Clinicopathological correlation of BLCAP expression in 2,197 sample TMA.

*<p>Kruskal-Wallis one way analysis of variance on ranks.</p>**<p>Mann-Whitney rank-sum test.</p

2D PAGE and 2D Western blot analysis of BLCAP protein spot patterns.

<p>(A) COS-1 cells transfected with pZeoSV2 empty vector and labeled with ³⁵S-methionine. (B) COS-1 cells transfected with pZeoSV2– BLCAP overexpressing construct and labeled with ³⁵S-methionine. Radioactive metabolic labeling (³⁵S-methionine) of COS-1 cells was used to ensure the highest detection sensitivity. (C) 2D Western blot of COS-1 cells transfected with pZeoSV2– BLCAP construct detected with anti-BLCAP antibody (10 sec film exposure). (D) 2D gel of proteins from breast tumor 63 stained with silver. (E) 2D Western blot of protein lysate from breast tumor 63 (see D) reacted with anti-BLCAP antibody (1 min film exposure). The positions of the BLCAP protein in the 2D-PAGE gels and corresponding 2D Western blots, are indicated by black arrows. The positions of several reference proteins are indicated by red arrows: ACTB – beta actin; ENO1 -alpha enolase 1; CANX – calnexin; PDI - Protein disulfide-isomerase; TUBA1A - tubulin alpha-1A chain; YWHAZ - 14-3-3 protein zeta/delta. The identity of all reference spots were confirmed by MS analysis.</p

Clinical characteristics of DCTB tissue specimens.

<p>Clinical characteristics of DCTB tissue specimens.</p

Immunohistochemical expression analysis of BLCAP in FFPE breast tissue samples.

<p>(A) No immunostaining was observed in a normal breast tissue section reacted with BLCAP antibody preincubated with immunizing peptide. (B) Immunohistochemical staining of BLCAP protein in a normal breast tissue sample demonstrated the presence of the BLCAP antigen in luminal epithelial cells with weak cytoplasmic expression (black arrow). Marked nuclear expression was also observed occasionally (red arrow). Yellow arrow points to a vessel with moderate immunoreactivity for BLCAP. (C) In a few cases IHC analysis of tumor samples showed that BLCAP was expressed in tumor cells with weak cytoplasmic expression (red arrow). (D) Most cases showed moderate to strong cytoplasmic expression with no detectable nuclear presence (red arrow) but in some cases (E) we could observe strong nuclear expression of BLCAP (red arrow). (F) In a few cases, samples were heterogenous with some cells showing distinct perinuclear immunoreactivity for BLCAP (red arrow). (G) Malignant cells showed stronger immunoreactivity (red arrows) than adjacent normal-looking ducts (black arrow), demonstrating up-regulation of this protein in tumor cells. Yellow arrows point to vessels with strong immunoreactivity for BLCAP. (H) We also observed up-regulation of BLCAP in early lesions where lobular carcinoma in situ cells showed overexpression of this protein (grey arrows) in relation to normal adjacent areas (black arrow), and at levels comparable to invasive carcinoma cells (red arrow).</p

Differential expression of BLCAP in tumor samples.

<p>The DCTB 123 patient set, including normal, tumor and lymph node metastasis samples (A), or a subset of matched 62 normal and tumor samples (B) were analyzed by quantitative IHC. Mean intensity scores for each group are indicated by red lines.</p

Expression analysis of BLCAP by quantitative IHC of normal specimens (blue bars) and corresponding tumor samples (red bars) of 62 matched cases from the DCTB dataset.

<p>Illustrative IHC images are shown for normal and tumor samples with weak immunoreactivity (N11 and T76, respectively), and for normal and tumor samples showing substantial immunoreactivity for BLCAP (N14 and T63, respectively). Magnification 20X.</p

FABP7 and HMGCS2 Are Novel Protein Markers for Apocrine Differentiation Categorizing Apocrine Carcinoma of the Breast

