between 1996-2003. The were present in ER+ tumors and less frequent in HER2+ (10%) and triple negative percentage of Ki67 positive nuclei among 500 tumor cells was evaluated for both (0%) BC in this cohort. There were no other recurrent histologic features predictive hot-spots (HS; highest proliferation) and cold-spots (CS; lowest proliferation) on full of a PI3K pathway mutation. sections. For standard mitotic count and PHH3, mitotic figures were counted in 10 Conclusions: Mutations in the PI3K pathway were more frequent in ST carcinomas. consecutive high power fields (HPF) in the most active area, and the number of mitoses NST tumors with lobular features were enriched for PIK3CA mutations. Following ER per mm2 was calculated. positivity, lobular histology was most predictive of a mutation. These findings suggest Results: The 3 proliferation markers were highly correlated. The upper quartiles PIK3CA mutations are a pathogenic driver in the low grade breast neoplasia pathway for Ki67-HS, Ki67-CS, PHH3 and mitotic count consistently showed the strongest consistent with previous studies portending an improved prognosis in this setting. associations with known unfavorable tumor features, including larger tumor diameter, high histologic grade, and negative estrogen receptor (ER) and progesterone receptor 201 C/EBPß Expression Is Predictor of Survival in Breast Cancer (PR) status. High proliferation was significantly associated with interval cancers as Patients compared with screen-detected cases. In a subset of cases (n = 190), follow-up data E Kurzejamska, K Jistrom, P Religa. Karolinska Institutet, Stockholm, Sweden; Lund were available (information is currently collected on all cases). Univariate survival University, Lund, Sweden. analysis of this subgroup showed comparable prognostic strength of Ki67-HS, PHH3 Background: C/EBPβ is a transcription factor playing a critical role in mammary count and mitotic count. In multivariate analysis, mitotic count was the strongest gland development and breast cancer progression. Recent studies show that loss of C/ prognostic factor among these. EBPβ contributes to progression of lymphoma In this study, we analyzed the prognostic Conclusions: By upper quartile, Ki67-HS, PHH3 and mitotic count all showed strong significance of C/EBPβ expression in metastatic breast cancer. associations with various unfavorable tumor features. In multivariate survival analysis of a subset of patients, mitotic count was the strongest proliferation marker. ANNUAL MEETING ABSTRACTS 51A Design: Tissue microarray (TMA) study was performed on 137 metastatic breast cancer clinicopathological characteristics, clinicians will have to face an increasing amount of patients. Using immunohistochemistry, the intensity of C/EBPβ staining was evaluated data derived from tumour molecular profiling. The aims of this study were to develop and compared with relevant clinicopathological characteristics. Cox proportional a new gene selection method based on fuzzy logic and classification algorithm, and hazards models and Kaplan-Meier analysis were used to assess the effect of C/EBPβ to validate the gene signatures obtained with this original method on breast cancer expression on overall survival (OS) in univariate and multivariate analysis, adjusted patient cohorts. for established prognostic factors. Design: Based on fuzzy logic and classification algorithm, our selection method Results: All parameters were assessed in univariate models using Cox proportional measures the contribution of each gene for each of two pre-defined classes in order hazards models and factors significant at p<0.05 were included in the final model. In to find the best discrimination. This algorithm extracts and ranks the most pertinent multivariate analysis, independent predictors of overall survival (OS) were C/EBPβ markers, since it is based on feature weighting according to optimal error rate, sensitivity expression (hazard ratio [HR] 0.2453; 95% confidence interval [CI] 0.08 to 0.72; and specificity. We applied the fuzzy logic selection on seven breast cancer microarray P=0.011), age (HR 1.1; 95% CI 1.06 to 1.46; P<0.0001) and chemotherapy (HR 6.3; databases to obtain new gene signatures. To validate these gene signatures, we designed 95% CI 1.82 to 21.82; P=0.004). Moreover, Kaplan-Meier survival analysis showed probes for the selected genes on Nimblegen custom microarrays and tested them on that C/EBPb expression is significantly associated with overall survival (P=0.004), but a series of 151 consecutive invasive breast carcinomas displaying clinicopathological also related to relapse-free survival (RFS). features similar to those observed in routine practice. Conclusions: Presented results indicate that C/EBPβ expression in breast cancer is an Results: Using fuzzy logic selection, we identified new gene lists that were included in independent predictor of OS in metastatic breast cancer patients. our Nimblegen in-house microarray (1100 genes,17000 probes). Analysis in the training public sets showed good performance of the 12 newly generated gene signatures for 202 Comparison of Automated and Manual Assessment of Ki-67 grade (sensitivity ranging from 80% to 100%, specificity 80% to 97%, error rate 3 to Proliferative Activity in Invasive Breast Carcinomas (IBCs) 12%). Similar results were obtained in our validation set of 151 breast cancer samples, with an error rate of less than 10% in the majority of the gene lists tested. Moreover, Y Kwon, J Ro, ES Lee, HS Kang, SW Kim, KJ Kim, GK Lee. National Cancer Center, when applying in particular 4 distinct lists among the 68 histological grade III and 20 Goyang, Republic of Korea. grade I tumours, 82 cases were correctly assigned in the two-class molecular grade. Background: Immunohistochemical detection of Ki-67 antigen has been widely used Interestingly, histological grade II tumours (n=63) were split in these two molecular to assess the growth fraction in human malignancies, including breast cancer. The Ki- grade categories with exception of 5 cases that showed an equivocal profile. 67 proliferative activity has been reported to be associated with clinical response to Conclusions: As a proof of concept, we confirmed the use of fuzzy logic selection chemotherapy, relapse, or survival in IBCs. The aim of this study is to determine whether as a new tool to identify gene signatures with good reliability and increased power of digital image analysis can be adopted in routine pathology practice for evaluating Ki-67 classification. This method based on artificial intelligence algorithms was successfully labeling index (LI) in IBCs. applied to molecular grade classification of breast cancers, but could be further Design: We included 210 IBCs histologically diagnosed and immunostained for Ki- developed for identification of any relevant prognostic or predictive multi-criteria 67 between Jun 2012 and July 2012. We assessed Ki-67 LI by manual and automated signature in breast cancer. counts, respectively. We compared Ki-67 values between manual and automated quantitation with St. Gallen criteria (low, intermediate, high). We intended to validate the accuracy of automated quantitation of Ki-67 LI using Aperio ImageScope (Aperio 205 Pathological Comparison of Breast Cancer Metastases with Technologies, Vista, CA, USA). Primary Tumours in a Large Prospective Trial Testing Whole Genome Results: This study demonstrated relatively high concordance rate (86.7%) with Approach substantial interrater agreement (κ=0.7975, p<0.001) between manual and automated M Lacroix-Triki, I Treilleux, M-C Mathieu, D Loussouarn, G Mac Grogan, A Vincent- Ki-67 values according to St. Gallen criteria. The difference of mean values between Salomon, F Penault-Llorca, T Filleron, T Dubois, T Bachelot, F Commo, M Campone, manual and automated Ki-67 LI was statistically significant (1.82±4.91%, p<0.001, M Jimenez, F Andre. Institut Claudius Regaud, Toulouse, France; Centre L Bérard, 95% CI=1.16-2.49%). However, manual Ki-67 LI was significantly correlated with Lyon, France; Institut Gustave Roussy, Villejuif, France; CHU, Nantes, France; automated Ki-67 LI with high correlation coefficient γ( =0.9791, p<0.001). Institut Bergonié, Bordeaux, France; Institut Curie, Paris, France; Centre Jean Perrin, Conclusions: Ki-67 values according to St. Gallen criteria have relatively high Clermont-Ferrand, France; Centre R Gauducheau, Saint Herblain, France; R&D concordance between manual and automated quantitation. Therefore, only if pathologists UNICANCER, Paris, France. confirm the tumor regions to be analyzed, digital image analysis can be adopted in Background: Owing to the report of biomarker switch between primary and recurrent routine practice for evaluating Ki-67 LI in IBCs. breast cancer, several groups recommend to re-sample metastatic sites. In the prospective SAFIR01 trial, systematic biopsies of metastatic sites were performed for genomic 203 Concordance of HER2 Gene Status between Dual-Color Silver analysis to direct patients to specific targeted agents. We report the practical feasibility of In Situ Hybridization (DSISH) and Fluorescence In Situ Hybridization (FISH) molecular characterization on metastasic biopsy specimens and pathological comparison in Invasive Breast Carcinomas (IBCs) after Preoperative Chemotherapy to matched primary tumours. Design: From 2011 to 2012, 423 patients with metastatic breast cancer were included. Y Kwon, HS Kang, ES Lee, IH Park, GK Lee, SY Park, J Ro. National Cancer Center, Biopsies of metastatic sites were performed: at least two samples were snap-frozen and Goyang, Republic of Korea. the remaining fixed in formalin (FFPE). DNA was extracted from the frozen sample, Background: HER2 FISH is a quantitative assay for selecting breast cancer patients for provided that it contained >50% cancer cells, and subjected to CGH array and sequencing anti-HER2 therapy. However, disadvantages of FISH assay include the requirement for a of PIK3CA and AKT. When sufficient FFPE material was available, hormone receptor specialized fluorescence microscope, the difficulty of preserving FISH signal for a long (HR) and HER2 status was re-assessed according to local institution protocols. Primary time, and the difficulty of evaluating the gene status in the context of tissue morphology. tumour characteristics were extracted from the initial pathology report and tumour Recently, a brightfield DSISH has been developed to overcome these disadvantages of collected for re-testing when available. FISH. The aim of this study is to determine the efficacy of DSISH for the evaluation Results: Metastasis biopsy was successful in 97% and involved the liver (40%), lymph of HER2 gene status in IBCs after preoperative chemotherapy. nodes (17.5%), skin (16%), lung (6.5%) and other sites (20%). Tumour cell content Design: Among 209 IBCs histologically confirmed and surgically resected after was sufficient to proceed to DNA extraction in 73% of the cases, allowing CGH preoperative chemotherapy between Jan 2002 and Dec 2005, we included 147 breast array and sequencing for at least 68% of the patients. Mutation of PIK3CA and AKT resection specimens with available formalin fixed paraffin embedded (FFPE) IBC were observed in 24% and 4.5% of the samples respectively. Most frequent genomic tissue. HER2 DSISH and FISH were performed in tissue microarray (TMA) sections alterations consisted of amplification of FGF4 (16%), HER2 (13%) and FGFR1 constructed from representative tumor blocks of IBCs. We compared HER2 DSISH to (11%), with a constellation of rarer (<5%) actionable genomic changes in about 20% FISH and also correlated it with HER2 immunohistochemistry (IHC). of the cases. Among the 204 cases available for comparison analysis with regards to Results: The concordance rate of HER2 status between DSISH and FISH was high HR status, 19% were discordant, with a loss of HR expression in 62% of these cases. (93.6%) with perfect interrater agreement (κ=0.8562, p<0.001) in 140 informative As for HER2, 8% of discordant cases were observed, mainly represented by a loss of cases. Seven (77.8%) of 9 discordant cases were HER2 IHC 2+ cases. All but one of 9 HER2 expression in 60% of these cases. exhibited equivocal DSISH with negative FISH results. The difference of mean value Conclusions: The discrepancy rates between primary and metastases for HR and between HER2 DSISH and FISH ratios was statistically significant (0.42±1.41, 95% HER2 in the SAFIR01 study are similar to those described in the literature. In this CI=0.19-0.66, p<0.001) but DSISH ratios were significantly correlated with FISH ratios large prospective trial, we have shown that whole genome DNA approach is feasible with high correlation coefficient γ( =0.8613, p<0.001). The result of HER2 DSISH was in the context of daily practice to identify actionable alterations and direct patients to significantly related to that of HER2 IHC (p<0.001), reflecting the association between specific targeted agents. HER2 gene amplification and protein overexpression. Conclusions: The overall concordance between HER2 DSISH and FISH is excellent with HER2/CEN17 ratio using ASCO/CAP criteria. DSISH can be a feasible alternative 206 Laminin ß1 and Laminin ß2 Expression in Invasive Breast to FISH for the evaluation of HER2 gene status in IBCs after preoperative chemotherapy. Carcinoma J-P Lai, X Fan, J Ljubimova, A Ljubimov, S Bose. Cedars-Sinai Medical Center, Los 204 Fuzzy Logic Selection as a New Reliable Tool To Identify Gene Angeles, CA. Signatures in Breast Cancer – The INNODIAG Study Background: Laminins are integral components of vascular and parenchymal basement membranes and play a primary role in cell adhesion, migration and differentiation. M Lacroix-Triki, T Kempowsky-Hamon, C Valle, L Hedjazi, S Lamarre, L Trouilh, L Previous studies on fresh frozen tumor sections have demonstrated that laminin isoforms Puydenus, L Mhamdi, F Dalenc, T Filleron, G Favre, M-V Le Lann, V Le Berre-Anton. are differentially expressed in blood vessels in gliomas and breast tumors. Preclinical Institut Claudius Regaud, Toulouse, France; CNRS, LAAS, Toulouse, France; INSA/ studies in mouse models bearing brain or breast tumors showed significant tumor growth CNRS/INRA - UMR5504/792, Toulouse, France. reduction when β1 laminin chain was blocked. These data show potential in using Background: For the last decade, breast cancer patient management has laminins for targeted delivery of therapeutic doses of anticancer drugs. This preliminary drastically evolved toward personalized medicine. In addition to the routinely used study was designed to examine the expression of β1 and β2 laminin chains in vascular 52A ANNUAL MEETING ABSTRACTS basement membranes of invasive breast cancer on formalin fixed paraffin embedded Results: Within our study, 11% (8 of 68) of the women with CS went on to develop (FFPE) sections in order to validate this test for standard clinical pathology practice. DCIS or invasive mammary carcinoma. Tumor was contralateral in 2 cases. The interval Design: Immunohistochemical staining for laminin β1 and laminin β2 chains was between benign biopsy and subsequent malignancy ranged from 6 to 31 years, with performed on 20 cases of FFPE breast cancers using monoclonal antibodies to laminin β1 a mean of 19 years. Atypical ductal hyperplasia or atypical lobular hyperplasia was (clone DG10) and laminin β2 (clone C4). Laminin β1 and laminin β2 staining intensities identified on the initial biopsy in 5 of the 68 cases. Yet, none of these women went on (0 to 3+) were evaluated in the intraneoplastic neovasculature and compared to that to develop mammary carcinoma. In the end, women with CS did not demonstrate a in the normal blood vessels in the surrounding parenchyma. Changes in expression statistically significant elevated risk of breast cancer (SIR 1.64%, 95% CI 0.71 – 3.23). were correlated to tumor characteristics including tumor size, grade, stage, ER, PR, Conclusions: Our findings show the presence of CS within a benign breast tissue biopsy HER2, Ki67 and p53 expression and HER2 amplification status. Statistical analysis does not with any statistical significance increase a woman’s risk for later development was performed using GraphPad Prism6. of mammary carcinoma. However, given the consistently observed presence of columnar Results: Laminin β1 expression was variably increased in intraneoplastic neo-vessels altered lobules, micropapillomas, and surrounding dense stromal fibrosis within the in all the cases. 9 cases showed marked overexpression (2 to 3+ increase). In contrast microenvironment of CS; it appears as though CS is a manifestation of an evolving laminin β2 expression was decreased in all the cases with 13 cases demonstrating a columnar cell lesion. marked reduction (2 to 3+ decrease). A significant reverse linear correlation was noted β β between laminin 1 and laminin 2 expression in 13 of the cases (p=0.0008). No 209 Correlation of Sentinel Lymph Node Metastases with significant correlations were observed between the expression of laminins and tumor Breast Tumor Grade, Harbor-UCLA Medical Center Experience: A characteristics with the exception of PR. Breast cancers with lower levels of PR showed Pathologic Cases Series with Evaluation of Tumor Size and Her2 and ER β higher levels of laminin 1 in the intra-neoplastic new vessels (p=0.0152). High grade Overexpression tumors had a higher rate (86%, 6/7) of reversal of vascular laminins from β2 to β1, J Lee, S French, R Venegas. Harbor-UCLA Medical Center, Torrance, CA. although statistical significance was not reached (p=0.057). Background: Metastases to sentinel lymph nodes is an important hallmark of breast Conclusions: Our results demonstrate a frequent switch in the expression of vascular carcinoma prognosis and staging. Although tumor grading is not currently factored into basement membrane laminins from β2 to β1 in the neo-capillaries of invasive breast the staging of breast cancer, in principle, higher grade breast carcinomas should exhibit cancers. Although no prognostic significance was observed, the differential expression a higher rate of metastases to sentinel lymph nodes. We report here the incidences of may be used for developing auxiliary diagnostic tests and targeted therapies. sentinel lymph node metastases in breast carcinomas according to their respective grades (I, II, and III) over a 2 year period from 2010 to 2012 and correlate such information 207 Carcinogenesis Pathways of Pleomorphic Lobular Carcinoma to estrogen receptor (ER) immunostain positivity and human epidermal growth factor I Lamzabi, R Ghai, V Reddy, P Bitterman, R Singh, P Gattuso. Rush University Medical receptor 2 (Her2) overexpression. Center, Chicago, IL. Design: We reviewed 135 consecutive cases of sentinel lymph node biopsies from our Background: Pleomorphic lobular carcinoma (PLC) is an aggressive variant of lobular institutional archives between 2010 and 2012. The presence of sentinel lymph node carcinoma. Several studies have compared the clinical and morphologic features of metastases, size of tumor, and Her2 and ER expression were evaluated and subsequently classical lobular carcinoma to that of PLC. However, sparse literature is available correlated with the tumor’s histologic grade. regarding the molecular characteristics of PLC. The aim of this study was to investigate Results: 135 consecutive sentinel lymph node biopsies were evaluated. 22, 52, and 23 the molecular pathways behind the biologic behavior of PLC. cases were scored by our institute’s breast pathologists as grade I, II, and III, respectively. Design: Cases negative for E-Cadherin and with morphologic features of PLC were 7 (31.8%), 21 (41%), and 2 (8.7%) cases of grade I, II, and III respectively, showed identified from our surgicsal pathology files for the period 2004 and 2012. Pertinent tumor metastases. The average tumor sizes were 1.59 +/- 1.01 cm, 1.86+/-1.12 cm, and clinicopathological features were reviewed. A panel of immunostains including estrogen 2.24+/- 1.19 cm for grade I, II, and III breast carcinomas respectively. 4.5%, 13.5%, receptors (ER), progesterone receptors (PR), P16, CD117, CK5, vimentin, EGFR, p53, and 45% of grade I, II, and III breast cancers were positive for Her2 overexpression. cyclin D1, and BCL-2 and fluorescence in situ hybridization (FISH) for HER-2 NEU Of the breast carcinomas that showed metastases to sentinel lymph nodes, no cases were performed in all cases. from the grade I tumors and 1 case each of the grade II and III tumors exhibited Her2 Results: A total of 35 cases were identified. The average age was 64 years. Twenty five overexpression. Finally, 9.1%, 25%, and 35% of grade I, II, and III breast cancers patients were Caucasian, 9 African-American and 1 Asian. T1, T2, T3, and T4 stages showed less than 80% IHC staining for ER. were seen 11, 16, 5, and 3 cases respectively. Twenty one patients (60%) had lymph Differences in metastatic potential, tumor size, Her2 and ER expression between different breast node metastases and 5 (14%) had distant metastases. HER-2 NEU was amplified by carcinoma grades Breast Cancer Percentage with Percentage with Her2 Percentage with <80% FISH in 4 cases (11%). Positivity for ER was seen in 31 cases (88%), PR in 22 cases Tumor size (cm) Grade SN* metastases over expression ER staining (62%), cyclin D1 in 30 cases (85%), P16 in 24 cases (68%), CD117 in 7 cases (20%), I 31.8 1.59 +/- 1.01 4.5 9.1 vimentin and EGFR in 2 cases (5%) and for BCL-2 in 33 cases (94%) including 7 cases II 41 1.86+/-1.12 13.5 25 with partial loss of expression. P53 was expressed in 18 cases with strong 3+ positivity III 5 2.24+/- 1.19 45 35 in only 7 cases (20%). Four triple negative cases were identified, of which only 2 were *SN-Sentinel Lymph Node positive for EGFR and vimentin, strongly positive for CK5, P53 and CD117, lost Conclusions: Unexpectedly, grade III breast carcinomas have a lower rate of sentinel BCL-2 expression indicating a basal-like type phenotype. Larger tumor size correlated lymph node metastases than observed in grade II breast carcinomas. Higher grade breast with loss of BCL-2 (p=0.003), and loss of PR (p=0.01). Lymph node metastasis was carcinomas were on average larger in size, more likely to exhibit Her2 overexpression, associated with lack of expression of CD117 (p=0.08), and larger tumor size (p=0.02). and less likely to exhibit ER expression. Finally, Her2 overexpression does not seem to Distant metastases were associated with higher P53 expression (p=0.03), high T stage correlate with an increased incidence of sentinel lymph node metastases. (p=0.03), loss of PR (p=0.03) and non Caucasian ethnicity (p=0.02). Tumors from non Caucasian patients also showed higher P53 expression (p=0.02). 210 BRST2, ER, e-Cadherin, CDX2, and NKX3.1 Utility in Conclusions: The carcinogenesis of PLC seems to involve aberrations in different Distinguishing Diffuse Gastric from Lobular Carcinoma pathways. BCL-2 appears to downregulate tumor growth. P53 mutation, CD117 GH Lewis, R West, RK Pai, TA Longacre, KC Jensen. Stanford University, Stanford, expression as well as loss of PR seem to promote metastases. Non Caucasian patients CA; University of Pittsburgh, Pittsburgh, PA; VA Palo Alto HCS, Palo Alto, CA. seem to have higher risk of distant metastasis. Basal cell type has a distinctive Background: Distinguishing diffuse gastric (DGC) from lobular breast carcinoma immunoprofile which suggests a distinct carcinogenesis pathway. We also report a lower in metastases is morphologically challenging. Few reports on the utility of frequency of HER-2 NEU amplification by FISH in PLC than most published literature. immunohistochemistry (IHC) are published. We studied a series of gastric (GC) and breast carcinomas (BC) by IHC, including NKX3.1, a prostate marker reportedly 208 The Definitive Microscopic Characterization and Long Term expressed in ductal (9%) and lobular (27%) carcinomas. Follow Up of Patients with Collagenous Spherulosis. A Benign Breast Design: BRST2, ER, e-cadherin (ECD), CDX2, and NKX3.1 IHC performed on Disease Cohort Study tissue microarrays containing 103 GC and 260 BC were divided based on infiltration MD Laudenschlager, DW Visscher, R Vierkant, M Frost. Mayo Clinic, Rochester, MN. pattern: diffuse (single file) vs clustered (tubules/clusters). Mixed cases were considered Background: Relegated into the realm of histologic distraction, the delicate eosinophilic diffuse. Any IHC labeling was considered positive and correlated by subtype: GC vs spider-like strands and radial deposits of collagenous spherulosis (CS) have not BC and diffuse GC (DGC) vs diffuse BC (DBC). P-values were calculated by 2-tailed been linked to risk of developing a breast malignancy. Yet, given their propensity to Fisher’s exact test. proliferate and calcify, CS foci are detected by mammography, subsequently biopsied Results: IHC are summarized in table 1; sensitivity, specificity, positive (PPV) and and observed microscopically. A prior study reported an associated premalignant or negative predictive values (NPV) of DGC vs DBC are in table 2. There were 37 DGC malignant process in over half of their cases of CS (56% - 33 of 59). This prompted and 99 DBC. ER positivity was seen in tumor cells of 3 GC all of which were DGC speculation that perhaps CS should be included in the category of proliferative breast (8.6%); however, ER positivity was present in stromal cells in 44.6% (45/101) of disease with atypia. Such a hypothesis had not previously been tested with a benign GC with negative tumor. Other than GC, the 3 patients with ER positive tumors (2 breast disease cohort. Furthermore, we sought to examine the microenvironment of CS females and 1 male) had no cancer history. Two cases of BC infiltrating in tubules/ in order to identify common attributes and definitively characterize this entity. This is clusters (BCTC) negative for NKX3.1, BRST2, and ER were positive for CDX2. The the largest series of CS cases to be reviewed and the only one with long term follow-up. 2 patients had no cancer history other than BC. NKX3.1 was present in comparable Design: We identified 68 CS cases from a benign breast disease cohort of 9,087 proportion of DBC (9.8%) vs BCTC (9.6%) (p=1.0) and in ECD positive (9.9%) vs women. This population is comprised of an institutional review board approved study ECD negative (5.9%) cases (p=0.7). of women (ages 18 – 85), who underwent benign breast biopsies during 1967 to 1991. Our intent was to ascertain how many women with CS went on to develop mammary carcinoma. Risk of subsequent breast cancer by CS and other covariates was assessed via standardized incidence ratios (SIR), using the Iowa SEER registry as the reference population. ANNUAL MEETING ABSTRACTS 53A

Table 1 IHC GC% BC% p DGC% DBC% p BRST2 0 (0/103) 74 (168/227) 0.0001 0 (0/37) 81.5 (66/81) 0.0001 ER 2.3 (3/101) 75.9 (173/228) 0.0001 8.6 (3/37) 89.9 (89/99) 0.0001 ECD 96.1 (99/103) 83.4 (176/211) 0.0009 91.9 (34/37) 67.5 (52/77) 0.0049 CDX2 87.2 (89/102) 0.9 (2/221) 0.0001 91.9 (34/37) 0 (0/79) 0.0001 NKX3.1 0 (0/103) 9.2 (21/228) 0.0004 0 (0/37) 9.8 (8/82) 0.0564

Table 2 DGC vs DBC BRST2 ER ECD CDX2 NKX3.1 Sensitivity % 81.5 89.9 91.9 87.2 9.8 Specificity % 100 91.4 32.5 99.1 100 PPV % 100 96.7 39.5 97.8 100 NPV % 71.2 76.2 89.3 94.4 33.3 Conclusions: BRST2, ER, and NKX3.1 are highly specific for BC, while CDX2 is highly specific and sensitive for GC. A panel including these markers is most useful in distinguishing metastatic GC from BC. However, ER should be interpreted carefully because ER can be positive in stromal cells in GC when tumor cells are negative. NKX3.1 may be useful, but is seen in less than 10% of BC. We found no correlation of NKX3.1 with either DBC or ECD negative BC.

