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Background: Multi-gene assays have informed the treatment of patients (pts) with estrogen receptor positive (ER+) and lymph node negative (LN-) breast cancer (BC). For pts with tumors in the 21-gene RS intermediate range (RS18-30), the benefit of adjuvant chemotherapy (chemo) added to endocrine therapy is uncertain. The 70-GS provides prognostic information by classifying LN+ and LN-, ER+ and ER- tumors as low or high risk though lacks predictive validation in phase III trials. Our primary objective was to perform the 70-GS on a large consecutive series of intermediate RS with known adjuvant treatment recommendations to evaluate the distribution of treatment recommendations by 70-GS risk category. Other objectives were to identify how many pts with intermediate RS are classified by the 70-GS as low or high risk; compare microarray-based assessment of ER, PR, and HER2 with ER, PR and HER2 by conventional immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and the 21-gene RS assay; and to compare clinical subtyping based on ER, PR, HER2 and Ki-67 with the 80-gene molecular subtyping signature. Methods: All consecutive ER+LN- BC cases with an intermediate 21-gene RS obtained at Loyola University Medical Center from 1/2005 - 9/2012 were analyzed. All adjuvant treatment recommendations were made after obtaining the 21-gene RS. The 70-GS, micro-array assessment of ER, PR and HER2, and molecular subtyping were performed on paraffin embedded tumor blocks. Standard descriptive statistical methods were employed. Results: 102 samples were analyzed. The 3 assays were successfully performed in 89 cases. Median RS was 21, range 18-30. 50 (56.2%) samples were 70-GS high risk; 39 (43.8%) were low risk. The table below shows the distribution of adjuvant chemotherapy recommendations made after obtaining the 21-gene RS. Of the 39 pts with low risk 70-GS results, 14 had been recommended chemo. Of the 50 high risk pts, 34 had been recommended no chemo. Concordance of microarray-based assessment of ER with the 21-gene ER test was 54/57 and 85/89 with ER by IHC. Concordance of microarray-based assessment of HER2 with the 21-gene HER2 test was 49/50, 87/89 with HER2 by IHC, and 87/89 by FISH. The 89 ER+HER2- clinical subtypes were re-categorized as 39 luminal A, 48 luminal B, 1 basal and 1 HER2 molecular subtypes. Conclusions: Within the 21-gene RS intermediate range, there is a mix of 70-GS high and low risk results. There is good concordance of microarray-based assessment of ER and HER2 with the 21-gene ER and HER2 test; with ER by IHC, and HER2 by IHC and FISH. The majority of the clinical ER+HER2- cases were categorized as molecular subtypes luminal A and B. The optimal adjuvant treatment recommendation for pts with intermediate RS but either 70-GS high risk or low risk is uncertain. Results of completed phase III prospective studies (TAILORx and MINDACT) will clarify this critical treatment dilemma for patients and their physicians. Adjuvant Chemotherapy Recommendations in pts with intermediate RS by 70-GS Risk Category Chemotherapy RecommendedChemotherapy Not RecommendedTotal21-gene Intermediate RS30598970-GS Low Risk14253970-GS High Risk16345070-GS Total305989 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-11-13.

Quantitative image analysis of histopathology slides is becoming an important technology in diagnostic pathology. To this end, it is essential to combine a robust image analysis software with the most commonly used immunohistochemical staining methods. In this investigation, we describe a practical application of NIH ImageJ software for quantitative vascular image analysis for diaminobenzene chromogen-based CD34 immunostain in breast cancer. CD34 immunostain is in a unique position to identify lymphangiogenesis and angiogenesis simultaneously in a given tumor tissue. This investigation aims at establishing a practical quantitative vascular image analysis solution for diagnostic pathologists by using ImageJ, and CD34 immunostain.Tissue microarray slides containing breast cancer tissue were immunostained for CD34 for simultaneous identification of lymphatic endothelial cells (LEC) and blood vessel endothelial cells (BEC). Digital images were analyzed using NIH ImageJ software. A CD34 score was quantified for each tissue core as a percentage (CD34-positive area/area of tissue core). The mean CD34 scores were 0.24%, 0.40%, 1.30%, 2.33%, 2.64%, and 3.44% for normal breast tissue, in stage IIA, IIB, IIIA, IIIB, and IIIC breast cancer tissue cores, respectively (p0.0001). The mean CD34 scores were 0.70% and 2.21% for lymph node-negative and lymph node-positive breast cancer patients, respectively (p0.0001).ImageJ software seems to be an attractive quantitative image analysis tool for diagnostic pathology for immunohistochemistry-based applications because of its capabilities, availability, and ease of use with most image formats. Our results show the feasibility, versatility, and ease of use of ImageJ and CD34 immunohistochemistry for vascular image analysis in breast pathology. Given the prospects of novel lymphatic and vascular endothelium-targeting therapeutics in breast oncology, the practical analysis of combined LEC and BEC density described in this report could enable diagnostic pathologists to apply quantitative vascular image analysis easily in their pathology practice and translational research.

