The mouse mammary gland is an excellent model system with which to study both the regulation of development and the functional differentiation of an organ. Most of the development occurs postnatally, when the gland undergoes a highly regulated cascade of invasive growth, branching, differentiation, secretion, apoptosis and remodelling during each pregnancy cycle [1,2]. Terminal differentiation of the alveolar epithelium is completed at the end of gestation with the onset of milk secretion (lactation). APR = acute-phase response; C/EBP = CCAAT/enhancer-binding protein; H&E = haematoxylin and eosin; IHC = immunohistochemistry; IL = interleukin; LIF = leukaemia inhibitory factor; LPS = lipopolysaccharide; OSM = oncostatin M; RT-PCR = reverse transcriptase polymerase chain reaction; SOM = self-organising map; TNF = tumour necrosis factor; WAP = whey acidic protein. Abstract Introduction: Involution of the mammary gland is a complex process of controlled apoptosis and tissue remodelling. The aim of the project was to identify genes that are specifically involved in this process.

Purpose: Microarray studies have linked Annexin A8 RNA expression to a ''basal cell^like'' subset of breast cancers, including BRCA1-related cancers, that are characterized by cytokeratin 5 (CK5) and CK17 expression and show poor prognosis. We assessed Annexin A8's contribution to the overall prognosis and its expression in normal, benign, and cancerous tissue and addressed Annexin A8's physiologic role in the mammary gland. Experimental Design: Using microarrays and reverse transcription-PCR, the Annexin A8 expression was studied during mouse mammary gland development and in isolated mammary structures. Reverse transcription-PCR on cultured human luminal and basal cells, along with immunocytochemistry on normal and benign breast tissues, was used for cellular localization. Annexin A8's prognostic relevance and its coexpression with CK5 were assessed on tissue arrays of 1,631cases of invasive breast cancer. Coexpression was further evaluated on a small cohort of 14 BRCA1-related breast cancers. Results: Annexin A8 was up-regulated during mouse mammary gland involution and in pubertal ductal epithelium. Annexin A8 showed preferred expression in cultured basal cells but predominant luminal expression in normal human breast tissue in vivo. Hyperplasias and in situ carcinomas showed a strong staining of basal cells. Annexin A8 expression was significantly associated with grade (P < 0.0001), CK5 (P < 0.0001), and estrogen receptor status (P < 0.0001); 85.7% BRCA1-related breast tumors coexpressed Annexin A8 and CK5. Conclusion: Annexin A8 is involved in mouse mammary gland involution. In humans, it is a luminally expressed protein with basal expression in cell culture and in hyperplasia/ductal carcinoma in situ. Expression in invasive breast carcinomas has a significant effect on survival (P = 0.03) but is not independent of grade or CK5.

Mammary morphogenesis in the mouse is driven by specialized structures at the ends of the developing ducts, the terminal end buds (TEB). The mechanisms controlling the precise branching and spacing of the ducts are, as yet, unknown. To identify genes that are associated with migration of TEB and differentiation of the subtending ducts, we developed a novel method of isolating TEB and ducts free of stroma, and compared the gene expression profiles of these two isolates using oligonucleotide microarrays. Ninety one genes were upregulated in TEB compared to ducts. Three of these genes, Sprr1A, Sema3B, and BASP1, are associated with axonal growth and guidance. Two additional members of the Sprr family, Sprr2A and 2B, not previously associated with axonal growth, were also highly expressed in TEB. Expression of these genes was confirmed by RT-PCR and Western blotting, and the cellular distribution of Sprr1A and BASP1 was demonstrated by immunohistochemistry. Other semaphorins, including Sema3C, 4A, 4F and the cancer invasion associated Sema 4D were also expressed in the mouse mammary gland along with the semaphorin receptors, Plexins A2, A3, B2, and D1, and Neuropilins 1 and 2. These results are discussed in the context of other proteins expressed in the developing gland that are known to be downstream effectors of these signaling molecules. We suggest that these genes may influence ductal growth and morphogenesis in the developing mammary gland.

Cell-matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2-3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell-matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.

The ACTH receptor (ACTH-R) is the second member of the melanocortin (MC-2) receptor family that includes five seventransmembrane G protein-coupled receptors and has been shown to be predominantly expressed in the adrenal cortex. It has been postulated that ACTH may regulate its own secretion through ultra-short-loop feedback within the pituitary. ACTH-secreting adenomas are characterized by resistance to glucocorticoid feedback, and they may have dysregulated ACTH feedback. We therefore investigated the ACTH-R in normal and adenomatous human pituitary tissue. We report here the identification of ACTH-R mRNA in the human pituitary gland, which was confirmed by direct sequencing. We studied the expression of the ACTH-R in 23 normal pituitary specimens and 53 pituitary adenomas (22 ACTHsecreting, nine GH-secreting, eight prolactin-secreting, one TSH-secreting, one FSH-secreting, 10 nonfunctioning, and two silent corticotroph adenomas), using the sensitive tech-