211 Analysis of Gene Mutations in Subtypes of Metaplastic Carcinoma of the Breast X Li, L Huo, SL Moulder, MZ Gilcrease. University of Texas MD Anderson Cancer Center, Houston, TX. Background: Metaplastic carcinoma is a particularly aggressive subtype of triple- negative breast cancer that not only lacks targeted therapies but is generally resistant to In all tumor groups, CM shows a statistically lower FM positivity rate than LM does standard chemotherapeutic regimens. The phosphoinositide 3-kinase (PI3K) signaling (*p < 0.05), with 58%, 24%, and 50% reduction comparing to LM, in InvCa, CIS, and & pathway is a potential therapeutic target in metaplastic carcinoma, as activating Inv CIS, respectively. Interestingly, in both LM and CM cases, CIS tumor groups mutations in the PI3K catalytic subunit (PIK3CA) have been reported in up to 50% of show highest positivity rate, whereas InvCa tumors reveal the lowest positivity rate metaplastic carcinomas. The frequency of PIK3CA and other gene mutations in specific among all tumor groups. subtypes of metaplastic carcinomas, however, is unclear. Conclusions: Our study demonstrates that taking separate CM in BCS render a better Design: Paraffin-embedded tumor blocks from 20 cases of metaplastic carcinoma of and cleaner overall FM status compared to taking LMs, therefore reducing the need of the breast were retrieved from the surgical pathology archives in the Department of re-excision. InvCa is more likely to be completely excised than CIS by both LM and Pathology at the UT MD Anderson Cancer Center. Eleven of these were spindle cell CM methods. Future studies will evaluate other potential predictors of margin status sarcomatoid carcinomas, and 9 were matrix-producing carcinomas with cartilaginous (e.g. tumor size, grade, lymph node status etc.), and corelate the local recurrence and matrix. DNA was extracted from each block following macrodissection, and a total survival data to define the role of LM and CM in patient management. 392 point mutations in 81 genes were analyzed using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry developed by Sequenom for detection of 213 The Expression Level of pThoc1 Is Associated with Her2 Status single nucleotide polymorphisms. and Tumor Size in Breast Cancer Results: Sufficient DNA was obtained in 19 (95%) of the 20 cases. Ten of the 19 Y Li, J Yang, JA Almenara, HT Richard, JP Bergeron, DW Goodrich, MO Idowu. Virginia cases were spindle cell sarcomatoid carcinomas and the remaining 9 cases were Commonwealth University Health System, Richmond, VA; Virginia Department of matrix-producing carcinomas. Gene mutations in the PI3K signaling pathway were Health, Richmond, VA; Roswell Park Cancer Institute, Buffalo, NY. detected in 3 (30%) of the spindle cell sarcomatoid carcinomas and in 1 (11%) of the Background: Human pThoc1 is a nuclear matrix protein originally isolated for binding matrix-producing carcinomas. These included a mutation in FLT3, PIK3CA or AKT1 to pRb, the product of RB1 tumor suppressor gene. Thoc1 protein is an essential in each of the 3 spindle cell sarcomatoid carcinomas and a mutation in PIK3CA in the component of transcription-elongation (TREX) complex, is upregulated in multiple matrix-producing carcinoma. In addition, TP53 mutations were observed in 2 (22%) of human cancers and cancer cell lines, and is required for survival of cancer cells but the matrix-producing carcinomas but in none of the spindle cell sarcomatoid carcinomas. not normal cells. We have previously shown that Thoc1 is required for early stage Conclusions: These findings suggest that the prevalence of specific therapeutic targets tumorigenesis in cell culture models (Li et al., Cancer Research, 2007) as well as in metaplastic carcinoma may vary according to histologic subtype. To our knowledge, prostate cancer mouse models (in press). However, the role of Thoc1 in human breast this is the first report of a FLT3 mutation in spindle cell sarcomatoid carcinoma of the cancer is largely unknown. The purpose of the study is to evaluate the association of breast. As FLT3 is an upstream activator of both PI3K and MAPK pathways, dual pThoc1 expression with clinicopathologic features in breast cancer. pathway inhibition might be important in some of these tumors. Additional studies are Design: Breast carcinoma lobectomy or mastectomy cases from 1992 to 2008 at needed to characterize more fully the range of mutations that occur in distinct subtypes VCUHS with at least 5 years follow up or local recurrence/distant metastasis were of metaplastic carcinoma. selected. Tissue microarrays were created with triplicate tumor cores from each case. Immunohistochemical staining was performed with a rabbit polyclonal antibody to 212 Comparison of Margin Status of Separate Cavity Margins and pThoc1 (Aviva, USA). Cases were scored for nuclear staining intensity (0-3) and Lumpectomy Margins in Breast Conserving Surgery percentage (0-100%). Thoc1 score was calculated by Intensity x Percentage. A score of Y Li, I Hughes, R Omman, W Roquiz, X Duan, A Szpaderska, C Perez, C Godellas, C >200 was considered strong. Statistical significance was determined using Chi-squared Ersahin. Loyola University Medical Center, Maywood, IL. test and Pearson correlation analysis. Background: The surgical margin status of breast-conserving surgery (BCS) predicts Results: Of 319 cases, 244 cases were scorable. Strong pThoc1 expression was local recurrence and determined the need for re-excision. Two widely used methods associated with small tumor size, Her2 negativity and low Ki67. No significant to evaluate the margins have been applied in BCS, the lumpectomy margins (LM) vs. correlation with tumor grade, stage, metastasis, local recurrence, age, ER, or PR was separate cavity margins (CM) as final margins (FM). Studies comparing the two types of observed. BCSs are limited, and differences between them are not well defined, but a few studies have shown that taking additional CMs reduces the need for re-excision. Design: In our study, a positive margin is defined as the tumor located less than 1 mm to the inked margin for both LM and CM. In cases with CM, 4 to 6 separate margins are evaluated as FM. The margin status of 582 patients who underwent BCS in our institution from 2005 to 2011 has been evaluated including 420 CM and 162 LM cases. The tumors identified in the specimens are divided into 3 categories, invasive cancer (InvCa) only, carcinoma in-situ (CIS) only and both (Inv&CIS). If one or more FMs are positive, it is counted as a case with a positive margin. The percentages of the cases with positive FMs are compared in each tumor category between LM and CM specimen. Conclusions: The association of strong pThoc1 expression with small tumor size, Her2 Results: In InvCa, CIS, and Inv&CIS categories, there are 30, 57, and 75 LM cases, negativity and low Ki67 is consistent with our observation that pThoc1 is required for respectively; and 69, 96, and 225 CM cases, respectively. Figure 1 compares the early tumorigenesis in cell culture and mouse models. Its unique mechanism in gene %positive-cases in LM and CM devided into 3 tumor groups. expression and tumorigenesis and differential requirement by tumor cells versus normal cells suggest that Thoc1 may be a good molecular target for cancer prevention or therapy. 54A ANNUAL MEETING ABSTRACTS 214 Correlation of Papillary Neoplasms Diagnosed on Core Needle Bhargava et al and Lewis et al. Metastatic breast carcinomas (BCA) are likely high Biopsy That Underwent Re-Excision at Danbury Hospital between 2009- grade with ER-negative status. It is practical to find a sensitive and breast organ specific 2011 immunomarker. GATA-3 is a sensitive and specific marker for breast and urothelial L Lippincott, R Vidhun. Danbury Hospital, Danbury, CT. carcinomas, as reported in our previous study (Liu H, et al. AJCP. 2012;138:57-64). Background: Papillary lesions of the breast consist of a heterogeneous group than span However, the reactivity of GATA-3 in ER-negative breast carcinomas (BCA) has not the spectrum of benign, atypical, and malignant (in situ, invasive) entities. Unfortunately, been reported. In the current study, we conducted immunohistochemical analysis of the morphologic spectrum of papillary lesions does not follow a stepwise progression GATA-3 expression in 96 cases of ER-negative BCA, including 49 tissue microarray of increasing proliferation and “atypical features” from benign to malignant examples. (TMA) and 47 core needle biopsy (CNB) cases. Interpretation of papillary lesions of the breast remains a challenging task because of Design: Immunohistochemical evaluation of the expression of GATA-3 (Santa Cruz the wide morphologic spectrum encountered in the benign, atypical, and malignant Biotech Inc, Cat. No. GATA3 [HG3-31]:sc-268) on 96 cases of ER-negative BCA subtypes. Data on clinical significance and outcome of papillary lesions, especially using TMA sections (49 cases) and CNB specimens (47 cases) was conducted. The those with superimposed atypia or areas similar to ductal carcinoma in situ, are sparse. results were recorded. Correlation with clinical, mammographic, and sonographic findings is crucial in all Results: GATA-3 expression in 96 cases of ER-negative BCA is recorded in Table 1. papillary neoplasms diagnosed on core biopsies. Whether or not all papillary lesions Overall, 59% of TMA and 79% of CNB cases showed nuclear staining for GATA-3, identified on core biopsies should be surgically excised remains a controversial issue. with majority (73%) being strong (S) and diffuse (4 or 3+). Design: We performed a retrospective study of breast core needle biopsy specimens Table 1. GATA-3 Expression in 96 Cases of ER Negative Breast Carcinomas with the finding of papillary neoplasm that underwent subsequent excisional biopsy GATA-3 at Danbury Hospital during a three year period (2009-2011), with the data retrieved Tumor Type GATA-3 Expression, TMA Expression, Core Biopsy from the Cerner databases. N Pos. % N Pos. % Results: 85 core needle biopsies performed during this period yielded a diagnosis of IDC, G2 21 13 62 8 7 88 papillary neoplasm of which 57 (67%) underwent subsequent re-excision. Of the 57 IDC, G3 22 15 68 30 24 80 re-excisions, 33 (58%) yielded a benign diagnosis, 15 (26%) yielded atypia (ADH IDC, HG, Metaplastic 1 0 0 4 1 25 and FEA) and 9 (16%) yielded invasive or in situ carcinoma. There was residual IDC, HG, Medullary Features 2 0 0 1 1 100 IDC, Apocrine Features 2 1 50 4 4 100 papilloma identified in 17 (51.5%) of the benign, 6 (40%) of the atypical and 8 (89%) Total 49 29 59 47 37 79 of the malignant cases. N=number of cases; Pos.=positive; IDC=Invasive ductal carcinoma Diagnosis Following Re-excision of Intraductal Papilloma Conclusions: GATA-3 is expressed in majority (66/96, 69%) of the ER-negative BCA, DCIS/Invasive Carcinoma Total (n=57) Benign (n=33) 58% Atypical (n=15) 26% which may serve as a sensitive and breast organ specific immunomarker in the workup (n=9) 16% Residual Papilloma 17 6 8 of tumors of unknown origin, with the exception of urothelial carcinomas as reported No Residual Papilloma 16 9 1 in our previous study, which has a positive rate of 86% (62/72). GATA-3 expression is Conclusions: Review of literature shows that papillary neoplasms identified on core less or none in carcinomas with medullary features or metaplastic BCA in the few cases biopsies yielded a higher lesion in up to 24% of cases. We found that a number of included in this study, which needs to be validated in larger studies. cases showed a malignant (DCIS/invasive carcinoma in 16%) or atypical (ADH, FEA, papilloma with atypia in 26%) diagnosis on excision specimens (p=0.06). Based on 217 Equivocal HER2 FISH Results on Breast Core Biopsy with our study, re-excision of intraductal papillomas may be prudent in order to capture Ratio <2.0: Repeat HER2 Testing on Excision Specimens Impacts Patient lesions that may harbor atypia or carcinoma within or in the surrounding breast tissue Management of a papillary neoplasm. C Livasy, B Calhoun, S Limentani. Levine Cancer Institute, Charlotte, NC. Background: In the first generation HER2 adjuvant clinical trials, a ratio of 2.0 was 215 Immunohistochemical Evaluation of TFF1 and TFF3 Expression used as the cutpoint to determine eligibility for trastuzumab therapy for patients whose in ER-Negative Breast Carcinomas tumors were tested by FISH. The ASCO/CAP guidelines subsequently defined cases H Liu, J Shi, J Prichard, F Lin. Geisinger Medical Center, Danville, PA. showing a ratio of 1.8-2.2 as equivocally amplified. HER2 equivocal tumors present a Background: The human trefoil proteins TFF1 and TFF3 belong to the trefoil factor challenge to oncologists who must decide whether or not to give HER2 targeted therapy. family, expressed primary in the epithelial cells of gastrointestinal tract. TFF1, also There are no clear evidence-based guidelines for how to resolve the HER2 status for called PS2, is frequently expressed in breast carcinomas (BCA) under the control of equivocally amplified tumors. The identification of HER2 genetic heterogeneity within estrogen. Studies reveal that TFF1 expression is associated with BCA with ER-positive tumors raises the possibility that sample size may be an important factor in accurately status and better prognosis. Our previous studies on tissue microarray (TMA) BCA, determining HER2 status, particularly for cases near the cutpoint. This study evaluates including invasive ductal carcinomas (IDC, G1, 2, 3 and other special types) and the potential clinical impact of HER2 retesting on excision specimens for patients with invasive lobular carcinomas (ILC, G1,2 and 3), demonstrated that 72% of IDC and 87% HER2 FISH equivocal results showing a ratio <2.0 on core biopsy, who would otherwise of ILC are reactive to TFF1; 84% of IDC and 94% of ILC, to TFF3. The ER-positive not be eligible for HER2 targeted therapy. rates in TMA BCA are 74% for IDC and 84% for ILC. In contrast, the vast majority of Design: A total of 35 breast core biopsy specimens were identified from our database lung adenocarcinomas, endometrial adenocarcinomas and ovarian serous carcinomas showing invasive mammary carcinoma and an equivocally amplified HER2 status were negative for TFF1. In the current study, we immunohistologically evaluated the (ratio <2.0) who had retesting of HER2 status by FISH on the subsequent excision expression of TFF1 and TFF3 in 49 consecutive ER-negative BCA on core needle specimen. The HER2 ratios from the core biopsy and excision specimen were compared. biopsy specimens using a single immunostaining system (Ventana). Changes in HER2 status between the core biopsy and excision specimen were noted Design: Immunohistochemical evaluation of the expression of TFF1 (Epitomics, AC- using ASCO/CAP criteria. 0045RUO) and TFF3 (Epitomics, AC-0103RUO) was performed on 49 consecutive Results: A definitive change in the classification of the HER2 status was observed in ER-negative BCA, including mainly IDC, G2, G3 and a few other special types, such 24 (68%) cases. Twenty cases (57%) reclassified as negative, 4 cases (11%) reclassified as metaplastic and medullary carcinomas on core needle biopsy specimens, using a as positive, and 11 cases (31%) remained equivocal. Three of the patients with HER2 Ventana immunostaining system. The staining intensity was graded as weak or strong. equivocal results on the excision specimen demonstrated a ratio >2.0. Using a ratio of 2.0 The distribution was recorded as negative (<5% of tumor cells stained), 1+ (5-25%), as the cutpoint for determining eligibility for HER2 targeted therapy, a total of 7 (20%) 2+ (26-50%), 3+ (51-75%), or 4+ (>75%). of patients became eligible for trastuzumab therapy. The four patients who showed a Results: Fourty-five percent (22/49) of ER negative BCA showed focal or diffuse definitive reclassification to HER2-positive status showed ratios of 4.0, 3.0, 2.5 and 2.4. positivity for TFF1. Eighty-six percent (42/49) of the ER-negative BCA were Conclusions: For patients with equivocally HER2 amplified results on core biopsy nonreactive to TFF3. Staining patterns of the positive cases were summarized in table 1. showing a ratio <2.0, repeat HER2 FISH testing on a larger volume of invasive Table 1. Staining patterns of TFF-1 and TFF-3 in 49 ER negative breast carcinoma carcinoma from excision specimens results in definitive reclassification of HER2 status Staining pattern TFF-1 TFF-3 for a significant number of patients. Approximately 1/5 of patients became eligible Weak Strong Weak Strong for HER2 targeted therapy based on results from the excision specimen. These results 1+ 0 1 0 0 support the ASCO/CAP recommendations for further testing of cases with HER2 2+ 11 2 1 3 equivocal results. 3+ 3 2 0 3 4+ 0 3 0 0 Percentage (%) 45% 14% 218 ER, PR, Her2, and Ki-67 Expression in Male and Female Breast Conclusions: These data suggest that TFF1 can be potentially used as a biomarker in Cancer differential diagnosis of ER-negative breast carcinomas from adenocarcinomas of the MG Lockyer, E Martinez-Blanco, G Verstovsek, I Bedrosian, C Albarracin, D Rosen. lung, endometrium and ovary. Baylor College of Medicine, Houston, TX; MD Anderson Cancer Center, Houston, TX. Background: Male breast cancer afflicts a small percentage of the population and is 216 Immunohistochemical Evaluation of GATA-3 Expression in usually advanced when treatment is sought due to late presentation. Treatment is based ER-Negative Breast Carcinomas on the readily available research done on female breast cancer with few examining the H Liu, J Shi, J Prichard, Y Gong, F Lin. Geisinger Medical Center, Danville, PA; immunophenotypic and clinicopathologic characteristics of male breast cancer. We University of Texas MD Anderson Cancer Center, Houston, TX. evaluated the expression of estrogen receptor (ER), progesterone receptor (PR), human Background: Currently, estrogen receptor (ER), GCDFP-15 and mammaglobin are epidermal growth factor 2 (HER2), and Ki-67 among male breast cancer specimens the most commonly used immunomarkers for identifying breast primary in tumors of from patients, and compared the results against results from a group of female breast unknown origin. The reported sensitivities for GCDFP-15 and mammaglobin are in cancer specimens that were previously evaluated. the ranges of 35-55% and 65-70%, respectively. Our un-published data are even lower, Design: Samples from men with primary breast cancer between 1995 and 2011 from 35% for GCDFP-15 and 50% for mammaglobin, which are similar to the reports by the Veterans Affairs Hospital and female primary sporadic breast cancer cases from ANNUAL MEETING ABSTRACTS 55A MD Anderson Cancer Center were selected through retrospective review of medical Conclusions: In this series, overall prognosis is good even for patients with LNm. records. Paraffin blocks and complete demographic and clinical follow-up was available Larger series and more molecular studies are needed to find out predictive factors for in 14 male patients and 39 female patients. Histopathologic diagnoses were based on these carcinomas. Our work highlights the need for an extended follow up as recurrences the ESBR grading system. A tissue microarray block was constructed and stained for occured as late as 152 months after diagnosis. ER, PR, Her2-neu, and Ki-67 immunohistochemical stains. Results: The average age at presentation was 67 in female and 66 in male. A total of 221 Radiological and Pathological Extent of Columnar Cell Changes 64% of male patients (9 cases) presented at stage 1, 21% (3 cases) at stage 2, and 14% with Atypia (CCC-A) Diagnosed on Core Needle Biopsy (CNB) Correlates (2 cases) at stage 3. No male patients presented at stage 4 disease or died of disease. with Carcinoma Upgrade on Surgical Excision (SE) A total of 6 female patients (15%) presented at stage 4 disease. Ductal carcinoma F-I Lu, D Giri. Sunnybrook Health Sciences Centre, Toronto, ON, Canada; Memorial was the predominant histologic pattern in both female (85%) and male (93%). Tumor Sloan-Kettering Cancer Centre, New York, NY. grade was similarly distributed among female and male breast cancers. The average Background: The management of pure CCC-A (i.e. flat epithelial atypia) diagnosed tumor size was 18 mm for female and 26 mm for male cancers. Female breast cancer on CNB continues to be problematic, with the current management consisting of SE had a higher proportion of positive nodes and lymphovascular invasion compared to to exclude coexisting ductal carcinoma in-situ (DCIS) and invasive carcinoma (IC). male. All male cases were ER positive, the majority were PR positive (64%) and all This study was designed to identify clinical, radiological and pathological features of were Her2 negative. ER, PR, and Her2 were positive in 74%, 46%, and 26% (2+/3+), CCC-A diagnosed on CNB that correlated with carcinoma upgrade on SE. respectively, in female cases. The Ki-67 proliferation index was high in 7% of male Design: We identified all CNB performed at our institution and signed out as CCC-A cases and 31% of female cases. between January 1, 2006 and October 31, 2011. Only cases diagnosed as pure CCC-A by Conclusions: In our cohort, male tumors presented at an earlier stage, have less positive 2 breast pathologists were included in this study. Clinical, radiological and pathological nodes, and have a similar tumor grade compared to female breast cancer. Male tumors features on CNB (see table) were evaluated blinded to the diagnoses on SE. Two-tail tend to express ER and PR, have a low Ki-67 proliferation index, and are Her2 negative. t-test was used to identify features that were significantly different between CCC-A cases with and without carcinoma upgrade. 219 Findings on Excision Following a Core Needle Biopsy Features of Columnar Cell Changes with Atypia (CCC-A) on Core Needle Biopsy Evaluated Age at diagnosis, menopausal status, personal &family history of breast Diagnosis of Lobular Neoplasia: A Single Institution Experience Clinical features A Lott Limbach, A Collie, E Downs-Kelly. Cleveland Clinic, Cleveland, OH. cancer, hormone replacement therapy &tamoxifen use Reason for biopsy (bx), bx modality, BI-RAD score, gauge of the bx Radiological features Background: Controversy exists with regard to appropriate follow-up and treatment needle, size, number (#) of cores bx, # of cores with calcification of patients with a core needle biopsy (CNB) diagnosis of lobular neoplasia; either # of cores with CCC-A, # of cores bx, number of terminal ducto-lobular atypical lobular hyperplasia (ALH) or lobular carcinoma in situ (LCIS). There have been units (TDLU) with CCC-A, size of CCC-A, calcification in& outside of CCC-A, morphology of CCC-A (dilated rigid TDLU, loss of polarity, numerous published studies attempting to address this issue with conclusions ranging Pathological features indistinct myoepithelial layer, nuclear enlargement, round nuclei, nuclear from only close clinical follow-up is needed to surgical excision should be performed in pleomorphism, nuclear hyperchromasia, vesicular chromatin, prominent all cases. These studies are limited by small numbers of patients and differing inclusion nucleoli, mitosis, necrosis) criteria, making them difficult to compare. We assessed the findings at our institution, Results: Thirty-four cases were diagnosed as pure CCC-A on pathology review. All of where virtually every CNB diagnosis of lobular neoplasia results in follow-up excision, these cases underwent SE. On SE, 4 of the 34 (11.8%) cases upgraded to carcinoma, to identify predictors of upgrade to a worse lesion. with 1 to IC and 3 to DCIS. Of the CNB features of CCC-A evaluated, only radiological Design: After obtaining institutional review board approval, we searched the anatomic size (26 mm vs 9 mm, p=0.03117), pathological size (7.4 mm vs 2.5 mm, p=0.000006) pathology database for breast CNBs with subsequent surgical excisions between 1993 and number of terminal ducto-lobular units (TDLU) involved (7.3 TDLU vs 2.5 TDLU, and 2011. Forty patients with pure lobular neoplasia (ALH or LCIS) on CNB with no p=0.00065) were significantly different between CCC-A cases with and without other high-risk lesions were identified. CNB and surgical excision histology, as well carcinoma upgrade. as radiologic data, were reviewed to determine if there were characteristics that could Conclusions: In this study, 34 cases of pure CCC-A diagnosed on CNB were identified, predict a worse lesion on excision. with 4 (11.8%) upgraded to either IC or DCIS on SE. Radiological size, pathological Results: We identified 40 CNB diagnoses of ALH/LCIS with subsequent surgical size and number of TDLU involved were the only CNB features of CCC-A found to excision and identified 10 cases (25%) (4 ALH and 6 LCIS) that were upgraded to a correlate with carcinoma upgrade on SE. worse lesion. Upgrade diagnoses included invasive ductal carcinoma (n=2), invasive lobular carcinoma (n=2), DCIS (n=5), and pleomorphic LCIS (n=1). All 10 cases that were upgraded had imaging findings highly suspicious for malignancy (BI-RADS 4), 222 Findings of Magnetic Resonance Imaging-Guided Breast including masses or pleomorphic calcifications. In contrast, 30 cases (75%) where there Biopsy: A Radiologic and Pathologic Correlation Study from a Single was no worse lesion identified on surgical excision did not have similar suspicious Institution imaging findings. B Lynch, S Seiler, G Moses, S Sahoo. UT Southwestern Medical Center, Dallas, TX. Conclusions: Similar to previous studies, we have shown a greater likelihood of upgrade Background: The role of Magnetic Resonance Imaging (MRI) in the diagnosis of from a core needle biopsy diagnosis of lobular neoplasia when a radiographic worrisome breast diseases has rapidly evolved from its initial use of screening high-risk women to finding, either mass or pleomorphic calcifications, is present. This data reinforces the a more widespread use in women with newly diagnosed breast cancer. The sensitivity importance of radiologic-pathologic correlation of suspicious breast lesions and may of breast MRI is high and many suspicious lesions detected by MRI will not have better identify those lesions requiring surgical excision. corresponding abnormality on mammography and ultrasound examinations. Therefore, a MRI-guided core biopsy is often performed to characterize the suspicious lesions. A detailed correlation of various suspicious categories of MRI findings with histopathology 220 Small Invasive Carcinomas of the Breast (T1a, T1b): The of the breast lesions is still lacking. Our goal is to study the histopathologic findings Importance of an Extended Follow Up that are frequently present as abnormal enhancing lesions. J Loureiro, P Monteiro, C Castro, MJ Bento, C Leal. Portuguese Oncology Institute, Design: A retrospective review was conducted on all patients undergoing MRI-guided Porto, Portugal. core biopsy during October 2007 to September 2012. Our study population consisted Background: Over the past years diagnosis of invasive breast carcinomas measuring of 140 MRI-guided needle core breast biopsies performed on 130 patients. Biopsies ≤ 1cm has increased. These women generally have good long-term outcomes, but not were performed using a 1.5-Tesla Phillips MR with a 9-gauge vacuum-assisted SUROS all. The aim of this study was to investigate whether women at high risk of breast-cancer biopsy device. Histopathologic findings of these biopsies were recorded. death could be identified based on clinical and pathological features. Results: Overall, 25 of 140 (18%) of our MRI-guided breast biopsies were malignant, ≤ Design: The cohort included 49 invasive breast carcinomas with 1cm, consecutively 16 of 140 (12%) were atypical epithelial hyperplasia, 99 of 140 (71%) were benign. diagnosed in 1995 and 1996. Clinical and morphological features were reviewed and Of the 30 malignant cases, 12 were invasive ductal carcinomas, 4 infiltrating lobular immunohistochemical studies were performed. Association of these variables with carcinomas, and 9 ductal carcinoma in situ. Of the 16 atypical epithelial hyperplasia, axillary lymph node metastases (LNm), recurrences, distant metastases (Dm) and 3 were ADH and 12 were atypical lobular hyperplasia/lobular carcinoma in-situ. All survival was analyzed. atypical hyperplasias were associated with other benign changes. Interestingly, columnar Results: Patients had between 26 and 83 year-old (mean 57.7 year-old), 39 (79.6%) had cell changes accounted for 40% of the benign lesions. stage I, 6 (12.2%) stage IIA, 1 (2.1%) stage IIIA and in 3 (6.1%) LN dissection was not Conclusions: Majority (71%) of our MRI-guided biopsy yielded benign diagnoses; performed. Morphologically, most tumors (n=39, 79,6%) were ductal and grade 1 or 2, only 18% of the sampled lesions were clinically significant (invasive carcinoma and and 28 (57.1%) had an extensive “in situ” component. All except one were hormone DCIS). Columnar cell changes are frequently identified within the enhancing lesions. receptors positive, HER2 was positive in 8 (16.3%) tumors and 19 (38.8%) tumors had Additional studies focusing on the pattern of enhancement seen in MRI on certain Ki-67>14%. In 4 cases material for immunohistochemical studies were not available. benign and malignant lesions may help to subcategorize lesions for MRI-guided biopsy. Using molecular classification, 22 (49.9%) tumors were Luminal A, 15 (30.6%) Luminal B (HER-), 7 (14.3%) Luminal B (HER+) and 1 (2.0%) Her+. Average time of follow- up was 171 months (50 up to 210): 36 (75.5%) patients are alive without evidence of 223 Detailed Characterization of Ductal Carcinoma In Situ disease, 9 (18.4%) died of other causes and 3 (6.1%) died of disease. Recurrences and Associated with a Mass on Imaging Dm occurred from 73 months up to 152 months after diagnosis (mean 119 months). A Mahajan, LZ Blanco, Jr., LC Wang, EB Mendelson, KP Siziopikou, ME Sullivan. Only stage I patients had recurrences (n=7, two of whom died) or Dm (n=1, dead). Northwestern University, Chicago, IL. None of the clinical or pathological parameters evaluated were statistically significant Background: With mammographic screening, it is uncommon for ductal carcinoma for these events. Only T1b tumors (≥0.7cm) had LNm. Patients with LNm (n=7, 14.3%) in situ (DCIS) to present as a mass. When a core biopsy (CB) targeting a mass yields had no recurrences or Dm and are alive without evidence of disease. only DCIS, pathologic-radiologic concordance can be challenging. We reviewed DCIS diagnosed by CB targeting a mass to determine how often a pathologic mass is present. 56A ANNUAL MEETING ABSTRACTS Myoepithelial markers (MEM) can be reduced or negative in some mass forming DCIS- (RT, XZ, VM). Ki -67 PI was calculated in a minimum of three randomly selected high like lesions such as encapsulated papillary carcinoma (EPC). MEM were evaluated to power fields (40X) including hot spots if present or a minimum of 500 cells, based on see if any specific histologic features were associated with reduced expression. the recommendation of the IBCWG. The inter-rater agreement was calculated using Design: The EMR was searched from 2007-10 for CB targeting a mass with a diagnosis intraclass correlation coefficient (ICC). Pearson corelation was used to compare the of DCIS. CB with ipsilateral invasive carcinoma (IC) or DCIS involving a benign mean Ki-67 PI with the recurrence score (Oncotype Dx).The RS classifies tumors into mass (e.g. fibroadenoma) were excluded. All slides were reviewed and pertinent data 3 risk categories: low (0-17), intermediate (18-30) and high (31-100). including grade, type and stromal charateristics were recorded. CB were categorized Results: In the 27 tumors, the Ki-67 PI assessment by the three pathologists had as 1) mass forming with normal ductal architecture; 2) mass forming with distorted significant differences in some cases yet the raters were 89.1% in overall agreement architecture (back-to-back or irregular); or 3) no clear mass. At least 2 MEM (p63, (ICC = 0.891, 95% confidence interval 0.806 - 0.945). There was no correlation between SMMHC, calponin and CD10) were performed on all CB. When the intensity of MEM Ki-67 and Oncotype Dx (r=0.081, p=0.694). Of the 26 cases (one case did not have around DCIS was less than in benign ducts, it was considered reduced. Results of the adequate material), there were 10 with low risk (mean group RS: 10.1; mean Ki-67: subsequent excision were recorded when available. 17.4; Ki-67 range: 3-33%); 14 with intermediate risk (mean group RS: 21.4; mean Ki- Results: 55 CB met inclusion criteria; 45% were BIRADS 4C/5. 93% (51/55) had 67: 19.5; Ki-67 range: 6-43%) and 3 with high risk (mean group RS: 53; mean Ki-67: high grade DCIS. 73% (40/55) had a pathologic mass on CB and of these 60% had 70; Ki-67 range: 55-91%). architectural distortion. 33% of CB with distortion had papillary type DCIS vs. 6% Conclusions: Microscopic evaluation of Ki-67 index is reproducible using the (1/16) of CB with normal architecture. No clear pathologic mass was identified in 15 method suggested by the IBCWG. The extremely wide range of Ki-67 PI in low and cases. 53 of the 55 cases had available excisions (Table 1). MEM were adequate on 50 intermediate risk groups, the unexpected low Ki-67 in intermediate risk groups, and CB and positive in 49, with reduced expression in 34. 53% with reduced MEM were the unexpected high Ki-67 PI in low risk groups, limits its use as a predictive measure upgraded vs. 27% with strong MEM. in these categories. The significance of Ki-67 in the high risk group needs further Table 1: Results evaluation in larger scale study. Papillary Features IC on Excision Pathologic Mass (N=40) 9 (23%) 16 (40%) No Mass (N=15) 0 (0%) 9 (64%) 226 Changes in Breast Cancer Reports after Pathology Second Positive MEM (N=15) 3 (20%) 4 (27%) Opinion Reduced MEM (N=34) 5 (15%) 18 (53%) V Marco Molina, T Muntal, J Cortes, B Gonzalez, I Rubio, F Garcia Hernandez. Hospital Negative MEM (N=1) 1 (100%) 0 Quiron Barcelona, Barcelona, Spain. Conclusions: CB of mass forming DCIS often show architectural distortion (60%) and Background: Breast cancer pathology reports contain valuable information about have a papillary component (23%). CB with no pathologic mass had the highest upgrade the histological diagnosis, prognostic factors and predictive indicators of therapeutic rate (64%) suggestive of undersampling. Reduced expression of MEM was seen more response. A second opinion may be requested by medical oncologists and surgeons, often in CB with papillary features and may indicate a possible EPC component. CB when a patient is referred from another institution for treatment. We report the experience with reduced MEM also had a higher upgrade rate. Complete negativity with multiple with pathology second opinion in selected patients referred to the Breast Oncology Unit. MEM in lesions that otherwise look like DCIS is a rare but problematic occurrence on Design: 205 cases referred to the Breast Oncology Unit were selected for second opinion CB and requires further study. after clinical evaluation, between 2002 and 2012. The cases reviewed included 102 core needle biopsies, 88 surgical specimens from the breast and 18 lymphadenopathies, 14 224 Claudin Expression in Triple Negative and ER-Positive Breast from the axillary region. Pathology second opinion was based on review of hematoxylin- Carcinoma eosin preparations, recuts of submitted paraffin blocks and written outside pathology MA Malik, C Cohen, A Adams, J Wang, GM Oprea. Emory University Hospital, reports. Immunohistochemical studies for hormone receptors, HER2, myoepithelial Atlanta, GA. cells, and other markers were performed in selected cases. A case was classified as Background: Tight junctions play an integral role in cell homeostasis. Claudins along major change in second opinion when the findings had the potential for significant with occluding and junctional adhesion molecules are the main membrane proteins change in treatment or prognosis. Otherwise, it was considered to represent minor which form these junctions. Reduced Claudin expression may be associated with change or concordant. decreased cell to cell adhesion resulting in a poor prognosis in various tumors. Claudin Results: In 51 cases (24.9%) the pathology review showed changes. Thirty-five (17%) expression has been variable in breast carcinomas according to the limited number of patients were classified as major changes and 16 (7.8%) as minor changes. In seven studies performed, including a recently described Claudin low phenotype. Here, we patients more than one major change was identified. The major discrepancies identified compare Claudins 1, 4 and 7 expression in Triple Negative breast carcinoma to that were related to the histological classification (13 cases), the presence or absence of of the ER+ phenotype. invasion in ductal carcinoma (17 cases), the results of hormone receptors (5 cases), and Design: Analysis was performed retrospectively on breast carcinomas using tissue HER2 (7 cases). Major changes in histological classification included 2 cases diagnosed microarrays of a total of 160 Triple Negative and 165 ER and/or PR+ tumors. CAP as ductal carcinoma and reclassified as benign, 3 cases with diagnosis of breast cancer standards were used to score immunohistochemistry for hormone receptor markers ER reclassified as metastatic lung cancer, 3 cases with diagnosis of breast cancer in the axilla and PR. Immunohistochemistry for Claudins 1 (1/70 polyclonal, Invitrogen), 4 (1/80 reclassified as primary cutaneous adnexal carcinomas (2) and metastatic melanoma (1). monoclonal 3E2CI, Invitrogen), and 7 (1/400 monoclonal 5D010F3, Invitrogen) was In 5 cases the histological type of the primary breast tumor was changed. performed. Identification of positivity was based on the intensity and percent of cells Conclusions: Second opinion in breast pathology may uncover significant discrepancies staining. Cell membrane staining was considered as positive. Strong membranous that impact in patient management and prognosis. Major discrepancies are most staining clearly visible at 4X magnification was considered 3+, staining visible only at frequently related to the assessment of the presence or absence of invasion in ductal high power was 1+ and mid-range staining as 2+. Tumors that scored 1-3+ for intensity carcinoma, the results of predictive makers of therapeutic response, and the differential with 5% of tumor cells staining were considered positive. diagnosis of breast cancer and non-mammary tumors in the breast, the axilla, and at Results: ER+ breast carcinoma more frequently showed Claudin 7 expression with distant sites. greater intensity of stain and increased number of cells staining when compared with Triple Negative carcinoma(p<0.001). Claudin 1 staining intensity was greater in Triple 227 Correlative Analysis of Immunohistochemical and OncotypeDX® Negative compared with ER+ breast carcinoma (p<0.001). Frequency of Claudins 1 and Derived PCR Data in Breast Cancer 4 expression, staining intensity of Claudin 4, and percent positive cells of Claudins 1 and Z McCroskey, G Venkataraman, P Robinson, S Lo, A Szpaderska, X Duan, C Ersahin. 4 were not significantly different between Triple Negative and ER+ breast carcinoma. Loyola University Medical Center, Maywood, IL. Conclusions: We conclude that Claudin 7 shows a statistically significant decreased Background: OncotypeDX® assay analyzes the expression of several metastasis expression in Triple Negative(Claudin low) compared to ER+ breast carcinoma, associated genes and estimates a distant disease Recurrence Score (RS). Testing is portending a worse prognosis for Triple Negative carcinoma. Claudin 1 expression was typically performed in estrogen receptor positive (ER+) breast cancer (BCA). We differentially more intense in Triple Negative breast carcinoma. Claudin 4 expression sought to correlate the concordance of immunohistochemical (IHC) stains with the in both populations was statistically similar. OncotypeDX® PCR derived data on these markers. Design: ER+ BCA cases (n=171) with available OncotypeDX® PCR data on ER, PR, 225 The Satisfactory Reproducibility of the Ki-67 Index in Breast and HER2 expression with concurrent available ER, PR Allred scores, HER2 and Ki67 Carcinoma, and Its Poor Correlation with the Recurrence Score IHC results were included. Ki67 and HER2 were modeled as ordinal covariates (0-10%, V Manucha, X Zhang, RM Thomas. Temple University Hospital, Philadelphia, PA. score 0; 10-20% score 1; and >20% score 2 for Ki67). Initial univariate correlations Background: Immunohistochemical assessment of Ki-67 proliferative index (PI) between RS, IHC data (ER, PR and Ki67) and PCR data were assessed. Univariable (u-) has been established as a valuable prognostic marker in early breast cancer; however, and multivariable (mv-) logistic regression (LR) with nested models were constructed its role in predicting response to neoadjuvant chemotherapy is still uncertain. Lack and Receiver Operating Characteristic curves (ROC) were examined. Model comparison of an accepted validated method of assessment of Ki-67 and wide variations in the was performed using likelihood ratio test. cutoffs has severely limited its clinical utility. In this study, we attempt to assess the Results: ER, PR and HER2 IHC were strongly correlated with their PCR expression intralaboratory interobserver variability in estimation of Ki-67 PI using the guidelines status (Pearson r=0,53, 0.72, 0.42 resply p<0.05) as expected. Concordance for ER and for analysis proposed by the International Breast Cancer Working Group (IBCWG). PR were 99% and 89% respectively between immunohistochemistry and qRT-PCR. We further assessed the relationship between the Ki-67 PI and the Recurrence Score Most discordant cases had low Allred scores. There was 100% concordance for HER2; (RS) obtained from Oncotype Dx to evaluate its value in predicting tumor recurrence/ however, all the cases were negative. In u-LR analyses, PR, HER2 and Ki67 explained response to neoadjuvant therapy. 5.5, 1, 20.7% of variance in RS, while via PCR PR and HER2 explained 19.8 and 5% of Design: 27 invasive breast carcinomas that were analyzed with the Oncotype Dx assay the variance in RS. Interestingly, by IHC Ki67 explained most of the variance, whereas over a 4 year period were included in the study. H+E sections and Ki-67 (MIB-1 clone, PR did not add any further information. Notably, PR explained most of the variance prediluted,Ventana) immunostaining was independently reviewed by three pathologists with small contributions by HER2 RT-PCR. We constructed a 2-parameter model using Ki67 and PR-PCR which explained nearly 34% of the variance in the RS (p<0.001) ANNUAL MEETING ABSTRACTS 57A or positive (score>0) ALDH1 cytoplasmic expression in tumor cells. Bivariate analysis was done via χ2 analysis and survivability data was calculated via the generation of Kaplan-Meier curves (SPSS v19). Statistical significance was assumed if P < 0.05. Results: In our study, Luminal A was 43.8%, Luminal B 14.4%, HER2 8.5%, and Triple Negative 33.3% of which 51% were basal-like phenotype. ALDH1 positivity was associated with high grade (0.03) and Her2 positivity (p=0.057). Expression was not associated with age, stage, or survival. In our study, ALDH1 was expressed in 40% of all the cases, but did not show a correlation with triple negative breast cancers, and was expressed in several ER+ breast cancers. Conclusions: Mammary stem cells, identified by cells expressing the marker aldehyde dehydrogenase 1 (ALDH1), have been associated with malignant transformation of breast tissue and have been reported in a subset of triple negative breast cancers. ALDH1 expression was found in 40% of breast cancers in our study with expression in all subtypes. We found statistically increased expression of ALDH1 in cases with HER2 positivity and high grade histology.