The walls of angiogenic blood vessel capillaries are composed of two principal cell types, blood vessel endothelial cells (BEC) and pericytes (PC), whereas the walls of lymphatic capillaries are composed of lymphatic endothelial cells (LEC). In this investigation we describe a practical application of NIH ImageJ software for quantitative image analysis for pericytes and endothelial cells in prostate cancer. We used a tissue microarray that contained 49 tissue cores (normal prostate tissue or prostatic carcinomas with Gleason scores of 6 through 10). These prostate cancer samples represented AJCC prognostic stages II, III, and IV. Slides were immunostained with anti-PDGFR-β antibody for identification of PC, and quantified as microvascular pericyte density (MVPD); they were also immunostained with anti-CD34 antibody for identification of LEC and BEC simultaneously, and quantified as microvascular endothelial density (MVED). CD31 and D2-40 immunostains were used to quantify BEC and lymphatic endothelial cells, respectively. Our results showed higher MVPD and MVED in prostate cancers with higher Gleason scores and higher stages, suggesting the prognostic utility of vascular image analysis in prostate pathology. This investigation demonstrates the feasibility, versatility, and ease of use of ImageJ software and pericyte-specific and endothelial-specific immunohistochemistry for quantitative image analysis in prostate pathology.

Background. Differentiating cervical intraepithelial neoplasia (CIN) from atrophy in postmenopausal women based on morphology alone is challenging. p16 and Ki-67 help distinguish CIN2/3 from atrophy. The goal of this study is to further characterize the utility of p16, Ki-67, and human papillomavirus (HPV) tests in women older than 50 years, particularly in CIN1. Design. The authors retrospectively identified cervical specimens from three, 1-year time periods. Included were cases from women older than 50 years with benign diagnoses, atrophy, and CIN. Slides were stained with p16 and Ki-67 and graded as positive or negative. Medical records were reviewed for cytology, HPV test, and histopathologic diagnoses from the time of biopsy to 2010. Results. A total of 97 cervical samples were included. In all, 34 (74%) CIN1 cases were negative for p16 and Ki-67. Of CIN1 cases with positive HPV tests, only 1/10 (10%) had positive p16 staining versus 2/2 (100%) of CIN2/3 cases. Of 39 women with CIN1 who had follow-up data available, 4 (10%) had subsequent histologic progression to CIN2/3 and none developed invasive disease. Conclusions. In our study, the majority of cases (74%) diagnosed as CIN1 in women ≥ 50 years are negative for p16 and Ki-67 and do not progress to high-grade dysplasia during 3- to 7-year follow-up. A combination of morphology, p16, and Ki-67 on cervical specimens in women older than 50 years, and furthermore, use of these stains on Pap tests in combination with HPV testing may help distinguish CIN from atrophy and reduce unnecessary invasive follow-up testing.

Triple-negative breast cancer (TNBC) demonstrates lack of expression of hormone receptors and human epidermal growth factor receptor. However, there is no targeted therapy for TNBC. The authors analyzed 29 TNBC cases for Notch-1 and Notch-4 biomarker expression and subcellular location, Ki67 proliferation rate, and relevant clinical/survival data. Results demonstrated an unfavorable Ki67 rate in 90% of cases, Notch-1 expression in tumor and endothelial cells in 100% of cases, and Notch-4 expression in tumor cells in 73% of cases and endothelial cells in 100% of cases. Additionally, subcellular localization of Notch-1 and Notch-4 was predominantly nuclear and cytoplasmic. In conclusion, ( a) the majority of TNBCs are high-grade infiltrating ductal carcinomas with high Ki67 proliferation rate and (b) both Notch-1 and Notch-4 receptors are overexpressed in tumor and vascular endothelial cells with subcellular localization different from that of hormone-positive breast cancer. Targeting Notch signaling with gamma secretase inhibitors should to be explored to further improve the survival rate of TNBC patients.

Background. Pseudoangiomatous stromal hyperplasia (PASH) of the breast is a benign lesion that can present as a palpable nodule or as an incidental finding in breast biopsies. Design. The study comprised 79 cases diagnosed at Loyola University Medical Center from 2002 to 2009. The pathology slides were reviewed to document the distribution, type, and association with preneoplastic or neoplastic epithelial lesions. Z-test for independent proportions is used for analysis. Results. A total of 76 patients were female and 3 were male. In all, 59.8% of patients presented with a breast mass and in 40.2% the lesion was an incidental finding. Classical PASH morphology was seen in 97.6% and proliferative PASH in 2.4%. Associated epithelial lesions were benign proliferative changes in 60.4%, atypical ductal and atypical lobular hyperplasia in 25.6% and infiltrating carcinoma in 11% of cases. Conclusions. Based on these results, extensive sampling of biopsy specimen with PASH and appropriate clinical and radiologic follow-up is recommended.

