Additional file 1: of Observational study on the prognostic value of testosterone and adiposity in postmenopausal estrogen receptor positive breast cancer patients

Figure S1. Workflow for the selection of TPM-ER-positive postmenopausal breast cancer patients. Shows the workflow for the selection of ER-positive postmenopausal breast cancer patients, starting from the 592 initial women recruited consecutively in the TPM cohort from December 2003 to December 2006, at Fondazione IRCSS Istituto Nazionale Tumori of Milan. (PDF 42 kb

Additional file 2: of Observational study on the prognostic value of testosterone and adiposity in postmenopausal estrogen receptor positive breast cancer patients

Figure S2. Boxplots of circulating level of testosterone (ng/mL) according to tumour histology (IDC = Invasive Ductal Carcinoma; ILC = Invasive Lobular Carcinoma), Grade (G), number of metastatic axillary lymph Nodes (N), progesterone receptor (PR) and HER-2 status of ER-positive postmenopausal breast cancer patients. Number and percentage of patients in each group are reported and p-values are given. The bar inside the box is the median value and the box upper and lower dimensions define the inter-quartile range. Shows the boxplots of circulating level of testosterone (ng/mL) according to the other tumour characteristics considered in the study (histology, tumour grade, axillary nodal status, PR and HER-2 status). On the whole, the results did not indicate an association between circulating level of testosterone and unfavorable tumour characteristics as high tumour grade, axillary involvement or HER2 overexpression. (PDF 22 kb

Glycosylation-dependent Trop-2 transport and signaling.

<p>(<b>A</b>) Binding of 2EF to fully-matured forms of Trop-2. Ample binding to the cell membrane (arrows) was revealed. Strong staining of the Golgi apparatus was also shown (arrowheads), consistent with recognition of glycosylated Trop-2. (<b>B</b>) Flow cytometry analysis of KM12SM cells stably transfected with glycosylation mutants. Living cells were analyzed for membrane-only staining. 2EF, T16 and 162–46.2: unconjugated anti-Trop-2 mAbs, followed by rabbit anti-mouse Alexa-488; control: irrelevant antibody stained cells. (<b>C</b>) Flow cytometry analysis of KM12SM stably transfected with wild-type Trop-2 or deglycosylated variant. Living cells were analyzed for membrane-only staining. 2EF: anti-Trop-2 Alexa-488 conjugated mAb; AF650: anti-Trop-2 goat pAb; control: irrelevant antibody-stained cells. Living cells were analyzed for membrane-only staining. (<b>D</b>) MTE 4–14 cells transfected with Trop-2 subjected to Ab-mediated capping. The T16 (left) and 2EF (right) mAbs were used for primary Ab incubation, followed by cross-linking with a secondary Ab conjugated with Alexa488.</p

Proportional hazard Cox regression analysis.

<p>Unadjusted and adjusted hazard ratios according to Trop-2 expression levels (at the cell membrane or intracellular, as detected by mAb or pAb) and corresponding P values.</p>a<p>: cause-specific hazard ratios. The adjusted models included age (continuous linear), grading, pT stage (pT2+pT3 versus pT1), number of positive lymph nodes (0, 1–3, 4–9, >9), ERα, HER-2/neu, p53 and E-cadherin (cut-off: 10% positive cells). Low; ≤5% positive cells; intermediate, 6–85%; high, ≥86%. Significantly different values and trends are in bold.</p

Impact of membrane versus intracellular Trop-2 on disease relapse.

<p>Crude cumulative incidence (CCI) estimates of disease relapse were obtained as 1-Kaplan-Meier curves for distinct Trop-2 expression sub-groups (cell membrane; mAb-detected intracellular; pAb-detected intracellular). Trop-2 expression was categorized according to (<b>top</b>) intensity scores (0, 1+, 2+, 3+), (<b>middle</b>) intensity grouping, i.e. positive scores 1–12 (+) versus score 0 (−), (<b>bottom</b>) percentage of stained cells (low, ≤5%; intermediate, 6–85%; high, ≥86%), as indicated in the panels. CCI were estimated accounting for death as a competing risk.</p

Immunohistochemistry analysis of Trop-2 expression in breast cancer.

<p>Breast cancer samples were analyzed by immunohistochemistry using the 162–46.2 anti-Trop-2 mAb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096993#pone.0096993-Lipinski1" target="_blank">[32]</a> for detection of the intracellular Trop-2 (<b>A</b>) and with the R&D AF650 goat pAb for detection of membrane-associated Trop-2 (<b>B</b>). Images are representative cases of ductal (top panels) and lobular (bottom panels) cancers. Arrows: normal breast ducts. Expression levels were classified as high and low/negative. Magnification is 40x.</p

Impact of membrane <i>versus</i> intracellular Trop-2 on patient survival.

<p>Cumulative incidence (CI) estimates of death from any cause were obtained as 1-Kaplan-Meier curves for distinct Trop-2 expression sub-groups (cell membrane; mAb-detected intracellular; pAb-detected intracellular). Trop-2 expression was categorized according to (<b>top</b>) intensity scores (0, 1+, 2+, 3+), (<b>middle</b>) intensity grouping, i.e. positive scores 1–12 (+) versus score 0 (−), (<b>bottom</b>) percentage of stained cells (low, ≤5%; intermediate, 6–85%; high, ≥86%), as indicated in the panels.</p

Association between Trop-2 functional states and tumor pathobiological parameters.

a<p>: variables analyzed.</p>b<p>: overall distribution for each of the variables included in the study.</p>c<p>: distribution of scores of cytoplasmic Trop-2 expression (low or high).</p>d<p>: distribution of scores of membrane Trop-2 expression (low or high). Both raw (N) and relative (%) frequencies are reported. OR: adjusted odds ratios for intracellular Trop-2 overexpression (high <i>versus</i> low), obtained by multiple logistic regression; categories used as reference have OR = 1. pT: pathological stage. Wald test P values for the mAb (P_M) and for the pAb (P_P) scores and associated numbers of cases analyzed (<i>n_M, n_P</i>) are indicated.</p