230 High Claudin-4 Expression in Triple Negative Breast Carcinomas from African-American Women TJ Naab, LJ Ricks-Santi, D Beyene, C Broome, AK Esnakula. Howard University, Washington, DC. Background: Progressive growth of breast cancer cells is associated with alterations Conclusions: There is some complementary information provided by combination of in the cell-cell and cell-matrix adhesions leading to disorganized growth with loss of IHC of which Ki67 explains significant proportion of variance in RS. Nearly 65% of polarity. The claudin family has an integral role in tight junctions, the apically located variance remains unexplained and it is most likely contributed by the other genes tested cell-cell adhesion complex. Claudin-4 binds to cytoplasmic tight junction proteins as part of the OncotypeDX® panel. However, discordant ER and PR results between interacting with signaling pathways, and to actin filaments, which alter cell polarity validated immunohistochemistry results and qRT-PCR raise the doubt whether qRT-PCR and cell motility. Overexpression of claudin-4 in primary breast carcinomas has been correctly quantitates the gene expression levels in some cases. associated with poor prognosis and high tumor grade. Our study is undertaken to analyze the expression of claudin-4 in the four main subtypes of breast cancer (Luminal 228 Automated and Visual Assessment of Ki-67 IHC in Breast A, Luminal B, HER2 positive, triple negative) and correlate with clinicopathological Carcinoma: Comparison of 3 Antibody Clones factors in African-American women. EC Minca, SA Elizabeth, T Koji, F Heather, TR Raymond. Cleveland Clinic Foundation, Design: Tissue microarrays (TMAs) were constructed from optimally-fixed formalin- Cleveland, OH; Ventana Medical Systems, Tucson, AZ; National Cancer Center fixed, paraffin-embedded tumor blocks from primary breast ductal carcinomas in Research Institute and Hospital, Tokyo, Japan. 203 African-American females. Two separate 1mm cores represented each case. Background: The expression of Ki-67 is widely used as a marker for tumor Tumors were subtyped based on expression of ER, PR, HER2, vimentin and 34βE12. cell proliferation and is an established prognostic factor in breast cancer. The Immunohistochemical evaluation for Claudin-4 was performed using rabbit monoclonal immunohistochemical (IHC) Ki-67 labeling index (LI) is commonly visually estimated antibody (3E2C1, Invitrogen, USA). The sections were evaluated for the intensity of as percentage of tumor cells with positive nuclear staining. Although the mouse MIB-1 reactivity (0-3) and the percentage of reactive cells; and an H-score was derived from is the most referenced antibody clone for Ki-67 IHC, several newer rabbit monoclonal the product of these measurements. Claudin-4 expression was scored as low (H-score antibodies are commercially available, with little data comparing their performance. <100), moderate (H-score 100-199) and high (H-score >200). Bivariate analysis was Furthermore, an automated platform has been recently FDA-approved for Ki-67 analysis done via χ2 analysis and multivariate analysis was done via Cox’s proportional hazards in breast cancer (Ventana). Here we compare 3 antibodies for Ki-67 LI by both visual model. Statistical significance was assumed if P < 0.05. and automated analysis, on a collection of breast carcinomas. Results: Among our study population, 43.8% were Luminal A, 14.4% Luminal B, Design: Three tissue microarrays containing 235 formalin-fixed paraffin-embedded 8.5% HER2 +, and 33.3% triple negative of which 51% were basal-like phenotype. breast cancer samples from 3 institutions (UK, Japan, Canada) were stained for Ki-67 High claudin-4 expression was associated with high grade (p=0.0045), ER negativity with the SP6 (Neomarkers/LabVision), 30-9 (Ventana), and MIB-1 (Dako) antibody (p=0.025), HER2 positivity (p<0.0001) and triple negative breast cancer (p<0.0001). clones using optimized protocols. The LI as percentage cells with positive staining was Claudin expression was not associated with age size, PR, stage, or survival. 70% of analyzed for each antibody on each array by using the Virtuoso Software and iScan triple negative cancers having basal-like phenotype showed high claudin-4 expression. Coreo Au scanner platform (Ventana) and manually by two independent pathologists. Conclusions: In our study, triple negative breast cancers in African-American women Statistical analysis was performed using Stata 12/SE software (StataCorp). were associated with high claudin-4 expression. High claudin-4 was also associated Results: MIB-1 antibody showed mean Ki-67 LIs of 9% (automated analysis), 7.3% with HER2+ and ER- cases. These markers may be useful in predicting progression and (manual #1) and 22.9% (manual #2). 30-9 and SP6 rabbit antibodies showed similar a poorer outcome in triple negative breast cancers which are more commonly found in and significantly higher Ki-67 LIs compared to MIB-1: automated 25% and 27.5%, African-American females. In our study, claudin-4 was also overexpressed in HER2+ manual #1 21% and 21.4%, manual #2 30.9% and 39.1%, respectively (p<0.001 in all breast cancers, which are also associated with poor outcome. six instances). Furthermore, the LI read variation was lower for all three antibodies with the automated analysis (SD 12.8% [MIB-1], 22.3% [30-9], 23.2% [SP6]) compared 231 Functional Characterisation of Fusion Genes in Micropapillary to the manual reads (SD 17.4% and 17.8% [MIB-1], 25.5% and 22.5% [30-9], 28.4% Carcinomas of the Breast and 25.4% [SP6]). R Natrajan, PM Wilkerson, C Marchio, MB Lambros, CKY Ng, C Topfer, I Kozarewa, Conclusions: The 30-9 and SP6 rabbit monoclonal antibodies show similarly higher J Hakas, K Mitsopoulos, D Hardisson, CJ Lord, B Weigelt, A Ashworth, A Sapino, A sensitivities for Ki-67 LI analysis in breast carcinomas compared to MIB-1 antibody. Chinnaiyan, CA Maher, JS Reis-Filho. Institute of Cancer Research, London, United Automated analysis for tumor proliferation index is a more accurate alternative to Kingdom; University of Turin, Turin, Italy; Memorial Sloan-Kettering Cancer Center, manual counts. Further correlation with the clinical outcome is required to validate New York, NY; Hospital Universitario La Paz, Madrid, Spain; University of Michigan, these tools as highly-sensitive and accurate for detecting tumor proliferation index. Ann Arbor, MI; Washington University, St Louis, MO. Background: Micropapillary carcinomas (MPCs) are a rare histological special type 229 Aldehyde Dehydrogenase 1 (ALDH1) Expression in Non-Lobular of breast cancer with an aggressive clinical behaviour. Some special types of breast Breast Carcinomas in African American Women cancer have been shown to be underpinned by recurrent fusion genes. The aims of this TJ Naab, AK Esnakula, LJ Ricks-Santi, Y Kanaan, B Gold. Howard University Hospital, study were to determine i) whether MPCs harbour recurrent fusion genes and ii) if the Washington, DC; Frederick National Laboratory for Cancer Research, Frederick, MD. fusion genes identified are of functional relevance. Background: Stem-cell like features have been linked to ALDH1 expression in Design: Five pure, microdissected MPCs were subjected to massively parallel RNA subpopulations of normal and neoplastic breast epithelial cells. ALDH1 expression sequencing (Genome Analyser IIx sequencer). Fusion genes were nominated using is associated with breast carcinomas having a poor prognosis. Previous studies have Chimerascan version 4.0 and validated using RT-PCR and Sanger sequencing. The linked the expression of ALDH1 with triple negative breast cancers, which are more genomic breakpoints of the fusion genes were identified by long-range PCR (Roche common in younger African-American women. The objective of our study is to correlate 20Kb Expand Plus) using custom primers. Validated fusion genes were interrogated the immunohistochemical expression of ALDH-1 in the four major subtypes of breast in an independent series of 11 MPCs, 16 grade and oestrogen receptor (ER)-matched carcinoma (luminal A, luminal B, HER2 positive, and Triple Negative) and other invasive carcinomas of no special type (IC-NSTs), 53 grade III IC-NSTs and in publicly clinicopathological factors in African American women. available datasets. The impact of forced expression of validated in-frame fusion Design: Tissue microarrays (TMAs) were constructed from optimally-fixed formalin- genes was investigated using growth assays and CellTiter Glo (Promega), and RNA fixed, paraffin-embedded tumor blocks from primary breast ductal carcinomas in interference (RNAi) mediated silencing of recurrent partner genes of out-of-frame 203 African-American females. Two separate 1mm cores represented each case. rearrangements was assessed in ER-positive breast cancer cell lines (i.e. MCF-7, BT474, Tumors were subtyped based on expression of ER, PR, HER2, vimentin and 34βE12. T47D, ZR75.1 and SUM44). Immunohistochemical evaluation for ALDH1 was performed using mouse monoclonal Results: RNA-sequencing resulted in the identification of eight fusion genes that were antibody (3E9, Sigma-Aldrich, USA). The sections were evaluated for the intensity of successfully validated by orthogonal methods, of which two were in-frame (SLC2A1- reactivity (0-3) and the percentage of reactive cells; and an H-score was derived from FAF1 and BCAS4-AURKA). Forced expression of these in-frame fusion genes resulted the product of these measurements. Cases were categorized as having negative (score=0) in increased proliferation in a subset of ER-positive breast cancer cell lines. No recurrent fusion genes were identified in the cases analysed.RAE1 (2%), CDK12 (2%) and LASP1 58A ANNUAL MEETING ABSTRACTS (1%) were identified in external datasets as recurrent partners in out-of-frame fusion above were detected in 14 cases (12.8%). Six cases can be attributed to florid biopsy genes. RNAi silencing of CDK12 and LASP1 (out-of-frame fusion partners) resulted site changes, or florid LCIS in areas of adenosis. In 8 cases a definite explanation in a significant increase in cell viability. could not be found. Conclusions: Our findings suggest that MPCs are not underpinned by a highly recurrent fusion gene. Although present at low frequency or seemingly private genetic events, 234 Characterisation of the Genetic Heterogeneity of Invasive some of the fusion transcripts found in MPCs may play a role in tumour cell growth Breast Cancers with Heterogeneous HER2 Overexpression and Gene and viability. Amplification CKY Ng, A Gauthier, PM Wilkerson, MB Lambros, L Arnoud, M Lacroix-Triki, F 232 Should Sentinel Lymph Node (SLN) Biopsy Be Performed in Penault-Llorca, MC Baranzelli, X Sastre-Garau, CJ Lord, M Zvelebil, C Mitsopoulos, Microinvasive Breast Carcinomas (MIC)? A Ashworth, R Natrajan, B Weigelt, O Delattre, P Cottu, JS Reis-Filho, A Vincent- A Nayak, SE Frost, CS Nagi, IJ Bleiweiss, S Jaffer. Mount Sinai School of Medicine, Salomon. Memorial Sloan-Kettering Cancer Center, New York, NY; Institute of Cancer New York, NY. Research, London, United Kingdom; Institut Curie, Paris, France; Centre Georges Background: The role of SLN biopsy in patients with MIC is questionable. To answer François Leclerc, Dijon, France; Institut Claudius Regaud, Toulouse, France; Centre this question, we examined the incidence of lymph node (LN) metastasis in patients Jean Perrin, Clermont Ferrand, France; Centre Oscar Lambret, Lille, France. diagnosed with MIC at our institution. Background: In a rare subgroup of breast cancers defined as HER2-positive by Design: A retrospective review of pathology database between 1994 and 2012 identified immunohistochemistry and/ or in situ hybridisation (ISH) according the ASCO/ CAP patients with MIC who underwent surgery at our institution. MIC was defined as per guidelines, HER2 overexpression and gene amplification are restricted to a subset of current AJCC definition including only patients with invasive foci <1mm in size. Clinical cancer cells (i.e. >30% but <100%). The aim of this study was to define the repertoire and pathologic variables were recorded including the SLN/axillary lymph node (ALN) of copy number aberrations and somatic mutations in the HER2-positive and HER2- status. Five additional levels and 2 immunohistochemical stains (CAM5.2 and AE1/ negative components of cases with heterogeneous HER2 overexpression and gene AE3) for cytokeratins (CK) were routinely done on all SLNs. LN metastasis were amplification. classified per current AJCC classification. Follow up data were recorded whenever Design: HER2-positive breast cancers with >30% but <100% of cells displaying HER2 available. overexpression were retrieved from the authors’ institutions, and the HER2 status was Results: Of 7000 patients operated for invasive breast carcinoma between 1994 and re-assessed using immunohistochemistry and ISH. The HER2-positive and HER2- 2012, 99 (1.4%) were classified as MIC. Median age was 56yrs (range, 31 to 83yrs). negative components from each case were microdissected from tissue sections stained Twenty eight patients underwent mastectomy and 71 had lumpectomy. Eighty one (82%) with an anti-HER2 antibody. DNA samples extracted from both components of each patients had SLN/ALN staging. Sixty four (65%) patients had SLN biopsy (avg. no. case were subjected to microarray-based comparative genomic hybridisation (aCGH). of SLN, 2). Seven (8.6%) of 81 cases showed isolated tumor (ITC)/epithelial cells in The HER2-positive and HER2-negative components of two cases were also subjected SLN. Three of these 7 cases showed reactive changes in LN (such as giant cells and to exome sequencing on a HiSeq2000 (60x) and the results were validated using an hemosiderin laden macrophages), papillary lesion in breast with or without displaced orthogonal method (i.e. Ion Torrent sequencer, >300x). epithelial cells within granulation tissue of biopsy site, or immunohistochemical (ER, Results: The HER2-positive and HER2-negative components of each case displayed PR and HER-2) discordance between the primary tumor in breast and LN, consistent gene copy number aberrations in common; however, genomic changes restricted to with iatrogenically displaced epithelial cells rather than true metastasis. The remaining either the HER2-positive or HER2-negative component of each case, in addition to 4 cases included 2 cases, each with a single CK+ cell in the subcapsular sinus detected 17q12 amplification encompassing the HER2 gene locus, were observed in ten of by IHC only; and 2 cases with ITCs singly and in small clusters (<20 cells per cross the twelve cases. Massively parallel sequencing of two cases demonstrated that the section) detected by H&E and IHC. The exact nature of CK+ cells in the former 2 cases HER2-positive and HER2-negative components from each case harboured somatic could not be determined and may still represent iatrogenically displaced cells. In the mutations in common. The HER2-negative components of the cases analysed harboured end, there were only 2 cases (2.5%) with minimal tumor volume (ITC) in axilla. Three somatic mutations of HER2 and ROR1, tyrosine kinase receptors, and PTTG1IP, a of these 4 cases had additional ALNs excised which were negative for tumor cells. proto-oncogene with putative ER elements, which were not present in the HER2- Follow up data did not reveal axillary recurrence in our cases. positive components. Conclusions: Our data suggests a very low incidence (2.5%) of SLN involvement Conclusions: Although the HER2-positive and HER2-negative components of breast only in the form of ITCs, in patients with MIC. None of our cases showed micro or cancers with heterogeneous HER2 gene amplification are clonally related, additional macrometastasis. We recommend reassessment of routine practice of SLN biopsy in genetic aberrations restricted to either the HER2-positive or HER2-negative component patients with MIC. of each case were observed, and may constitute potential drivers in the absence of HER2 overexpression/ gene amplification. 233 Correlation between MRI Findings and Gross and Microscopic Pathology in Total Mastectomy Cases: A Review of 109 Cases 235 Are Breast Cancer Metastases Enriched for Stem Cells? KG Neill, J Liu, B Cliff, B O’Hea, M Singh, C Tornos. Stony Brook University Hospital, DN Nguyen, A Subhawong, CG Kleer, P Argani, A Cimino-Mathews. Johns Hopkins Stony Brook, NY. Hospital, Baltimore, MD; University of Michigan, Ann Arbor, MI. Background: Bilateral breast MRI’s are often done to evaluate the extent of disease Background: The existence of tumor-initiating cells is one hypothesis for tumor preoperatively in patients recently diagnosed with invasive carcinoma or DCIS. Based initiation and progression. The presence of a stem cell population might imply a major on the MRI findings, total mastectomy may be recommended if there is multifocal/ shift in the therapeutic approach to cancer, as current therapies do not target stem multicentric disease, or if the main lesion is large compared to the breast size. cells. This is particularly relevant in breast carcinoma, where no targeted therapies are Design: We reviewed 109 cases of total mastectomies performed at our institution, available for triple negative carcinomas. In addition, few therapeutic options remain in which MRI’s were completed and available before the surgery. We compared the for metastatic breast carcinoma resistant to initial therapy. Here, we evaluate the stem number of biopsy proven malignant lesions and highly suspicious lesions found on MRI, cell profile of matched primary (PBC) and metastatic (MBC) breast carcinoma sampled including size, location and MRI diagnosis, with the findings on gross and microscopic at surgery or at autopsy from 31 different patients. examination of the same areas. A copy of the MRI report was available in all cases at Design: Tissue microarrays (TMAs) from matched PBCs and surgically-resected MBCs the time of grossing to establish correlation between MRI and gross findings. Samples (n=15), as well as from matched PBCs and multiple MBCs sampled at rapid autopsies were taken from all areas of concern on MRI to correlate with histologic diagnosis. (n=16), were subjected to immunohistochemistry for ALDH1, EZH2, and CD44/ Patients treated with neoadjuvant chemotherapy were excluded. CD24. Tumor cell cytoplasmic ALDH1 and nuclear EZH2 were scored by percentage Results: In 9 cases the size of the carcinoma was overestimated by the MRI by more labeling and intensity, with >1% considered positive. Percentage CD44+/CD24- cells than 1 cm (Table). In two cases the size of the carcinoma was underestimated by the (pink membranous staining) was recorded, with >1% cells considered positive. A 20% MRI by more than 1 cm (1.2 cm to 2.5 cm invasive ductal carcinoma, and 2.4 cm to 3.9 change in the percentage labeling for a given stem cell marker between the PBC and cm IDC). In 3 cases the MRI did not detect any carcinoma, only biopsy site changes, MBC was considered an increase or decrease. Carcinomas were subdivided into the but there was invasive carcinoma (one case had 0.9 cm invasive lobular carcinoma, following subtypes: luminal (ER+/PR+/Her2-), luminal loss (ER/PR loss from PBC to one case had 2 foci of IDC 1.7 cm and 0.3 cm, and one case had 3 foci of IDC 1 cm, MBC), Her2 (ER-/PR-/Her2+), and triple negative (TNC) (ER-/PR-/Her2-). 0.7 cm, and 0.15 cm). Results: Overall, ALDH1 labeled 8/31 (26%) PBC of MBC; EZH2 labeled all PBC Cases of Breast Carcinoma Overestimated by MRI and MBC; and there were CD44+/CD24- cells in 28/31 (90%) PBC or MBC. Forty-two Case MRI MRI MRI Pathology Pathology Pathology percent MBC showed no change in stem cell marker expression compared to the matched #Lesions SIZE DIAGNOSIS #LESIONS SIZE DIAGNOSIS OBC; 23% MBC showed a decrease; 35% MBC showed an increase (see table). There 1 1 8.3cm DCIS 1 5.4cm DCIS was a trend towards increased stem cell marker expression in the surgically resected 2.6cm DCIS + 2 1 8cm Inv + DCIS 1 0.2cm IDC MBC, with a decrease in the autopsy MBC. 3 2 3.5cm, 2.9cm Invasive both 1 3.1cm IDC Change in stem cell profile between matched PBC and MBC 6cm DCIS + 4 1 1 3.5cm IDC Decrease in 1 Decrease in 2 Increase in 1 Increase in 2 3.7cm inv Tumor type (n) No change stem cell stem cell stem cell stem cell 5 Multifocal 7cm area Inv lobular 2 1.3cm + 0.8cm ILC + LCIS marker markers marker markers 6 1 3.7cm Invasive 1 1.6cm ILC + LCIS Luminal (13) 8 0 0 0 5 IDC+ florid 7 2 4.8cm + 1cm Both invasive 1 2.8cm Luminal loss (4) 2 1 1 0 0 Bx site Her-2 (3) 0 2 0 0 1 8 1 4.8cm Invasive 1 3.2cm IDC TNC (11) 3 3 0 1 4 9 1 4.9cm Invasive 1 3.5cm IDC Total (31) 13/31 (42%) 6/31 (19%) 1/31 (3%) 1/31 (3%) 10/31 (32%) Conclusions: MRI is an accurate method of detecting and measuring foci of invasive carcinoma and ductal carcinoma in situ. In this series “major discrepancies” as defined ANNUAL MEETING ABSTRACTS 59A Conclusions: Stem cell markers are frequently expressed in PBC and MBC, however 238 Utilizing the Oncotype DX Recurrence Score in the Adjuvant the degree of labeling may increase or decrease in the MBC compared to the matched Treatment Management of Node-Positive, Early-Stage Breast Cancer PBC. The trend of increased stem cell marker expression in early MBC versus end-stage Patients MBC may reflect selection of this population in initial MBC, or alternatively reflect MT Nguyen, Z Chen, T D’Alfonso, A Stessin, H Nagar, M Hayes, SJ Shin. Weill Cornell fixation artifact in the autopsy MBC. Medical Center, New York, NY; New York Presbyterian, Weill Cornell Medical Center, New York, NY. 236 How Much Is Oncotype DX Recurrence Score Impacting the Background: The 21 gene-RT-PCR assay Oncotype DX (Genomic Health) stratifies Adjuvant Treatment Management of Node-Negative Early-Stage Breast patients into low, intermediate, and high risk groups for developing systemic disease Cancer Patients? recurrence. While this assay has been used in the adjuvant treatment planning of node- MT Nguyen, Z Chen, T D’Alfonso, A Stessin, H Nagar, M Hayes, SJ Shin. Weill Cornell negative, ER/PR positive, HER-2 negative, early-stage breast cancer patients, clinicians Medical Center, New York, NY; New York Presbyterian, Weill Cornell Medical Center, have begun to order this test in clinically similar breast cancer patients who are node- New York, NY. positive. Recent studies have shown that the utilization of this assay in the setting of Background: Oncotype DX (Genomic Health) is a commercially available 21 gene node-negative early-stage breast cancer has led to a decrease in adjuvant chemotherapy RT-PCR assay that is widely used to determine the risk of distant disease recurrence use in these patients. However, whether this trend is also true when utilized in node- and the benefit of adjuvant chemotherapy in patients with early-stage breast cancer. We positive breast cancer patients is unknown and represents the aim of the current study. sought to investigate the clinical impact of Oncotype DX testing compared to traditional Design: 543 breast cancer patients that had Oncotype DX testing performed as part clinicopathologic features in the planning of adjuvant treatment (chemotherapy) in of their routine clinical care between 2005-2012 at a single large academic medical node-negative, ER/PR positive, Her2neu-negative early-stage breast cancer patients. center were retrospectively identified. Age, tumor size, tumor grade, nodal status, ER/ Design: A single-center retrospective study (2005-2012) was conducted on 543 patients PR status, Her2neu status, Ki-67 index, Oncotype DX recurrence score (ODxRS), and who had Oncotype DX testing performed as part of their routine clinical care. Age, treatment plan were obtained by electronic medical record for each patient. Fisher’s exact tumor size, tumor type, tumor grade, lymphovascular invasion, nodal status, ER/PR test was performed to determine the association between ODxRS and the utilization status, Her2neu status by IHC and FISH, proliferation index (Ki-67), Oncotype DX of adjuvant chemotherapy. recurrence score (ODxRS), and treatment were obtained by electronic medical record Results: 42 of 543 (8%) patients had node-positive, ER/PR positive, HER-2 negative, for each patient. Logistic regression was performed to determine the association early-stage breast cancer. Nodal positivity was defined as metastasis > 2mm in size. between age, tumor size, tumor grade, present of lymphovascular invasion and lymph 23 of 42 (55%) patients did not receive adjuvant chemotherapy. Of these 23 patients, node micrometastasis, Ki-67 index, and ODxRS; and the utilization of adjuvant 16 had a low ODxRS while the remaining 7 had an intermediate ODxRS. 19 patients chemotherapy in these patients. received adjuvant chemotherapy and of these, 8 had a low ODxRS, 8 had an intermediate Results: 313 of 543 patients were identified to have node-negative, ER/PR positive, ODxRS and 3 had a high ODxRS. No significant association was identified between Her2neu-negative early-stage invasive breast cancer. Among these 313 patients, 175 ODxRX and adjuvant chemotherapy utilization (p=0.067). (55.9%) had low risk ODxRS, 126 (40%) intermediate risk ODxRS, and 12 (3.8%) had Conclusions: When Oncotype DX testing is performed, the resulting ODxRS does high risk ODxRS. 68 of 313 (21.7%) patients received adjuvant chemotherapy of which not appear to significantly influence the adjuvant treatment management of this subset 12 of 68 (17.6%) patients had low risk ODxRS, 45 of 68 (65.2%) had intermediate of patients. risk ODxRS, and 11 of 68 (11.3%) had high risk ODxRS. The remaining 245 (78.3%) patients did not receive adjuvant chemotherapy of which 163 of 245 (66.5%) had low 239 ARD1 Expression Correlates with Favorable Prognostic risk ODxRS, 81 of 245 (33.1%) had intermediate risk ODxRS, and 1 of 245 (0.4%) Variables in Invasive Mammary Carcinoma (MC) had high risk ODxRS. A statistically significant association between high risk ODxRS G Niu, BVS Kallakury, AB Boguniewicz, CB Sheehan, CE Sheehan, JS Ross. Albany and adjuvant chemotherapy utilization was identified (OR 84.6; 95% CI 24.5, 292; Medical College, Albany, NY; Georgetown University Hospital, Washington, DC. p-value <0.001) and this association was more significant than any association using Background: The arrest defective 1 (ARD1) protein is an acetyltransferase linked to clinicopathologic parameters. proliferation and apoptosis in mammalian cells and recently associated with several Conclusions: Our results indicate that when Oncotype DX is performed, high risk human tumors. Although ARD1 expression has been previously demonstrated in MC ODxRS significantly favored adjuvant chemotherapy utilization more than known by IHC, the prognostic significance of ARD1 in clinical MC specimens has not been clinicopathologic factors in the management of node-negative, ER/PR positive, previously studied. Her2neu-negative early-stage breast cancer patients. Design: Formalin-fixed, paraffin-embedded tissue sections from 115 cases MC [76 ductal carcinomas (IDC) and 39 lobular carcinomas (ILC)] were immunostained 237 Does Clinicopathologic Features Influence the Use of Adjuvant by automated method (Ventana Medical Systems Inc., Tucson, AZ) using mouse Chemotherapy in Early-Stage Breast Cancer Patients Who Have an monoclonal ARD1 antibody (Abnova, Jhongli, Taiwan). Nuclear and cytoplasmic Oncotype DX Intermediate Risk Score? immunoreactivity were scored based on intensity (weak, moderate, intense) and MT Nguyen, Z Chen, T D’Alfonso, A Stessin, H Nagar, M Hayes, SJ Shin. Weill Cornell percentage of positive cells (focal <= 10%, regional 11-50%, diffuse >50%) in tumor Medical Center, New York, NY; New York Presbyterian, Weill Cornell Medical Center, (T), in situ disease (I) when present, and adjacent benign epithelium (B). An additive New York, NY. index was calculated and cases were assessed as T=0/B=0, T=B, T>B, T < B; and Background: Oncotype DX (Genomic Health) is a commercially available 21 gene I=0/B=0, I=B, I > B, I < B). Results were correlated with clinicopathologic variables. RT-PCR assay that is widely used to determine the risk of distant recurrence and the Results: Nuclear immunoreactivity was noted in 97/115 (84%) cases overall with T < benefit of adjuvant chemotherapy in patients with early stage breast cancer. This assay B [73/115 (63%)], T=B [24/115 (21%)], T=0/B=0 [18/115 (16%)]; I=B [27/54 (50%)], calculates a recurrence score (RS) that is stratified as low, intermediate or high. Patients IB [1/54 (2%)] cases. Loss of nuclear with low RS have been shown to gain little to no benefit from adjuvant chemotherapy ARD1 immunoreactivity correlated overall with tumor type [T < B in 72% ILC vs while those with a high RS derive large benefits from adjuvant therapy. Currently, it 59% IDC, T=B in 8% ILC vs 28% IDC, T=0/B=0 in 21% ILC vs 13% IDC p=0.04]; is unclear whether patients with an intermediate RS (18-30) benefit from adjuvant overall with tumor stage [T < B in 65% early vs 56% advanced, T=B in 28% early vs chemotherapy. The objective of this study was to determine if routinely available 13% advanced, T=0/B=0 in 7% early vs 31% advanced, p=0.003] and within the IDC clinicopathologic parameters impact the decision for adjuvant chemotherapy utilization subgroup [T < B in 60% early vs 54% advanced, T=B in 36% early vs 17% advanced, in breast cancer patients found to have an intermediate RS. T=0/B=0 in 4% early vs 29% advanced, p=0.007]; and overall with ER status [T < B Design: A single-center retrospective study (2005-2012) was conducted on 543 in 69% ER+ vs 54% ER-, T=B in 22% ER+ vs 20% ER-, T=0/B=0 in 9% ER+ vs 27% patients with invasive breast carcinoma who had Oncotype DX testing performed ER-, p=0.046]. Within the 54/115 (47%) cases with in situ disease present, in all Iglands in 100% and treatment were obtained by electronic medical record for each patient. Logistic cases with T=B 56/115 (49%), T>B 55/115 (48%), T < B 4/115 (3%); I=B 42/54 (78%) regression was performed to determine the association between clinicopathologic and I>B 12/54 (22%) cases with no significant correlations. On multivariate analysis, parameters (age, tumor size, tumor grade, presence of lymphovascular invasion, lymph disease recurrence (p=0.002) independently predicted overall survival. node micrometastasis, and Ki-67 index) and the utilization of adjuvant chemotherapy. Conclusions: Loss of nuclear ARD1 expression is associated with favorable prognostic Results: 154 of 543 (28.3%) early-stage breast cancer patients (ER/PR positive, Her2neu variables in MC including ILC subtype, early pathologic stage and ER+ status. Further negative, node negative) was found to have an intermediate RS by Oncotype DX testing. study of ARD1 in MC appears warranted. 56 of 154 (36.4%) received adjuvant chemotherapy while 98 of 154 (63.4%) did not. Twelve patients refused adjuvant chemotherapy (recommended) and thus, excluded 240 Role of Androgen Receptor (AR) as a Prognostic Factor in from the analysis. A statistically significant association between high tumor grade and Primary Breast Cancer (BC) the utilization of adjuvant chemotherapy was identified (OR 10.2, 95% CI 2.97, 35.1; S Nofech-Mozes, C Parra-Herran, R Dent, C Villarreal, MA Quintayo, JMS Bartlett, p-value <0.0001). Associations of remaining clinicopathologic parameters with the J Starczynski, P Sun, S Narod, W Hanna. University of Toronto, Toronto, Canada; utilization of adjuvant chemotherapy were not significant. Sunnybrook Health Sciences Centre, Toronto, Canada; Ontario Institute for Cancer Conclusions: Our results suggest that high tumor grade is a clinicopathologic feature Research, Toronto, Canada; Women’s College Hospital, Toronto, Canada. that significantly favored the use of adjuvant chemotherapy in early stage breast cancer Background: There is evidence that androgen signaling pathway may play a role in patients classified in the intermediate risk group by Oncotype DX testing. breast carcinogenesis through the activation of estrogen-responsive genes. In BC cell lines, when lacking estrogens, androgens stimulate tumor growth by binding to estrogen receptor (ERα). When estrogens are present, however, androgens act as their antagonists and inhibit the growth of the tumor; this is probably mediated by AR and can be blocked by antiandrogens. Our aim is to determine the frequency of AR expression in BC and 60A ANNUAL MEETING ABSTRACTS correlate it with established clinicopathological prognostic factors and outcome. Conclusions: This sysetmatic analysis of the breast cancer interactome identified 10 Design: We studied 1008 primary BC from the Henrietta Banting database, with a mean novel candidate epithelial-stromal cross-talk partners, including 3 pairs of markers follow up of 9.1 yrs, treated in a single institution. Immunohistochemistry for AR, ER, associated with breast cancer survival. These findings provide new insights into the progesterone receptor (PR) and HER2 was applied to 1 mm core tissue microarrays biology of the breast cancer microenvironment and represent novel candidate therapeutic in triplicates. Interpretation of AR was performed using the Ariol platform (Leica targets for manipulating epithelilal-stromal interactions in breast cancer treatment. Biosystems 2012) which produced a histoscore [(% cells staining 1+)x1 +(%staining 2+)x2 + (%staining 3+)x3 for a total ranging from 0 to 300]. 152 cases were also scored 243 HSET Expression in Triple Negative and Non-Triple Negative by two pathologists and manual and automated readings were compared. For analysis, Breast Cancers cases were divided in AR-rich (histoscore ≥200) and AR-poor (histoscore <200). The GM Oprea-Ilies, V Pannu, Y Liu, D Nickleach, SE Pambuccian, R Aneja. Emory association between AR expression and standard prognostic factors (age, tumor size, University, Atlanta, GA; Georgia State University, Atlanta, GA; Winship Cancer nodal status, ER, PR, and HER2 status) was examined. Relative risk of death was Institute, Atlanta, GA; Loyola University Chicago, Maywood, IL. calculated for age, tumor size and AR expression in univariate and multivariate analyses. Background: Chromosomal instability arising from centrosome amplification and Estimates were considered statistically significant for two-tailed values ofP <0.05. mitotic aberrations has long been associated with tumorigenesis. Although amplified Results: The interclass correlation coefficient for the manual vs the automated reading centrosomes would be expected to compromise cell viability by compelling cells to was 0.9195. Among 1008 cases, 256 (25.4%) were AR-poor and 752 were (74.6%) form multipolar spindles resulting in death-inducing aneuploidy, cancer cells turn AR-rich. AR-rich status was significantly associated with older age (57 vs 52 years, things in their favor by employing clever mechanisms to suppress multipolarity by p<0.0001), smaller tumor size (2.54 vs 2.96 cm, p=0.001), ER+ (90.4 vs 45.5%, clustering their supernumerary centrosomes. As a result, cancer cells, with the aid of p<0.0001) and PR+ (72.4 vs 26.8%, p<0.0001), but not with nodal status (p=0.67) or clustering mechanisms, maintain bipolar spindle phenotypes that are accompanied HER2+ (p=0.53). AR rich status was associated with better BC specific survival (75% by low grade aneuploidy, an edge to their survival. HSET is a kinesin-like minus-end vs 61.3%, p<0.0001). Multivariate Cox-regression revealed a relative risk of 0.67 directed motor and centrosome clustering molecule/factor that plays an important role (95%CI 0.52-0.85, p=0.001) for AR expression. in key cellular functions, including mitosis. The aim of the study was to assess the Conclusions: AR is frequently expressed in our population. A histoscore cut-off value HSET frequency in TNT breast cancers (BC) as compared with BC expressing ER/ of >200 demonstrated significant association with multiple prognostic clinicopathologic PR and/or HER (non-TNT). factors and BC specific survival.Our study indicates that high AR expression is a Design: BC diagnosed during a 7-year period were reviewed. The BC markers ER, PR, prognostic factor in BC. and Her-2 scored by the new CAP standards were used to classify the tumors into TNT and non-TNT. Tissue microarrays were stained with an anti-Kinesin-related protein 241 Relationship between the Insulator Factors and the Methylation HSET monoclonal antibody (a gift from Claire Walczak, Indiana University). HSET Status in Breast Neoplasia expression was evaluated separately in nuclei and cytoplasm and was semiquantitavely K Nomoto, J Imura, K Abe, Y Uchida, T Nakajima, S Miwa, S Hayashi, K Tsuneyama. scored as positive on a scale from 1-3. Tumors with more than 1+ staining in >10% Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, of nuclei and more than 1+ and cytoplasmic staining in >50% of tumor cells were Toyama, Japan; Ibaraki Prefectural Central Hospital, Kasama, Ibaraki, Japan. considered positive. Background: DNA methylation is related in each process of multistep carcinoma Results: Of the 193 BC from 143 African American (AA) and 50 non-African American genesis and is regulated by a complex machinery that includes DNA methytransferases (non-AA) women aged 24-90, 120 were TNT and 73 were non-TNT. HSET showed (DNMTs). Another regulated factor includes CCCTC-binding factor (CTCF). CTCF cytoplasmic positivity in 65% TNT vs 27% non-TNT, and in 92% African-American is a chromatin insulator with a conserved zing finger phosphoprotein that shows (AA) vs 8% non-AA patients. HSET showed nuclear positivity in 63% TNT vs 31% methylation-sensitive binding to near a promoter region. We studied the relationship non-TNT, and in 91% AA vs 9% non-AA patients. All differences were staistically between methylation status and the expression of DNMT and CTCF. significant (p<0.05). There were no statically differences in age, tumor size and lymph Design: Surgically resected tumors and corresponding normal tissues were collected node metastases between tumors that showed nuclear or cytoplasmic positivity for from 91 patients diagnosed with 30 cases of invasive ductal carcinoma: Ca, 21 cases HSET and tumors that did not. of intraductal papilloma: Pap, 31 cases of fibroadenoma: FA and 10 cases of fibrocystic Conclusions: 1. HSET shows statically significantly higher nuclear and cytoplasmic disease: FD. CTCF-1, DNMT-1 and -3b were examined immunohistochemically. MSP expression in TNT vs non-TNT. method was used for the search of methylation status. 2. HSET is expressed in a significantly higher percentage of AA vs non-AA women. Results: We observed weak CTCF-1 positive reaction in FD, nuclear reaction in FA and 3. Further studies may be useful to determine if HSET represents a useful therapeutic Pap, strong cytoplasmic reaction in Ca. DNMT-1 and -3b showed a tendency to strong target. positive reaction in comparison with FA and Pap in Ca. The cases of Ca that CTCF-1 and DNMT-3b expressed showed the hypermethylation status. 