INTRODUCTION: Ductal carcinoma in situ (DCIS) is a non-invasive breast cancer, which often grows discontinuously within the breast tissue. Younger age, residual mammographic microcalcifications, positive surgical margins, tumor size, nuclear grade and architectural type are consistently related to ipsilateral breast tumor recurrence in DCIS. To re-excise or not when a lumpectomy margin is close is a controversial issue for breast pathologists, surgeons and oncologists, and often no residual disease is found when a re-excision is performed. The aim of this study is to investigate the clinico-pathological features of DCIS that might predict residual disease in the re-excision specimen in patients treated by lumpectomy with either positive or close margins. METHODS: Consecutive patients with DCIS who required postlumpectomy re-excision either for positive or for close margins were selected in this retrospective analysis. Close margin was defined as less than 0.1 cm. The initial lumpectomy specimens were examined for tumor size, architectural type, nuclear grade and margin status, and patient age was considered. The subsequent re-excision specimens were analyzed for residual disease. The clinico-pathological features of original lumpectomy specimens were correlated with the presence or absence of residual DCIS in the re-excision specimens. Logistic regression statistical test was used to determine if any of these clinico-pathological features predicted the presence or absence of DCIS in the subsequent re-excision specimens. RESULTS: There were 37 cases of DCIS without accompanying invasive carcinoma that had positive or close resection margins (positive margin n=8; 21.6%, close margin n=29; 78.4%). Age of the patients ranged from 30 to 93 years. DCIS present in the original lumpectomy specimen was comedo type with necrosis n=8; 21.6% and non-comedo type n-29; 78.4%. Twenty-one lumpectomy specimens (57%) exhibited more than one type of DCIS. Four tumors were nuclear grade 1 (10.8%). 17 were nuclear grade 2 (45.9%), and 16 were nuclear grade 3 (43.2%). Residual DCIS was present in 20 out of 37 (54.1%) re-excision specimens. Six out of 8 (75%) comedo DCIS had residual disease, compared to 14 out of 29 (48.3%) non-comedo DCIS. Eleven out of 20 patients (55%) with residual disease were younger than 60 years. The correlation of patient age and type of DCIS to residual disease in the re-excision specimens was short of statistical significance due to small size of study sample (p value = 0.178 and 0.175 respectively). There was no statistically significant association between nuclear grade and distance to margin with residual disease in re-excision specimens (p value = 0.757 and 0.734 respectively). CONCLUSION: Younger age and comedo type DCIS may be important factors in predicting residual disease in the re-excision specimens. If this can be confirmed in a larger, multi-institutional data set, there could be a more tailored selection of who needs a re-excision. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-15-09.

Basal-like (BL) carcinoma, distinguished by the expression of keratins that are a characteristic of myoepithelial cells, is 1 of the 5 distinct subtypes of breast tumors identified by gene microarray technologies. BL cancers have been well described in women 40 years of age were evaluated. The study demonstrated that there is a subset of patients >40 years of age with breast cancers, manifesting features of BL carcinoma. There has been a significant increase in BL cancers among women >40 years of age having larger tumors and lymph node metastasis in comparison with younger women

The basal-like phenotype (BLP) subtype of breast carcinoma has been identified as 1 of 5 tumor subtypes first revealed by microarray profiling. This phenotype tends to be more aggressive, is more often associated with BRCA1 mutations, and carries a poor prognosis. Few studies have morphologically characterized BLP on resected breast specimens (RS), and no studies have evaluated these diagnostic parameters in core needle biopsies (CNB) of breast. We identified a group of 35 RS that demonstrated BLP by morphology and/or immunophenotype based on the criteria used in the literature. Retrospectively, we reviewed the CNB of these RS for the following morphologic features: growth pattern, nuclear grade, mitotic rate, presence of ductal carcinoma in situ, necrosis, and lymphocytic response. Of these histologic features, solid growth pattern [88.6% (31/35)] with nuclear grade 3 [100% (35/35)], marked lymphocytic infiltrate [74.3% (26/35)], and absence or

Aminopeptidase P (APP) isoforms specifically remove the N-terminal amino acid from peptides that have a proline residue in the second position. The mRNA levels of three different isoforms, each coded by a different gene, were determined in 16 human tissues and in peripheral blood mononuclear cell (PBMC) fractions by RT-PCR. The cytosolic isoform, APP1, and the cell surface membrane-bound isoform, APP2, are expressed in all of the human tissues and PBMC fractions examined. The very high expression of APP2 mRNA in kidney compared to other tissues was confirmed by enzyme activity measurements. Among the PBMC fractions, APP2 expression is highest in resting CD8 + T cells, but decreases in these cells following their activation with phytohemagglutinin; in contrast, expression of APP2 increases in CD4 + T cells upon activation. The third isoform, APP3, is a hypothetical protein identified by nucleotide sequencing. A detailed analysis of its amino acid sequence confirmed that the protein is an aminopeptidase P-like enzyme with greater similarity to Escherichia coli APP than to either APP1 or APP2. Two splice variants of APP3 exist, one of which is predicted to have a mitochondrial localization (APP3m) while the other is cytosolic (APP3c). Both forms are variably expressed in all of the human tissues and PBMC fractions examined.