SUMMARYPolymorphonuclear neutrophils (PMNs) are capable of synthesizing various pro-inflammatory cytokines which may indirectly influence specific immune responses. PMNs may also have the capacity to present foreign peptides to helper T cells (Th cells). In support of this hypothesis, recent studies have shown that neutrophils, when activated by the correct combination of cytokines, can be induced to express cell surface major histocompatibility complex (MHC) Class II (DR) antigen, CD80 (B7.1) and CD86 (B7.2): molecules required for antigen presentation and subsequent T-cell activation. In this study we have used normal 'resting' human peripheral blood neutrophils and demonstrated, using a mild fixation and permeabilization protocol, significant cytoplasmic 'stores' of these molecules known to be important in antigen presentation. Cytoplasmic MHC Class II antigen was found with two out of 20 normal donors tested whereas cytoplasmic CD80 and CD86 were found to a variable extent within all normal donors. Surprisingly, we also found several other neutrophil cytoplasmic CD antigens more commonly associated with B cells, i.e. CD20, CD21 (CR2/EBV-R) and CD22 (BL-CAM). All of these antigens were confined to the 'resting' cell cytoplasm and were never found to be expressed on the cell surface. To exclude the possibility that these antigens were absorbed from plasma and to provide evidence for active synthesis, we used a novel whole blood in situ hybridization flow cytometry assay method to detect mRNA specific for these antigens within normal PMNs. We also conducted real-time polymerase chain reactions to confirm these findings using CD22 as a good example of an 'inappropriately expressed' CD antigen. These observations therefore provide support for the hypothesis that human PMNs have the potential to express molecules required for antigen presentation and cell signalling.

We have previously shown that Annexin A8 (ANXA8) is strongly associated with the basal-like subgroup of breast cancers, including BRCA1-associated breast cancers, and poor prognosis; while in the mouse mammary gland AnxA8 mRNA is expressed in low-proliferative isolated pubertal mouse mammary ductal epithelium and after enforced involution, but not in isolated highly proliferative terminal end buds (TEB) or during pregnancy. To better understand ANXA8’s association with this breast cancer subgroup we established ANXA8’s cellular distribution in the mammary gland and ANXA8’s effect on cell proliferation. We show that ANXA8 expression in the mouse mammary gland was strong during pre-puberty before the expansion of the rudimentary ductal network and was limited to a distinct subpopulation of ductal luminal epithelial cells but was not detected in TEB or in alveoli during pregnancy. Similarly, during late involution its expression was found in the surviving ductal epithelium, but not in the apoptotic alveoli. Double-immunofluorescence (IF) showed that ANXA8 positive (+ve) cells were ER-alpha negative (−ve) and mostly quiescent, as defined by lack of Ki67 expression during puberty and mid-pregnancy, but not terminally differentiated with ∼15% of ANXA8 +ve cells re-entering the cell cycle at the start of pregnancy (day 4.5). RT-PCR on RNA from FACS-sorted cells and double-IF showed that ANXA8+ve cells were a subpopulation of c-kit +ve luminal progenitor cells, which have recently been identified as the cells of origin of basal-like breast cancers. Over expression of ANXA8 in the mammary epithelial cell line Kim-2 led to a G0/G1 arrest and suppressed Ki67 expression, indicating cell cycle exit. Our data therefore identify ANXA8 as a potential mediator of quiescence in the normal mouse mammary ductal epithelium, while its expression in basal-like breast cancers may be linked to ANXA8’s association with their specific cells of origin.

Background: Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival of only 3%. Surgery is the only curative option but only 15-20% of patients are suitable and post-operative recovery is long, with significant early mortality and a 5-year survival of around 20%. Thus not all patients benefit from surgery and prognostication and subsequent stratification of patients to surgical or non-surgical management could be better. Initial staging currently relies on imaging; the main pathological prognostic factors are not known until after resection. Initial diagnosis usually involves pancreatic cytology samples. Prognostication is possible in such samples using biomarkers such as miR-21. Prognostication on cytology is not yet clinically routine, perhaps because previous research methodology has involved tissue extracts e.g. RT-PCR and not in situ techniques, as often preferred by pathologists. Our aim was to study the expression of miR-21 in resected PDAC by in situ hybridisation and its relationship to clinico-pathological parameters including prognosis. Methods: We studied PDACs from 69 patients, using tissue microarrays (TMAs). Chromogenic in situ hybridisation (CISH) for miR-21 was performed using an EXIQON kit. Staining was scored based on intensity as: negative or weak (low expression); or moderate or strong (high expression). Kaplan-Meier survival curves were generated for progression free survival (PFS) and overall survival (OS) and correlated with miR-21 scores using univariate and multivariate COX proportion hazard models. Results: miR-21 expression on CISH was cytoplasmic and mainly in neoplastic PDAC epithelium and stroma; further analysis used epithelial expression data only. miR-21 was highly expressed in 39 (56%) patients; miR-21 was low or absent in 30 (44%) patients. Median PFS was 9 months (3.8-14.2, 95% CI) and 19 months (12.1-26.9, 95% CI) in the high and low expression groups respectively (P&lt;0.002). Median OS was 15 months (12.9-18.1, 95% CI) and 24 months (18.5-28.8, 95% CI) in the high and low expression groups respectively (P&lt;0.001). On univariate analysis with standard clinico-pathological factors, miR-21, tumor grade and lymph node invasion were prognostic for PFS and OS; on multivariate analysis, only high miR-21 was prognostic, being associated with poor PFS (HR 1.8, p&lt;0.01) and OS (HR 2.1, p&lt;0.01). Conclusion: High epithelial expression of miR-21 assessed by CISH is independently predictive of poorer overall and progression-free survival in patients with PDAC. Prognostication using miR-21 in diagnostic cytology samples is theoretically possible. CISH enables tissue localisation of the biomarker, in this case miR-21. Use of CISH might facilitate adoption of such stratification methods and enable improved allocation of patients with into surgical or non-surgical treatment groups. Note: This abstract was not presented at the meeting. Citation Format: Asif Ali, Elisa Giovannetti, Niccola Funel, Roderick Ferrier, Jennifer Morton, Karin A. Oien. miR-21 overexpression assessed by in situ hybridisation is an independent predictor of survival in patients with resected pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2860. doi:10.1158/1538-7445.AM2014-2860