244 How Luminal Are Triple Negative Breast Cancers? A Cytokeratin Conclusions: The mechanism of methylation and insulator might participate mutually Co-Expression Study in the multistep carcinogenesis of the breast tissue. In this process, it is suggested that GM Oprea-Ilies, Y Liu, D Nickleach, LA Mclendon, SE Pambuccian. Emory University, CTCF moves to cytoplasm from nucleus by methylation. Atlanta, GA; Loyola University, Maywood, IL; Winship Cancer Insitute, Atlanta, GA. Background: Triple negative breast cancers (BC)(TNT), defined by the lack of 242 The Breast Cancer Epithelial-Stromal Interactome expression/amplification of ER, PR and Her2, and basal phenotype (BP) BC, defined E-Y Oh, NM Knobluch, AH Beck. Beth Israel Deaconess Medical Center & Harvard molecularly, only partially overlap. Although BP BC frequently express “basal” Medical School, Boston, MA. cytokeratins, such as CK5, in addition to EGFR, p-cadherin and p63, while BC Background: The significance of tumor microenvironment in breast cancer expressing ER, PR or Her2 (“non-TNT”) usually express “luminal” cytokeratins such tumorigenesis and invasion is increasingly recognized. It is, however, largely unknown as CK7, there is significant overlap in the expression of these markers. The aim of the how tumor epithelial cells interact with the stromal cells to make their microenvironment study was to compare the frequency of “luminal” CK7 and “basal” CK5 expression in more advantageous for tumor growth and invasion. Our aim is to discover epithelial- TNT and non-TNT using a dual staining approach. stromal cross-talk partners in breast cancer. Design: BC diagnosed during a 7-year period were reviewed and divided into TNT Design: Using the Gene Expression Omnibus, we identified 2 LCM expression profiling and non-TNT based on ER, PR, and Her2 expression/amplification, scored by the new datasets. Study 1 consisted of 5 samples captured from normal epithelium (NE1) and CAP guidelines. Tissue microarrays were constructed with two 1 mm representative stroma (NS1), and 28 samples captured from breast cancer epithelium (BrE1) and stroma cores from each BC and were stained, using a sequential staining protocol, on a Leica (BrS1). Study 2 consisted of 14 samples from each of: normal epithelium (NE2), normal Bond III staining platform using CK5 (Clone XM26, Novocastra)/brown chromogen stroma (NS2), breast cancer epithelium (BrE2), and breast cancer stroma (BrS2). We and CK7 (CK7 Clone RN7, Novocastra)/red chromogen. This allowed the evaluation performed a genome-wide epithelial-stromal expression association study to identify of the expression/coexpression of CK5 and CK7 in individual tumor cells. pairs of genes showing highly correlated expression (either positive or negative) between epithelial and stromal compartments. This analysis was implemented using the MatrixEQTL package in R. We performed 4 sets of analyses (NE1vs NS1, NE2 vs NS2, BrE1 vs BrS1, and BrE2 vs BrE2) and identified epithelial-stromal cross-talk pairs consistently identified as showing highly correlated expression in breast cancer epithelial and stromal compartments but not in the normal breast epithelial and stromal compartments. Results: We identified 10 breast cancer epithelial-stromal cross-talk pairs at an FDR of < 0.02%. 9 of the interactions indicated a positive association between epithelial and stromal expression levels, and 1 of the pairs indicated an inverse association. For genes in these 10 pairs, we assessed their association with survival using a publicly available breast cancer expression profiling meta-dataset of 2898 breast cancer patients with clinical follow-up. This analysis shows that for the pairs NPM1(epi)-FOSL2(str), S100A8 (epi)-S100A8(str), and CYP4F8(epi)-PAX1(str), both members of each of the gene pairs are significantly associated with survival (all P’s < 0.02), further suggesting that these epithelial-stromal cross-talk partners may play important roles in breast cancer invasion and progression. ANNUAL MEETING ABSTRACTS 61A Design: This is a retrospective study of 206 invasive breast carcinoma cases in which HER2 testing was done by using 6 different anti-HER2 antibodies on immunohistochemistry(IHC) and results were interpreted by three specialist breast pathologists and compared to FISH as a gold standard. Results: Comparison of HER2 status by IHC using 6 different antibodies, US FDA approved HercepTest had the highest sensitivity, specificity, positive and negative predictive values with lowest false positive (11.7%) and false negative (1%) rates. Considering only Score 3+ as positive, concordance rates with FISH were Hercep (93.29%) >Pathway Ventana (90.6%) >Biocare (89.5%) >NCL CB11 (89.4%) >Biogenex (89.13%) >Dako A0485 (88.6%). With all six antibodies, absence of staining was highly predictive of non-amplified cases (NPV in the range of 92.2%-98.7%) and strong, complete membrane staining was highly predictive of amplified cases (PPV in the range of 84.9%-88.8%). Among the six antibodies, highest concordance of HercepTest was seen with Dako Cerb2 - A0485 (Kappa value 0.82) and lowest concordance was seen with Biocare Cerb2 antibody (Kappa value 0.76). There was almost perfect agreement between Hercep, Dako A0485, Biogenex antibodies and substantial agreement between antibodies Biocare, NCL CB11 and Pathway Ventana. Substantial agreement was noted among the three pathologists for IHC interpretation (kappa value: 0.6-0.79). Pathologists involved in both, routine IHC and FISH reporting of HER2 detection had maximum agreement especially with Pathway Ventana antibody. Apparent causes of inter-observer variations in IHC interpretation in the present study are cytoplasmic staining with membrane accentuation, mimicking the membrane positivity, edge artifact especially IHC results were scored semiquantitatively, based on the percentage of tumor cells in biopsy samples, poor fixation and technical processing and heterogeneity of staining. staining. Conclusions: We conclude theranostic IHC viz. HER2 which has implication for Results: 193 BC from women aged 24-90 years, 120 TNT and 73 non-TNT. were targeted therapy in breast cancer needs to be performed with clinically validated kits included. CK7 was expressed in the vast majority of TNT and non-TNT (91.09% along with stringent quality control. Thus there is no economical solution for Her2neu vs. 84.29%, p = 0.173). CK5 was expressed in 11% of non-TNT and 59% of TNT testing and none of cheaper antibodies compred as well as the FDA approved antibodies. (p<0.001) and coexpression of CK5 and CK7 was present in all of the latter. While the expression of CK7 tended to be uniform in all tumor cells, the expression of CK5 was more heterogeneous and almost all cells expressing CK5 also expressed CK7. 247 Hormonal and Her-2 Receptor Status in DCIS and Concurrent No expression of either CK5 or CK7 was present in a minority of TNT and non-TNT Invasive Carcinoma (16% vs. 2%, p<0.001) DM Pandya, CD Patel, S Zee, J Liu, C Tornos. Stony Brook University Medical Center, Conclusions: While “luminal” CK7 was expressed in both TNT and non-TNT, “basal” Stony Brook, NY. CK5 expression and CK5/CK7 co-expression was found in a significantly higher % Background: Information on hormone status is useful in determining prognosis of TNT. The fact that BC differ largely in the acquisition of “basality” and not in the and therapy of invasive breast carcinomas. There are instances in which the amount loss of “luminality” suggests that they may represent a form of incomplete metaplasia, of invasive tumor is inadequate to render accurate results. The aim of this study with acquisition of CK5 expression. was to compare the ER, PR, and Her2/neu immunohistochemistry of invasive carcinomas and its concurrent DCIS present at the immediate periphery, to determine whether the immunoprofile is similar enough to extrapolate and apply the DCIS 245 Analysis of FOXP3 mRNA Expression in Subtypes of Breast immunohistochemical profile towards therapy of the invasive tumor. Carcinoma and Breast Cancer Cell Lines Design: We reviewed the H&E slides and ER, PR and Her-2 immunostains from 50 F Ortiz-Martinez, A Perez-Balaguer, JM Sempere-Ortells, M Rodrigo, J Ponce, P cases of invasive ductal carcinomas and concurrent DCIS in the same paraffin block. Martinez-Peinado, E Lerma, J Sanchez-Paya, FI Aranda, G Peiro. University General The histological characteristics, and the percentage and intensity of the immunostaining Hospital, Alicante, Spain; University of Alicante, Alicante, Spain; Hospital de la Santa of both the in situ and invasive component were reviewed and compared by the same Creu i Sant Pau, Barcelona, Spain. pathologist. Background: The X-linked gene FOXP3 (Xp11.23) plays a key role in the immune Results: The ER was concordant in 47 cases (94%), positive in 40, and negative in suppressive function of regulatory T-cells (Tregs). More recently, it has been identified 7 cases. The PR was concordant in 42 cases (84%), positive in 35 and negative in 7 as a tumor suppressor gene that regulates tumor development. Here, we analyzed the cases. The Her-2 was concordant in 48 cases (96%), negative in 42 and positive in 6 expression of FOXP3 mRNA in breast cancer (BC) cell lines representing different cases. The three markers were concordant in 40 cases (80%). ER was negative in one phenotypes together with a series of BC patients. invasive tumor (grade 2) with 30% positivity in the DCIS (high nuclear grade without Design: BC cell lines consisted of MCF-7, T-47D, BT-474, SK-BR-3, MDA-MB-231, necrosis). Two cases had ER positive in invasive carcinoma but negative in DCIS. MDA-MB-468 and a normal epithelial breast cell line, 184A1. Quantitative RT-PCR was The invasive carcinoma in both cases was moderately differentiated with DCIS being performed using TaqMan® Assays. Mammary tissue and 184A1 were used as calibrators either intermediate or high grade with necrosis. Two cases of moderately differentiated for tumor samples and BC cells, respectively; PUM1 and ACTB were the reference invasive carcinoma where PR was negative, while the DCIS was positive in both cases, ∆∆ genes. Expression was determined using the CT method and was expressed as fold with 60-80% positivity, low to intermediate nuclear grade with necrosis. Six cases were change (FC). 189 BC tumors were classified immunohistochemically into Luminal positive for PR in the invasive component (grade 2) but negative in the DCIS (low to (32%), HER2+ (36%) or TN/BL (32%). intermediate grade). Two of the fifty cases showed discordance in Her2/neu staining. Results: FOXP3 mRNA expression was higher in Luminal subtype cell lines (MCF-7 The invasive tumor was negative for Her-2 in both cases (one grade 1 and one grade FC=4.87; T-47D FC=12.11) and in Luminal/HER2+ cells (BT-474 FC=4.77) compared 2) while the DCIS was high grade with necrosis and positive for Her-2. with HER2+ cells (SK-BR-3 FC=2.83) and TN/BL cells (MDA-MB-231 FC=0.11; Conclusions: 80% of the tumors were concordant for all three markers. However, 20% MDA-MB-468 FC=0.31). Median patient’s age was 57 years (range 26-89) and the of cases had at list one marker discordant. There were no cases of DCIS negative/invasive median follow-up was 85 months (range 6-259). Tumors showed vascular invasion in tumor positive, therefore one may assume that when the DCIS is Her-2 negative the 51% (85/167), necrosis in 45% (74/164) and positive lymph node-status in 34% (62/181). invasive component is also negative. Other assumptions regarding ER and PR cannot Low FOXP3 mRNA expression (FC<0.5) (35%, 67/189) predominated in Luminal be justified since we found ER or PR positive DC IS with ER or PR negative invasive (42%, 27/65) and TN/BL subtypes (39%, 25/65; p=0.004), with lower histological grades cancer, and vice versa ER or PR negative DCIS with ER or PR positive invasive cancer. (1+2 vs 3) (51%, 34/67; p=0.013). Patients with decreased FOXP3 tumor levels had longer overall survival (85% vs 69%; p=0.012). Phenotype stratification also showed a trend to better prognosis in those with Luminal (96% vs 79%), HER2+ (77% vs 63%) 248 NIK- and IKK2-Binding Protein: A Novel Marker of Breast Cancer and TN/BL (76% vs 68%) tumors (p=0.11; Kaplan-Meier, log-rank test). S Parajuli, H Wang, W Hu, X Zhang. Temple University Hospital, Philadelphia, PA; Conclusions: Low level of FOXP3 mRNA correlated with good prognostic Temple University School of Medicine, Philadelphia, PA. factors. Moreover, we identified a subgroup of patients with better prognosis in all Background: Nuclear factor of κB (NF-κB) has been shown to modulate expression of immunophenotypes of BC. The results of FOXP3 mRNA expression in BC cell lines and genes involved in cell proliferation, differentiation, apoptosis and metastasis. The IκB tissues were comparable only in TN/BL phenotype, showing low levels of expression. kinase-2 (IKK2) and NF-κB–inducing kinase (NIK) are proteins involved in classical Supported by Grants FIS 10/00082, AP-172/10, FCVI-HGUA (2010/PC-04 and 2011/ and alternative pathways of NF-κB activation. NIK- and IKK2- binding protein (NIBP) PC-03). is a novel protein which bridges these two pathways and enhances cytokine-induced NF-kB activation in various cancer cell lines. Upregulation of NIBP mRNA expression in breast cancer cell lines and tumor tissues has been identified through bioinformatics. 246 Variability in Her2neu Testing: Validation Study in Search of This study is undertaken to evaluate the NIBP protein expression in benign breast Economic Solution for India tissue and in breast cancer and to explore the clinical correlation of its expression with T Pai, T Shet, A Patil, S Desai. Tata Memorial Hospital, Parel, Mumbai, Maharashtra, tumor grades and stages. India. Design: Immunohistochemistry for NIBP was performed on a high density breast cancer Background: The two FDA approved antibodies Hercep from DAKO and Ventana tissue array (616 cores from 322 cases). Cytoplasmic staining of glandular epithelial Her2neu are expensive as screening method for Her2neu evaluation in breast cancer or cancer cells in each core was assessed by two pathologists blind to the clinical patients in institutes across India. As no EQAS exists there is a need to validate locally information and it was scored semi-quantitatively as negative (0), weekly positive (1+), available inexpensive antibodies before their generic use. moderately positive (2+) or strongly positive (3+). Mann-Whitney two-tailed U test was employed for the statistical analysis using GraphPad Prism software. 62A ANNUAL MEETING ABSTRACTS Results: Materials from 26 benign breast tissue and 258 carcinomas were available Conclusions: We report the concordance of axillary lymph node FNAC with SLN FS for the study, including 242 invasive ductal carcinomas, not otherwise specified (IDC- and ALND in the management of breast cancer at our institution. In our experience, NOS), 4 invasive lobular carcinomas, 6 mucinous carcinomas, 3 medullary carcinomas, FNAC and SLN FS show good concordance with each other and with ALND findings. 2 tubular carcinomas and 1 ductal carcinoma in situ. Among the invasive carcinomas, We propose that in cases of discordant FNAC and radiographic findings, nodal status staging was available in 257 cases (24 Stage I, 198 Stage II and 35 Stage III) and assessment via SLN-FS should be done prior to performing ALND, thereby reducing grading in 237 cases (13 Grade I, 178 Grade II and 46 Grade III). NIBP expression patient morbidity. Our findings also suggest that in concordant cases and for patients was found significantly upregulated in carcinomas, in comparison to benign breast treated neo-adjuvantly prior to surgery, SLN FS can be forgone for routine histologic epithelium (p<0.001). Its expression was correlated partially with the tumor staging: evaluation. higher level of protein expression was noted in Stage III carcinomas than that in Stage I carcinomas (p<0.05), although correlation with tumor grading was not significantly 251 Differences in Tumor Grade of Invasive Breast Carcinomas in established in this cohort of invasive carcinomas. Its expression was significantly higher Core Biopsy and Excision: An Analysis of 142 Cases (p<0.05) in IDC-NOS than that of the special types of invasive carcinomas that have CD Patel, DM Pandya, J Liu, C Bernstein, C Tornos. Stony Brook University Medical relatively better prognosis. Center, Stony Brook, NY. Conclusions: The results suggest that NIBP is a novel tumor marker of breast cancer Background: Nottingham histologic score (NHS) is widely used to determine the tumor and its expression may be associated with patient’s prognosis. Further study to validate grade of invasive breast cancers. Some studies done in other tissues support the idea these findings is required in a larger scale of routine tissue samples. of mitosis being dependent of ischemia time. The aim of this study was to compare the tumor grade in the core biopsy (ischemia time less than 10 minutes), and surgical 249 ER and PR Immunohistochemistry and HER2 FISH vs. Oncotype excision (ischemia time greater than 1 hour). DX®: Implications for Breast Cancer Treatment Design: 142 cases of invasive breast cancer that had core biopsies and surgical resections MM Park, JJ Ebel, DL Zynger. Ohio State University Medical Center, Columbus, OH. done at out institution were retrieved from our files. The same pathologist reviewed Background: Hormone receptor and human epidermal growth factor (HER2) are all 142 cases, and determined the NHS examining 50 high power fields in each case potent clinical biomarkers to predict response to adjuvant therapy for breast cancer. using the same microscope. Architectural score, nuclear grade and mitotic count were Immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) are compared in all cases. validated techniques while reverse-transcriptase polymerase chain reaction (qRT-PCR) Results: 119 cases (83.80%) had a similar NHS in the core and excision. 20 cases is a relatively new assay for this purpose. Our aim is to evaluate ER, PR, and HER2 (14.08%) had a higher NHS in the excision, and 3 (2.12%) in the core. A higher grade concordance between IHC and FISH, and a commercially available qRT-PCR-based in the excision was due to higher mitotic count in 16 cases, higher nuclear grade in one assay, Oncotype DX® (Genomic Health, Redwood City, CA, USA). case, and more than one parameter in 3 cases. Despite the fact that all core biopsies had Design: A retrospective review of invasive breast carcinoma resections performed at an ischemia time of 10 minutes or less, they all had poor nuclear detail with homogeneous our institution from 2008 to 2012 was conducted to isolate cases in which an Oncotype chromatin pattern, and pyknotic – like nuclei. This was seen in all cases regardless of DX® assay was utilized. ER and PR by IHC and HER2 by FISH had been previously the radiological method used (stereotactic, ultrasound or MRI guided). This affected assessed. Cases were organized by whether biomarkers were performed on: biopsy not only nuclear grading but also mitotic count (mitoses were difficult to separate from only, resection only, or biopsy and resection. When biomarkers were performed on both pyknotic nuclei). The nuclear detail was better in most excision specimens despite a the biopsy and resection, analysis was done on the resection data as this was the same longer ischemia time. tissue block used for Oncotype DX® testing. For discordant cases in which the biopsy Conclusions: NHS in core biopsy and excision is concordant in 83.8% of the cases. was used, markers were newly performed on the resection block and this data was used 14.08% of cases have a higher score in the excision due to better preservation of nuclear for analysis. Statistical methods to calculate correlation and agreement included the detail. Despite a longer ischemia time, practically all excisions had surprisingly better Pearson correlation coefficient (r) and Cohen’s kappa (κ), respectively. nuclear detail, allowing easier mitotic counts and nuclear grading. The reason for the Results: ER showed a concordance of 98.9% (262/265), r=0.42 (95% confidence poor nuclear detail in all cores is uncertain, but could be related to the technique used interval (CI): 0.31-0.51). Positive percent agreement (PPA) for ER was 98.9% (262/265). by the radiologists to retrieve the tissue. This finding deserves further investigation. Negative percent agreement (NPA) and κ could not be calculated because Oncotype DX® testing was not ordered for IHC ER-negative cases, precluding true negative 252 Correlation of RANKL, IKKalpha/beta and Maspin Expression results. PR was concordant in 91.3% (242/265), r=0.80 (95% CI: 0.75-0.84), and with FOXP3 Regulatory T-Cells in Immunophenotypes of Breast Carcinoma κ=0.63 (95% CI: 0.50-0.77). PPA for PR was 100% (218/218) and NPA was 51.1% G Peiro, F Ortiz-Martinez, A Perez-Balaguer, P Martinez-Peinado, J Ponce, FI Aranda, (24/47). HER2 results were concordant in 99.2% (245/247), r=0.34 (95% CI: 0.23- E Lerma, J Sanchez-Paya, JM Sempere-Ortells. University General Hospital, Alicante, 0.45), and κ=0.12 (95% CI: -0.08-0.31). PPA for HER2 was 0% (0/2) and NPA was Spain; University of Alicante, Alicante, Spain; Hospital de la Santa Creu i Sant Pau, 100% (245/245). Of the 3 FISH HER2-amplified cases, 2 (66.7%) were negative and Barcelona, Spain. 1 (33.3%) was equivocal by Oncotype DX®; all FISH HER2-equivocal cases (n=3) Background: RANKL, the receptor activator of NFkB (RANK/TNFRSF11A) ligand, were negative by Oncotype DX®. has been reported to activate IKKalpha (kappaB kinase inhibitor alpha), which in Conclusions: While our results exhibited high to moderate concordance rates between turn controls Maspin expression. On the other hand, IKKbeta is a key regulator of IHC and Oncotype DX® for ER and PR biomarkers, our data showed poor PPA for FOXP3 expression during development of natural regulatory T-cells (Tregs). Recent HER2. Oncotype DX® may not accurately identify HER2-positive breast carcinomas studies suggest their role in the pathogenesis of cancer and metastasis. In this study we in comparison to FISH. We recommend exercising caution in the interpretation of evaluated the FOXP3+ Tregs content in tumor microenvironment of breast carcinoma discordant results. (BC) and correlated the results with RANKL, IKKalpha/beta and Maspin expression; and patient’s outcome. 250 Concordance of Fine Needle Aspiration, Sentinel Lymph Node, Design: We performed an immunohistochemical (IHC) study of FOXP3, RANKL, and Axillary Dissection in Assessing Nodal Metastasis in Breast Cancer phospho-(p)-IKKalpha/beta and Maspin on paraffin-embedded tissue microarrays Patients (1mm cores) containing 245 BC, stratified by immunophenotypes: 39% Luminal LN Parsons, B Behmaram, Z Basir. Medical College of Wisconsin, Milwaukee, WI. A/B (ER/PR+, HER2-), 40% HER2+ (>30% cells 3+ by IHC and/or FISH/CISH Background: Assessment of axillary lymph node metastasis is essential in the amplification), and 21% TN/basal-like (ER/PR/HER2- +/-CK5/6+/-EGFR+). FOXP3+ management of patients with breast cancer. While axillary lymph node dissection Tregs within the tumor and/or immediately adjacent stroma were counted in 3 high (ALND) provides a definitive histologic diagnosis it can result in significant morbidity. In power fields (x400). Cytoplasm or nuclear staining were semiquantitatively scored addition, recent studies indicate that patients with limited nodal disease may not benefit based on intensity and distribution (0-300). IHC results, clinicopathological factors from ALND. Fine needle aspiration cytology (FNAC) and sentinel lymph node (SLN) and outcome were correlated. biopsy with frozen section analysis (FS) are alternative methods for determining nodal Results: Median patients’ age was 50 years (range 20-85 years) and median follow-up status which are significantly less invasive. We sought to examine the concordance of 99 months (range 6-302 months). High number of Tregs (median threshold >15 FOXP3+ FNAC, SLN FS, and ALND in our institution to determine the utility of these methods. cells) (22%) correlated positively with tumors presented at younger ages (31%), of Design: A database search was performed for cases of axillary lymph node FNACs grade 3 (33%) with necrosis (40%) and overexpression of RANKL (38%) and nuclear from 01/07 to 08/12; of these, cases with corresponding ALND and/or SLN FS were or cytoplasmic Maspin (34% and 30%; respectively); and negatively with p-IKKalpha/ selected. Pathologic data including histologic type, grade, and hormonal status were beta (38%) (all p<0.05). TN/basal-like tumors showed increased Tregs (39%), and obtained. Clinical data including radiographic findings were acquired from the patient overexpressed RANKL (43%) and Maspin (43% nuclear and 51% cytoplasm), but medical record. Luminal tumors overexpressed p-IKKalpha/beta (72%) (p=0.001). Overall survival was Results: Forty-seven FNAC results from 41 patients with corresponding SLN FS/ALND better for patients whose tumors overexpressed RANKL (94%; p=0.01) or p-IKKalpha/ were identified. Cases in which FNAC demonstrated metastatic disease proceeded beta (86%; p=0.05). However, neither Tregs content nor Maspin subcellular location directly to ALND without further assessment of SLN status. Thirty-six (77%) of the showed prognostic significance (p=ns) (Kaplan-Meier; log rank test). 47 FNACs were concordant with the ALND and radiographic findings. Of the 11 Conclusions: We demonstrated a correlation of FOXP3+ Tregs with RANKL and discordant cases, three cases were diagnosed as “negative for malignancy” by FNAC Maspin expression that were specifically increased in TN/basal-like tumors, supporting with subsequent positive SLN-FS and were attributed to sampling error. Six cases that their role as markers of aggressive BC. However, IKKalpha/beta was detected in Luminal were diagnosed as “positive for malignancy” by FNAC were observed in patients who phenotypes. Nevertheless, only RANKL and IKKalpha/beta correlated with outcome. underwent neo-adjuvant chemotherapy prior to surgery; ALND in these patients showed Supported by Grants FIS 10/00082, AP-172/10, FCVI-HGUA (2010/PC-04 and 2011/ no metastatic disease. Two cases diagnosed as “positive for malignancy” by FNAC PC-03). showed no metastasis on ALND; in both, radiologic findings were of low suspicion for metastatic disease. FNAC and SLN-FS were concordant in 17/19 cases (89%), and SLN FS and ALND findings were congruent in 18/19 cases (95%). ANNUAL MEETING ABSTRACTS 63A 253 Recurrence in Ductal Carcinoma In Situ (DCIS): A Clinico- results were correlated with clinicopathological factors and outcome of the patients. Pathological Study Results: GLI3 was expressed in all cell lines, whereas GLI1 and GLI2 mRNA F Penault-Llorca, L Tixier, A Cayre, F Kwiatkowski, G Lebouedec, N Radosevic-Robin, C expression was only detected in TN/BL cell lines subtype. Expression of GLI1 was seen Abrial, F Mishellany, M-M Dauplat. Centre Jean Perrin and EA 4677 ERTICa University in 48% (63/130) of BC, GLI2 in 46% (60/130) and GLI3 in 100% (130/130). Among of Auvergne, Clermont-Ferrand, France; Centre Jean Perrin, Clermont-Ferrand, France. immunophenotypes, HER2 expressed less frequently GLI1 (24%, 15/47; p<0.000) or Background: Ductal carcinoma in situ account for approximately 15-20 % of all newly GLI2 (28%, 16/46; p=0.22). Age at diagnosis, tumors size, histologic grade, necrosis, diagnosed breast cancers corresponding to 7000-8000 new cases every year in France. lymph-vascular invasion or lymph-node status showed no significant association (p=ns). Currently, therapeutic decision is taken according to different factors: size of the lesion, Disease-free survival (DFS) analysis showed that patients whose tumors expressed GLI1 surgical possibilities, and the patient’s will. But, DCIS appears to be as complex and had worse prognosis among all immunophenotypes: Luminal (63% vs. 83%), HER2 heterogeneous disease at the biological and morphological level as invasive carcinoma. (64% vs. 68%) and TN/BL (76% vs. 89%) (p=0.048). However, GLI2 results correlated Our aim was to define clinical and biological risk factors for recurrence in order to only in TN/BL (75% vs. 90%, p=0.03; Kaplan-Meier; log-rank test). provide an optimized and personalized treatment. Conclusions: The Hh pathway is activated in all phenotypes of BC, reflected by the Design: Retrospective study of 87 patients (mean age 53 yrs) treated by demonstration of GLI genes expression. Interestingly, GLI1 and GLI2 expression conservative treatment (surgery and radiation therapy) for pure DCIS. Clinical and stratified patients at higher risk of recurrence specifically in TN/BL, making those immunohistochemical data were studied. For each lesion, we analysed several markers genes good candidates for a potential therapy target. Our study shows a correlation of (ER, RP, HER-2, EGFR, CK 5/6, P-Cadherin, p53, Ki67, RA, p63). Eight local expression of GLI1 and GLI2 in the TN/BL cell lines and BC subtype. recurrences (LR) occurred (9.2 %): 5 in situ (62 %) and 3 invasive. Furthermore, 6 Supported by Grant FCVI-HGUA 2012/C-08. contralateral events were observed. Results: Mean size 1,4cm, grade 2: 55%, ER+: 88,5% PR+: 41,8%,HER2+: 17%. 256 Relationship of Sentinel and Axillary Lymph Node Status with Risk of recurrence was significantly associated with tumour size > 5 mm (p= 0.032 Locoregional Recurrence/Metastasis: A Seven-Year Clinical Follow-Up for LR), necrosis (p=0.048 for LR and p=0.03 for any new events (ANE)), Ki67>5% Study (p=0.015 for ANE), dense inflammatory infiltrate p=0,025, low p-cadherin membrane J Petersen, ME Vergara-Lluri, Y Omidvar, R Prati, SK Apple. David Geffen School of staining (p=0,016), decrease in p63 staining (p=0,049). On multivariate analysis for Medicine at UCLA, Los Angeles, CA. tumors>0.5cm, low membrane p-cadherin and Ki67>5% were the only significant Background: Lymph node status is known to be the most important prognostic factor markers, associated to a risk of relapse of 7.7 and 6.5 respectively. in breast cancer. More recently, the American College of Surgeons Oncology Group Conclusions: Ki67 and P-cadherin evaluations in DCIS should be tested in larger (ACOSOG) Z0011 trial study suggested that axillary lymph node dissection (ALND) is series to confirm their potential use in the evaluation of DCIS aggressiveness. More not necessary in T1 and T2 breast carcinoma regardless of sentinel lymph node (SLN) data will be presented at the meeting (molecular signature for DCIS and comparison status. The Z0011 trial advocated conservative management as disease-free survival and with a group of micro-invasive carcinoma). recurrence rates did not appear to differ between SLN alone versus SLN with ALND. We sought to examine SLN and ALND positive rates and to evaluate the probability of 254 Prediction of Response to the Anti-EGFR Antibody locoregional recurrence and/or metastasis (LRM) with regard to SLN and ALN status. Panitumumab Combined with Standard Neoadjuvant Chemotherapy in Design: A retrospective analysis of all breast cancer patients at our medical center from Triple Negative Breast Cancer (TNBC): The Immune and IGFR Pathways 2003-2007 who underwent both SLN and ALND was performed (n=112), regardless of F Penault-Llorca, C Abrial, M-M Dauplat, M Privat, N Uhrhammer, A Desrichard, Y pathologic tumor stage. The median clinical follow-up was 7.33 years. Patients lost to Bidet, N Radosevic-Robin, A Cayre, C Aube, F Kwiatkowski, Y-J Bignon, P Chollet, follow-up were excluded from study. We then compared multiple clinicopathological J-M Nabholtz. Centre Jean Perrin and EA 4677 ERTICa University of Auvergne, parameters of patients who had LRM against those that were disease free (DF) using Clermont-Ferrand, France. Fischer’s exact test on GraphPad Prism statistical analysis software. Background: EGFR overexpression is one of the hallmarks of the “basal-like” TNBC Results: SLN was positive in 70% of the total (78/112); including 8 (7%) with isolated definition by immunohistochemistry (IHC) (Nielsen, 2004). A phase II neoadjuvant tumor cells (ITC); 11 (10%) with micrometastases; and 59 (53%) with macrometastases. clinical trial targeting EGFR in TNBC was conducted in our institution. We investigated ALND was positive in 35% of the total (39/112); including 0% with ITC; 3 (3%) with various biomarkers in order to better identify an EGFR-sensitive population for potential micrometastases, and 36 (32%) with macrometastases. Thirty-six of 78 patients with further regimen development. positive SLN had positive ALND (46%), while 31 of 34 patients with negative SLN Design: Sixty patients (pts) with stage II-IIIA TNBC were prospectively included. The had negative ALND (91%). The risk that a patient with positive SLN having a positive systemic treatment (ST) consisted of panitumumab combined with FEC 100, followed ALN was approximately nine times higher than the risk of a patient with negative SLN by 4 cycles of docetaxel. All pts underwent surgery after the ST completion. Patient having a positive ALN, with an odds ratio of 8.88 (p<0.0001, 95% CI 2.497 to 31.42). characteristics: median tumor size: 40 mm [20-120]; invasive ductal carcinoma: 96.7%; With LRM as a clinical endpoint, all clinical parameters including age, menopausal SBR grade III: 71.7%; complete pathological response (pCR) rate: 46.8% (Chevallier’s status, tumor grade, tumor stage, tumor biomarkers, lymphovascular invasion, types classification). Paraffin-embedded and frozen tumor samples were collected before and of surgery, radiation, chemotherapy, and neoadjuvant therapy did not predict LRM. after the ST for biologic studies. Germinal BRCA1 mutations, FC-gamma receptor Conclusions: While positive SLN predicts positive ALND, nodal status did not polymorphism and EGFR, KRAS, BRAF and PI3KCA somatic mutations were analyzed predict locoregional recurrence/metastases. Our study appears to support conservative by NGS. Biomarkers evaluated by IHC were: EGFR, IGF-1R, MET, cytokeratins 5/6 management of breast cancer patients with positive sentinel lymph nodes. and 8/18, PTEN, P-cadherin, ALDH1, Ki-67, p53, tumoral FOXP3 expression and the number of FOXP3+ or CD8+ tumor-infiltrating lymphocytes (TILs). 257 Phyllodes Tumors: Histologic Features in Relation to Clinical Results: By univariate analysis, high CD8+ TILs and tumor FOXP3-positivity were Follow-Up response-predictive (pCR rates: CD8 TILs: 84% high vs 16% low; FOXP3: 80% high MR Phillips, Y Omidvar, SK Apple. UCLA, Los Angeles, CA. vs 30% low; p=0.000004). Similarly, high IGF-1R expressors responded better than Background: Phyllodes tumors (PTs) are rare fibroepithelial neoplasms of the breast the low ones (67% vs 28%, respectively, p=0.012). In multivariate analysis, only CD8+ which are subdivided into benign, borderline and malignant based on histologic criteria. TIL count remained significant (p=0.0002). Sequencing revealed BRCA1 mutations Surgical excision with a wide margin (>1cm) is the standard treatment to minimize in 10% of pts. Somatic mutations of PI3K were observed in 6 pts. No mutations were recurrence, however, the histologic classification of PTs does not reliably predict clinical observed in BRAF, KRAS, or EGFR, although numerous EGFR SNPs were found. behavior despite adequate excision. This study aims to examine the clinicopathologic Conclusions: Interestingly, the CD8+ TIL count and tumor FOXP3 expression seem features and outcome of PTs in order to identify risk factors for local recurrence and to predict the response to panitumumab. The IGF-1R tumor levels seem to play a distant metastasis. determinant role in TNBC response to anti-EGFR antibodies, which is in concordance Design: A retrospective review of 62 patients with PTs from 1996-2012 was analyzed with our recent observations in a head-and-neck cancer cohort (Clin Canc Res 2012). with regard to histologic features and clinical follow-up. We included only patient Confirmatory and mechanistic studies of those biomarkers are warranted. samples with slides available for review and excluded all cases that had no or loss of clinical follow-up. Analysis of histologic features included tumor size, mitotic rate/10 255 Study of GLI1, GLI2 and GLI3 Transcripts Levels of Expression HPF, stromal overgrowth, periglandular cuffing, cellularity, necrosis, heterogeneous in Immunophenotypes of Breast Carcinoma and Breast Cancer Cell Lines differentiation, borders, margin width and co-existing fibroadenoma (FA). Clinical A Perez-Balaguer, F Ortiz-Martinez, M Niveiro, FI Aranda, E Lerma, G Peiro. features included age, follow-up duration, type of surgery, local recurrence, distant University General Hospital, Alicante, Spain; Hospital de la Santa Creu i Sant Pau, metastasis and death rate. Barcelona, Spain. Results: Benign, borderline and malignant PTs comprised 31/62 (50%), 14/62 (23%) Background: The Hedgehog (Hh) signaling pathway is a key developmental regulatory and 17/62 (27%) with mean age of 38, 44 and 50 years, respectively. Median follow- pathway, the deregulation of which has been implicated in several types of cancers, up was 54 months. including breast carcinoma (BC). The GLI genes are effectors of the Hh signal in the cell. Our aim was to analyze the expression of GLI1, GLI2 and GLI3 genes in cell lines representing the different BC phenotypes, as well as in tumor samples to determine the role of the Hh pathway in the pathogenesis of the subtypes of BC. Design: BC cell lines consisted of: MCF-7 and T-47D (Luminal type); BT-474 (Luminal with HER2+); SK-BR-3 (HER2+); MDA-MB-231 and MDA-MB-468 (triple negative/ basal-like -TN/BL-), and normal epithelial breast cell line (184A1). Relative expression ∆∆ was determined using the CT method and was expressed as relative quantification (RQ) unit. Samples of 130 BC were classified immunohistochemically into Luminal A and B (26%), HER2 (36%) or TN/BL (38%) phenotypes. The expression analysis 64A ANNUAL MEETING ABSTRACTS