Background: The incidence of the metabolic syndrome (MS) has been increasing in the United States and elsewhere. The interaction of MS with breast cancer (BC) incidence, tumor biology and outcomes are under study. We hypothesized that the presence of MS would predict BC recurrence to a variable degree across the diverse BC biology as defined by the risk categories of the 21-gene recurrence score (RS) assay. Patients and Methods: We studied consecutive patients (pts) with newly diagnosed, estrogen receptor (ER) positive, lymph node (LN) negative BC treated in our institution between 2006–2011 who had a 21-gene RS assay done on their tumors. All pts were treated with standard systemic and local therapy. The electronic medical record was queried for key diagnoses including MS and its constituent parts. The WHO definition was used to categorize pts as having MS defined as diabetes mellitus (DM) or glucose intolerance, plus at least 2 of the following: hypertension (HTN), dyslipidemia (HL), central obesity and microalbuminemia. Tumor characteristics including Ki67 index, grade, tumor size, HER2/neu status; and pt characteristics including age, race, menopausal status, body mass index were recorded. The association of MS and the tumor and patient characteristics with the RS tertiles of low, intermediate and high risk was analyzed. Results: We identified 332 pts, median age 62 years, of whom 88 (27%) had MS. There was no significant association between the MS and any of the patient or tumor variables including the 21-gene RS assay, except for race (p = 0.004). Eleven of 21 (52%) African-American women had MS, 68 of 284 (24%) Caucasian women had MS, and 9 of 21 (43%) others including Hispanic and Asian women had MS. However, there was a significant association between recurrence and MS (p = 0.0002) independent of other factors. Of the 21 pts who recurred, 13 (61.9%) had MS. There was an association of recurrence and MS within RS tertiles. For pts with low risk scores, 7/44 (15.9%) with MS vs. 1/126 (0.79%) without MS had recurrence (p = 0.0003). For pts with intermediate risk scores, 5/30 (16.67%) with MS vs. 4/83 (4.82%) without MS had recurrence (p = 0.05). For patients with high risk scores, 1/9 (11.11%) with MS vs. 2/15 (13.33%) without MS had recurrence (p = 1). Conclusion: MS is an independent risk factor for BC recurrence among women with LN negative, ER positive BC treated with standard adjuvant therapy. There is a striking impact of MS on recurrence in pts with tumor biologies defined by low (and to a lesser degree) intermediate risk 21-gene RS assay scores. However, there is no difference in recurrence risk by MS among those pts with high RS. This implies that interventions directed at modifying MS in newly diagnosed pts with early BC may potentially favorably impact survival in those with specific tumor biologies as defined by multigene assays. Thus, long-term prospective studies should be conducted to further evaluate both the short and long term effects of MS on BC outcomes. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD10-02.

Background Bradykinin is a potent cardioprotective hormone, the beneficial role of which in vivo appears to be limited by its rapid metabolism. Inhibitors of peptidases that degrade endogenously formed bradykinin are themselves cardioprotective, presumably by increasing local bradykinin concentrations. As bradykinin-degrading peptidases are potential therapeutic targets, it is important to identify these enzymes in different animal models of cardiac function. Objective To determine the mechanism of bradykinin degradation in the coronary circulation of the rabbit, using an isolated perfused heart preparation. Design and methods [ 3 H]Bradykinin (16 nmol/l) was perfused as a bolus through the isolated rabbit heart in the presence and absence of specific peptidase inhibitors. The effluent was collected and the radiolabeled metabolites of [ 3 H]bradykinin were separated by high performance liquid chromatography, identified, and quantified. Results [ 3 H]Bradykinin was metabolized to the extent of 62 ± 3% in a single passage through the rabbit coronary circulation at a physiological flow rate. The metabolites were identified as [ 3 H]bradykinin (1-5) and [ 3 H]bradykinin (1-7) , accounting for 50 ± 4 and 12 ± 2% of the radioactivity, respectively. Co-perfusion with the angiotensin converting enzyme inhibitor, ramiprilat, completely blocked formation of these metabolites. Conclusions Angiotensin-converting enzyme fully accounts for the metabolism of [ 3 H]bradykinin in the rabbit coronary circulation. This result contrasts with data obtained using rat heart, which demonstrated a prominent role for aminopeptidase P in bradykinin metabolism in this species.

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

Background. Myeloid sarcoma is a tumor of myeloblasts or immature myeloid cells occurring in an extramedullary site. Myeloid sarcoma of female genital tract is very rare with no cases of vulvar location reported in English-language literature. Case. A 73-year-old female presented with an indurated mass encompassing her left labia majora and vulva. The mass was diagnosed as vulvar myeloid sarcoma. The patient's peripheral blood smear revealed Auer rods and other findings consistent with a diagnosis of acute myeloid leukemia (M-2 type, FAB classification). Conclusion. To the best of our knowledge this case is the first report in the English-language literature of the myeloid sarcoma of the vulva. Correct diagnosis of myeloid sarcoma in an otherwise asymptomatic patient is crucial for early administration of antileukemic chemotherapy.

Bradykinin (Bk), which is produced locally in the heart, exhibits potent cardioprotective effects. However, these effects appear to be limited by rapid degradation of the peptide. To determine the mechanism of Bk metabolism in the coronary circulation, [3H]Bk was perfused through the isolated rat heart via the aorta in the presence and absence of specific peptidase inhibitors. The radiolabeled metabolites were collected from the pulmonary artery and then separated, identified, and quantified by reversed-phase high-performance liquid chromatography (HPLC) by using a radioactive flow detector. In the absence of inhibitors, only 45 +/- 2% of the radioactivity eluted from the coronary circulation as intact [3H]Bk. The chromatograms suggested that Bk was being hydrolyzed at the Arg1-Pro2 bond by aminopeptidase P and at the Pro7-Phe8 bond by angiotensin-converting enzyme. When the aminopeptidase P inhibitor, apstatin (200 microM), was coperfused with [3H]Bk, cleavage at the Arg1-Pro2 bond was blocked and the amount of intact [3H]Bk in the perfusate increased to 57 +/- 5% (p < 0.05 vs. control). Coperfusion with the angiotensin-converting enzyme inhibitor, ramiprilat (0.5 microM), alone blocked cleavage at the Pro7-Phe8 bond and increased intact [3H]Bk to 75 +/- 3% (p < 0.001 vs. control). When both apstatin and ramiprilat were present, almost all of the radioactivity (96 +/- 1%) eluted as intact [3H]Bk (p < 0.01 vs. ramiprilat alone). The results indicate that the degradation of Bk in the rat coronary circulation can be fully accounted for by aminopeptidase P (approximately 30%) and angiotensin-converting enzyme (approximately 70%).

