250 pro- and anti-tumorigenic effects of MCs in different cancers. The accumulation of MCs has been associated with enhanced growth and invasion of several human cancers [10]. On the other hand, MC inﬁltration has been associated with good prognosis in breast [11, 12] ovarian [13], lung [14] and colorectal [15] carcinomas. Murine models have shown that tumor incidence and growth are inversely correlated with the MC density in MC-deﬁcient mice compared to normal [16]. Our previously published pilot report of 348 case series of breast carcinomas showed that the presence of stromal MCs correlated with a good prognosis (P = 0.0036) in invasive breast cancer [11].We present here a large tissue microarray (TMA) study of 4,444 cases of invasive breast carcinomas with clinical outcome data. This study vali- dates the independent prognostic signiﬁcance of stromal MCs in breast cancer. Materials and methods Patient selection A total of 4,620 archival samples from patients with invasive breast carcinoma referred to the British Columbia Cancer Agency between January 1986 and September 1992 were used for TMA construction. The Clinical Research Ethics Board of the University of British Columbia approved the study. Patients with in-situ disease, metastatic disease at presentation, and male breast cancer were excluded from analysis, thus bringing the ﬁnal tally to 4,444 cases. This represents 34% of all patients diagnosed with breast cancer in the province of British Columbia during this time period. This large, well characterized co- hort is derived from a consecutive series of patients who were referred to the BC Cancer Agency for consultation and had tumor samples sent to a central laboratory at the Vancouver General Hospital for estrogen receptor (ER) status. Consequently, for all of these patients we have available detailed demographic and outcome data, as well as formalin ﬁxed parafﬁn embedded (FFPE) primary tumor samples for immunohistochemical analysis. Available clinical information includes age, histology, tumor grade, tumor size, lymph node status, type of local and adjuvant systemic therapy, and dates of ﬁrst recurrence and death. Breast Cancer Res Treat (2008) 107:249–257 Clinical and pathological variables were determined following well-established criteria. A portion of this cohort of patients was recently used in a population study validating the on-line breast cancer prognostic calculator ADJUVANT! Online [17]. TMA construction The Vancouver Hospital ER laboratory retained single archival tumor blocks from each case in this patient cohort. The material had been frozen prior to neutral buffered formalin ﬁxation. All the parafﬁn sections were ﬁrst stained with H&E and reviewed by a pathologist. Representative tumor areas were circled and matched with the donor blocks. 0.6-mm cores were punched out from the donor blocks and embedded 1 mm apart in 17 recipient blocks using Tissue Microarrayer (Beecher instruments Silver Springs, MD). These tissue arrays have been used to test the new ER SP1 antibody that improves the sensitivity for detecting ER by IHC [18]. Immunohistochemistry (IHC) Sections from TMA were cut at 4 lm and immunostained with antibody to KIT (CD117), a transmembrane tyrosine kinase acting as a type III receptor for MC growth factor. Slides were also concurrently stained for ER and Her2 using standard immunoperoxidase techniques. The anti- bodies and antigen retrieval methods are summarized in Table 1. Immunohistochemical image processing and scoring The stained slides were digitally scanned with a BLISS automated digital imaging microscope (Bacus Laboratories, Lombard, IL) which consists of a microscope with a scanning stage, video camera and software designed for scanning TMAs. A relational database was constructed using identiﬁcation information and immunohistochemistry scores for each tissue core in the microarrays. An internet website was then constructed using this database and a WebSlide- Viewer Java applet provided by the manufacturer to view the microarray images and allow for an image zooming func- tionality. This website is publicly accessible through http:// www.gpecimage.ubc.ca/tma/web/viewer.php. Table 1 Details of antibodies used for immunohistochemistry 123 Antibody Isotype Company Antigen retrieval Concentration c-kit Rabbit polyclonal Dako Ventana 1:100 Her2 SP3 Rabbit monoclonal Lab vision Steam 30 min, 0.05 M TRIS buffer (pH 10) 1:100 ER SP1 Rabbit monoclonal Lab vision Citrate buffer (pH 6) 1:250