Table 1. Clinicopathologic features & outcome of PT Super Sensitive ISH Detection Kit (BioGenex, DF400-YAX). The signal was amplified Benign PT Borderline PT Malignant PT Mean Age (yr) 38 44 50 with anti-fluorescein antibody and poly-HRP labeled secondary antibody,and visualized Mean size (cm) 3.1 4.8 6.7 with DAB chromogen. Scramble probes served as negative control. Avg mitoses/10 HPF 1 5 17 Stromal overgrowth 16% focal 43% focal 88% diffuse Results: TN BR CA showed up-regulation of miR-21 (2/4 cases), miR-221 (4/8 cases), Necrosis (coagulative) 0% 0% 41% miR-222 (5/8 cases) and down-regulation of miR-205. miR-150 was up-regulated in Heterologous diff 0% 0% 41% Her2+ BR CA but reduced in ER/PR+ BR CA. Normal breast tissue showed low to Co-existing FA 61% 64% 53% Borders negative expression of miRNAs. Pushing 71% 28.5% 12% Figure1: BR CA positive for miR-221 (left panel), negative with scramble probe Irregularly pushing 29% 43% 0% Infiltrative 0% 28.5% 76% (right panel) Unknown 0% 0% 18% Local excision vs mastectomy 0% vs 100% 86% vs 14% 47% vs 53% Recurrence rate 13% 14% 18% Margin: recurrence rate @: 4/9 (44%) 1/4 (25%) pushing 3/14 (14%) 0 <1mm: 3/9 (33%) 1/4 (25%) pushing 0 2/3 (67%) 2mm: 1/9 (11%) 1/4 (25%) irreg pushing 0 0 neg (>1cm): 3/9 (33%) 1/4 (25%) pushing 2/10 (20%) 0 Distant metastasis 0% 0% 12% Death rate 0% 0% 12% Conclusions: PTs are seen within and/or adjacent to FA in 60% of patient samples which supports the hypothesis that FA is a precursor lesion to PT. Recurrence, death rate and distant metastasis are directly related to the histologic findings of stromal overgrowth, necrosis and heterologous differentiation in malignant PTs. The status of the resection margin was not associated with recurrence free survival in benign PTs, while close margin status in borderline and malignant PTs was related to recurrence rate. The type of surgery was not a significant factor for local recurrence. Conclusions: miRNA profiling is feasible in FFPE samples of BR CA using chromogenic ISH. Although no significant difference was noted in the expression of the various miRNAs in the different molecular subtypes, the presence of differential 258 Frozen Section Evaluation of Breast Carcinoma Sentinel Lymph staining warrants additional studies on larger number of cases to determine prognostic/ Nodes: A Retrospective Review of 1940 Cases predictive value. JS Poling, P Argani, AM Cimino-Mathews. Johns Hopkins Hospital, Baltimore, MD. Background: Many sentinel node biopsies (SLNB) are evaluated intraoperatively by frozen section, which may impact the need for further axillary dissection (AD). However, 260 Immunogenotypic Signatures Predict Pathologic Complete the need for AD in patients with small metastases has been recently called into question, Response (pCR) in HER2 Positive Breast Carcinoma Patients Treated with raising the possibility that frozen SLNB may be unnecessary. Furthermore, frozen Neoadjuvant Trastuzumab-Based Therapy section can compromise tissue for further study. As a compromise at our institution, BP Portier, Z Wang, E Minca, GT Budd, C Lanigan, L Morrison, RR Tubbs. Cleveland we grossly evaluate all SLNB and freeze half of the node. Here, we evaluate the frozen Clinic, Cleveland, OH; Ventana Medical Systems, Inc., Tucson, AZ. SLNB discrepancy rate at our institution, focusing on cause of discrepancy and need Background: Neoadjuvant breast cancer treatment, primarily used for “downstaging” for further surgery. patients, may achieve a pathologic complete response (pCR), especially non-luminal B Design: We reviewed surgical pathology records for all breast cancer resections with HER2 positive cases. Determination of which patients are more or not likely to achieve frozen section of SLNB examined from 01/2003 (n=1940). For cases with a frozen pCR would allow a more personalized, predictive approach to neoadjuvant management. section discrepancy, we compiled clinicopathologic data including age, tumor type, Design: Biopsy specimens from 34 patients with invasive ductal carcinoma treated tumor size, metastasis size, reason for discrepancy, and follow-up including AD and with Trastuzumab-based therapy prior to definitive resection and pathologic staging survival. were evaluated by dual color bright field in situ hybridization (dual ISH) using repeat Results: In ninety-five cases (4.9%), the SLNB was called negative on frozen but depleted locus specific probes and reference centromeric probes (CEN). Probe pairs positive on final examination (false negatives), as detailed below. Overall frozen SLNB included MET+CEN7, TOP2A+CEN17, PTEN+CEN10, PIK3CA+CEN3; and Ki-67 sensitivity was 76.3%; specificity was 99.9%; positive predictive value was 99.7%; expression was assessed by immunohistochemistry (IHC). Values of parameters for negative predictive value was 94.0%. Overall 42% of patients with false negative frozen absolute and relative genomic gain, loss, and collective gain or loss were determined SLNB were taken back to surgery for an AD; 29% of patients from 2009-12 were taken for each locus and CEN for all specimens. A range of cutoffs were applied to each back to surgery, whereas 53% of patients from 2003-08 were taken back. parameter and Receiver Operator Characteristics (ROC) curves were generated. Area Under the Curve (AUC) was calculated as one measure of a parameter’s ability Clinicopathologic data of false negative frozen SLNB 2 Age Median 52 years, Range 22-82 years to distinguish pCR. Sensitivities, specificities, and X probabilities calculated from Metastasis Size Median 1.05 mm, Range 0.05-16 mm contingency tables were used to select optimal cutoffs and establish clinical relevance. Primary Tumor Type Ductal 64 (67.4%), Lobular 20 (21.1%), Mammary 11 (11.6%) Combinations of genomic parameters, and combinations of genotype with KI67 IHC Primary Tumor Size Median 1.7 cm, Range 0.1-6.0 cm results (“immunogenotype”), were also evaluated. Survival (as of 10/1/2012) Alive 88 (92.6%), Dead (7.4%) IHC Needed 19 (20%), Block Sampling 33 (34.7%), Tissue Results: High level of MET/CEN7 gain or loss was predictive of pCR (AUC=0.824, Discrepancy Reason Sampling 37 (38.9%), Interpretation 6 (6.3%) N=25; 100% sensitivity, 69% specificity, p=0.00089 at optimal cutoff), and combined Additional Axillary Dissection Yes 39 (41.9%)* with low PIK3CA/CEN3 gain provided further improvement (AUC 0.937, N=24; 89% Performed sensitivity, 93% specificity, p=0.000056). Combining low PTEN/CEN10 gain or loss * positive nodes found in 6/39 additional dissections with high MET/CEN7 gain and high CEN7 gain also provided improvement (AUC Conclusions: The protocol of freezing one half of a SLNB is a reasonable method, with 0.994, N=19; 100% sensitivity, 91% specificity, p=0.000089). The most robust signature a low false negative rate. The majority of missed metastases are isolated tumor cells for predicting pCR was high MET/CEN7 gain or loss + low PIK3CA/CEN3 gain + or micrometastases. The main adverse outcome is the need for separate AD; however, high Ki-67 (AUC=1.0, N=24; 100% sensitivity, 100% specificity, p=0.00000096). additional positive nodes are uncommonly found. The trend over time of fewer patients Conclusions: Immunogenotypic signatures of moderate complexity generated from getting additional AD after a discrepant frozen SLNB suggests that clinicians may be dual ISH and IHC predict pCR in HER2 molecular class breast carcinoma patients using this information differently in recent years. treated with Trastuzumab-based preoperative therapy. These findings require validation in additional patient cohorts. 259 Differential Expression of miR-21, miR-205, miR-221, miR-222, and miR-150 in Molecular Subtypes of Breast Cancer 261 Prognostic Significance of Fibroblast Growth Factor Receptor A Poongothai, X Ling, P Sudipta, K Kalra, RK Pillai, MB Amin, S Bose. Biogenex Lab, 1 (FGFR-1) Overexpression in Invasive Mammary Carcinoma (BRCA) Freemont, CA; Cedars-Sinai Medical Center, Los Angeles, CA. MJ Presta, RN Al-Rohil, CB Sheehan, CE Sheehan, AB Boguniewicz, JS Ross. Albany Background: microRNAs (miRNAs) are short RNA molecules that are involved in Medical College, Albany, NY. many critical cellular processes including oncogenesis. Different cancer types and Background: FGFR-1 is a multifunctional polypeptide growth factor receptor that has subtypes at different stages of progression display unique miRNA profiles that may be a well established role in angiogenesis and tumorigenesis. Although amplification of used as diagnostic, prognostic and therapeutic tools. The detection of miRNA in clinical the FGFR1 gene has been linked to adverse prognosis in human BRCA, the prognostic samples has been difficult, requiring total RNA extracts which lack critical spatial significance of FGFR-1 protein expression is not well characterized. information. In situ hybridization (ISH) allows direct assessment of malignant cells. Design: Formalin-fixed, paraffin-embedded tissue sections from 129 cases of BRCA Evaluation of miRNA profiles in breast cancer (BR CA) holds promise for improving [102 ductal carcinomas (IDC) and 27 lobular carcinomas (ILC)] were immunostained understanding of pathogenesis and therapeutic outcome. This pilot study is designed to by a manual method using rabbit polyclonal Flg (FGFR-1) (sc 121; Santa Cruz Biotech, assess the feasibility of miRNA profiling in formalin fixed paraffin embedded (FFPE) Santa Cruz, CA). Cytoplasmic immunoreactivity was semiquantitatively scored based BR CA samples using in-situ hybridization (ISH) for miR-150, -21, -205, -221 and -222. on staining intensity and distribution and the results were correlated with morphologic Design: 18 samples of FFPE BR CA and 10 normal breast samples were studied. BR and prognostic variables. Ò CA were molecularly subtyped using immunostains for Estrogen Receptor (Insite ER), Results: FGFR-1 immunoreactivity was present in a predominately cytoplasmic Ò TM Progesterone Receptor (Insite PR), and HER-2/neu (Her2)) with SuperSensitive pattern. Cytoplasmic FGFR-1 overexpression was observed in 85/129 (66%) tumors Polymer-HRP IHC Detection System (all BioGenex). Sub categorization resulted in 5 and correlated overall with advanced stage [79% advanced stage vs 55% early stage, ER/PR positive (+), 5 Her2+, and 8 triple negative (TN) BR CA. Cases were subjected p=0.005], disease recurrence [79% recurrent vs 60% non-recurrent, p=0.03], and survival to ISH using FAM-labeled Hsa-miR-1 and Hsa-miR-145 probes (BioGenex) followed by on Cox univariate analysis (p=0.05). Within the IDC subgroup, FGFR-1 overexpression correlated with advanced stage [81% advanced stage vs 58% early stage, p=0.011]. ANNUAL MEETING ABSTRACTS 65A Within the ER negative subgroup, FGFR-1 overexpression correlated with early age observer study, borderline ER expression cases were considered rare representing at diagnosis [92% <45 years of age vs 43% diagnosed between 45-55 years of age <1% of routine cases. The surprisingly high prevalence brought to light in this study vs 47% >55 years, p=0.021] and advanced stage [75% advanced stage vs 44% early is clinically significant given patient treatment and possible survival implications. stage, p=0.037], while showing a trend toward correlation with disease recurrence [74% recurrent vs 46% non-recurrent, p=0.066]. On multivariate analysis, early age at 264 Stability and Prognostic Value of Slug, Sox9 and Sox10 diagnosis (p=0.006) and advanced stage (p<0.0001) independently predicted disease Expression in Breast Cancers Treated with Neoadjuvant Chemotherapy recurrence; and advanced stage (p<0.0001) independently predicted overall survival. C Riemenschnitter, I Teleki, V Tischler, W Guo, Z Varga. Institute of Surgical Pathology, Conclusions: Cytoplasmic FGFR-1 overexpression correlates with advanced stage and University Hospital Zurich, Zurich, Switzerland; Semmelweis University, Budapest, disease recurrence in BRCA. In an ER negative subgroup, cytoplasmic overexpression Hungary; Albert Einstein College of Medicine, New York, NY. correlates with early age at diagnosis and advanced stage. Expression also shows a trend Background: Expression of transcription factors as Slug and Sox9 was recently used towards correlation with disease recurrence. On multivariate analysis, diagnosis at an to determine mammary stem cell state. In this study we addressed the prognostic role early age and advanced stage independently predicts disease recurrence, and advanced of Slug, Sox9 and Sox10 expression in neoadjuvant treated primary breast cancer and stage independently predicts overall survival. Further study of FGFR-1 expression as a their correlation to pathological response and overall survival. Furthermore, we tested prognostic factor and potential target of therapy for BRCA appears warranted. stability of these markers during chemotherapy. Design: We analyzed immunohistochemical expression of Slug, Sox9 and Sox10 in 262 MRI-Guided Breast Core Biopsies: Pathologic Features of Newly tissue microarrays of 96 breast cancers prior to and after neoadjuvant chemotherapy. Diagnosed Malignancies Expression was evaluated in invasive tumor cells and in tumor stroma and scored as ES Reisenbichler, SC Lester. Brigham and Women’s Hospital, Boston, MA. 0, 1+, 2+ 3+. Expression profile prior to and after chemotherapy was correlated to Background: Magnetic resonance imaging (MRI) of the breast is used for select overall survival (Kaplan Meier) and with established clinico-pathological parameter. groups of patients. The likelihood of detecting malignancy is related to the reason for Results: Sox9, Sox10 and Slug were expressed in 82-96% of the tumor cells prior to the examination. This study reviewed a large series of MRI-guided breast core needle chemotherapy. Slug was expressed in 97% of the cases in tumor stroma before therapy. biopsies to determine the incidence and types of cancers found and to correlate the Change in expression profile after chemotherapy occurred only in Slug expression in cancers with the MRI findings and the indication for the study. tumor cells (decreased from 82 to 51%, p=0.0001, Fisher’s exact test). The other markers Design: MRI-guided breast core biopsies, performed at our institution over 1 year, were showed no significant change after chemotherapy. Stromal Sox9 expression (0 to 2+) identified with review of corresponding clinical, diagnostic radiology, and pathology correlated to better overall survival after chemotherapy (p=0.004) and reached almost reports. Patients were categorized according to MRI indication: high risk (family history, statistical significance prior to chemotherapy (p=0.065). There was no correlation germline mutation, prior chest radiation, or contralateral breast cancer), evaluation of between Sox9 and hormone receptor expression. In multivariate analysis, the stromal disease extent in patients with current cancer, surveillance for recurrence of prior cancer, Sox9 expression after chemotherapy proved to be an independent and better prognostic and as a problem solving method. Lesion characteristics by MRI and final pathologic marker than hormone receptor status. Other clinicopathological parameter (as Her2 diagnoses were recorded. status or pathological stage) showed no correlation to the studied markers. Results: One hundred-thirty-six consecutive core biopsies were performed on 120 Conclusions: Strong stromal Sox9 expression in breast cancer after chemotherapy patients. Most patients (n=75, 63%) underwent MRI due to a high risk of breast cancer. was found to bear negative prognostic information and was associated with shortened Within this screening group, the most common risk indicator was family history overall survival. without a germline mutation. Malignancies (atypical ductal hyperplasia (ADH) with ductal carcinoma in situ (DCIS) upgrade at excision, DCIS, or invasive carcinoma) 265 Relapsed Classic E-Cadherin (CDH1) Mutated Invasive Lobular were identified in 29 (22%) of the biopsies. Analysis of the types of MRI lesions did Breast Cancer (ILC) Features IHC/FISH Negative HER2 (ERBB2) Gene not identify any lesions with a likelihood of malignancy over 50%. All cases of DCIS Mutations Sensitive to Anti-HER2 Targeted Therapies: A Next Generation presented as non-mass-like enhancement (NMLE). Invasive carcinomas presented as Sequencing (NGS) Study both mass forming lesions (58%) and NMLE (42%). The highest malignancy rate (5 JS Ross, C Sheehan, A Boguniewicz, G Otto, S Downing, J Curran, G Palmer, S Ali, of 8 biopsies, 62.5%) was in the group with prior ipsilateral breast cancer. The lowest R Yelensky, D Lipson, V Miller, P Stephens. Albany Medical College, Albany, NY; malignancy rate was in the group undergoing screening (15 of 86 biopsies, 17%). Of Foundation Medicine Inc., Cambridge, MA. the 9 invasive carcinomas identified by screening, 8 (89%) were small (< 1 cm) and all Background: Although ILC generally has a favorable prognosis, relapsed ILC may showed a favorable prognostic receptor profile (estrogen receptor (ER) positive, HER2 follow an aggressive clinical course. In this study, we queried whether NGS could negative). Of the 6 cases of DCIS identified in the high risk group, all demonstrated identify novel and unanticipated targets of therapy for patients with relapsed ILC. intermediate or high nuclear grade with ER negativity in 3 (50%) of cases. Design: NGS was performed on hybridization-captured, adaptor ligation based libraries Conclusions: MRI-guided core biopsies demonstrated a rate of malignancy (in this high using DNA extracted from 4 FFPE sections cut at 10 microns from 12 ILC that had risk population) similar to that seen for mammographic lesions in the general population. relapsed after primary surgery and adjuvant hormonal therapy. The exons of 182 cancer- The type of MRI lesion did not correlate well with the likelihood of malignancy or the related genes were fully sequenced using the Illumina HiSeq 2000 to at an average type of malignancy. The most common types of carcinomas identified by screening were depth of 983X and evaluated for genomic alterations (GA) including point mutations small ER positive invasive tumors and high grade ER negative DCIS. (mut), insertions, deletions, copy number alterations (amp), and select gene fusions/ rearrangements. Actionable GA were defined as those identifying anti-cancer drugs on 263 An Interobserver Concordance Study Reporting Estrogen the market or in registered clinical trials (CT). Receptor (ER) in Invasive Breast Tumors Reveals a High Prevalence (4.9%) Results: The 12 ILC (mean age 54) included 10 (83%) grade II and 2 (17%) grade of Discordant Positive Versus Negative Results, Despite Excellent Overall III tumors. Three ILC (25%) were Stage III and 9 (75%) were Stage IV at the time of Concordance NGS. NGS was performed on: primary ILC 4 (33%), lymph node metastases 3 (25%), ES Reisenbichler, A Ly, SC Lester, JE Brock. Brigham and Women’s Hospital, Boston, liver metastases 2 (18%), pleural fluid cell blocks 2 (18%), a skin recurrence 1 (1%) MA. case(s). NGS found a total of 38 GA in the ILC series with an average of 3.2 GA per Background: 2010 ASCO/CAP guidelines for reporting ER set a 1% threshold for a tumor. 17 GA (1.4 per tumor) were potentially associated with clinical benefit of targeted positive result which is based upon a 1990s study showing improved outcome with therapies and 1 (0.1 per tumor) GA associated with targeted therapy resistance. Eleven/ hormone therapy with 1% weak staining (Allred score 3). Specifically, one observer twelve (92%) of patients’ tumors harbored at least one GA potentially associated with reviewed 1,982 cases with 11% of cases reviewed by a second observer for a concordance clinical benefit of targeted therapies. The GA generated potential entry into a total of score of 0.87 using 6F11 ER antibody. A group of six dedicated breast pathologists report 47 CT (average 3.9 per tumor). ILC GA included mut in CDH1 (100%); TP53 (42%); the concordance in ER by Allred and H-score using two widely used ER antibodies and PIK3CA (42%); RB1 (18%); and KRAS and RUNX1 each at (9%) and amp in myc reveal a hitherto unappreciated discordance reporting cases around the 1% threshold. (18%), FGFR1 (18%) and ESR1 (9%). Two (18%) of ILC had mut in HER2 (ERBB2) Design: Routinely processed consecutive cases of invasive breast carcinoma were in tumors that were HER2 negative by IHC and FISH. Both patients demonstrated stained with ER-SP1 and ER-1D5 and slides were scored for Allred (0-8) and H-score significant disease regression during treatment by anti HER2 targeted therapy with (0-300) by 3 observers. Cases with positive versus negative discordance between the trastuzumab and lapatinib. original 3 observers were reviewed by 3 additional observers. Conclusions: NGS of CDH1 mutated ILC uncovers an unexpectedly high frequency of Results: For 264 cases evaluated, the two antibodies resulted in a difference in Allred GA including HER2 mut sensitive to anti-HER2 targeted therapies. Deep sequencing of score of ≤1 point in 254 (96%), 250 (94%) and 252 (95%) cases for each respective genomic DNA can provide a broad cancer-related gene survey at a depth of coverage observer and < 50 point difference between H-scores in 232 (88%), 242 (92%) and that provides sensitive detection for all classes of GA, and when applied to ILC patients 232 (88%) of cases. Pairwise kappa agreement between observers ranged from 0.863 can reveal actionable GA that inform treatment decisions. to 0.924 with SP1 and 0.892 to 0.943 with 1D5 when dividing cases as either positive ≥ ≤ (Allred >2; H-score 1) or negative (Allred 2; H-score <1). Thirteen cases (4.9%) 266 Pattern of Tumour Cell Migration as a Prognostic Parameter showed discrepant positive/negative results between the original 3 observers with for Lymph Node Metastasis in HER2-Enriched Breast Cancer one or both antibodies and were evaluated by 3 additional observers. Within these I Roxanis, J Russell, B Lavery. Oxford University Hospitals NHS Trust and Oxford 13 cases, the interobserver H-scores were discrepant by up to 49 points with SP1 and Biomedical Research Centre, Oxford, United Kingdom; Oxford Medical Genetics 50.5 points with 1D5. Allred scores were discrepant by up to 4 and 5 points with SP1 Laboratories, Oxford, United Kingdom; , Oxford, United Kingdom. and 1D5 respectively. All 6 observers found a higher rate of positive cases with SP1 Background: As standardisation of the definitions and methodologies of molecular than with 1D5. No statistically significant intra- or inter-observer difference was seen classification is still suboptimal, histological features with prognostic significance are between the 2 antibodies in either Allred score or H-score across all tumors reviewed. of interest not only for potential application in clinical practice but also as a tool for Conclusions: Despite excellent inter-observer concordance in evaluating ER expression formation of testable hypotheses concerning the biology of breast cancer progression. levels, we find almost 5% of routine cases evaluated have a low level of ER expression In contrast to the relatively homogeneous morphology of special types, variation in difficult to classify as ER positive or negative by manual counting. Prior to this multi- 66A ANNUAL MEETING ABSTRACTS the architectural arrangement of invasive tumour cells characterises invasive ductal Risk Stratification by Modified Mandard TRG carcinomas not otherwise specified. Such variability conceivably corresponds to MODIFIED differences in the transcriptomic profile. Recurrence Risk RCB Class Mandard TRG+/- LN Mandard TRG Design: In the current study, we aimed to assess morphologically the potential prognostic Minimal (0%) pCR 1 1 significance of migration patterns in sections of HER2-enriched breast tumours. Aiming RCB 1 1 + LN 2 to estimate the propensity of invasive tumour cells to form large confluent sheets rather 2 2 than invade as single cells or smaller clusters, we employed a semiquantitative collective migration (CM) scoring system that ranged from 0 to 10. Significant (22-36%) RCB 2 2 + LN 3 3 3 Results: Our main finding was that the subgroup with CMhigh migration pattern had 4 4 significantly higher propensity for regional lymph node metastasis (61% vs 19%, RCB 3 2 + LN (>10) 3 p=0.0025). This difference was even more significant when cases with basal-like features 3 + LN 4 and lobular type were excluded (80% vs 12.5%, p=0.000009). 4 + LN 5 5 + LN 5 Conclusions: A modified Mandard TRG, incorporating lymph node status is rapid, reproducible, easily applicable and clinically relevant in determing response to neoadjuvant treatment in breast cancer. Employing one simple rule, this system reliably separates cases into validated RCB classes offering a viable alternative for evaluating pathologic response in routine practice.

268 Analysis of Histopathologic Features Predictive for the Diagnosis of “Basal” like Breast Carcinoma MN Sable, TM Shet, AJ Patil, BG Ramnani, SB Desai. Tata Memorial Hospital, Mumbai, Maharashtra, India. Background: Breast cancer is classified into five subgroups based on gene expression profiling. Of the five subgroups, Basal like breast cancer (BLBC) is a poor prognostic subgroup having limited treatment options. Majorities of triple negative (ER/PR/ CerbB2) breast cancers (TNBC) are BLBC, but both entities are not synonymous. The aims and objectives of this study were to assign breast tumours to “basal” category using histopathologic criteria documented in published literature, to study expression of various “basal” markers described in published literature in these tumours using IHC, and to compare the diagnosis based on surrogate “basal” markers on IHC with that offered on histopathologic features. Design: Two hundred and five-naïve TNBCs (2005-2008) were studied for The pattern of migration was a significant prognostic predictor for lymph node metastasis histopathological features viz. grade, architectural pattern, stromal responses, independent of the proliferative status or the size of the tumours. cytomorphology, duct carcinoma in situ (DCIS) and metaplastic differentiation. They Conclusions: The pattern of tumour cell migration in HER2-enriched breast cancers is were classified as BLBC-M (morphology) and non- BLBC. Simultaneously, they were significantly correlated with lymph node status. Confirmation of this finding would have evaluated for basal cytokeratins (CK5/6, CK 14 and CK 17) and EGFR to classify them significant clinical implication in planning axillary treatment. Furthermore, as nodal as BLBC-I (immunohistochemistry) or core basal phenotype by using criteria of any metastasis indicates both tumour chronology and tumour aggressiveness, our finding basal cytokeratin and/or EGFR positivity. Remaining TNBCs were considered as nBLBC may facilitate formation of hypotheses regarding differential biological behaviour of (7 markers negative). Using logistic regression (SPSS16 software) both BLBC-M and tumours grouped within same histological and molecular subtype. The relevance of BLBC-I were analyzed for each histological feature. Multiple logistic regression was epithelial-mesenchymal transition and TGFβ signaling will be discussed. employed to identify the features which could predict the BLBC. Results: Among 205 TNBC, 91% cases were BLBC (core). Histological features viz. mixed type of margin (p= 0.046), moderate inflammatory response (p= 0.031), 267 Modified Mandard Tumour Regression Grade Predicts Residual absence of intraduct component (p= 0.034) and prominence of nucleoli (p= 0.042) were Cancer Burden Score in Neoadjuvant Treated Breast Cancer frequently associated with BLBC than nBLBCs. All other morphological features were C Ryan, J White, F O’Connell, TJ Browne, L Feeley, MW Bennett. Cork University insignificant to distinguish between BLBC (core) and nBLBC. On multiple logistic Hospital, Cork, NA, Ireland. regression, absence of DCIS (p= 0.041), was the only one feature which could predict Background: Evaluation of pathologic response to neoadjuvant treatment in breast BLBC (core) in TNBC study group. cancer is an important prognostic factor, however, criteria for its classification have Conclusions: Morphology and immunohistochemistry do not assist in correct not been standardized. The Residual Cancer Burden (RCB) system is frequently used identification of BLBC (Core basal) phenotype from non-BLBC in TNBC study group. and combines several histologic parameters, including nodal status, into a complex In the present study, absence of DCIS was the only histopathologic feature, which could 0.17 LN 0.17 logarithmic equation (RCB=1.4(finvdprim) +[4(1-0.75) )dmet] ) yielding four categories predict BLBC independently. Well-characterized and standardized IHC signature would - pathologic complete response (pCR) and RCB scores 1-3 - which correlate with distant help accurate identification of BLBC and impart uniformity among various studies. relapse-free survival (0-36% at 5 yrs). The Mandard Tumour Regression Grade (TRG) is a simple, easily applied qualitative comparison of relative proportion of tumour bed 269 CCN Expression in Ductal Proliferations; a Preliminary Study residual carcinoma and fibrosis. It is frequently applied in gastrointestinal cancers, but its use has not been studied in breast cancer. O Saglam, G Toruner, KG Haines. Yale University, New Haven, CT; UMDNJ, New Design: We aim to Jersey Medical School, Newark, NJ. 1) Compare Mandard TRG to RCB Background: CCN (Cysteine rich protein 61/Connective tissue growth factor/ 2) Modify Mandard TRG to incorporate nodal status Nephroblastoma overexpressed gene) family has several biologic roles including cell 3) Compare modified system to validated RCB score in the same cohort survival, differentiation, adhesion, migration and angiogenesis. Expression of CCN Fifty consecutive neoadjuvant-treated breast cancer cases (2011-12) were classified genes can enhance or suppress tumor cells, depending on the specific cancer. Aberrant using the Mandard TRG. This system was then modified to incorporate nodal status. expression of CCN1 is observed in many cancers, including infiltrating ductal carcinoma The same cases were also classified into RCB categories using the online calculator. of the breast. The expression of CCN1 in pre-invasive ductal lesions, however, has not Two pathologists classified each case. been studied in detail. Results: Mandard TRGs and RCB classes are compared in Table 1. Design: 6 cases of Usual Duct Hyperplasia (UDH), 7 cases of Atypical Intraductal Hyperplasia (AIDH), 11 Ductal Carcinoma In Situ (DCIS) grade 1, 9 DCIS grade 2 and Mandard TRG and Equivalent RCB Class RCB Class 10 DCIS grade 3 were studied for CCN1 expression by immunohistochemistry. Solid, Mandard TRG pCR 1 2 3 cribriform, micropapillary and papillary subtypes of DCIS were represented. All 11 1 - Complete regression 10 1* 0 0 DCIS-1, 4 of 9 DCIS-2 and 5 of 10 DCIS-3 were ER+/PR+. 3 DCIS-2 were ER+/PR-. 2 - Isolated cell nests 0 3 8* 2* The remaining cases were ER-/PR-. Staining for CCN1 in > 50% of cells was reported 3 - More residual cancer but fibrosis > tumour 0 0 1 6* as “diffuse”, staining in 1-50% of cells was considered “focal”, and staining in <1% of 4 - Residual cancer > fibrosis 0 0 1 6* 5 - No regression 0 0 0 1 tumor cells was “negative”. Cytoplasmic and nuclear staining were recorded separately. Results: CCN1 immunostaining was primarily cytoplasmic, with focal nuclear staining * positive lymph nodes limited to DCIS-3. UDH and surrounding benign breast tissue were negative for CCN1. Applying the simple rule - to predict RCB class, increase the Mandard TRG by one AIDH was negative in 4, focal in 2 and diffusely positive in 1 case. DCIS-1 was negative when there is lymph node positivity - accurately stratifies 43/50 cases by RCB class. in 3, focal in 3 and diffusely positive in 5 cases. DCIS-2 was focal in 2 and diffusely Collapsing those RCB classes with minimal (pCR and RCB1; 0%) versus significant positive in 7 cases. DCIS-3 was diffusely positive in all 10 cases. (RCB2-3; 22-36%) 5-year recurrence risk, the modified Mandard TRG accurately stratifies 100% of cases into clinically relevant prognostic categories (Table 2). ANNUAL MEETING ABSTRACTS 67A 271 Columnar Cell Hyperplasia with Atypia Is Biologically Similar to Ductal Carcinoma In Situ, Not Columnar Cell Hyperplasia S Sanati, C Allred. Washington University in St Louis, School of Medicine, St Louis, MO. Background: Columnar cell hyperplasia with atypia (CCH-A) is diagnostically challenging on H&E-stained slides. Although its clinical significance is unclear, compelling evidence suggests that it is associated with a high-risk for developing breast cancer, and may be a direct precursor. We hypothesize that CCH-A is biologically different than columnar cell hyperplasia without atypia (CCH), and similar to low- grade ductal carcinoma in situ (DCIS) which, if true, could provide novel tools (i.e. biomarkers) to improve the diagnosis of CCH-A. The purpose of this study was to compare molecular features of CCH, CCH-A, and DCIS at the RNA and protein levels, predicting that there are unique features shared between CCH-A and DCIS. Design: Gene expression profiling was performed using microarrays (Affymetrix U133-A) utilizing RNA isolated from luminal cells within normal terminal duct lobular units (TDLUs), CCH (n=8) and DCIS (n=11) obtained by laser-capture microdissection. Selected transcripts expressed at significantly different levels are being re-evaluated at the protein level by immunohistochemistry on independent samples of TDLUs, CCH, CCH-A, and DCIS. Results: Microarray analyses identified a large number of significantly differentially expressed gene transcripts (representative examples in Table 1): Examples of significantly differentially expressed transcript genes in TDLU vs CCH and CCH vs DCIS (all p <0.001) Gene TDLU vs CCH (fold change) CCH vs DCIS (fold change) AFP +4X -110X AREG +10X -12X CD44 - -9X SERPINB5 - -12X GABBR1 -4X -9X WIF1 - -69X In preliminary IHC studies, expression of CD44 and WIF1 at the protein level was similar across all comparisons. SERPINB5 (maspin) protein expression, which was primarily restricted to myoepithelium in TDLUs, was substantially decreased in CCH-A and DCIS, consistent with the RNA results. SERPINB5 was also expressed (and mislocalized) in the cytoplasm of a varying proportion of CCH-A and DCIS luminal epithelial cells, which was not observed in TDLUs and CCH. Similar IHC studies are ongoing involving additional genes/proteins. Conclusions: Over 200 differentially expressed gene transcripts (RNA) were identified between CCH and DCIS. Preliminary IHC studies suggest that some of these transcripts (e.g. SERPINB5) are also differentially expressed at the protein level, and that DCIS and CCH-A are similar. Biomarkers of this nature could be useful in improving our ability to diagnose CCH-A.

272 Multicentric Comparative Study between One-Step Nucleic Acid Amplification (OSNA) Whole Node Assay and Standard Histology for Axillary Lymph Node Assessment Conclusions: 1. CCN1 is not expressed in normal or hyperplastic breast ductal I Sansano, B Vieites, M Sancho, C Garcia, I Amendoeira, L Bernet, JM Perez, M epithelium. Espinosa-Bravo, IT Rubio, S Ramon y Cajal, V Peg. H. U. Vall d Hebron, Barcelona, 2. The frequency of CCN1 expression correlates with the grade of the in situ lesion, Spain; H. U. Virgen del Rocio, Sevilla, Spain; H. de Salamanca, Salamanca, Spain; H. being present in all DCIS-3 cases examined. de Sao Joao, Porto, Portugal; H. Lluis Alcanyis, Xativa, Valencia, Spain; H. U. Vall 3. CCN1 expression is independent of ER or PR expression. d’Hebron, Barcelona, Spain. Background: Automated molecular detection of cytokeratin 19 mRNA by one-step 270 NKX3.1 Expression in Primary Breast Carcinoma nucleic acid amplification (OSNA) has demonstrated to be an accurate and standarized MA Samols, A Subhawong, R Sharma, PB Illei, P Argani, AM Cimino-Mathews. Johns method to assess sentinel lymph node (SLN) biopsies. The introduction of this tool Hopkins Hospital, Baltimore, MD. has resulted in an upstaging rate in SLNs between 9 and 47% due mainly to the whole Background: NKX3.1 functions as a tumor suppressor gene that is regulated by analyses of the node. However, little is known about the rest of axillary nodes. In this androgens and is downregulated in prostate cancer. Immunohistochemistry (IHC) study we compare the OSNA method with histopathology of single tissue sections for for the NKX3.1 has been shown to be highly specific for prostatic-derived tumors the staging of axillary non SLNs in patients with early breast cancer. and tissue; however, NKX3.1 expression has also been reported in a small number of Design: A cohort of 79 patients who underwent complete axillary lymph node dissection breast carcinomas. Furthermore, NKX3.1 has been shown to inhibit estrogen receptor (cALND) after identification of SLN metastasis by the OSNA assay were included. signaling (Cancer Res. 2008; 68:7380-5). Here, we investigate labeling of NKX3.1 in Axillary dissections were sent without fixation to the Pathology Department, where all invasive ductal and lobular carcinomas of the breast with full characterization of ER, non-SLNs were identified and isolated. A central 1-mm slice of each node was fixed PR and Her-2 status. and embedded in paraffin for histological examination while the remaining tissue was Design: Tissue microarrays (TMAs) of primary invasive breast carcinomas were labeled frozen and stored at -80ºC before analysis by OSNA. by IHC for NKX3.1. The TMAs included 86 cases of invasive ducal carcinoma (IDC) Results: A total number of 1084 axillary nodes were obtained, mean 13,7 (range 8-32). and 37 cases of invasive lobular carcinomas (ILC). The IDC consisted of 20 luminal 763 met weight criteria (> 50 mg) to be analyzed both by histology and OSNA. The A, 7 luminal B, 14 Her-2 positive/ER negative, 32 basal like, and 13 unclassified ER/ molecular assay detected 59 macrometastases and 66 micrometastases while histological PR/Her2 triple negative carcinomas. The ILC consisted of 34 cases of luminal A and analysis revealed 54 macrometastases and 12 micrometastases (p<0,05). Regarding 3 cases of luminal B. The ILC included 3 cases with pleomorphic LCIS and 1 with cALND, 50 out of 79 patients (63,3%) were diagnosed as positive by OSNA while only histiocytoid features. NKX3.1 expression was scored as percentage nuclear positivity 29 (36,7%) by histology (p<0,05). Furthermore, 14 patients would have changed staging and staining intensity. Any amount of staining was considered positive. from pN1 to pN2 if the diagnoses provided would have been the OSNA one (p<0,05). Results: Nuclear NKX3.1 labeling was seen in 2 of 86 IDCs (2%), and 10 of 37 ILCs Conclusions: These results show that, compared to molecular detection, histopathology (27%). All cases were luminal A, one case of the positive ILC had pleomorphic features. of single tissue sections significantly underestimates the frequency of axillary node Staining intensity was weak in all cases with variable percentages of cells demonstrating metastases and thus pN status. However, it is still uncertain the clinical value of occult expression (1-100%). NKX3.1 positivity was seen only in ER- and PR-positive breast metastasis in non-SLNs. Prospective studies will be carried out to determine the clinical carcinomas, which showed a significant correlation (p=0.0002, Fisher exact test). impact of this finding. Positive staining was also significantly associated with ILC (p<0.0001, Fisher exact test). Expression was not correlated with tumor stage, size, Her2 expression, presence 273 Pogesterone Receptor (PgR) Protein but Not mRNA Is of lymph node metastases or patient age. Associated with Better Outcome in Tamoxifen Treated Breast Cancer Conclusions: Our study is the first to evaluate NKX3.1 expression in breast carcinomas Patients with known ER, PR and Her-2 status. NKX3.1 immunolabeling was significantly KA Schalper, K Li, E Zarrella, DL Rimm. Yale University, New Haven, CT. associated with ILC and positive hormone receptor status. Further studies are needed Background: ERα and PgR protein are frequently co-expressed in breast carcinomas to evaluate what role if any NKX3.1 plays in ER signaling and hormonal response in and their levels are positively associated with better patient outcome. PgR protein these neoplasms. expression has been shown to be independently predictive of response to hormonal therapy, but some studies suggest its specific for ERα. Less is known about mRNA. 68A ANNUAL MEETING ABSTRACTS Data using both RT-PCR (Kim et al, 2011 JCO) and quantitative in situ mRNA 10b expression was reduced in the metastatic brain tumors as compared to primary hybridization (qISH) (Bordeaux et al., 2012, PLoS One) show that ESR1 mRNA levels breast cancer tumors. Statistical analysis using the Kruskal-Wallis test across all 3 may be predictive of response to endocrine therapy in a manner that is different from groups was significant (p < 0.001). ERα protein. Here we assess the prognostic and predictive properties of PgR mRNA. Design: PgR protein and mRNA were measured on formalin fixed paraffin embedded (FFPE) tumor samples on two cohorts represented on tissue microarrays (TMA). Cohort 1 represents a retrospective cohort of 404 breast carcinomas collected from the Yale Pathology archives between 1975 and 2005. Cohort 2 was constructed from 260 breast carcinomas, most of them between 2000 and 2009. PgR protein was measured using the mouse monoclonal clone PGR636 (DAKO). ERα was measured with the rabbit monoclonal clone SP1 (Thermo scientific). The RNAscope paired-primer method was used for PgR mRNA qISH with Ubiquitin C and DapB ISH probes were used as positive and negative controls for each run. PgR protein and mRNA were measured using the AQUA method of quantitative fluorescence. Results: In both cohorts PgR protein scores showed a positive non-linear association with PgR mRNA levels (Cohort 1 R2=0.12, P<0.001; Cohort 2 R2=0.59, P= P<0.0001). PgR protein and mRNA levels were also concordant to pathologist readings of PgR status. Neither PgR protein or mRNA levels were prognostic in Cohort 1. However, positive PgR protein using a visually defined threshold was associated with better outcome only in Tamoxifen treated patients and this effect was independent from ERα levels (Log Rank p=0.009). PgR mRNA was not predictive for response to Tamoxifen. Conclusions: Our data show that elevated PgR protein but not in situ PgR mRNA levels may be predictive of favorable response to endocrine therapy in breast carcinomas. Validation in larger independent cohorts is required.