We describe the pathologic and clinical presentation of a very rare, fatal case of luteinized thecoma with sclerosing peritonitis in a 40-year-old woman, who had a history of total abdominal hysterectomy and a left salpingo-oophorectomy in 2003. The patient presented with abdominal pain, and radiologic examinations revealed a 10-cm heterogenous right pelvic mass with partial necrosis. The patient eventually underwent an exploratory laparotomy, which revealed an ovarian tumor with multiple implants in the peritoneal cavity. The ovarian lesion was made up of spindle cells among clusters of luteinized stromal cells that expanded to the ovarian cortex. Tumor cells were positive for vimentin, estrogen and progesterone receptors, and CCD68 (focally) and negative for CD34, α-smooth muscle actin, β-catenin, and desmin by immunohistochemical studies. Luteinized cells were positive for α-inhibin and calretinin. Tumor cells exhibited low Ki-67 proliferation indices. The patient died because of the sclerosing peritonitis component of the disease.

The term "triple-negative breast cancer" (TNBC) is a heterogeneous subtype of breast cancer. Unfortunately, due to the lack of expression of hormone receptors and human epidermal growth factor receptor-2, therefore the lack of US Food and Drug Administration-approved targeted therapies, TNBC has the worst prognosis of all subtypes of breast cancer. Notch signaling has emerged as a pro-oncogene in several human malignancies and has particularly been associated with the triple-negative subtype of breast cancer. This chapter explores the role of Notch signaling in triple negative and other subtypes of breast cancer, the relationship of Notch with other breast cancer biomarkers, prognostic indicators associated with Notch, and potential therapeutic strategies targeting Notch inhibition. Hopefully, better understanding of this signaling pathway in the future will lead to optimal molecular therapeutic treatments for TNBC patients, improving their quality of life and outcome.

The walls of angiogenic blood vessel capillaries are composed of two principal cell types, blood vessel endothelial cells (BEC) and pericytes (PC), whereas the walls of lymphatic capillaries are composed of lymphatic endothelial cells (LEC). In this investigation we describe a practical application of NIH ImageJ software for quantitative image analysis for pericytes and endothelial cells in prostate cancer. We used a tissue microarray that contained 49 tissue cores (normal prostate tissue or prostatic carcinomas with Gleason scores of 6 through 10). These prostate cancer samples represented AJCC prognostic stages II, III, and IV. Slides were immunostained with anti-PDGFR-β antibody for identification of PC, and quantified as microvascular pericyte density (MVPD); they were also immunostained with anti-CD34 antibody for identification of LEC and BEC simultaneously, and quantified as microvascular endothelial density (MVED). CD31 and D2-40 immunostains were used to quantify BEC and lymphatic endothelial cells, respectively. Our results showed higher MVPD and MVED in prostate cancers with higher Gleason scores and higher stages, suggesting the prognostic utility of vascular image analysis in prostate pathology. This investigation demonstrates the feasibility, versatility, and ease of use of ImageJ software and pericyte-specific and endothelial-specific immunohistochemistry for quantitative image analysis in prostate pathology.

Background. The purpose of this study was to evaluate whether breast carcinoma biomarkers vary among separate tumor foci of multifocal/multicentric (MF/MC) breast carcinomas and whether this variation correlates with morphological features and tumor grade. Design. We reviewed the biomarker profiles of MF/MC invasive breast carcinomas diagnosed between January 2001 and June 2010 at our institution. Estrogen receptor (ER), progesterone receptor (PR), and human epidermal receptor protein (HER2) results were classified as positive or negative. Results. Out of the 51 patients included in the study, 6 cases had 2 tumor foci with different morphologies, 7 cases had 2 foci with similar morphology but different grades, and 38 cases had 2 tumor foci with similar morphologies and grades. Out of the 38 patients who had MF/MC tumors with the same morphology and grade, only 1 patient had a difference in ER and PR status between foci. Out of the 7 patients who had morphologically similar tumors with different grades, 4 had similar results in both tumor foci, 3 had different results for ER and PR, and another had differing results for HER2 between the foci. All 6 patients who had MF/MC foci with different morphologies exhibited similar ER, PR, and HER2 results between the foci. Conclusion. Regardless of the similarity in tumor morphology or grade, a small number of cases included foci that exhibited different tumor marker expression, which might affect the treatment strategy. Therefore, our results suggest that the evaluation of tumor markers in different foci should be considered in MF/MC tumors for accurate treatment strategies.

Background Heart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective tissue growth factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited. Methods and Results Left ventricle (LV) tissue from patients undergoing cardiac transplantation for ischemic (ICM; n = 20) and dilated (DCM; n = 20) cardiomyopathies and from nonfailing (NF; n = 20) donor hearts were examined. Paired samples (n = 15) from patients undergoing LV assist device (LVAD) implantation as “bridge to transplant” (34–1,145 days) also were analyzed. There was more interstitial fibrosis in both ICM and DCM compared with NF hearts. Hydroxyproline concentration was also significantly increased in DCM compared with NF samples. The expression of CTGF, transforming growth factor (TGF) β1, collagen (COL) 1-α1, COL3-α1, matrix metalloproteinase (MMP) 2, and MMP9 mRNA in ICM and DCM were also significantly elevated compared with NF samples. Although TGF-β1, CTGF, COL1-α1, and COL3-α1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading. Conclusions CTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.