<div><p>Apocrine carcinoma of the breast is a distinctive malignancy with unique morphological and molecular features, generally characterized by being negative for estrogen and progesterone receptors, and thus not electable for endocrine therapy. Despite the fact that they are morphologically distinct from other breast lesions, no standard molecular criteria are currently available for their diagnosis. Using gel-based proteomics in combination with mass spectrometry and immunohistochemistry we have identified two novel markers, HMGCS2 and FABP7 that categorize the entire breast apocrine differentiation spectrum from benign metaplasia and cysts to invasive stages. Expression of HMGCS2 and FABP7 is strongly associated with apocrine differentiation; their expression is retained by most invasive apocrine carcinomas (IAC) showing positive immunoreactivity in 100% and 78% of apocrine carcinomas, respectively, as compared to non-apocrine tumors (16.7% and 6.8%). The nuclear localization of FABP7 in tumor cells was shown to be associated with more aggressive stages of apocrine carcinomas. In addition, when added to the panel of apocrine biomarkers previously reported by our group: 15-PGDH, HMGCR and ACSM1, together they provide a signature that may represent a golden molecular standard for defining the apocrine phenotype in the breast. Moreover, we show that combining HMGCS2 to the steroidal profile (HMGCS2+/Androgen Receptor (AR)+/Estrogen Receptor(ER)-/Progesteron Receptor (PR)- identifies IACs with a greater sensitivity (79%) as compared with the steroidal profile (AR+/ER-/PR-) alone (54%). We have also presented a detailed immunohistochemical analysis of breast apocrine lesions with a panel of antibodies against proteins which correspond to 10 genes selected from published transcriptomic signatures that currently characterize molecular apocrine subtype and shown that except for melanophilin that is overexpressed in benign apocrine lesions, these proteins were not specific for morphological apocrine differentiation in breast.</p></div

Expression profile of FABP7, HMGCS2 and other markers among breast cancer subtypes.

<p>(A) The diagram presents immunohistochemical profiles of ADCIS, IACs and invasive ductal carcinomas (TNBC, Luminal A Luminal B and HER2) reacted with antibodies against HMGCS2, FABP7, AR, ER, PR and HER2. The rows indicate the expression of particular protein in every core in breast cancer TMA (BRC1501, 1502 and 1503; Pantomics INC, USA): red box – positive staining, white box – negative staining, grey box – parameter was not determined. (B) Frequencies of positives for HMGCS2 and FABP7 among breast cancer subtypes. ADCIS =  apocrine ductal carcinoma <i>in situ</i>; IAC =  apocrine carcinoma; IDC =  invasive ductal carcinoma; TNBC =  triple negative breast cancer. The cut-off values for HMGCS2 and FABP7 are specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112024#s4" target="_blank">Materials and Methods</a>. The cut-off values for ER, PR, AR and HER2 are specified in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112024#pone.0112024.s005" target="_blank">Table S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112024#pone.0112024.s006" target="_blank">S4</a>.</p

Immunohistochemical analysis of FABP7 and HMGCS2 expression in benign breast lesions with apocrine differentiation.

<p>FFPE sections of normal breast and benign breast lesions with apocrine differentiation adjacent to tumor were stained with antibodies against FABP7 (upper panel) and HMGCS2 (low panel). (A) and (E) shows serial sections of normal breast tissue. Luminal and basal/myoepithelial cells are indicated by red and black arrows, respectively. (B) and (F) show sections of large normal ducts. (C) and (G) show serial sections of breast lesions with benign apocrine differentiation (apocrine adenosis). Positive and negative luminal cells are indicated by black and green arrows, respectively. (D) and (H) show serial sections of lesions with apocrine cysts. Apocrine cysts with apical snouts and normal small ducts are indicated with black and green arrows, respectively. Magnification: x10. Representative areas for each staining are shown in higher magnification (x20). The cut-off values for FABP7 and HMGCS2 are specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112024#s4" target="_blank">Materials and Methods</a>.</p

2D PAGE analysis of FABP7 and HMGCS2 expression among breast cancer subtypes.

<p>The representative images of IEF 2D PAGE of protein lysates prepared from frozen sections of 4 breast tumor subtypes: IAC (A), TNBC (B), Luminal B (C) and Her2+(D). HMGCS2 and FABP7 have been identified by MS and indicated by blue arrows. The positions of HMGCS2 and FABP7 on the 2D gels of TNBC, Luminal B and HER2 were determined by matching of corresponding images by PDQUEST software. Alpha-enolase variants, identified by MS, are co-migrated with HMGCS2 and indicated by black arrows. Two other IAC markers, 15-PGDH and ACSMS1 described in our previous studies are shown for reference. IAC =  invasive apocrine carcinoma; TNBC =  triple negative breast cancer. Tumors have been stratified as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112024#s4" target="_blank">Materials and Methods</a>.</p