274 Characteristics of Breast Cancer in Patients with PTEN Hamartoma Tumour Syndrome AP Sciallis, A Nassar, LA Boardman, VS Chandan, SS Shah. Mayo Clinic, Rochester, MN. Conclusions: MicroRNA miR-10b was significantly increased in the primary breast Background: Phosphatase and tensin homologue (PTEN) is a tumor suppressor protein cancers which metastasized to the brain. Its espression was reduced in the metastatic important to the (PI3K)-AKT pathway of tumor cell growth and survival. Germline brain tumors indicating ‘homing’ effect. Our findings are clinically relevant since they mutations of PTEN causing haploinsufficiency are central to the PTEN hamartoma have potential clinical use in prognosis and targetted anti-metastatic therapy in breast tumour syndrome, of which Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes cancer patients. MicroRNA miR-10b could be silenced by antagomirs (chemically are members. Cowden syndrome (CS) is also a hereditary breast cancer syndrome, and modified anti-miRNA oligonucleotides) to treat and prevent brain metastasis. These it is well-known that affected patients have an increased lifetime risk of developing molecular microRNA signature-based prophylactic and therapeutic strategies could breast cancer. However, analysis of specific tumor characteristics, including type, improve patients’ quality of life and overall survival in the era of personalized medicine. grade, hormone receptor status, and stage at presentation, has been subject to only a few published studies. Our institution has a well-characterized database of patients affected with a PTEN hamartoma tumor syndrome. We wish to present our experience 276 Clinicopathologic Profile and Outcomes of Microinvasive Breast with these patients focusing on breast cancer characteristics. Cancer – An Analysis of 42 Cases Design: After obtaining IRB approval, we accessed a departmental database of patients LA Shatat, N Gloyeske, R Madan, O Tawfik, F Fan. University of Kansas, Kansas with either Cowden or Bannayan-Riley-Ruvalcaba syndromes. Syndrome diagnosis was City, KS. reached through established clinical criteria with occasional supplementation through Background: The clinical implication of microinvasive breast carcinoma, especially PTEN gene mutation analysis. Patient characteristics were then reviewed. when multiple foci are identified, is still not clearly defined. This study is designed Results: The study population consisted of 20 patients (10 M, 10 F; average age 53.6 to collect clinicopathologic and follow up data on patients with microinvasive breast years), 17 with CS and 3 with BRR. Breast cancer was present in 6 CS patients (all carcinoma. female; 60% of women; average age at presentation 44.8 years). Benign breast disease, Design: Microinvasive breast carcinoma cases were identified from our surgical typically manifested as proliferative fibrocystic changes, affected 3 additional women, pathology files between 1998 and 2012. Patients treated with neoadjuvant therapy were 2 of whom required bilateral mastectomy due to symptom severity. In breast cancer excluded from the study. Histologic data including invasive breast cancer type, nuclear patients, 3 tumors were invasive vs 3 in situ, 3 patients had bilateral disease, and all were grade, number of microinvasive foci and lymph node status were collected. The type, ductal phenotype of various grade. Patients with invasive disease presented with a breast size, and grade of the associated in-situ component were also recorded. Biomarkers mass (average size 2.1 cm, range 1.2-3.5) and had either Stage IIA (2) or Stage IIIA were recorded from the in-situ component due to the inconsistent availability of markers (1) disease. In 2 patients (1 bilateral invasive tumors; 1 DCIS) immunohistochemistry on the microinvasive component. Clinical follow up data such as incidence of local was performed and all were ER/PR positive but did not overexpress HER2 if invasive. recurrence, distant metastasis as well as survival was also noted. Conclusions: Our analysis demonstrates the high rate of breast cancer occurring in Results: Forty two microinvasive carcinoma cases were identified among 1180 women with CS. Breast cancer was manifested as either invasive or in situ disease; all invasive breast cancer cases for the same time period, representing 3.6% of the overall tumors were ductal and expressed ER and PR when performed. Our study highlights invasive cases. The mean age of the 42 patients was 58 years (range 33-90 years). The the phenotypic tumor similarities between CS patients and the general population, and clinicopathologic profiles of these cases are shown in Table 1. especially given the absence of breast cancer in affected males, may suggest a potential Table 1 interaction between tumor development in CS patients and hormone status. Pathologic Features Number of Cases (n=42) Number of Microinvasive Foci ≤3 foci: 29 4-9 foci: 6 275 Molecular MicroRNA Signatures of Breast Cancer with Brain ≥10 foci: 3 Metastasis Unknown: 4 S Sethi, A Ahmad, R Ali-Fehmi, S Mittal, W Chen, FH Sarkar. Wayne State University Type of Microinvasive Carcinoma Ductal: 39; Mucinous: 3 Nuclear Grade Nuclear grade 1: 5 School of Medicine, Detroit, MI. Nuclear grade 2: 10 Background: Breast cancer is the most common non-skin cancer among women in Nuclear grade 3: 27 the US. Breast cancer-related mortality and morbidity are primarily due to metastatic Associated Ductal Carcinoma In-situ Comedo: 26; Non-comedo: 13; unknown: 3 disease especially brain metastasis which also impacts patients’ quality of life. Molecular In-situ Grade Low to intermediate: 7 mechanisms of brain metastasis of breast cancer is largely unknown. Molecular High: 34 Unknown: 1 signatures in the primary breast cancers that identify prediliction for brain metastasis In-situ Size (cm) 0.4-6.5 (mean: 2.3) would be very important for prognosis and designing novel targetted therapies to prevent Biomarkers of In-situ Component ER+/Her2+: 5 and eliminate brain metastasis. MicroRNAs (miRNAs) are recently described novel ER+/Her2-: 10 molecules with great promise in this regard. ER-/Her2+: 13 Design: RNA was extracted from formalin fixed paraffin embedded tumor tissues from ER-/Her2-: 5 Unknown: 9 a cohort of breast cancer patients with brain metastasis, and age, stage and follow-up Lymph Node Status No nodal metastasis: 32 matched breast cancer cases without brain metastasis. We interogated microRNA Positive isolated tumor cells: 3 expression status by profiling using EQIXON assay. Quantitative real-time PCR (qRT- Positive micrometastasis: 1 PCR) was used to validate abnormal expression of miRNAs. Data was statistically Positive macrometastasis: 1 analyzed using Kruskal-Wallis test to determine the clinical significance of the findings. Unknown: 5 Follow up (1-112 months) Alive with no local recurrence or distant metastasis: 36 Results: Of the over 120 microRNAs profiled, expression of microRNA miR-10b was Death: 1 (respiratory failure, no evidence of metastasis) highly increased in the primary breast cancers of patients who subsequently developed Unknown: 5 brain metastasis as compared to those who did not develop brain metastasis. The miR- ANNUAL MEETING ABSTRACTS 69A Conclusions: Microinvasive breast carcinoma represents a small portion of invasive 279 Evaluation of Breast Pathologies Using Multi-Photon carcinomas. The majority of cases is associated with high grade ductal carcinoma in- Microscopy situ and have high nuclear grade. There is no consistent ER/Her2 expression pattern Y Sheykin, YK Tao, OO Ahsen, D Shen, D Schmolze, N Johnson, JG Fujimoto, JL in the associated in-situ carcinoma. The number of microinvasive foci, the extent of Connolly. Beth Israel Deaconess Medical Center, Boston, MA; Massachusetts Institute the in-situ component and the biomarker expression pattern do not appear to impact of Technology, Cambridge, MA. the overall excellent outcome. Background: Multi-photon microscopy (MPM) enables high-resolution imaging of intact tissue specimens using both endogenous and exogenous fluorescent and harmonic 277 Breast Cancer with Brain Metastases: Risk Factors and Survival generation contrast. MPM is well-suited for visualizing cytologic and architectural Q Shen, NK Ibrahim, KD Aldape, A Sahin. University of Texas MD Anderson Cancer markers used to differentiate between benign and neoplastic tissues including nuclear Center, Houston, TX. size and shape, cellular arrangement in tissue, and characteristics of intercellular stroma, & Background: The aim of our study was to identify clinicopathologic parameters all of which are assessed when examining hematoxylin and eosin (H E)-stained associated with increased risk for breast cancer brain metastases (BCBM) and to assess histology slides. The advantage of MPM over standard histology is its ability to rapidly survival rates after treatment for BCBM. image freshly excised specimen surfaces without the need for histological processing, Design: We retrospectively analyzed clinicopathologic data from 142 patients who had which could enable intraoperative margin assessment. Here, we investigate the utility been histologically diagnosed as having BCBM between 2002 to 2009 at The University of MPM for evaluating breast pathologies. of Texas MD Anderson Cancer Center. Design: In a prospective study, 179 fresh human breast tissue specimens from 50 Results: The median age at diagnosis of breast cancer was 46 years (range, 24-73 patients were imaged using a 100 fs laser source at 740 nm wavelength. Specimens years). Most patients had invasive ductal histology (91%), grade 3 tumors (68%), were rapid stained with a nuclear stain Acridine Orange (AO). Dual channels were used and positive axillary lymph nodes (64%). A total of 56% of the tumors were estrogen to detect exogenous AO fluorescence nuclear contrast and intrinsic second-harmonic µ 2 receptor (ER)-negative, 45% were Her2 positive, and 27% were triple negative (TN). generation stromal contrast. Multiple overlapping 480 m fields were automatically Brain metastasis was solitary in 52% of patients. The median interval from breast imaged and mosaicked in post-processing, and MPM color channels were remapped to & cancer diagnoses to brain metastases was 46 months (range, 0-266 months), and the a colormap resembling H E staining. Specimens were subsequently fixed in formalin & overall median survival duration after brain metastases was 14.5 months (range, 0-107 and processed for corresponding H E sections. & months). Overall median survival times were shorter for ER-negative (11 months) Results: Of 179 specimens, 138 MPM and corresponding H E datasets were than for ER-positive (24.5 months) patients and were shorter for TN (9 months) than randomized and scored by 3 pathologists in a double-blinded study. The study for non-TN (18 months) patients. Patients who were Her2-positive lived longer than included 59 normal (NBT), 5 fibroadenoma (FA), 11 usual ductal hyperplasia (UDH), did those with Her2-negative disease (17 months versus 11 months). Biomarkers were 10 fibrocystic changes (FC), 20 ductal carcinoma in situ (DCIS), 5 lobular carcinoma evaluated in paired primary tumors and brain metastases in 36 patients for ER status, in situ (LCIS), 38 invasive ductal carcinoma (IDC), and 9 invasive lobular carcinoma in 34 patients for progesterone receptor (PR) status, and in 39 patients for Her2 status. (ILC) specimens (figure). Analysis of the read showed that MPM had a sensitivity and Her2 expression was highly concordant (90% concordance rate) between the breast specificity of 95.0% and 92.9%, respectively, for distinguishing between benign (NBT, and brain tumors, and altered expression was noted in 28% of pairs for ER and in 20% FA, UDH, and FC) and malignant (DCIS, LCIS, IDC, and ILC) breast tissue as compared & of pairs for PR. Three patients with Her2-positive primary tumors had Her2-negative to standard H E histology with an interobserver agreement (Fleiss’ kappa) of 0.85. brain metastases, and only one patient with a Her2-negative breast carcinoma developed Conclusions: MPM provides high sensitivity and specificity for distinguishing a Her2-positive brain tumor. between benign and neoplastic breast tissue. The advantage of MPM imaging is that it Conclusions: Patients with ER-negative, Her-2-positive and TN breast cancers were potentially allows rapid nondestructive intraoperative margin assessment of resected more likely to develop BCBM. Expression of breast cancer biomarkers did not differ breast specimens without the need for frozen section exam. significantly between the primary breast tumors and the corresponding brain metastases. 280 Clinicopathological Significance of CADM4 Expression in 278 Phyllodes Tumor in Young Women – Focus on Tubular Invasive Ductal Carcinoma of the Breast Adenomatous Tumors with Propensity for Phyllodes like Recurrences J Sim, H Han, SS Paik. College of Medicine, Hanyang University, Seoul, Republic T Shet, B Ramnani, S Sethi. Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India. of Korea. Background: The diagnosis of Phyllodes tumor in women < 20 years poses a problem Background: Cell adhesion molecule 4 (CADM4) is a novel tumor suppressor involved for most pathologist. There are tumors that straddle between Fibroadenoma variant in cell adhesion. It has been suggested that the loss or decreased expression of CADM4 (Juvenile fibroadenoma) and phyllodes tumor, where histopathology criteria for is associated with the development of some cancers. However, the CADM4 expression diagnosis are not well defined. in breast cancer has not been investigated. The purpose of this study was to investigate Design: A retrospective review of all non-conventional firboepithelial neoplasms or the clinocopathological significance of CADM4 expression in breast cancer. juvenile fibroadenomas or phyllodes tumor in women < 24 years from 2003 to 2011 Design: We used the tissue microarrays consisting of 45 cases of ductal carcinoma in was conducted. Many of fibroepithelial neoplasms in younger women were found to situ (DCIS) and 256 cases of invasive ductal carcinoma (IDC). We evaluated CADM4 have a tubular or adenomyoepithelioma like epithelial compoenent and hence a review expression in the tissue microarrays by immunohistochemistry, reviewed the patients’ classifcation was devised. medical records, and performed an adjustment of the clinicopathological data with a Results: Of the 41 cases, 7 were diagnosed as phyllodes tumor upfront while remaining statistical analysis. 35 were an interesting spectrum of lesions that were classified as 1) Tubular adenoma Results: CADM4 was expressed in 37 of 45 DCIS cases (82.2%) and 173 of 256 IDC and sclerosing adenosis type (TA/SA) characterized by multiple capsulated nodules cases (67.6%). The expression rate of CADM4 was significantly higher in DCIS than resembling adenosis with cellular intervening stroma.2) Tubular adenomyoepithelioma IDC (p = 0.049). Loss or decrease of CADM4 expression was strongly correlated with with cellular stroma(TAMECS) where the epithelial component resembled an higher histologic grade (p = 0.020), absence of estrogen receptor (p < 0.001), absence adenomyoepithelioma with myoepithelial proliferation but the intervening stroma was of progesterone receptor (p = 0.024), and overexpression of c-erbB-2 (p = 0.018). In cellular. 3) Juvenile fibroadenoma (JFA) like with TDLU like structure and fair amounts univariable and multivariable Cox regression analyses for all 256 IDC cases, CADM4 of paucicellular stroma. A summary of differences is given - expression was not significantly associated with overall and disease-free survival. However, CADM4 expression showed a significant positive association with longer Clinical differences in three groups TA/SA type( n = 15) TAMECS( n= 9) JFA type( n=8) disease-free survival in 187 cases of early stage IDC (p = 0.039, log-rank test). Age < 11 years 3 0 0 Conclusions: Loss or decrease of CADM4 expression seems to play an important role Age 12- 18 years 7 7 5 in breast cancer progression, especially related to the invasiveness, and its aggressive Age 19-24 years 5 2 3 biological behavior. CADM4 can be applied to a novel predicting marker for recurrence Bilateral Breast affected 9 4 6 possibility or outcome of disease in an early stage IDC. Multiple tumors 11 2 3 Size < 5cm 10 4 3 Size 5- 10 cm 4 3 5 281 Computational Image Analysis To Identify a Morphological Size > 11 cm 1 3 0 Signature of Claudin-Low Breast Cancer Recurrences 9 6 4 Recurrences as phyllodes tumor 3 3 4 K Singh, L Collins, N Knoblauch, M Hefti, A Beck. Beth Israel Deaconess Medical Center, Boston, MA. Ten of the 35 patients developed phyllodes tumors. A thorough histologic evaluation Background: The Claudin-low molecular subtype of breast cancer has recently been revealed that MIB1 >10% and stromal overgrowth at 20X magnification served to identified as a distinct subset of triple negative breast cancers that tends to show predict likelihood of development of phyllodes tumor in this group. Stromal cellularity, decreased expression of the Claudin genes and metaplastic morphologic features, tumor size CD10 and CD 34 did not predict these recurrences. One patient was put on suggestive of an epithelial-mesenchymal transition (EMT) phenotype. Our study aims imatinib and had 50% reduction in her bilateral breast lumps but patient died due to to further characterize morphologic differences between Basal-like and Claudin-low acute liver failure. In the last two patients MRI was used to choose excision of the most breast cancers. cellular tumor. One patient with metastatic phyllodes tumor succumbed to the disease. Design: To characterize the morphologic signature of this breast cancer subset, we Conclusions: The histopathology criteria for defining phyllodes tumor in young performed an adaption of the C-Path (Computational Pathologist) method for whole- women needs to be redefined. The tumors described above represent a distinct group slide images. In brief, the C-Path method segments a histologic image into 3 regions with propensity for multiplicity and phyllodes like recurrences and a relook of all (epithelium, stroma, and an indeterminate region termed “EMT-like”). C-Path then fibroepithelial neoplasms in children with these findings is required. computes thousands of quantitative morphologic characteristics from these regions, characterizing the size, shape, texture, intensity, variability, and relationships between objects within the tissue regions. C-Path was performed on a total of 57 breast cancers that had undergone molecular profiling and subtyping as part of The Cancer Genome 70A ANNUAL MEETING ABSTRACTS Atlas Project and had been classified as Basal-like (n=52) or Claudin-low (n=5). We Ki67 r=0,72). When comparing automated image analysis results with conventional performed an unpaired two-class Significance Analysis of Microarrays to identify histological evaluation of IHC markers, there were only 3 cases misclassified for morphologic features significantly associated with the Claudin-low subtype. We set ER positivity or negativity, and 4 cases misclassified for PR positivity or negativity. a significance threshold of an FDR < 15% for identifying significantly differentially Correlation of RT-qPCR data for ER and PR with quantitative immunohistology was expressed morphologic features. highly significant (p<0.0001) with few cases misclassified as negative by image analysis. Results: We identified 95 morphologic features significantly associated with the Correlation coefficients between image analysis and RT-qPCR on fresh frozen tissue, Claudin-low molecular subtype at the FDR threshold of 15% and 2 features at a threshold were 0.72 for ER, 0.73 for PR, and 0.51 for Ki67. In the multivariate model, the RT- of <1%. 73 (77%) of these features are morphologic characteristics of the breast cancer qPCR value on FFPE tissue for Ki-67 was the only significant parameter (p<0.05) using stroma (including: variability of stromal matrix intensity, with less variability in complete pathologic response as the endpoint. This was confirmed with a multivariate Claudin-low), 20 (21%) of the features are of the EMT-like tissue (including: textural recursive partitioning model. Also, thresholds for RT-qPCR ans image analysis were correlation, with higher values seen in the Claudin-low cancers), and only 2 (2%) are calculated with this model. morphologic characteristics of the epithelium (both features are measures of the darkest Conclusions: Image analysis of ER, PgR, and Ki-67 is an accurate tool for the pixel intensity in a region, with higher values observed in Claudin-low, potentially measurement of tumor proliferation and shows good correlation with RT-qPCR of reflecting the influence of intra-epithelial inflammatory cells). FFPE and FF tumor tissue. However, in the neoadjuvant setting, RT-qPCR of Ki-67 on Conclusions: In this broad analysis of quantitative morphologic differences between FFPE tissue was superior to conventional or automated IHC analysis for the prediction Claudin-low and Basal-like breast cancer, we identified morphologic features of complete pathologic tumor response. significantly associated with the Claudin-low molecular subtype. The differentially expressed features are almost exclusively from the stroma and EMT-like tissue with 284 Hormone Receptor Expression in HER2+ Breast Cancer and few significant differences identified in the epithelial compartment. Relationship to Ki67 and p53 Expression D Smith, D Xiang, A Christie, G John, Y Peng, S Sahoo, Y Fang, V Sarode. UT 282 Comparison of HER2 Testing by IHC/FISH and RT-PCR in Southwestern Medical Center, Dallas, TX. Estrogen Receptor (ER) Negative or Borderline Patients (pts) with Early Background: HER2+ invasive breast cancer (IBCA) is a heterogeneous group less Stage Breast Cancer (ESBC) likely to be estrogen receptor (ER) positive than HER2- IBCA. Bidirectional crosstalk B Singh, N Ziguridis, S Butler, F Jamshidian, D Cherbavaz, A Sing. New York University between HER2 and ER signaling pathways has been demonstrated in vivo, and a School of Medicine, New York, NY; Genomic Health, Inc., Redwood City, CA. subset of HER2+ IBCA appears to be driven by ER. In addition to ER, PR and HER2, Background: Accurate determination of HER2 status is critical for making HER2 proliferation and p53 alteration play important roles in defining molecular subtypes. targeted therapy treatment (Tx) decisions. HER2 concordance between analytical Further characterization of the HER2+ subset is important for appropriate targeted methods (IHC, FISH & RT-PCR) is ≥95% (Baehner JCO 2012, Baehner ASCO Breast therapies. The relationship between HER2 and ER, PR, Ki67 and p53 expression has 2008). Lack of agreement between IHC/FISH and RT-PCR has been recently reported not been well documented. (Dabbs JCO 2011). A study to evaluate new CAP-mandated IHC positive cutoffs for ER Design: Using a set of 482 HER2-amplified IBCA tumors, we examined the relationship protein expression using ER mRNA expression assessed by RT-PCR was undertaken between ER, PR, Ki67 and p53 expression and HER2 amplification. Estimation of in consecutive ESBC pts from a single institution. Here we report the agreement of biomarkers was performed using routine immunohistochemistry. HER2 FISH assay was HER2 assessments by standardized IHC, FISH and RT-PCR. performed with a PathVysion kit according to manufacturer’s guidelines. Biomarker Design: Consecutive IHC-defined ER-negative (<1% positive cells) or ER-borderline semi-quantification was performed by image analysis. Positive cut-offs were as follows: (1-10% positive cells) ESBC pts were identified at NYU School of Medicine. HER2 ≥1% for ER, PR; FISH ratio >2.2 for HER2. For each parameter among the subgroups, protein assessment used standard IHC (4B5, Ventana) with reflex testing of HER2 1-way ANOVA was used for overall mean comparison and Tukey-Kramer tests were 2+ cases by FISH (defined as ≥2.0) using the FDA-approved HER2 DNA Probe Kit used for pairwise comparisons. (Vysis). Hybridization results were recorded and analyzed by BioView Duet system Results: Of the HER2+ tumors, 200/482 (41.49%) were ER+ and PR+; 193 (40.04%) (Allegro Plus Automated Scanner) with HER2 application software (BV-HER2-AF). were hormone receptor (HR) negative; and 89 (18.46%) were ER+ (85) or PR+ (4). Quantitative HER2 mRNA assessment used the Oncotype DX® assay, reference- HER2 amplification was lower in triple positive compared to double positive and normalized expression measurements ranged from 0 to 15, where each 1-unit increase HR- tumors. ER and PR expression levels were higher in triple positive than in double reflects about a two-fold increase in RNA and HER2 categories used pre-specified positive tumors. HR- tumors showed higher Ki-67 and p53 expression compared to reference-normalized values (positive≥11.5, equivocal ≥10.7 to <11.5, negative <10.7). HR+ tumors. Mean values for each parameter in each of the subgroups are shown below. Results: Of 140 evaluable samples, 106 (76%) were ER(-) and 34 (24%) were ER- Variable ER+/PR+/HER2+ ER+ or PR+/HER2+ ER-/PR-/HER2+ p-value HER2 Amplification borderline by IHC. Pts characteristics included: 69.3% were >50 yrs; 83.6% had poor 4.60 5.45 5.38 0.0027 differentiation; 31.2% had >1 positive nodes; 34.3% were > 2.0 cm; 26.4% were (FISH Ratio)∗§ ER (%)∗§¶ 79.31 45.81† 0.00 <0.0001 HER2(+) by IHC/FISH. There was 83.8% positive agreement (PPA) between HER2 PR (%)∗§¶ 40.24 8.25‡ 0.00 <0.0001 (+) pts by IHC/FISH and RT-PCR (88.6% PPA by ASCO/CAP guidelines). Percent Ki67 (%)§¶ 39.56 41.75 51.81 <0.0001 negative agreement (PNA) was 99.0% between HER2 (-) cases by IHC/FISH and RT- p53 (%)§¶ 27.81 28.94 49.45 <0.0001 PCR. Of the 32 HER2-IHC 3+ pts, 93.8% were HER2 (+) and 6.2% were equivocal by ∗Triple positive and double positive significantly different (p<0.05) §Triple positive and HR- RT-PCR. Table shows cross classification of HER2 status by RT-PCR versus IHC/FISH. significantly different (p<0.05) ¶Double positive and HR- significantly different (p<0.05) †HER2+/ Conclusions: Accurate and precise measurement of HER2 is important for Tx decision ER+/PR- tumors ‡HER2+/ER-/PR+ tumors making. In this subset analysis of ESBC pts with borderline or negative ER by IHC Conclusions: High levels of ER expression and lower levels of HER2 amplification in there is a high degree of PPA and PNA between HER2 assessment by standardized triple positive compared to HR- tumors may indicate dominance of ER signaling in this methods by IHC/FISH and quantitative RT-PCR. subset of HER2+ IBCA. Selected patients in this subgroup may benefit from inhibition HER2 Status by RT-PCR vs HER2 Status by IHC/FISH of both ER and HER2. On the other hand, tumors with absent HR showed higher HER2 IHC/FISH amplification, proliferation and p53 expression indicating a more aggressive subset. RT-PCR (-) (+) Total (-) 101(98.1%) 4(10.8%) 105(75.0%) Equivocal 1(0.9%) 2(5.4%) 3(2.1%) 285 P16 Expression and Its Correlation with Ki67 in Triple-Negative (+) 1(0.9%) 31(83.8%) 32(22.9%) Breast Carcinoma Total 103 37 140 JZ Sugianto, V Sarode, S Sahoo, Y Fang, D Oliver, Y Peng. University of Texas Southwestern Medical Center, Dallas, TX. Background: Triple-negative breast carcinoma (TNBC) characterized by negativity of 283 Image Analysis and Quantitative Real-Time PCR of Nuclear ER, PR, and HER2 is associated with a poor prognosis due to its aggressive behavior Antigens (ER, PgR, Ki-67) in Breast Cancer and lack of conventional therapeutic targets. Many TN tumors resemble squamous cell P Sinn, M Keller, V Elke, A Schneeweiss, R Wirtz. University of Heidelberg, Heidelberg, carcinoma morphologically. Diffuse p16 expression is a surrogate marker for high risk Germany; STRATIFYER Molecular Pathology GmbH, Köln, Germany; National Center HPV-related squamous cell carcinoma. High Ki67 expression is associated with worse of Tumor Diseases (NCT), Heidelberg, Germany. prognosis in TN cancer patients. P16 expression in TNBC and its correlation with Ki67 Background: The exact determination of hormone receptors and tumor proliferation expression have not been well characterized. We investigated p16 expression in TNBC is of critical importance for clinical decision making in breast cancer. Recently, cases to explore its potential prognostic value. molecular methods have emerged as an alternative to quantitative estimation of Design: Sixty high grade invasive TNBC cases were identified at our institution (2004 immunohistochemistry results. - 2011). Tissue microarrays (TMAs) were constructed with duplicate representative Design: Matched fresh and formalin fixed pretreatment biopsy samples were available cores of each tumor and adjacent normal breast ducts. The TMAs were immunolabled from 90 patients participating in neoadjuvant clinical trial. RT-qPCR data were available for p16, Ki67 and p53. P16 stain was scored based on intensity and extent of nuclear after extracting RNA using Qiagen kits. RNA was isolated from fixed tissue samples and cytoplasmic staining as follows: negative (0), weak/1-30% (1), moderate/31-60% by using coated magnetic particles.Multiplex RT-qPCR was performed by TaqMan® (2), moderate to strong/61-90% (3), strong/91-100% (4). Immunostained slides were based primer probe sets for ESR1, PGR, Ki67, as well as CALM2 as reference gene. All reviewed and scored by two pathologists (YP, JS). A final average score of each case cases were also used for quantitative immunohistology by automated image analysis on was used for statistical analysis. Ki67 and p53 stains were evaluated by image analysis. a virtual microscopy system (Aperio Technologies, Vista, CA, USA).For determination We compared p16 expression of tumors and adjacent normal tissue. Correlation analysis of the biologically most significant measure of tumor proliferation, a multivariate linear was performed between p16 and Ki67 or p53 expression. model was used to compare RT-qPCR and imaging data with the residual tumor burden Results: TN tumors were found to have significantly higher expression of p16 compared score (RBC) according to Symmans (2007). with adjacent normal breast ducts (2.075 ± 0.216 vs.0.146 ± 0.044, p<0.0001; paired Results: We observed a significant correlation of mRNA data from independent, T-test). 42% of the tumors demonstrated diffuse or near diffuse p16 expression (score matched fresh and fixed biopsy samples by using RT-qPCR (ER1 r=0,91; PR r=0,79, of 3 or 4). All the tumors showed high Ki67 expression, but only 37 of 60 (62%) were ANNUAL MEETING ABSTRACTS 71A p53-positive (>1%). Among p53-negative tumors (23, 38%), p16 expression (2.44 ± 288 Pathological Appraisal of Low Nuclear Grade Ductal Carcinoma 0.309) positively correlated with Ki67 (65.22% ± 7.23; r=0.739, p<0.001; Pearson In Situ of the Breast correlation). No correlation was found between p16 and Ki67 expression among the PH Tan, AA Thike, JS-L Wong, GH Ho, S Thilagaratnam. Singapore General Hospital, p53-positive tumors (r=0.157). Singapore, Singapore; National Cancer Centre, Singapore, Singapore; Health Promotion Conclusions: P16 expression is significantly increased in TN tumors compared with Board, Singapore, Singapore. paired normal breast ducts. Its expression correlates with Ki67 in p53-negative but not Background: Ductal carcinoma in situ (DCIS) of the breast is increasingly diagnosed p53-positive TN tumors, suggesting p16 may have a prognostic value in p53-negative TN as a result of mammographic screening. Debate regarding the true biological potential tumors. Further study is needed to better define the role of p16 as a potential prognostic of low grade DCIS continues to challenge therapeutic considerations. In this study, we marker. Primary TNBC can resemble HPV-related sqaumous cell carcinoma not only review the clinicopathological characteristics of a series of low nuclear grade DCIS morphologically but also immunophenotypically with strong diffuse p16 expression; and correlate with clinical outcome. this can be helpful in identifying metastatic TNBC. Design: Cases of DCIS of low nuclear grade were retrieved from the files of the Department of Pathology, Singapore General Hospital, between 1994 and 2010. Clinical 286 Significance of GATA3 Expression in Outcomes of Breast presentation was correlated with histological parameters. Associated microscopic Cancer Patients Who Received Systemic Chemo and/or Hormonal Therapy changes were documented. Follow-up was obtained from patient casenotes. Statistical and Clinicopathological Features of GATA3 Positive Tumors associations with a p value of <0.05 were deemed significant. M Surowiecka, C Blair, D Knapp, L Varghese, E Gulbahce. University of Minnesota, Results: Of 237 cases of low grade DCIS in our files, 36 had to be excluded due to Minneapolis, MN; University of Alabama at Birmingham, Birmingham, AL; VAMC, presence of invasive disease in 3 cases, 2 cases were lobular carcinoma in situ and not Minneapolis, MN; Fairview Southdale Hospital, Edina, MN. DCIS on histological review, 10 cases contained intermediate grade foci, and no material Background: GATA3 and ER are involved in a positive cross-regulatory loop and was available in 21 cases. From the final 201 cases, 128 (63.7%) were screen detected are frequently coexpressed in breast cancers. GATA3 expression was shown to be an and 73 (36.3%) were symptomatic. Median age of affected women was 50 years and independent predictor of response neoadjuvant chemotherapy, and overall and disease median DCIS size was 9 mm. The commonest architectural pattern was cribriform with free survival in some studies, while others showed no difference. We investigated the 78 (39%) cases, followed by combined cribriform and micropapillary with 24 (12%) association of GATA3 expression with clinicopathologic variables and outcome in cases. Presence of solid papillary carcinoma elements was noted in 28 (14%) cases. patients who received systemic chemo and/or hormonal therapy. There was statistically significant association of presence of calcifications with screen Design: Breast cancer tissue microarray blocks from 516 women who received systemic detected DCIS (p < 0.001). Flat epithelial atypia (FEA) was observed in 18 (9%) cases. chemo and/or hormonal therapy were used for GATA3 immunohistochemistry. Patients’ Recurrent disease occurred in 10 (5%) women during a median follow-up of 69 months. clinicopathologic findings and outcomes were correlated to GATA3 expression. Nuclear Deaths were reported in 3 women, with one being breast cancer related, while the other staining ≥1% was considered positive for GATA3, ER, and PR. 2 died from carcinoma of the vagina and larynx respectively. Results: 436/516(84.5%) of the cases were GATA3 +. GATA3 + tumors were more likely Conclusions: Low nuclear grade DCIS has a small risk of recurrence, though there to be higher stage (p=0.01), and have Grade 1 or 2, ER+, PR + and non-triple negative can be breast cancer specific mortality related to subsequent diagnosis of invasive phenotypes (all p<0.0001). ER -/GATA3 + tumors, compared to ER-/GATA3- tumors, breast cancer. Longer follow-up is needed to understand the true biological nature of had worse breast cancer survival (BCS) (p=0.02), and trend for worse overall survival these lesions. (OS) (p=0.05) in univariate analysis. There was no difference in OS and BCS between patients who received chemo and/or hormonal therapy among GATA3 + and – cases. 289 The OncoType DX® Ductal Carcinoma In Situ (DCIS) Score™ Conclusions: When positivity is evaluated similar to ER/PR (i.e. ≥1% is +), GATA3 + Assay and Quantitative Gene Expression for ER, PR, and HER2: Experience tumors are correlated with lower grade, ER+, PR +, and non-triple negative phenotypes. with 1,071 Patients Although there was no difference in OS and BCS between GATA3 + and – groups, V Tan, H Bailey, AC Tsiatis, J Anderson, J Liu, SM Butler, FL Baehner, C Millward. there was an adverse effect of GATA3 expression among the ER subgroup of patients Genomic Health, Inc., Redwodd City, CA; University of California, San Francisco, CA. who received systemic therapy. Background: The DCIS Score has been validated as a predictor of the 10 year risk of any local recurrence (DCIS or invasive) (Solin et al, SABCS 2011). Here we report the 287 Folate Receptor alpha Is Frequently Expressed in ER/PR Genomic Health Clinical Laboratory experience with DCIS patient samples. Negative and Triple Negative Breast Cancers Design: 1,071 patient samples from 12/28/11 through 9/28/2012 that passed pathology D Tacha, R Bremer. Biocare Medical, LLC, Concord, CA. review and RT-PCR quality measures were included. The 21-gene assay was performed, Background: Folate receptor alpha (FRA) is a membrane-attached protein that and the DCIS Score was calculated based on the validated algorithm using 7 of the facilitates transport of folate, and has been found to be overexpressed in several cancers, 16 cancer-related genes, measured from 0 to 15 (relative to reference genes, one unit including lung, ovarian and breast cancers. In a previous immunohistochemical study, increment associated with a 2-fold change in expression). Low, intermediate, and overexpression of FRA in breast cancer strongly correlated with early recurrence and high risk categories are defined as DCIS Scores of <39, 39-54, and >54, respectively. ≥ decreased median survival. As a result, FRA has emerged as a potential therapeutic target. Descriptive statistics for the DCIS Score, ER, PR, and HER2 were obtained. 6.5 is ≥ ≥ A humanized monoclonal antibody to FRA (Farletuzumab) in currently in development the ER cut-off; 5.5 is the cut-off for PR and for HER2, 11.5 is positive, 10.7 to 11.4 and may be an attractive treatment strategy either alone or combined with chemotherapy. is equivocal, < 10.7 is negative. Design: A new, highly specific clone of an anti-FRA antibody, [26B3.F2], suitable for Results: Of 1,071 samples, 785 (73%) were excisions and 286 (27%) were core biopsies, immunohistochemistry, has recently been developed and characterized on formalin-fixed median age was 59.0 (range 23.0-91.0), 1,069 females and 2 males. The histologic ≥ paraffin-embedded tissue. 26B3 was optimally tittered and applied to 67 cases of breast subtype distribution was: cribriform 38%, solid 37%, DCIS with 50% comedonecrosis cancer, with previously determined ER, PR and Her2 status. Only membrane staining 10%, papillary 9%, micropapillary 5%, apocrine 0.4%, and other 0.5%. The mean of FRA was considered positive, with a cut-off point of 10% of tumor cells staining. DCIS Score was 30.8 (range 0-96.7) with a mean score result of 30.9 for excisions Results: Of the 67 cases of invasive ductal carcinoma stained with 26B3, 20 were and 30.4 for cores. 