Tumors or chronic inflammatory lesions of the occipital condyle may cause occipital pain associated with an ipsilateral hypoglossal nerve injury (occipital condyle syndrome). We describe a young woman with recurrent otitis media and occipital condyle syndrome associated with a limited form of Wegener's disease.

The number of positive axillary lymph nodes (LNs) is the only node-related factor for prognostic evaluation of breast cancer recognized by AJCC (TNM staging). However, N staging may not completely reflect LN tumor involvement due to the erroneous count of LNs in the presence of matted LNs and different tumor volume in LNs. Additionally, the positive/total LN ratio (LNR) has been shown to outperform N staging in survival prediction. In our study, to better quantify the tumor involvement of axillary LNs, we measured the cross-sectional cancer area (CSCA) of the positive LNs in 292 breast cancer patients diagnosed between 1998 and 2000 in our institution and compared its prognostic value to that of number of positive LNs (metLN)/N stage and LNR. Statistical analyses of these three LN-related factors were performed by Kaplan-Meier method and multivariate Cox's regression model. Patients were divided into three groups based on the different LN CSCA (500 mm2), or LNR (0.65), or N stage (N1–N3). Multivariate analysis demonstrated LNR was the most significant LN-related survival predictor with hazard ratio (HR) 25.0 (), compared to the metLN (HR 0.09, ) and CSCA (HR 2.24, ).

Aims. To compare the results of estrogen and progesterone receptors (ER, PR), and human epidermal growth factor receptor-2 (HER2) expression status on biopsy and excision specimens and to evaluate the effect of cold ischemia time and/or formalin fixation on these biomarkers.Methods. Breast carcinomas that were diagnosed between 2007 and 2009 by core needle biopsy, and subsequently excised in our institution, were included in the study. Data regarding the tumor morphology, grade, and ER, PR, and HER2 status were retrospectively collected from the pathology reports.Results. Five out of 149 (3.4%) cases with ER-positive receptor status in the biopsy specimen became ER-negative in the subsequent excision specimen. Nine out of 126 (7.1%) cases with PR-positive receptor status in the biopsy specimen became PR-negative in the excision specimen. Receptor status change was predominantly seen in tumors with low ER and PR receptor expression. HER2 results were consistent between biopsy and excision specimens in all cases tested.Conclusions. Cold ischemia time and/or formalin fixation affect mainly ER and PR testing with low Allred scores and support the implementation of the ASCO/CAP guidelines. HER2 results, however, were not affected in our limited number of patients.

Small cell carcinoma of the breast is a very rare entity that is histologically indistinguishable from small cell carcinomas of other organs. The presence of an in situ component is the most important feature that indicates the primary nature of the breast tumor. Thyroid transcription factor-1 (TTF-1) is a marker specific to lung and thyroid but is also expressed in small cell carcinoma of pulmonary and extrapulmonary origin. TTF-1 expression in breast small cell carcinoma has been reported only rarely. This reported case is unique because of the characteristic morphological features and immunohistochemical profile. The invasive tumor demonstrated neuroendocrine differentiation morphologically and immunohistochemically, expressed by TTF-1, EGFR, and basal-type cytokeratins. An unequivocal in situ component was identified intimately admixed with the invasive carcinoma. To the authors’ knowledge, this is the first report of breast small cell carcinoma showing the expression of “basal markers.”

A 25-yr-old female with complaints of infrequent, heavy periods and burning of skin during urination came for an annual gynecologic examination and Pap test. The patient’s medical history is significant for genital herpes treated with topical acyclovir, and irregular periods that were nonresponsive to oral contraceptive treatment. We observed a fungal organism with ‘‘snowshoe’’-like characteristic appearance consistent with the conidia of Alternaria spp. (Fig. C-1A) in her ThinPrep (Cytyc Corporation, Boxborough, MA). Figure C-1B of a growing culture of Alternaria shows muriform conidia, septate and brown hyphae. The organism has a natural brown pigment due to the production of a characteristic melaninlike pigment. Alternaria spp. are ubiquitous and are saprophytes commonly isolated from plants, soil, food, and indoor environment. This fungus is usually believed to be a stain contaminant in Pap test. Although there are several cases of human infection caused by Alternaria spp., the majority of cases occurred in patients with severe underlying disease or in those receiving immunosuppressive drugs. The reported lesions were mostly cutaneous involving dorsal part of the hands, fingers, elbows, knees, face, and pretibial areas. Rare cases of visceral infections and osteomyelitis due to Alternaria have been reported. Alternaria alternate is the most common species isolated from human infections. To the best of our knowledge, there are no reported cases of this organism isolated from the uterine cervix. Our findings in this Pap test most likely represent a contamination.

A cardiac transplant candidate with ischemic cardiomyopathy developed bilateral small parenchymal opacities in lower lobes of the lung. A fine-needle aspiration (FNA) was performed that revealed changes characteristic of amiodarone toxicity. Subsequently performed lung biopsies and electron microscopic studies confirmed the initial FNA diagnosis. The patient has been successfully transplanted with marked improvement in his clinical findings. This is the first case of amiodarone lung toxicity where the diagnosis was initially suggested based on the FNA findings. We also describe the clinical, cytological, histological, and electron microscopic (EM) findings of amiodarone-related pulmonary toxicity and provide a review of the literature. Diagn. Cytopathol. 2006;34:351–354. © 2006 Wiley-Liss, Inc.

Background. Mesonephric adenocarcinoma of the vagina is exceedingly rare, with only one well-documented case in the literature. Little is known regarding clinical presentation, pathological characteristics, therapy, or prognosis of the vaginal mesonephric adenocarcinoma. Case. A 55-year-old woman presented with a polypoid mass at the right vaginal apex, extending to the right paravaginal tissue. The tumor was an adenocarcinoma with ductal and tubular pattern arising in a background of mesonephric remnants. Tumor cells showed immunoreactivity for pankeratin, epithelial membrane antigen (EMA), and calretinin. The right fallopian tube and one paravaginal lymph node were positive for metastases. The patient is disease-free 3 years after surgery, radiation therapy, and chemotherapy. Conclusion. We report the second case of mesonephric adenocarcinoma of the vagina with metastasis to the right fallopian tube and to one paravaginal lymph node.

Verruciform xanthoma (VX) is a rare lesion with a predilection for oral mucosa. Only 16 cases of VX of the penis have been reported. Histologically, VX lesions in different locations are identical; however, the etiology is controversial. Previous studies have reported the presence of human papillomavirus (HPV) in VX of the skin. The purpose of this study was to determine whether HPV is a causative agent in this rare case of VX of the penis. Microscopically, the lesion demonstrated prominent verrucoid squamous hyperplasia with hyperkeratosis, parakeratosis, and acanthosis. Histiocytes, a hallmark of VX, were identified in the elongated dermal papillae. Nested polymerase chain reaction was performed on the DNA with the commonly used primer sets MY9/MY11 and GP5+/GP6+, which identify more than 40 HPV types. The results failed to identify HPV DNA in the sample, although HPV could be readily detected in genomic DNA extracted from paraffin-embedded condyloma acuminatum, a known HPV-associated lesion. Additionally, we tested a VX lesion of the palate for HPV DNA and obtained negative results. Our results indicate that VX can arise without HPV infection and suggest other possible origins may be involved.

Membrane-bound aminopeptidase P (mAPP) is a highly specific exopeptidase that removes the N-terminal amino acid only from a peptide (three amino acids or longer) that has a prolyl residue in the second position. mAPP can inactivate bradykinin, a potent vasodilating and cardioprotective peptide hormone, by hydrolyzing the Arg1–Pro2 bond. Studies on the rat have shown that the metabolism of bradykinin is an important physiological role of this enzyme. We report here the complete coding sequences for rat and mouse mAPP determined from mRNA isolated from lung tissue. Key structural features that determine post-translational processing and substrate recognition and catalysis were identified based on sequence homologies and the crystal structure of Escherichia coli aminopeptidase P complexed with Pro–Leu. The tissue-specific expression of mAPP was studied using the polymerase chain reaction. The mAPP gene is widely, but variably, expressed in adult tissues of the rat and mouse and in mouse embryos.

47 Background: Multigene assays and risk-prediction models are used to provide prognostic information in the management of patients with estrogen receptor positive (ER+) lymph node negative (LN-) breast cancer (BC). Some have predictive utility in determining the value of chemotherapy added to endocrine therapy. We previously reported a mix of high and low risk 70-GS within the intermediate risk 21-gene RS. The Magee equations provide an estimate of the 21-gene RS using standard pathological findings. AOL provides a 10-year risk of BC recurrence or death. The objective of this study is to evaluate correlations between the 70-GS as a nondichotomous continuous variable, the Magee equations, and AOL within patients (pts) with 21-gene intermediate RS. Methods: The 70-GS as a continuous variable (highest risk of -1 to lowest risk of +1) was obtained on all consecutive ER+LN- BC cases with an intermediate 21-gene RS at Loyola U Med Ctr from 1/2005 to 9/2012. AOL recurrence risk and the Magee equations were calculated from clinical-pathologic findings. Pearson pairwise correlations among all four recurrence measures were generated. Results: Results of the 70-GS as a continuous variable and the 21-gene RS were available in 89 cases. A higher risk 70-GS correlates with a higher 21-gene RS (r = -0.31; p = 0.003). A higher risk of recurrence as estimated by AOL is associated with a higher 21-gene RS (r = 0.22; p = 0.036), higher Magee equation 1 (r = 0.36; p = 0.001) and 2 (r = 0.32; p = 0.004), and a higher risk 70-GS (r = -0.40; p < 0.001). Magee equations 1 and 2 are statistically correlated to the 21-gene RS (r = 0.24; p = 0.028 and r = 0.23; p = 0.036 respectively), but not to the 70-GS (p = 0.064 and p = 0.057 respectively). Magee equation 3 is not statistically correlated with 70-GS, 21-gene RS or AOL. Conclusions: There is a continuum of highest to lowest risk 70-GS among LN- ER+ BC within intermediate risk RS. The prospective PROMIS study will assess if the 70-GS adds clinical utility regarding adjuvant therapy choice for these pts.