687 (64%) were low risk, 190 (18%) were intermediate risk, and positive for FRA expression. Significantly, a higher incidence of expression of FRA 194 (18%) were high risk. The mean DCIS Score per histologic subtype was: apocrine ≥ was observed in hormone receptor negative patients (ER-/PR-), independent of Her2 23.1, cribriform 26.0, DCIS with 50% comedonecrosis 48.5, micropapillary 23.5, status (Table 1, p=0.03). Only 4/27 (14.8%) specimens positive for ER and PR were papillary 19.1, and solid 35.1. The mean ER, PR, and HER2 expression levels were also positive for FRA; whereas 16/40 (40.0%) specimens negative for ER and PR 9.5, 7.4, and 9.9, respectively, and were similar between excisions and cores. The mean expressed FRA. ER and PR for each histologic subtype were: apocrine (6.9, 5.1), cribriform (9.7, 7.6), DCIS with ≥50% comedonecrosis (8.4, 6.3), micropapillary (9.4, 7.5), papillary (10.9, Table 1: FRA Expression and ER/PR/Her2 Status ER+/PR+, Her2+/- ER-/PR-, Her2+/- 8.6), and solid (9.4, 7.2) and for HER2: apocrine (9.2), cribriform (9.8), DCIS with FRA + 4 16 ≥50% comedonecrosis (10.0), micropapillary (9.9), papillary (9.7), solid DCIS (9.9). FRA - 23 24 Conclusions: A wide DCIS Score distribution was observed for each histologic % FRA + 14.8% 40.0% subtype. Similar results were observed in DCIS Score value, ER, PR, and HER2 gene Importantly, half of all triple negative specimens (9/18, 50.0%) were positive for FRA, expression between excisions and cores. The majority of samples submitted were ER/ a significant increase over FRA expression in specimens that were ER/PR and/or Her2 PR+ and HER2-negative. positive (11/49, 22.4%) (Table 2, p=0.04). Table 2: FRA Expression in Triple Negative Breast Cancers 290 Immunohistochemical Expression of Androgen Receptor in ER+/PR+ or Her2+ ER-/PR- Her2- (Triple Negative) Triple Negative Breast Cancer: Significant Association with Improved FRA + 11 9 FRA - 38 9 Disease Free Survival % FRA + 22.4% 50.0% AA Thike, L Chong, PY Cheok, J Iqbal, PH Tan. Singapore General Hospital, Singapore, Conclusions: FRA expression has been identified in 30% (20/67) of breast cancers, Singapore. with an increased incidence in ER/PR negative cases. Significantly, 50% (9/18) of Background: Triple negative (TN) invasive breast cancers are defined by the absence triple negative breast cancers were positive for FRA expression. Determining positive of estrogen receptor, progesterone receptor and c-erbB2 expression. Treatment of TN FRA expression in breast cancers may identify patients that would benefit from an breast cancers remains challenging. Androgen receptor (AR) is a member of the nuclear anti-folate targeted therapy, including hormone receptor or Her2 positive patients that receptor superfamily. It is known to be involved in signaling pathways that regulate cell have failed to respond to prior treatment, or triple negative patients, for whom limited proliferation and has been implicated in breast tumorigenesis. This study investigates the treatment options are available. immunohistochemical expression of AR in a large series of triple negative invasive breast cancers diagnosed at our institution, correlating its expression with clinical outcome. Design: The cohort comprised 699 invasive TN breast cancers diagnosed from 1994 to 2010 for which tissue microarrays were constructed. Antibodies to basal markers 72A ANNUAL MEETING ABSTRACTS (CK14, 34βE12, EGFR) and AR were applied to sections cut from tissue microarray Results: Patients (pts) with TNs and non-TNs had similar age (mean 56 +14 vs 54 +15 blocks, using the streptavidin-biotin method. Positive AR expression was defined as years (y)). Significantly more TNs than non-TNs were MES(+) (82/226, 36% vs 14/88, staining of 1% or more of invasive tumor cell nuclei. Disease free survival (DFS) 16%; p= 0.0006). In TNs, MES reactivity significantly correlated with CK5/6 (56/80, and overall survival (OS) were defined as time from diagnosis to recurrence or death 70%; p= <0.00001) and CK14 (29/72, 40%; p=0.017), but not with EGFR (57/78, respectively, and correlated with protein immunohistochemical expression. A p value 73%; p=0.87). Histologic grade (p=0.49) and subtype (p=0.29), median size (2.3 cm+ <0.05 defined statistical significance. 2.9 vs 2.5 cm+ 2.4; p=0.70), lymphovascular invasion (44% vs 33%; p=0.11), and LN Results: AR expression was observed in 38% of tumors and the proportion of AR metastases (50% vs 42%; p=0.26) did not differ in MES(+)TNs vs MES(-)TNs. Pts positive tumor cells ranged from 1% to 95% (mean 29, median 10). About 54% of tumors with MES(+)TN were older than pts with MES(-)TN (58+14 vs 54+14 y; p=0.04), had harbored AR immunoreactivity in more than 10% of tumor cells. AR immunoexpression significantly lower OS and DFS than pts with MES(-)TN. They developed more distant was inversely associated with histologic grade (p=0.002) and mitotic score (p=0.009). metastases (16/69, 23% vs 12/126, 9.5%) with shorter interval (19+11 vs 35+18 months). Among 147 triple negative tumors in this series with known metastatic disease on Significance persisted in log-rank tests of basal-like phenotype and LN status. [Table 1] follow-up, 67% of primary tumors did not exhibit AR immunoreactivity. CK14, 34βE12 Table 1. 5-Year OS and DFS in MES(+) TN (KM Estimates, 95% CI) and EGFR confirmed 85% of cases to be basal-like, without significant association of MES(-) TN, n=144 MES(+) TN, n=82 p value basal-like phenotype with AR expression. DFS was significantly better in AR positive OS 0.91 (0.83-0.95) 0.66 (0.52-0.77) 0.0001 TN breast cancer (p=0.05), with a trend observed for improved OS, as well as better DFS 0.86 (0.79-0.92) 0.67 (0.54-0.77) 0.0003 Basal keratin(+) 0.92 (0.80-0.97) 0.62 (0.45-0.75) 0.0002 DFS and OS, in AR positive basal-like TN breast cancers. Basal keratin(-) 0.90 (0.78-0.96) 0.90 (0.47-0.98) Conclusions: Our study demonstrated that loss of AR expression in invasive TN LN(+) 0.87 (0.70-0.94) 0.57 (0.35-0.73) 0.0003 breast cancers was significantly associated with worse pathological parameters while LN(-) 0.94 (0.85-0.98) 0.76 (0.57-0.87) its presence augured an improved prognosis. More work in elucidating the relationship Conclusions: MES expression is higher in TNs than non-TNs. MES identifies CK5/6 of AR with mechanisms of progression, as well trials in harnessing the potential of and/or CK14 positive TNs, but shows no significant correlation with EGFR. MES(+) targeted treatment for AR expressing TN tumors, need to be performed. TNs have significantly increased risk of distant metastases and reduced survival. Our results show that pts with MES(+)TNs are an enriched population for trials of MES- 291 Borderline Ductal Epithelial Lesions: Interobserver targeted therapies. Reproducibility in 43 Cases with Original Diagnosis of Atypical Ductal Hyperplasia Bordering on Ductal Carcinoma In-Situ 293 Tumor Budding Is Significantly Associated with Lymph Node G Tozbikian, E Brogi, J Catalano, S Patil, KJ Van Zee, M Kadivar, CE Vallejo, YH Wen. Metastasis in Invasive Ductal Breast Cancer Memorial Sloan-Kettering Cancer Center, New York, NY; Rasool-Akram Hospital, M Trippel, K Pfaltz, I Zlobec, C Tapia. University of Bern, Bern, Switzerland. Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran. Background: Tumor budding is a phenomenon associated with epithelial mesenchymal Background: The distinction between atypical ductal hyperplasia (ADH) and ductal transition and correlates with tumor invasiveness and metastasis. Whether tumor budding carcinoma in situ (DCIS) has substantial clinical impact. In most cases use of established is a histomorphological feature of invasive breast cancer has not yet been determined. diagnostic criteria (Page DL, Rogers LW. Hum Path 1992) allows one to solve this Therefore, we investigated the prognostic value of peritumoral (PTB) and intratumoral differential, but occasionally pathologists render a diagnosis (dx) of ADH bordering (ITB) budding in invasive ductal breast cancer. on DCIS (ADH/DCIS). We studied diagnostic interobserver reproducibility in these Design: Two pathologists counted tumor buds within the tumor (ITB) and at the borderline lesions. periphery (PTB) of 68 primary invasive ductal breast cancers as follows: Whole tissue Design: We identified patients (pts) who had breast surgical excision (EXC) at our center sections were stained for PanCK (AE1/AE3) and hot-spots of tumor buds (i.e., isolated between 1997 and 2008 and received dx of ADH/DCIS. Patient age and specimen type tumor cells or small groups of up to 5 cells) were counted at 400x. A cut-off of 10 buds (core biopsy (CBX) or EXC) were noted. A study pathologist identified the diagnostic was established to assign “high-grade” PTB or ITB. PTB and ITB were then correlated slides. Four pathologists (aware of the index dx) independently scored each case as with clinico-pathological features. benign, ADH, ADH/DCIS or DCIS. They assessed histologic pattern, lesion size (≤2 or Results: Fifty-three percent (n=36) and 56% (n=38) were high grade for ITB and PTB, >2 mm), number of involved (#) ducts (≤ 2 or >2), and nuclear grade (NG) (1 or >1). respectively. High grade PTB was significantly (p=0.0262) associated with lymph Interobserver variance was calculated by k-statistic. Complete scores (available for 3 node metastasis and was more frequent in hormone receptor positive (ER: 62%, PR: reviewers) were averaged into a consensus score (CDx) and correlated with pathologic 57%), her2 negative (59%) and larger breast cancers (>T3: 61%). ITB was significantly variables by Fisher test. (p<0.008) associated with hormone receptor positive tumors and was more frequent Results: Slides from 43 pts with EXC and dx of ADH/DCIS were available for study. in her2 negative (56%) and larger tumors (>T3: 62%). PTB and ITB didn’t show an Pt median age was 52+11 years. Indications for EXC were Ca2+ (25), mass (10) and association with tumor grading. PTB and ITP were significantly correlated (p<0.0001). unknown (8). 28 pts had prior CBX. ADH/DCIS dx pertained to 12 CBXs, 30 EXCs, Conclusions: For the first time, we could show that PTB is a strong (p=0.0262) and to both for 1 pt. The lesion median size was 2.4 mm (95%CI: 1.8-2.8). ADH/DCIS predictive tumor feature for lymph node metastasis in invasive ductal breast cancer. was the most frequent CDx (25/43; 58%), followed by ADH (14/43; 33%) and DCIS Larger tumors have more tumor buds but only PTB seems to play a role in invasiveness. (4/43; 9%). CDx of DCIS correlated significantly with # ducts, but not with size, NG, This may be due to the lymph vessels at the tumor periphery being more numerous. or histologic pattern. [Table 1] There was complete (4/4) agreement in 14% of cases Whether tumor budding should be implemented in the diagnostics of breast cancer (4 ADH, 1 ADH/DCIS, 1 DCIS) and near agreement (3/4) in 35% (9 ADH, 4 ADH/ should be determined by further review in the future. DCIS, 2 DCIS). Interobserver agreement was slight for diagnosis (k= 0.11), # ducts (k =0.09), NG (k= 0.13); and moderate for size (k= 0.4). 294 Mucinous Breast Carcinoma Associated with Lobular Table 1: Correlation Between CDx and Pathologic Variables ADH/DCIS Neoplasia: A Potentially Characteristic Subset with Abnormality of ADH (n=14) DCIS (n=4) p value k (n=25) Cell Adhesion and Polarity Molecules, and Lack in Neuroendocrine # Ducts ≤ 2 12 19 1 0.045 0.09 Differentiation >2 2 6 3 H Tsuda, M Yoshida, Y Katsurada, AM Maeshima. National Cancer Center Hospital, Size (mm) ≤ 2 9 16 2 0.6 0.40 >2 5 9 2 Chuo-ku, Tokyo, Japan. NG 1 14 22 4 0.7 0.13 Background: Mucinous carcinoma (MUC) accounts for about 2% of all breast >1 0 3 0 carcinomas, whereas lobular neoplasia (lobular carcinoma in situ/atypical lobular Conclusions: Our study shows substantial interobserver variability in the dx of hyperplasia) is found in 0.5-4% of otherwise benign breast biopsies. The intraductal challenging borderline ductal proliferations. On re-review, ADH/DCIS was upgraded component of MUC is usually common ductal carcinoma in situ (DCIS), but the to DCIS only in 9%. Concern for DCIS was significantly higher in lesions involving component may be endocrine (or solid-papillary) subtype DCIS. Since we have >2 ducts. Correlation with clinical follow-up is in progress. experienced a case of MUC that accompanied with extensive LN component, we had the speculation that a subset of MUC occurs in association with LN and may show unique biological properties. 292 Mesothelin Expression in Triple Negative Breast Carcinomas Design: In surgically resected consecutive 41 cases of MUC, the presence of LN Correlates Significantly with Basal-Like Phenotype, Distant Metastases component or the coexistence of LN foci around the MUC lesion was examined and Decreased Survival histopathologically. In the MUC and LN components, the expressions of E-cadherin, G Tozbikian, E Brogi, K Kadota, J Catalano, A Muzaffar, S Patil, PS Adusumilli, L β-catenin, chromogranin A, and synaptophysin were immunohistochemically examined. Norton, YH Wen. Memorial Sloan-Kettering Cancer Center, New York, NY. The correlation between endocrine differentiation and coexistence with LN was also Background: Mesothelin (MES) is an antigen expressed in some human malignancies. analyzed in these MUC cases. Immunotherapies targeting MES are being developed. The clinicopathologic and Results: LN was detected in the background of MUC in 12 cases (29%), varying 1 to prognostic significance of MES expression in triple negative breast carcinomas (TNs) 85 mm in diameter: 6 with LN only and other 6 with both LN and DCIS components. has not been fully assessed. In 11 of these 12 LNs, both E-cadherin and β-catenin were negative. In MUC Design: We evaluated the expression of MES (clone 5B2, Vector Laboratories) and component, E-cadherin and β-catenin expressions were abnormal (heterogeneous or basal markers (CK5/6, CK14, EGFR) in tissue microarrays of 226 TNs and 88 non-TNs. markedly decreased) in 3 (25%) and 8 (67%) of 12 cases of MUC with LN component, We scored percentage (score 0=0%, 1=1-50%, 2=51-100%) and intensity (score 0=no respectively, whereas they were abnormal in only 2 (7.6%) and 5 (19%) of 26 MUCs signal, 1=weak, 2=moderate, 3=strong) of MES staining in tumor cells and regarded without LN component, respectively. The percentage of neuroendocrine feature in cases with sum score >3 as positive (MES(+)). Statistical analysis used t-test to correlate MUC was not different between MUCs with and without LN component (6/13, 46% MES(+) with basal phenotype and clinicopathologic features, and Kaplan-Meier method and 14/27, 52%), but in 6 cases of MUC with LN without DCIS component, 5 (83%) (KM) and log-rank test to evaluate overall survival (OS) and disease-free survival were negative of neuroendocrine features. (DFS) based on MSLN positivity, basal-like phenotype and lymph node (LN) status. ANNUAL MEETING ABSTRACTS 73A Conclusions: Although it is still undetermined whether there is sequence from LN to expression by targeting mRNAs. They have been implicated in regulation of cellular MUC or there is a common carcinogenic pathway for LN and MUC, MUC associated growth and differentiation and have been found to dysregulate cell proliferation in with LN had characteristic molecular features including derangement of cell-cell human tumors, including breast cancer. We evaluated the phenotypic and molecular adhesion and cell polarity molecule expressions as well as lack in neuroendocrine characteristics of mucinous tumors and performed miRNA profiling searching for differentiation. similarities/differences between the two types and the DCIS associated with type B. Design: We studied 30 cases of MC. IHC for ER, PR, Her2, and MiB1 was performed 295 BreastPRS Prognostic Score Is Comparable to Oncotype DX in all cases. Twelve cases representing high and low cellularity tumors, and associated Recurrence Score DCIS were microdissected and RNA was extracted. MiRNAs expression was profiled with miRNA array (nCounter Human v2 miRNA assay, Nanospring) and the data was R van Laar, TM D’Alfonso, R Flinchum, N Brown, L Saint John, SJ Shin. Signal Genetics, analyzed using nSolver and Partek. The relative miRNA levels were indicated as median LLC, New York, NY; Weill Cornell Medical College, New York, NY. fold changes and a cut off of 2 folds was used. Background: BreastPRS (Signal Genetics) is a new molecular characterization assay Results: Patients age range from 36-86 years.Five patients (B type) had nodal metastasis developed and validated from a meta-analysis of publically available genomic datasets. (1-4 + nodes). All cases were ER+, 26 were PR+ and 2 were Her2+ but only one BreastPRS is unique in that the 200 genes utilized in its algorithm (validated in a large had FISH amplification. We identified 14 (9 up / 5 down) differentially expressed series of breast cancer patients) were significantly associated with recurrence free miRNAs associated with type B mucinous tumors (including upregulated miR-21-5p survival, independent of traditional prognostic variables including age, tumor grade, and miR-125b-5p and downregulated miR-494 and miR-720). 26 miRNAs (11 up and ER status, tumor size, and nodal involvement (J Mol Diagn 2011;13:297-304). One 15 down) were found to be aberrantly expressed in the high cellular tumors, relative of the criticisms of the widely used Oncotype DX (Genomic Health) assay is that the to their associated DCIS (including upregulated miR-188-5p and miR-363-3p and 21 gene signature largely consists of genes for which surrogate (less expensive and downregulated miR-125a-5p and miR-1283). There were 9 commonly deregulated more expedient) means such as immunohistochemistry (e.g. ER, Ki-67) can be used miRNAs (6 up and 3 down) in carcinomas with high cellularity when compared to to evaluate their expression. Also, the Oncotype DX assay stratifies patients into 3 risk low cellularity tumors and DCIS (including upregulated miR-144-3p, miR-149-5p, groups where the benefit of adjuvant chemotherapy in the intermediate risk group is miR-320e, miR-338-3p, miR-631, and miR-2682-5p and downregulated miR-200c-3p, uncertain. In contrast, BreastPRS is a binary assay which stratifies patients into low and miR-1260a, and miR-4454). high risk groups. In this study, we sought to correlate BreastPRS prognostic score with Conclusions: Our study demonstrates that invasive type B mucinous carcinoma has known Oncotype DX recurrence score (ODxRS) as well as clinicopathogic features a different miRNA expression pattern from those with low cellularity and from the in breast cancer patients. associated DCIS. This microRNA signature most likely reflects unique molecular Design: 307 patients with invasive breast cancer and known ODxRS performed as part changes for each group of lesions, and it can not only prove to be useful in diagnosis of their routine clinical care were identified in our files. Unstained formalin-fixed paraffin and prognosis, it can also have great impact in the development of new molecular embedded slides from representative tumor blocks were used for gene array analysis. targets and therapy. RNA was isolated after manual microdissection from tissue slides using Prelude FFPA RNA Isolation Module (NuGen). The isolated RNA was converted to cDNA, amplified, and then hybridized to Affymetrix 133 Plus 2.0 Whole Genome microarray. Results 298 A High Number of Chromosomal Breakpoints Identifies Poor were analyzed using the Signal Genetics ResultsPX platform and R (www.r-project. Prognosis N0 Luminal HER2 Negative Early Invasive Breast Carcinomas org). The BreastPRS prognostic score was compared with known clinicopathologic Better Than Proliferation variables (age, tumor size, nodal status, ER/PR/HER-2 status, Ki-67, lymphovascular A Vincent-Salomon, V Benhamo, E Gravier, Y De Rycke, O Mariani, F Reyal, P Cottu, invasion) and ODxRS for each patient. A Fourquet, X Sastre, O Delattre. Institut Curie, Paris, France; INSERM U830, Paris, Results: The correlation of the recurrence score and assignment to a risk group (ODxRs: France. High, Intermediate, or Low; BreastPRS: High or Low) generated by the BreastPRS and Background: Our aim was to validate a DNA genomic complexity signature in early Oncotype DX was statistically significant (P<0.001). In comparison to the ODxRS, luminal ERBB2-ve node negative invasive carcinomas. the BreastPRS prognostic score also showed stronger association with tumor grade, Design: A training set of 214 cases (30 cases with a metastatic event within less than 4 presence of lymphovascular invasion, and Ki67 index. years; 79 controls with no metastastic event at 5 years) and a validation set composed Conclusions: The BreastPRS recurrence score is comparable to ODxRS and therefore, of 105 consecutive patients (30 relapses and 8 metastatic events) were genotyped may have similar prognostic value in this clinical setting. Prospective studies would be with SNPs array. Proliferation was assessed in the validation series both with Ki67 necessary to confirm this observation. (immunohistochemistry) and the genomic grade index (transcriptomic analysis). Results: In the training set, the threshold (Younden index) was 34 breakpoints with a 296 Determining the Frequency of MAGI3-AKT3 Gene sensitivity of 0.57 and a specificity of 0.94 (AUC: 0.81[0.71;0.91]. In the validation Rearrangements in Triple Negative Breast Cancer set, the outcome of patients with > 34 breakpoints was poorer (19 events/83 patients) than that of patients with < 34 breakpoints (11 events/22 patients) with a median S Varma, JM Mosquera, TY MacDonald, MA Rubin, SJ Shin. Weill Cornell Medical time to progression of 108 months (logrank test p<0.001; RR: 3.7 [1.73; 7.92]). In College, New York, NY. multivariate analysis, the number of breakpoints remained the only significant parameter Background: Discovery of recurrent gene rearrangements is a relatively new avenue for prediction of outcome (RR: 3.12, CI[1.33; 7.31], p= 0.009). Ki67 and the Genomic in breast cancer. Recently, MAGI3-AKT3 fusion has been reported to be present in 7% Grade Index were not significant in multivariate analysis but were correlated with the of triple negative-basal like subtype of breast cancer (5 out of 72 cases in Banerji et number of breakpoints. al. Nature 2012). The clinical implications are significant since the confirmation of Conclusions: Here, we demonstrated that patients with small N0 luminal ERBB2-ve this discovery may offer novel treatments with small molecule AKT kinase inhibitors. invasive carcinomas harbored a shorter disease and metastatic free intervals when > We assessed MAGI3-AKT3 gene rearrangements in triple negative breast cancer using 34 breakpoints were identified on genomic profiles assessed by SNPs. This genomic fluorescencein situ hybridization (FISH). complexity signature in this series of early luminal ERBB2-ve carcinomas does better Design: 154 triple negative breast cancer cases predominantly from a Caucasian than proliferation to determine the outcome. population were studied. Three cores from each case were represented in tissue microarrays (TMAs) and interrogated using locus-specific, dual-color break-apart FISH assays for MAGI3 and AKT3 genes, and a fusion assay for MAGI3-AKT3. 299 Reduced Expression of TGF-Beta Receptor III Is a Specific Results: No MAGI3 or AKT3 break apart signals were identified by FISH in any of the Event in Triple Negative Breast Carcinomas 154 cases. At least 50 nuclei per core were evaluated using a fluorescence microscope. N Virani, C Kleer. University of Michigan, Ann Arbor, MI. Conclusions: The absence of MAGI3-AKT3 gene rearrangements by FISH in our Background: TGF-β receptor III (TBR3) is a member of the TGF-beta signaling patient population suggests a possibility that the recently described MAGI3-AKT3 gene pathways, with functions in cell proliferation and migration in many human tissues, fusion occurs at the transcript level only. Our study has the statistical power to detect including regulation of breast carcinogenesis. However, the specific role of TBR3 is gene rearrangements that occur at a frequency of as low as 3% with 95% confidence. poorly understood and has not yet been extensively studied. Recent studies propose Therefore we can reliably deduce that if these genetic alterations are present, they are that it may function as a tumor suppressor, based on the observation that loss of TBR3 much less frequent than that reported in the above study (7 %). Alternatively, it may be correlates with disease progression. population-specific since the patient cohort where this gene fusion was identified was Design: Tissue microarrays (TMAs) were used from primary breast carcinomas in comprised of Mexican and Vietnamese ethnicities only. Our findings have important 203 female patients. Three separate cores represented each case. Sections were stained clinical implications: Further studies are needed to determine whether MAGI3-AKT3 with a rabbit polyclonal anti-TBR3 antibody and evaluated for intensity of reactivity gene rearrangements are recurrent events at the genomic level and enriched in triple- based on a previously validated scoring system (1 to 4, TBR3 low= scores 1-2; TBR3 negative breast cancer, which may translate in benefit from ATP-competitive AKT high= scores 3-4). inhibitors. Results: Of the 203 invasive carcinomas, 103 were luminal A, 25 luminal B, 9 HER2, and 64 triple negative (TN). TBR3 was high in 109 (54%) and low in 94 (46%) cases. 297 Mucinous Carcinoma of the Breast, One or Two Diseases? Low TBR3 was statistically significantly associated with the TN subtype. TBR3 was MiRNAs and Molecular Profiling of Invasive and In Situ Mucinous low in 61 (95%) and high in 3 (5%) of 64 TN carcinomas. The opposite was observed Carcinoma in luminal A tumors, where TBR3 was high 80 (78%), and low in 23 (22%) tumors. In luminal A tumors low TBR3 was statistically significantly associated with high L Vicioso, A Li, A Matilla, K Lara, MJ Merino. NCI, Bethesda, MD; Hospital Clinico, histological grade. TBR3 was not associated with HER2 or LB subtypes. Malaga, Spain. Conclusions: We discovered a significant association between loss or reduction of Background: Mucinous carcinoma (MC) is a rare subtype of ductal carcinoma of TBR3 expression and TN breast cancers. We also identified that TBR3 is reduced in a breast generally associated with good prognosis. Recently, two types of tumor have subset of luminal A tumors where it is associated with high histological grade. These been recognized, type A or paucicellular, and type B hypercellular with neuroendocrine data suggest that TBR3 loss might be causally involved in the development of TN breast differentiation. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene cancers, and warrant further investigation. 74A ANNUAL MEETING ABSTRACTS 300 Correlation of Oncotype DX Recurrence Score with IRS1 and Our recent findings further demonstrated that NgBR can bind and recruit farnesylated IRS2 Protein Expression and Cellular Localization in Breast Cancer Ras to plasma membrane, which is a critical step required for the activation of Ras V Walavalkar, D Kandil, S Lu, K Edmiston, L Shaw, A Khan. UMass Memorial Medical signaling in human breast cancer cells and tumorigenesis. Center, Worcester, MA; Rhode Island Hospital, Providence, RI. Design: Here, we use immunohistochemistry (IHC) and real-time PCR approaches to Background: Insulin receptor substrate 1 and 2 proteins (IRS1, IRS2) are signaling examine the expression and localization of Nogo-B and NgBR in two cohorts of 400 proteins that impact breast cancer cell function. IRS1 promotes cell proliferation and cases of breast invasive ductal carcinoma (IDC) arranged on tissue microarray slides. IRS2 regulates cell motility, invasion and glycolysis. Our previous study has shown Expressions of ER-alpha,PR,HER2,CK5,CK5/6,CK14,EGFR and Survivin were also a significant correlation between cellular localization of IRS2 and biologic behavior examined using IHC in these tumors. Data were analyzed using statistical software in breast cancer (Breast Cancer Res Treat 2011 Dec;130(3):759-72). Oncotype DX SPSS 16.0 for Windows. The relationship was tested using Pearson Chi-square tests. (Genomic Health, Redwood City, CA) is a 21 gene test used to stratify breast cancers A P-value<0.05 is defined as statistical significance. with low, intermediate and high risk recurrence scores (RS) which predicts response Results: NgBR and Nogo B proteins were detected in the most of epithelial and to chemotherapy. In this study our aim is to correlate IRS1 and IRS2 expression and myoepithelial cells in the normal breast tissues, as well as in interstitial blood vessels. localization in breast carcinoma cells with their Oncotype DX RS. Expression of Nogo-B and NgBR in tumor cells is much stronger than in normal breast Design: 97 breast carcinomas sent for Oncotype DX testing from 2006-2009 were epithelial cells. The breakdown of the distribution of NgBR in breast tumors was as collected from our files and IHC for IRS1 and IRS2 was performed on them. Follow- follows: 36.5% negative, 30.3% weak and 33.3% strong, respectively. The breakdown up data was acquired from our tumor registry. For IRS1 a nuclear staining pattern of the distribution of Nogo-B in tumors was as follows: 8.2% negative, 23.5% weak was defined as diffuse nuclear staining without cytoplasmic staining. IRS2 staining and 68.2% strong, respectively. The results further indicated that NgBR and Nogo-B patterns were defined as; Diffuse (D):even cytoplasmic staining without demarcation are highly expressed in ER alpha-positive, HER2-negative and CK5-negative IDC, of cell borders, Punctate (P):distinct cytoplasmic puncta with or without background and strongly associated with luminal A sub-type breast cancer (P<0.001). Striking, staining of the cytoplasm, Membrane (M):clear staining of cell borders with or without Nogo-B and NgBR are also strongly associated with the expression of survivn, which background staining of the cytoplasm. Statistical correlation for IRS1 and IRS2 with is a well-known apoptosis inhibitor (P<0.0001). The correlation between NgBR and RS was performed. survivin was further confirmed by real-time PCR analysis using normalized breast Results: cancer cDNA arrays from Origene. Staining pattern Low RS (n=55) Intermediate RS (n=30) High RS (n=12) p-value Conclusions: Nogo-B and NgBR are new molecular markers for breast cancer, IRS1 Nuclear 23 19 5 0.5645 particularly for luminal A sub-type breast cancer. The data suggest that the expression Negative 32 11 7 of Nogo-B and NgBR may be essential in promoting tumor cell proliferation via survivin in breast cancer. Therefore, Nogo-B and NgBR may be a novel target for IRS2 Diffuse(D) 11 5 5 0.0371 treatment of breast cancer. Membrane(M) 5 2 3 Punctate(P) 37 17 3 Negative 2 6 1 303 Mucinous Carcinoma of the Breast: Clinicopathological IRS1 staining did not correlate with RS (p=0.5645). The distribution of D, P, M and Analysis with Emphasis on Biomarker Expression and Incidence of negative patterns for IRS2 was significant among the different RS groups (p=0.0371). Metastasis and Recurrence The D pattern was seen more in the high(41.67%), versus the intermediate(16.67%) N Webster, OW Tawfik, F Fan. University of Kansas Medical Center, Kansas City, KS. and low RS groups(20%). The M pattern was seen more in the high(25%), versus the Background: Mucinous carcinomas are typically ER and PR positive and do not usually intermediate(6.67%) and low RS groups(9.09%). The P pattern was seen more in the overexpress the HER2 protein. They are generally associated with low rates of local and low(67.27%), versus the intermediate(56.67%) and high RS groups(25%). systemic recurrence. The goal of this study is to assess the clinicopathologic features Conclusions: The expression and cellular localization of IRS2 protein plays an and biomarker expressions of breast mucinous carcinoma cases and their association important role in the biology of breast carcinoma cells. Diffuse and membranous with axillary lymph node metastasis and systemic recurrence. patterns of staining for IRS2 in breast carcinoma cells significantly correlate with high Design: Cases of pure mucinous carcinoma (>90% mucinous) and mixed mucinous RS, whereas punctate patterns of staining correlate with low RS and may be useful in carcinoma (<90% mucinous) of the breast were identified from our surgical pathology a clinical setting where Oncotype DX testing is unavailable. files between 1997 and 2012. Histopathologic data, including histologic grade, nuclear grade, and lymph node metastases was collected. The tumor biomarkers expression 301 Follow-Up of Breast Papillary Lesion on Needle Core Biopsy: including ER, PR, and HER2 status was also collected in addition to clinical information Experience in African-American Population such as age, tumor size, and incidence of local recurrence and distant metastasis. H Wang, C D’Cruz, A Mahmood, P Tsang. Newark Beth Israel Medical Center, Results: 36 cases of mucinous carcinoma and 13 cases of mixed mucinous carcinoma Newark, NJ. were identified. The clinicopathologic data and biomarker profile of these cases are Background: The best clinical follow-up after a needle core biopsy diagnosis of shown in the following table. breast papillary lesions remains inconclusive. Our study addresses this question by Table 1 retrospectively reviewing our experience in a large urban medical center on a primarily Pure Mucinous Carcinoma (n=36) Mixed Mucinous Carcinoma (n=13) Mean Age (years) 67 (range 42-88) 56 (range 41-74) African American population. Mean Tumor Size (cm) 1.5 (range 0.1-6.8) 3.0 (range 0.8-10.5) Design: A search of our medical center’s database for breast papillary lesions diagnosed Nuclear Grade 1: 19 1: 0 by core needle biopsy between September 2002 and September 2012 was conducted. 2: 17 2: 10 Cases were reviewed and categorized into benign, atypical and malignant. Immunostains 3: 0 3: 3 of CK5/6 and CK903 were performed when necessary. Histologic Grade 1: 28 1: 0 2: 8 2: 10 Results: A total of 40 breast papillary lesions were diagnosed on needle core biopsy, 3: 0 3: 3 including 33 (82.5%) benign papillary lesions, 4 (10%) atypical lesions, and 3 (7.5%) Axillary Lymph Node* Positive: 3 Positive: 7 intraductal papillary carcinomas. Of these 40 patients, 14 patients (12 African- Negative: 27 Negative: 5 Americans, 2 Hispanics) underwent lumpectomy within 6 months after core needle Follow-up (months) 41 (range 0-99) 46 (range 9-116) biopsy. Pathology of the lumpectomy showed: 2 of the 11 (18%) benign papillary Recurrence* Yes: 2 Yes: 3 No: 27 No: 3 lesions on needle biopsy were upgraded to intraductal papillary carcinoma; the 2 atypical Death* Yes: 0 Yes: 0 papillary lesion cases on needle biopsy were both upgraded, one into intraductal papillary No: 29 No: 13 carcinoma, the other microinvasive carcinoma; the only case of malignant papillary ER/PR/HER2 Status ER+/PR+/HER2-: 34 ER+/ PR±/HER2-: 10 lesion on core needle biopsy remained as intraductal papillary carcinoma on lumpectomy ER +/PR -/HER2-: 2 ER+/ PR±/HER2+: 3 examination. Combining the benign and atypical papillary lesion cases on needle biopsy, * Complete clinical and/or pathologic data was not available for all cases. the upgrading rate in the lumpectomy was 29%. As part of this study, immunostains Conclusions: While all pure mucinous carcinomas and the majority of mixed mucinous of CK5/6 and CK903 were performed on all four core needle biopsies that were later carcinomas were ER+/Her2-, some cases of mixed mucinous carcinomas were ER+/ upgraded to intraductal papillary carcinoma. The diagnosis of only one core could Her2+. PR expression was variable. Despite having an overall good prognosis, a small potentially be upgraded into malignant after reviewing immunohistochemical results. percentage of pure mucinous carcinoma cases had positive axillary lymph nodes and Conclusions: Our results showed in a predominantly African-American population, a exhibited late distant metastases. Mixed mucinous carcinomas had a worse prognosis large percentage (29%) of benign or atypical breast papillary lesions diagnosed on needle with more frequent lymph node metastases and systemic recurrence. Negative PR and/ core biopsy will be upgraded in the final excisional examination. Early excision of all or positive Her2 expression appear to be associated with a worse outcome. papillary lesions diagnosed by core needle biopsy is thus justified in this population. 304 Analytical Variables That Affect the Prognostic Utility of Ki67 302 Nogo-B and NgBR Are Strongly Associated with Luminal A in Invasive Breast Cancer Sub-Type Breast Cancer Y Wu, B Li, G Pond, AM Mulligan, MN Levine, T Whelan, A Bane. McMaster University, B Wang, B Zhao, J Huang, RQ Miao. China-Japan Friendship Hospital, Beijing, China; Hamilton, ON, Canada; University of Toronto, Toronto, ON, Canada. Medical College of Wisconsin, Milwaukee, WI. Background: Ki67 has been demonstrated to be a prognostic marker in invasive breast Background: NgBR is a type I receptor with a single transmembrane domain and was cancer; a high Ki67 is associated with poorer disease free survival (DFS) and overall identified as a specific receptor for Nogo-B. Our previous work has shown that axon survival (OS). There are however several analytical variables; including scoring methods guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and appropriate cut points for assigning a tumor as Ki67 high or low, that remain to and morphogenesis of endothelial cells in vitro and blood vessel formation in zebrafish. be established. The goals of this study were to objectively determine the number of tumor nuclei requiring evaluation to reliably assign a tumor as Ki67 high or low and to determine an appropriate cut point. ANNUAL MEETING ABSTRACTS 75A Design: 300 full sections of T1/T2 lymph node negative, invasive breast cancers with 12 yr DFS and OS available were stained for Ki67 using the SP6 clone. 200 tumor nuclei were evaluated in each of four randomly selected regions of interest (ROI) and one hotspot ROI for every tumor. The Ki67 labelling index (LI); defined as the % of tumor nuclei positively stained for Ki67 was calculated for each ROI separately and in combination. Tumors were classified as Ki67 high or low based on cut points ranging from 5% to 35%. Likelihood ratio statistics were calculated using Cox proportional hazards regression models, with DFS and OS as the outcome and Ki67 status based on each cut point as the prognostic factor, to define a potentially optimal cut point based on discriminatory ability. Results: The Ki67 LI was strongly correlated between different ROI (Spearman’s r>0.78 for all comparisons), regardless of whether the ROI was randomly selected or a hotspot. Despite this, discrepancies in classification of Ki67 status were common; amongst randomly selected ROI, ∼[/underline]15% of tumors would be classified differently when LI was calculated from 200 nuclei compared with 600 nuclei. Patients with tumors classified as Ki67 high had a worse DFS and OS than those classified as Ki67 low. Greatest discrimination after combining all randomly-selected ROI (800 nuclei) was observed at the 14% cut point; HR=1.70 (95% CI: 1.10-2.64), p=0.017 for DFS and HR=1.73 (95% CI: 1.05-2.84), p=0.031 for OS. When the hotspot evaluation was analysed alone or in combination with randomly selected ROI it was not associated with DFS or OS, regardless of the cut point applied. Conclusions: In this series of breast cancers evaluation of at least 800 tumor nuclei across multiple ROI for Ki67 is required for prognostic utility. The hotspot Ki67 LI does not appear to be valuable. A Ki67 LI cut point of 14% had the strongest discriminatory ability for DFS and OS in this data set.

305 Computational Discovery of Breast Cancer Morphologic Subtypes and Their Molecular Underpinnings Y Xia, M Hefti, N Knoblauch, AH Beck. Beth Israel Deaconess Medical Center, Boston, MA. Background: The Cancer Genome Atlas Network (TCGA) recently performed 306 Mesothelin, a Targetable Surface Protein, Is Highly Expressed comprehensive molecular profiling on∼ 500 cases of invasive breast carcinoma. Several in Breast Carcinomas in African American Women data types (mRNA expression profiling, miRNA sequencing, DNA copy number R Xian, T Julia, Z Amy, R Vonderheide, C June, Z Paul. Hospital of the University of variations, DNA methylation profiling, and protein expression profiling) were analyzed Pennsylvania, Philadelphia, PA. by the TCGA to define breast cancer molecular subtypes. The goal of our research is: Background: Even when adjusted for cancer stage and treatment, African American 1) to perform comprehensive and quantitative morphologic analyses of TCGA samples (AA) women with breast cancer (BRCA) have worse prognosis than non-AA women. to define breast cancer morphologic subtypes and 2) to identify associations between Recent studies have shown that AA women are also more likely to have triple negative morphologic and molecular subtypes of invasive breast carcinoma. breast cancers (TNBC), a poor prognosis BRCA subtype that lacks targeted therapy. Design: We adapted the C-Path platform for use on downloaded whole slide images We have previously demonstrated that mesothelin, a cell surface glycoprotein, is from 425 breast cancer cases previously analyzed by the TCGA, and quantitated a total preferentially expressed in TNBC, as compared to ER/PR+ and HER2+ BRCA. Since of 2370 nuclear, cytoplasmic, epithelial-mesenchymal transition, and global features AA women are likely to have TNBC, we set out to evaluate the disparity, if any, of per image at 20X magnification. We performed k-means clustering on the quantitated mesothelin expression in BRCA derived from AA and Caucasian (CC) women, as morphologic data to identify robust morphologic subtypes. After estimating the optimal anti-mesothelin therapy may be an effective strategy in decreasing the cancer burden number of morphologic subtypes, we performed chi-square analyses to evaluate in AA women. for associations between morphologic subtypes (identified in our analysis) and the Design: Expression of Mesothelin was evaluated in 118 patients with BRCA - 65 previously determined molecular subtypes. TNBC (26 CC, 39 AA), 24 ER+/HER2- (18 CC, 6 AA), and 29 HER2+ (21 CC, Results: We identified 5 robust morphologic subtypes (figure 1). Subtypes 1, 2, 3, 4, 8 AA). We used an anti-mesothelin antibody (5B2, NeoMarker, 1:20) by standard and 5 were comprised of 95 (21%), 81 (19%), 141 (33%), 65 (15%) and 47 (11%) of immunohistochemical methods on paraffin sections of the above cases retrieved from the breast cancer cases, respectively. On chi-square analyses, morphologic subtypes our institutional files from 2009-2011. Positive staining was scored as a product of were significantly associated with molecular subtypes defined by mRNA (p =1.5 x staining extent (%) and intensity (1+, 2+, 3+) with a maximal score of 300. A score of 10-7) and protein expression (p = 0.006), DNA methylation profiling (p = 0.007) and 5 or greater was considered positive. miRNA sequencing (p = 0.04). Morphologic subtypes may also be associated with Results: Mesothelin is expressed in 44% of all BRCA cases with this breakdown: 75% molecular subtypes defined by DNA copy number variations (p = 0.08). The standardized of TNBCs, 0% of ER+/HER2- (p=0.0004), and 10% of HER2+ (p=0.0018) BRCA. No residuals from the chi-square analyses show that individual associations between specific mesothelin reactivity is noted in non-neoplastic mammary epithelium. When stratified morphologic and molecular subtypes were important for rejecting the null hypotheses by race, differences in mesothelin expression are found between AA and CC patients (i.e. no association between morphologic and molecular subtypes). (see Table 1). Conclusions: Morphologic profiling of comprehensively annotated cancer specimens Table 1. Expression of mesothelin AA and CC patients (n=118). facilitates the discovery of novel morphologic subtypes and characterization of their Mesothelin positivity in AA patients Mesothelin positivity in CC molecular underpinnings. (% of cases/mean IHC staining patients (% of cases/mean IHC p-value score) staining score) Figure 1 All Cases 52/43.3 28/14.5 0.003 TNBC 82/58.3 65/28.6 0.052 ER/PR+ 0/0 0/0 0.575 HER2+ 25/3.1 5/9.3 0.690 Conclusions: We report here that mesothelin is expressed in a significantly larger proportion of BRCA in AA women as compared with BRCA in CC. Our finding supports the hypothesis that AA women develop distinct BRCA given known differences in clinical presentation, treatment, and outcome. As novel cell based immunotherapy targeting mesothelin in mesothelioma, and other mesothelin positive adenocarcinomas, is underway, our results suggest that AA patients may be more likely to be eligible for this novel therapy, which could potentially close the disparity gap in treatment outcomes.

307 Negative HER-2 by Immunohistochemistry Correlates Strongly with Negative HER-2 Amplification by Fluorescence In Situ Hybridization in Triple Negative Breast Cancers R Xian, P Raghunath, Z Paul. Hospital of the University of Pennsylvania, Philadelphia, PA. Background: Immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) are the two most commonly applied techniques for determining HER-2 status on routine breast cancer (BRCA) specimens. Due to its cost and technical complexity, many laboratories reserve FISH as a confirmatory test for equivocal IHC results. Although, in general IHC and FISH have been shown to have excellent concordance in determining HER2 status by some investigators, others still contend that FISH should be performed in all BRCA regardless of IHC results, as occasional IHC-/FISH+ cases have been found. However, it is unclear if these cases are luminal A type BRCA or 76A ANNUAL MEETING ABSTRACTS triple negative breast carcinomas (TNBC). Our institution performs FISH routinely on slides. Nodes containing only isolated tumor cells were excluded from the positive all cases of TNBC. This allowed us to evaluate a large number of TNBC cases for the lymph node status group. probability of HER-2 amplification by FISH when IHC staining is negative. Results: We regarded 64 IDCNOS cases as having IMPC as a component by reviewing Design: Predictive markers were tested by Dako HercepTest TM and Dako ER/PR HE slides. Among these, 35 cases showed the reversed apical expression of EMA pharmDXTM on all primary invasive breast carcinomas on the Dako autostainer. FISH and/or MUC1 at the whole circumference of at least one tumor nest. The lymph node was performed by standard techniques using the Vysis Her-2 DNA probe kitTM. HER- status of both the cases with and without IMPC component is summarized in Table 2 is considered amplified when the HER-2/CEP17 ratio is >2.2 or HER-2/cell is >6. 1. Chi-square test revealed statistically significant difference between the two groups HER-2/CEP17 ratios between 1.8-2.2, and HER-2 copies/cell of 4-6, are considered (p=0.011). IDCNOS with IMPC component had the lymph node metastasis more equivocal. TNBC were identified from our laboratory database, and the corresponding frequently than IDCNOS without it. IHC and FISH results were tabulated. As controls, we also performed the same analysis Table 1. Correlation between the presence of IMPC component and lymph node status on all equivocal HER-2 cases by IHC from the same time period. Node (+) Node (-) Results: From 2009-2011, a total 112 TNBC were identified, which had follow-up IMPC component (+) 13 22 35 HER2 FISH analysis. FISH was also performed on 72 IHC equivocal cases from the IMPC component (-) 42 186 228 55 208 263 same period. See Table 1 for IHC and FISH correlation. IMPC: invasive micropapillary carcinoma, p=0.011 (Chi-square test) Table 1. HER-2 status by IHC correlates with FISH results, with a p-value of <0.0001 (n=206). Conclusions: When IMPC is noted as a component in pT1c IDCNOS, its presence FISH NEG (%) FISH Equivocal (%) FISH POS (%) IHC NEG 109 (97.3%) 2 (1.8%) 1 (0.9%) affects the lymph node status, even when the IMPC component is minimal. We should IHC Equivocal 61 (84.7%) 4 (5.6%) 7 (9.7%) account for the presence of IMPC, even if it is small amount. The HercepTest was repeated on IHC-/FISH+ cases and remained negative by IHC. Conclusions: Our results indicate a strong correlation between IHC and FISH analysis 310 Histological Features Differentiating Cellular Fibroadenoma of HER-2 expression in TNBC. HER2 gene amplifications by FISH is very rare in TNBC from Benign Phyllodes Tumor on Core Needle Biopsies (<1%). In addition to possible technical failure in IHC (true false IHC results), this S Yasir, RG Gamez, DW Visscher, A Nassar. Mayo Clinic, Rochester, MN. discrepancy could also be explained by various translational events. Our data suggests Background: Fibroepithelial lesions (FEL) of the breast are a heterogeneous group of that HER2 amplification is very uncommon in TNBC. tumors ranging from cellular fibroadenoma (CFA) to benign phyllodes tumor (BPT). Distinction between the two is challenging on core needle biopsy (CNB) and has a great 308 Toll-Like Receptor 3 Signaling in Breast Cancer impact on appropriate clinical management. The objective of this study is to evaluate M Yamashita, PE Bomeisl, GC Sen, HL Gilmore. University Hospitals Case Medical the histological features of FEL on CNB that can help differentiating BPT from CFA Center, Cleveland, OH; Case Western Reserve University, Cleveland, OH; Cleveland on core needle biopsy. Clinic, Cleveland, OH. Design: All patients diagnosed with FEL on CNB between 2002 and 2012 were Background: Toll-like receptor 3 (TLR3) is a sensor for double-stranded (ds) RNA, retrieved from our anatomic pathology data base. Patients with subsequent excision a common byproduct of viral replication, and RNA from necrotic cells. A randomized were selected for further evaluation. Histopathological features were evaluated on CNB clinical trial showed TLR3 stimulation therapy is effective for TLR3 positive breast blinded to the excision diagnoses. These features included stromal overgrowth, mitoses cancer: it significantly decreased the risk of metastatic relapse in TLR3 positive breast per 10 high power fields (HPF), increased stromal cellularity, stromal fragmentation, cancer. Recently our group reported that 1) Epidermal Growth Factor Receptor (EGFR) adipose tissue infiltration, stromal heterogeneity, subepithelial condensation and is essential for TLR3 signaling (Sci Signal 2012), 2) TLR3 signaling suppresses cell stromal pleomorphism. migration and cell proliferation through TRIF-independent Src branch of TLR3 (J. Results: See table #1 Immnol 2012). EGFR and Src are essential components of TLR3. Theoretically, triple Table #1: Clinicopathologic Characteristics that Differentiate Cellular Fibroadenoma from Benign Phyllodes Tumor positive (TLR3, EGFR, and Src positive) breast cancer is good indication for the TLR3 Fibroadenoma Phyllodes (N=25) p-value stimulation therapy. However, the expression profile of these proteins in breast cancer, (N=43) especially the relationship of these proteins in four breast cancer classes: Luminal A, Age, mean (SD) 39.0(14.5 ) 43.9(12.8) 0.1499** Luminal B, HER2-enriched(HER2), and Basal-like (Basal), has not been studied yet. Stromal overgrowth 17(39.5%) 14(56.0%) 0.1887 Design: The expression levels of TLR3, EGFR, and Src in twenty-nine breast cancer Mitosis =0 21(48.8%) 4(16.0%) <0.0001 Mitoses≤2 21(48.8%) 1(4.0%) <0.0001 tissues (10 Luminal A, 3 Luminal B, 2 HER2, and 14 Basal) were examined by Mitoses>2 1(4.0%) 20(80.0%) <0.0001 immunohistochemistry on paraffin sections, and evaluated in blind manner. Increased stromal Cellularity 38(88.4%) 24(96.0%) *0.4024 Results: TLR3 is positive in 21/29 of total breast cancer cases (72.4%); 7/10 of Luminal Stromal Fragmentation 24(55.8%) 19(76.0%) 0.0960 A (70%), 2/3 of Luminal B (66.7%), 1/2 of HER2 (50%), and 11/14 of Basal (78.6%). Adipose tissue infiltration 9(20.9%) 10(40.0%) 0.0911 EGFR is positive in 5/29 total cases (17.2%); 0/10 of Luminal A (0%), 1/3 of Luminal Heterogeneity 16(37.2%) 16(64.0%) 0.0328 Subepithelial Condensation 10(23.3%) 16(64.0%) 0.0009 B (33.3%), 1/2 of HER2 (50%), and 3/14 of Basal (21.4%). Src is positive in 28/29 Stromal pleomorphism 20(46.5%) 17(68.0%) 0.0863 of total cases (96.6%); 10/10 of Luminal A (100%), 3/3 of Luminal B (100%), 2/2 of * Fisher’s exact test, ** t-test, all of the rest chi-square test. HER2 (100%), and 13/14 of Basal (92.9%). Triple positive staining was observed in Conclusions: The presence of subepithelial condensation, stromal heterogeneity and 0/10 of Luminal A (0%), 1/3 of Luminal B (33.3%), 1/2 of HER2 (50%), and 2/14 of more than 2 mitotic figures per 10 HPF on core needle biopsy are more predictive of Basal (14.3%). benign phyllodes tumor than of cellular fibroadenoma. Other features studied, including Conclusions: Majority of Luminal A expressed TLR3, but not EGFR. Some of other stromal overgrowth, increased stromal cellularity, stromal fragmentation, adipose classes showed triple positive (TLR3, EGFR, and Src), suggesting TLR3 stimulation tissue infiltration and stromal pleomorphism are not helpful in differentiating these therapy is effective for some cases of Luminal B, HER2, and Basal, but not Luminal two entities on needle core biopsy. A. Now we are accumulating cases, and will be testing the effect of TLR3 stimulation on cell migration, a functional assay of cancer progression, using appropriate cell culture system with TLR3, EGFR, or Src know-down. This project will describe the 311 Cellular Fibroepithelial Lesions of the Breast: Histologic detailed mechanism of TLR3 signaling in breast cancer, and will contribute to clinical Evaluation and Long Term Follow Up development for a personalized anticancer therapy. S Yasir, TM Allers, RG Gamez, M Frost, A Nassar, DW Visscher. Mayo Clinic, Rochester, MN. Background: Cellular fibroepithelial lesions (CFL) comprise a spectrum of breast 309 Does the Presence of Invasive Micropapillary Carcinoma tumors characterized by prominent stromal cellularity, minimal or mild atypia and Component Truly Affect the Lymph Node Status of Invasive Ductal focal mitotic activity. CFL represent a mixture of cellular fibroadenoma (CFA), Carcinoma? Review of 263 Cases of pT1c Breast Cancer benign phyllodes tumors (BPT) and juvenile fibroadenoma. As a group histological S Yamazaki, E Konishi, N Shinkura, T Taguchi, A Yanagisawa. Kyoto Prefectural classification is often problematic. Further CFL have been insufficiently studied for their University of Medicine, Kyoto, Japan; Kyoto Breast Center Sawai Memorial Clinic, local recurrence potential. The aim of the study was to evaluate the histologic features Kyoto, Japan. of CFL and characterize their long term follow up and recurrence rate. Background: Invasive micropapillary carcinoma (IMPC) of the breast is thought to have Design: Forty one CFL were recovered from the benign breast disease cohort at Mayo frequent lymph node metastasis and result in a worse outcome. IMPC may be found as a Clinic (1967-2000). Cases with a combination of mitotic activity, intracanalicular component of invasive ductal carcinoma, not otherwise specified (IDCNOS). However, growth and mild atypia were classified as BPT (23 of 41). Eleven of 41 the lesions were whether the presence of even a minor IMPC component, e.g., a single micropapillary classified as CFA, and 7 of the 41 cases had overlapping morphology (indeterminate nodule affects the lymph node status of IDCNOS, or not, is still unclear, especially when category). None of these patients were widely excised. Size of the tumors and follow the tumor size (pT) is the same. We elucidate the lymph node status of pT1c IDCNOS up on the patients were retrieved from the clinical records. Mean follow up is 20.78 cases that have IMPC as a component and compare the lymph node status between the years (range: 3 to 44 years). cases with and without IMPC component. Results: Design: Two hundred and sixty three surgical cases of pT1c IDCNOS, were studied. Table #1: Demographic and Follow up characteristics of Cellular Fibroepithelial Lesions None of the patients had any neoadjuvant chemotherapy and/or hormonal therapy. We Number of patients Mean Age Mean Tumor Size reviewed all HE slides to detect the cases with IMPC component. For the detected recurred cases, we performed immunohistochemistry against EMA (epithelial membrane antigen) Benign Phyllodes Tumor (23 of 41) 42 years 2.2cm (0.9-4.0 cm range) 0/23 and MUC1 (Muc-1 glycoprotein), in order to elucidate whether the expression was Cellular Fibroadenoma (11 of 41) 31 years 2.2cm (1.5-3.5 cm range) 2/11 Indeterminate (7 of 41) 48 years 2.3cm (1.2-3.0 cm range) 0/7 noted at the reversed apical membrane or not. When the expression of EMA and/or MUC1 was found at the whole circumference of at least one tumor nest, we regarded Of all the FEL, two cases (4.9%) with an original diagnosis of cellular fibroadenoma it as having IMPC component. Lymph node status was checked by reviewing the HE recurred as fibroadenomas after one and five years of follow up. The size of the recurrence was 1.2 and 0.9 cm. respectively. ANNUAL MEETING ABSTRACTS 77A Conclusions: As a group, CFL have a low proclivity for recurrence, even when 314 Breast Carcinomas with Low ER Expression Share Similar enucleated with close or positive margins. Presence of histological features of benign Clinicopathological Features with ER Negative Tumors phyllodes tumor did not correlate with increased recurrence potential. Z Zhang, J Wang, P Tang. University of Rochester Medical Center, Rochester, NY; RTI Health Solution, Research Triangle Park, NC. 312 Histopathologic Characteristics and Molecular Classification Background: Positive ER expression is prognostic and predictive for breast cancer of Breast Cancer in Young Lebanese Women patients. Recent ASCO/CAP guideline recommends that the cut off value for ER ≥ C Youssef Massad, F Fedda, F Boulos. American University of Beirut Medical Center, positivity drops to 1%. Here, we analyzed various clinical and pathologic factors Beirut, Lebanon; University of Arkansas for Medical Sciences, Little Rock, AR. in different subgroups of breast cancer with different levels of ER expression, with Background: Breast cancer in young women (<40 years) is known to have more specially emphasis on the low ER expression subgroup. aggressive biology, higher grade at presentation and more frequent ER receptor Design: One thousand and fifty-one cases of invasive breast carcinomas diagnosed at negativity than in older patients. Most available data is from western populations University of Rochester Medical Center between 1999 and 2012 were evaluated. The where about 7% of patients present at a young age. Studies have shown that in Lebanon clinical and pathological data including patient age, tumor focality, tumor size, nodal and the Mideast, almost 50% of patients present before the age of 50, and that up to status, histologic grade, status of lymphovascular invasion (LVI) and perineural invasion 22% present before age 40. It is not clear whether this difference in epidemiology is (PNI), and expression of ER, PR, HER2 and Ki-67 from each case were recorded. associated with a difference in the histopathologic and molecular features of breast These cases were divided into five subgroups based on the levels of ER expression: cancer in our younger population. Our aim is to study the overall characteristics of ER<1%, ER1-10%, ER11-50%, ER51-70%, and ER>70%. The relationship of clinical breast cancer in young women from Lebanon in comparison to those of the same age and pathologic features between these subgroups was evaluated. group in the western population. Results: The clinicopathological characteristics (patient age, tumor focality, tumor Design: 192 cases of breast cancer diagnosed in patients younger than 40 years between size, nodal status, histologic grade, status of LVI and PNI, and expression of ER, PR, 1996 and 2010 with full available clinicopathologic information were reviewed. HER2 and Ki-67) were significantly different when we compared all the five subgroups Variables including cancer type, tumor grade, estrogen and progesterone receptor status, together. However, the comparison between ER<1% and ER1-10% subgroups showed and HER2/neu overexpression were analyzed. HER2/neu FISH testing results were no statistical difference in tumor size, nodal status, tubular formation, mitosis, expression available for equivocal cases diagnosed after 2009. The tumors were divided based of HER-2 and Ki-67, and status of LVI and PNL, with exception for patient age, on their receptor status into the molecular categories luminal A and B, Her2 amplified histological grade and nuclear grade. Comparison between ER1-10% and ER>11% and triple negative/basal-like. showed significant difference in nodal status, histological grade, tubular formation, Results: Tumor types were as follows: Ductal (183/192, 95.3%), Lobular (5/192, 2.6%), nuclear grade, mitosis, expression of HER2 and Ki-67, and status of LVI, with exception Mucinous (4, 2.1%). Grades of tumors were distributed as follows: Grade 1 (18/192, of patient age, tumor size, status of PNI. Significant difference were also found between ≤ 9.4%), grade 2 (64/192, 33.3%), and grade 3 (110/192, 57.3%). The molecular subtypes ER>70% and subgroups with ER 70%. No significant difference existed between ER of 165/192 cases were determined, and were as follows: Luminal A (ER+, Her2-, low 11-50% and ER51-70%. Ki-67): n= 61/165, 37%; Luminal B (ER+, Her2 +/-, high Ki-67): n= 43/165, 25.5%; Conclusions: Breast carcinomas with Low ER expression (1-10%) represent distinctive Triple negative/Basal-like (ER-, Her2-): n= 39/165, 23.6%; Her2 amplified (ER-, subgroup that is associated with more aggressive clinicopathological features in spite Her2+): n= 22/165, 13.3%. In 14.1% (n= 27/192) of cases, the molecular type could of being ER positive. It is more similar to ER negative tumors than to the rest of ER not be determined because of equivocal Her2 results. 23/27 of these cases were of a positive groups, and should be managed accordingly. There are significant clinical luminal subtype (ER+). pathological difference among three groups of breast carcinomas based on levels of ≤ Conclusions: Despite a greater proportion of breast cancer in young women from ER expression (ER 10%, ER11-70%, and ER>70%). Further studies are needed to Lebanon, the histopathologic characteristics and distribution of luminal subtypes is investigate the clinical significance among these three groups. comparable to the western world, but with a significantly lower rate of triple negative/ basal-like neoplasms (23.6% vs. 42%). Our results suggest that, although more frequent, 315 Endothelial Mesenchymal Transition Gene Signatures in breast cancers in young Lebanese women may have a different biology and a less Metastatic Early Stage Breast Cancer aggressive phenotype than their western counterparts. P Zhang, J He, R Zreik, A Rao. Scott and White Memorial Hospital, Temple, TX; Texas A&M University Health Science Center, Temple, TX. 313 Does Morphologic Apocrine Differentiation (AD) in Ductal or Background: The etiology of distant metastasis in early stage breast cancer patients Lobular Breast Cancer Correlate with “Molecular Apocrine” Subset? remains unclear. Recent studies have demonstrated that dynamic interactions between D Zakhia, NS Gupta, DA Chitale. Henry Ford Hospital, Detroit, MI. the neoplastic cells and the tumour microenvironment can play an important role in Background: Apocrine carcinoma (AC) is a rare morphologically defined subtype of metastasis of many malignant neoplasms. We performed multivariate analysis in an early invasive ductal carcinoma (IDC) which lacks estrogen (ER) and progesterone receptors stage breast cancer cohort and identified matched specimens with and without metastasis (PgR) but expresses androgen receptors (AR). However, the histological criteria to identify further discriminators. A gene profiling study involving mesenchymal of AC are subjective and not reproducible. In addition, many pleomorphic lobular transition (EMT) genes was performed to search for genetic alterations in early stage carcinomas (PLC) show apocrine features. Our aim was to define morphologic & HER2 negative breast cancer patients. immunohistochemical criteria for AD and explore whether there is a positive correlation Design: Retrospective chart review of patients diagnosed with Stage I and II Breast of morphologic AD with AR expression. Cancer from 2001 to 2003 was performed. Multivariate analysis Covariates evaluated Design: Cases of AC and PLC (invasive and in-situ) were identified from electronic included age, Nottingham combined grade, Estrogen receptor (ER) and progesterone records of a single institution from 1999 -2012. Additional cases were selected from receptor (PR) status. Cox Proportional Hazards (PH) regression modeling and Kaplan review of ER- PgR- negative IDC from 1996 - 2009. AD was histologically defined Meier Survival Curves were used to determine independent risk factors of tumor free as more than 90% of the tumor cells having large round nuclei, prominent nucleoli, survival. Based on the results, we matched cases with distant metastasis and without eosinophilic, abundant, granular cytoplasm with sharp cell borders, and occasional apical metastasis by stage, age, ethnicity, ER/PR, and HER2 status. Total RNA was extracted snouting. Immunohistochemical stains were done using antibodies against ER, PgR, from micro-dissected formalin fixed paraffin embedded tissue using the RNeasy Her2, AR, MIB-1, GCDFP-15. ER, PgR, Her2 were scored using published ASCO/ extraction kit. RNA was converted to cDNA using RT^2 PreAMP cDNA Synthesis Kit CAP guidelines. AR was semi-quantitatively scored using an H-score with score >10 (Qiagen, CA, USA). cDNAs were analyzed for EMT gene expression profiling, using considered a positive result. MIB-1 was scored as % positive tumor nuclei and were RT^2 Profiler PCR ARRAY (Rotor-Gene Format) Human EMT kit on the Rotor-Gene grouped into low (<25%) and high (>=25%). Q (Qiagen, CA, USA). Results: All cases were females. Of 21 AC cases, 13/21(62%) were invasive AC and Results: With 10 year follow-up, of 366 stage I and II breast cancer patients, 28 patients 8/21(38%) apocrine DCIS (ADCIS). 20/21(95%) were positive for GCDFP-15 and AR were identified with distant metastasis. Stage, age, ethnicity, ER/PR status persisted as with all cases showing >80% reactivity. All AC were ER and PR negative. 6/21(29%) important risk factors. EMT gene expression profiling study revealed seven genes with showed HER2 overexpression, (3 ADCIS; 3 AC). 4/21(19%) showed high MIB-1 at least 170-fold overexpression in metastatic cancers. The top 3 most overexpressed labelling, one of which was HER2+. Of 10 cases of PLC, there were 8/10 (80%) invasive genes are AKT1, JAG1 and SMAD2. The overexpressed genes were involved in core PLC and 2/10(20%) pleomorphic lobular carcinoma in-situ (PLCIS). All PLC and PLCIS signaling pathways of tumor genesis including PIK3CA signaling, Wnt/Notch signaling β were negative for GCDFP-15, 9/10 (90%) showed >80% immunoreactivity to AR. and TGF- signaling etc. These results may support these signaling pathways and 8/10 (80%) were ER+, 7/10(70%) PgR+, 4/10(40%) expressed high MIB-1 labelling. processes aberrations also contribute to breast cancer metastasis. 2/10(20%) were HER2+, one of which was ER- PR-, high MIB-1. Conclusions: Our study suggests upregulation of EMT gene expression plays an Conclusions: We did not find morphologic differences in triple negative and Her2+ important role in metastasis in early stage breast cancer patients. These results may AC. AD, irrespective of the hormonal status or differentiation (ductal or lobular), support these signaling pathways and processes aberrations also contribute to breast significantly& consistently correlated with high AR positivity and relatively low MIB1 cancer metastasis. Further investigation in a larger cohort to verify the results is needed. labeling. Thus AR+ /low MIB1 phenotype would help identify a relatively low risk group among aggressive triple negative breast cancers which may also identify cost effective 316 Different Biomarkers May Play Critical Roles in Cancer therapeutic strategy for AR inhibitors in the management of patients with ‘molecular Progression of Different Subgroups of Breast Carcinomas apocrine’ breast cancers. GCDFP-15 was strongly associated with AC but not PLC. Z Zhang, J Wang, P Tang. University of Rochester Medical Center, Rochester, NY; RTI Health Solutions, Research Triangle Park, NC. Background: Ductal carcinoma in situ (DCIS) is considered to be a non-obligatory precursor with varying potential for progression to invasive ductal cancer (IDC). The factors underlying this progression are poorly understood. We compared the expression of a panel of biomarkers between pure DCIS and the DCIS component of IDC in order to better define key molecules that distinguish these two groups of breast cancer. 78A ANNUAL MEETING ABSTRACTS

Design: We identified 101 cases of pure DCIS and 225 cases of DCIS with co-existing Univariate Analysis of Clinicopathologic Factors IDC at University of Rochester Medical Center between 1997 and 2008. The clinical Variables OR 95% CI p-value (patient age) and pathological (tumor size and nuclear grade) features were recorded, No. of re-excisions (1 vs. 2&3) 1.1 [0.1, 11.8] 1.0 tissue microarray from these cases were constructed, and immunohistochemical (IHC) Family history of cancer 0.4 [0.03, 3.9] 0.4 Endocrine therapy 2.1 [0.2, 22.5] 0.6 analyses were performed for ER, PR, AR (androgen receptor), HER2, Ki67, IMP3 ER+ 0.4 [0.04, 3.0] 0.3 (Insulin-like growth factor II mRNA-binding protein 3), Bcl2, Cox2 and Cyclin D1. ER, PR+ 2.4 [0.2, 25.9] 0.5 PR and AR were scored positive with Allred scores ≥3; HER2 was scored according to Nuclear grade (3 vs. 1&2) 2.9 [0.3, 24.3] 0.3 2007 ASCO/CAP guidelines; Ki67 was scored positive with ≥15% of nuclear staining; Necrosis 5.1 [0.5, 55.9] 0.2 any strong cytoplasmic staining was considered positive for IMP3; ≥10% strong nuclear Close margins (2 mm or less) 2.6 [0.2, 33.2] 0.5 staining was considered as positive for Cyclin D1, Bcl2; and ≥25% cytoplasmic stain Conclusions: As management of localized DCIS has become more standardized to was defined as positive for Cox2. include BCS and RT, it may become more difficult to reliably predict 5-year LR risk. Results: Comparison between these two groups showed only tumor size, nuclear grade, Large-scaled studies may be needed to accurately evaluate the DCIS recurrence risk in and IMP3 with significant difference. However, when we compared different subgroups patients who received standardized therapy to assist clinical decision making in pursuit by patient age, tumor size, nuclear grade, and expression of ER/PR, HER2 and Ki67, of individualized medicine. significant difference were observed for all these factors in different subgroups. The higher expressions of ER, PR, AR, HER2, Cyclin D1 and Cox2 were associated with pure DCIS; while higher expressions of Ki67, IMP3 and Bcl2 were observed in DCIS Cardiovascular with co-exisiting IDC. Nuclear Age (y) Size (cm) ER/PR HER2 Ki67 318 Preclinical Evaluation of Plaque Morphology and Development Grade Expression levels in DCIS > IDC by Flat-Panel Computed Tomography ER+, PR+ <40 3 I Aboshady, LM Buja. Texas Heart Institute, Houston, TX; University of Texas HSC, AR+ >4.0 3 Houston, TX. HER2+ >60 ER-/PR- Background: Current multidetector computed tomography (MDCT) imaging of plaque Cyclin D1+ 3 ER-/PR- HER2+ >15% components within lesions will require additional improvement to the instrumentation Cox 2+ HER2- Expression levels in DCIS < IDC in order to accurately predict their vulnerability. We have previously shown in an Ki67+ 40-60 3 HER2- acute pilot study that Flat-panel computed tomography (FpCT) provides better spatial IMP3+ >4.0 resolution than 64-MDCT and better assesses plaque components in vivo in animal aortas Bcl2+ ER+/PR+ similar in size to human coronary arteries. In this phase, we quantitatively evaluated Conclusions: Breast carcinoma is a heterogeneous group of tumors, different biomarkers the maximum capabilities and limitations of FpCT in longitudinal studies of plaque may play critical roles in cancer progression of different subgroups. development as correlated to histopathology as a gold standard. Design: The commercially available amorphous silicon fluoroscopic FpCT x-ray detectors offer 200µ native resolution. With an automated tool; multiplanar reformatted 317 Stromal Cells in Phyllodes Tumors of Breast Exhibit Stem Cell images were created perpendicular to the center line of the aorta along its entire imaged Marker Expression length. Lesions in 184 aortic histology sections from 6 WHHL rabbits and 30 NZW Y Zhang, E Chung, KA Toy, LJ Pierce, CG Kleer. UC Davis Medical Center, Sacramento, hyperlipidemic rabbits were quantitatively compared with 64-CT (image thickness, CA; University of Michigan, Ann Arbor, MI. 0.625 mm) and FpCT (image thickness, 0.150 mm) images. In the long-term phase of Background: Phyllodes tumors (PT) of breast are fibroepithelial neoplasms with the study, rabbits received a high-fat diet (0.5% cholesterol) and angiotensin II (0.4 stromal hypercelullarity, which is the basis for the diagnosis of benign, borderline µg kg1 min1 , through SC osmotic pumps) for 6 months. Lesions are monitored and and malignant. The histologic diagnosis of PT is often difficult, and the pathological correlated through monthly serial scanning sessions over 6 months. features may not always predict clinical behavior. Previous studies suggest that EZH2 Results: FpCT was more sensitive in detecting eccentric lesions (42% vs. 0%; P=0.000). epigenetically regulates cell-type identity and cellular differentiation. We hypothesized In detecting plaques with ≤10% lipid (low-attenuation foci), FpCT was more sensitive that in PT, EZH2 and the stem cell marker ALDH1 may be expressed in stromal cells than 64-CT (24% vs. 0.7%; P<0.00) and had a greater AUC (0.6 vs. 0.5; P<0.006). and associated with their degree of differentiation. Additionally, FpCT was more sensitive (65% vs. 0%; P<0.00) in detecting plaques with Design: Fourty-one PTs were histologically classified following WHO criteria, and ≤5% calcium (high-attenuation foci) but not in detecting branch points. Both FpCT and blinded to outcome. Immunohistochemistry for EZH2 and ALDH1 was routinely histology could detect low-attenuation foci as small as 0.3 mm, whereas 64-CT could performed on formalin-fixed, paraffin-embedded whole tissue sections. The percentage detect only low-attenuation foci ≥1.5 mm in diameter. Of the 48 lesions classified to of ALDH1 expressing stromal and epithelial cells was recorded. A low or high EZH2 AHA criteria as type II by histology, 42 were also classified as type II by FpCT and expression was identified based on a cutoff of 10% positive staining cells. 35 by 64-CT. FpCT was significantly more sensitive than 64-CT in detecting all types. Results: Of the 41 PTs, 25 (61%) were benign, 8 (19.5%) borderline, and 8 (19.5%) Conclusions: The study confirms our pilot study data that FpCT seems to have more malignant. EZH2 expression was low and rare in the epithelial cells, but significantly potential in quantitative screening for low-risk small atherosclerotic lesions than MDCT overexpressed in the stromal cells of borderline and malignant PT (4 of 8, 50% of each, in a rabbit model of atherosclerosis. FpCT have also the capacity for quantitatively p<0.005). ALDH1 was exclusively expressed in the stromal cells of PT, and significantly assessment the development of calcific and lipid components of atherosclerotic plaque. associated with the histological classification. ALDH1 positive cells were present in 40% of benign PT, 75% of borderline and 75% of malignant PT respectively (p<0.005). Conclusions: EZH2 expression in PT may be helpful to distinguish benign from 319 Detection of Soluble Lectin-Like Oxidized Receptor (sLOX-1) borderline and malignant tumors in challenging cases. Our data suggest that the as a Biomarker To Predict Sickle Cell Disease Vasculopathy stromal cells in PT exhibit a mesenchymal stem cell component, which increases with M Chen, X Lin, H Archibald, R Green. UC Davis Medical Center, Sacramento, CA. the degree of malignancy. Background: Sickle cell disease (SCD) crisis is associated with significant morbidity and mortality due to vascular occlusion. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is identified as a major endothelial receptor for oxidized low-density 1586 Predicting 5-Year Recurrence of Ductal Carcinoma In Situ lipoprotein (ox-LDL) as well as aged erythrocytes. Enhanced expression of LOX-1 is (DCIS) Following Initial Breast Conserving Surgery and Radiation Therapy implicated in atherosclerosis, inflammation and diabetic vasculopathy. The pathogenesis CP Kragel, S Wei. University of Alabama at Birmingham, Birmingham, AL. of SCD vasculopathy mechanistically resembles atherosclerosis. The purpose of this Background: A large number of previous studies have identified various study is to investigate the role of LOX-1 as a biomarker to predict SCD vasculopathy. clinicopathologic factors significantly associated with local recurrence (LR) for DCIS. Design: We analyzed LOX-1 gene expression by qPCR in human coronary endothelial However, the LR rate has been greatly reduced since breast-conserving surgery (BCS) cells following static incubation with sickle RBCs. We studied in vivo LOX-1 expression with radiation therapy (RT) became a standard treatment option in women with localized in autopsy SCD patient’s vasculatures by immunohistochemistry. We measured DCIS. Yet, there is still a benefit to identifying patients at lower risk for LR in order to circulating soluble LOX-1 (sLOX-1) concentrations by sandwich ELISA in the plasma avoid overtreatment. In this study, we sought to establish factors significantly associated of SCD patients under sickle cell crisis compared with steady-state. with LR and to examine the utility of the recently published Memorial Sloan-Kettering Results: LOX-1 mediated binding and internalization of sickle RBCs in endothelial (MSK) nomogram in predicting LR rates in patients with DCIS after BCS and RT. cells was inhibited by pre-incubation with Ox-LDL. Design: The Surgical Pathology files of the authors’ institution were searched to identify patients with DCIS who underwent BCS and subsequent RT from 2003-2007. Those with coexisting invasive carcinoma or receiving mastectomies following BCS with positive margins were excluded. The odds ratios (OR) were calculated, and Fisher Exact Probability or t test was utilized for statistical analysis, when appropriate. The clinicopathologic factors populated the MSK DCIS nomogram. Results: A total of 31 cases meeting the inclusion criteria were identified in the study period, of which only 4 patients (13%) had LR within 5 years, 3 with DCIS and 1 with invasive carcinoma. While the presence of necrosis in DCIS showed the highest odds ratio for LR, univariate analysis failed to identify any of the clinicopathologic factors examined significantly associated with LR. Further, there was no clear association between the MSK nomogram-predicted and observed 5-year LR rates. The mean probability of LR was 3.75% in the recurrence group and 2.81% in the non-recurrence LOX-1 gene expression in human endothelial cells was significantly increased by group (p=0.16). incubation with sickle RBCs. Upregulation was detected after 1 hour of incubation,