BACKGROUND Obesity is an apparent risk factor for postmenopausal breast cancer (BC), estrogen receptor (ER) positive BC, larger tumors, lymph node (LN) involvement, as well as recurrence of BC and BC death despite treatment. However, the specific mechanism of these increased risks remains unclear. Our prior work described the negative influence of metabolic syndrome on BC recurrence in patients assessed by a 21 gene recurrence score (RS) in the low risk and intermediate risk tertiles (Lakhani et al PSABCS 2012). The objective of this study was to analyze the interaction of obesity and BC biology in patients assessed by the 70 gene signature (70-GS) for BC recurrence and the 80 gene molecular subtyping. METHODS We studied consecutive patients with newly diagnosed ER positive, LN negative BC treated at Loyola University Medical Center between 2005 -2012 who had an intermediate RS. The 70-GS was done on these same paraffin-embedded tumor blocks for risk level and molecular subtype. Standard descriptive statistics are reported. Wilcoxon rank sum tests were performed for the comparison of BMI by groups defined by RS scores (≤24 vs ≥25) and the 70 gene signature assay (high risk vs low risk). In addition, Chi-squared test, or Fisher's exact test as appropriate, was used to examine the association between BMI tertiles and the 70GS or molecular subtype. RESULTS From 102 patients with intermediate RS, the 70-GS was successful in 89 samples. The average age was 61 years (range 41-79). The median body mass index (BMI) was 29 (range 18-53). There was a significant association between BMI and RS (p = 0.0110): median BMI = 28 in patients with RS ≤24; median BMI 33 in patients with RS ≥25. There was a significant association between BMI and the 70-GS (p = 0.0116). Median BMI 27 in low risk group; median BMI 32 in high risk group. There was a significant association between the 70-GS and BMI (p = 0.0190). Association between the 70-GS and BMI70-GS RiskHealthy (18-25)Overweight (26-30)Obese (>30)TotalHigh (n pts)1292950Low (n pts)15131139 There was a significant association between molecular subtype and BMI tertiles (p = 0.0803). Association between molecular subtype and BMI tertiles HealthyOverweightObeseTotalLuminal A (n pts)14131239Luminal B (n pts)1392648 CONCLUSION There is a striking interaction of obesity and BC biology as defined by the RS and 70-GS assays. Our data suggest that BC in obese women may have more aggressive tumor biology and higher risk of recurrence than BC in those with a low BMI. Further molecular characterization of the BC from obese patients may elucidate the role obesity plays in BC development and progression, as well as provide rationale for targeted therapeutic trials. Clinical trials targeting known carcinogenic pathways related to obesity (such as metformin and statins) are ongoing. Several genes in the 70-GS are the same genes expressed in obese patients. Additional studies examining the prognostic and predictive value of the 70-GS need to be conducted for this patient population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-50.

Lung cancer is the second most common cancer in the United States. Since therapeutic regimens target specific mutations, molecular testing has become routine practice. Molecular analysis is expensive and poses a burden on rising health care costs. The present study was undertaken to see if pathologic features are predictive of molecular alterations in lung cancers. A retrospective search of …

e15538 Background: Ovarian cancer is one of the leading causes of increased morbidity and mortality worldwide. The fluid content of the cystic ovarian tumors has never been thoroughly analyzed. It is hypothesized that the fluid contents of the ovarian cystic tumors have procoagulant effects. Methods: Under an institutionally approved protocol, cystic fluids from ovarian neoplasms were collected (n=28) and analyzed to determine their procoagulant potential. Assays such as Thrombelastography (TEG, Helige, Germany), Platelet aggregometry (BioData, Horsham, PA), Fibrinopeptide A (FPA, Dade Behring, Marburg, Germany), Thrombin Antithrombin Complex (TAT, Dade Behring), and Zymuphen Platelet Microparticle Activity Assay (Hyphen BioMed, Neuville-Sur-Oise, France). Cerebral Array II (Randox, Crumlin, CO) was used to determine the levels of C-reactive protein (CRP), D-Dimer, Neuron Specific Enolase (NSE), Neutrophil Gelatinase-Associated Lipocalin (NGAL), soluble Tumor Necrosis Factor Receptor I (TNFRI) and Thrombomodulin. Results: The TEG parameters such as r- time, rk-time, k-time, MA, and angle were measured to be 17 , 22 , 5 , 54 mm, 55° with clear cell carcinoma, 18 , 24 , 6 , 58 , 54° with benign serous cyst compared to saline control values of 23 , 30 , 7 , 41 mm and 48° respectively. Platelet aggregometry showed increased aggregations when cystic fluids were supplemented in donor platelet rich plasma. The levels of Fibrinopeptide A were in the range of 24.1 and 56.6 ng/ml compared to a control value of 1.8 ng/ml. The TAT complexes were in the range of 6.7- 175 µg/ml compared to a control value of 0.7 ug/ml. The platelet microparticle measurements were markedly increased and were in the range of 10-45 nM for benign and malignant cystic fluids, compared to control values in the range of 2.3-6.6 nM. The results of CRP, D-Dimer, NSE, NGAL, TNFRI and TM were 9.28 mg/m (Normal=5.03) , >2000 ng/ml (N=227.34) , >120 ng/ml (N=10.50), >1229 ng/ml (N=228.16), 3.98 ng/ml (5.91) and 13.55 ng/ml (N=13.55) respectively. Conclusions: Benign ovarian tumors have prothrombogenic activity as much as the malignant tumors. Early surgical evaluation of clinically silent benign ovarian tumors should be considered.