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

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

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

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

Protein expression profile of transcriptome-derived apocrine markers among breast cancer subtypes.

<p>The diagram presents immunohistochemical profiles of four breast tumor subtypes, TNBC, Luminal A Luminal B and HER2, reacted with antibodies against ten transcriptome-derived apocrine markers: XBP1, TSC22D3, ABCA12, SIDT1, FOXA1, RHOB, BLVRA, SLC2A10, SLC7A8 and MLPH. 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. Corresponding frequencies of positives are shown on the right side of the diagram. Samples were considered as positive if 10% or more of the cells showed a clear positive staining with the antibodies. IDC =  invasive ductal 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

MLPH is expressed by non-malignant apocrine cells but is lost in IACs.

<p>Representative images of FFPE sections immunostained with antibody against MLPH. (A) normal ducts, (B) benign apocrine cysts (mainly luminal membranous immunoreactivity), (C) sclerosing adenosis with apocrine differentiation, (D) IAC showing positive immunostaining in pseudo-glands structures (cytoplasmic and luminal membranous immunoreactivity) and (E) IAC with negative immunostaining. Magnification: x20.</p

FABP7 and HMGCS2 are colocalized in lesions with apocrine differentiation.

<p>Indirect double-label immunofluorescence analysis of normal breast lesion with apocrine metaplasia (left panel) and apocrine cyst sections (right panel) reacted with FABP7 (subpanels B and F) and HMGCS2 (subpanels C and G). Sections were counterstained with the nuclear stain DAPI (blue channel). Merge images are shown on subpanels (D) and (H), respectively.</p

Immunohistochemical analysis of molecular apocrine markers derived from trancriptomics data within the apocrine cyst and carcinoma dataset.

<p>Representative staining of FFPE of apocrine cysts and IACs with antibodies against SLC2A10 (Glucose transporter type 10, sections A and B) - cytoplasmic immunoreactivity; SLC7A8 (L-type amino acid transporter 2, sections C and D) - mainly cytoplasmic and membranous immunereactivity; MLPH (Melanophilin, sections E and F) - cytoplasmic and luminal membranous immunoreactivity; FOXA1 (Hepatocyte nuclear factor 3-alpha, sections G and H) – nuclear staining; XBP1 (X-box-binding protein 1, sections I and J) – mainly nuclear and cytoplasmic immunereactivity; BLVRA (Biliverdin reductase A, sections K and L) - cytoplasmic immunoreactivity which combined with rare nuclear positivity; RHOB (Rho-related GTP-binding protein RhoB, sections M and N) - cytoplasmic immunoreactivity; TSC22D3 (TSC22 domain family protein 3, sections O and P) - mainly nuclear and cytoplasmic immunereactivity; ABCA12 (ATP-binding cassette sub-family A member 12, sections Q and R) - cytoplasmic immunoreactivity and SIDT1 (SID1 transmembrane family member 1, sections I and G) - cytoplasmic immunoreactivity. Magnification: x10. Representative areas are shown in higher magnification (x20) and examples of positive staining are indicated by red arrows.</p

Immunohistochemical expression analysis of FABP7 and HMGCS2 among breast cancer subtypes.

<p>The representative IHC images of FFPE sections from 5 breast tumor subtypes immunostained with antibodies against FABP7 (A,C,E,G and I) and HMGCS2 (B,D,F,H and J). ADCIS  =  apocrine ductal carcinoma <i>in situ</i>; IAC =  invasive apocrine carcinoma; TNBC =  triple negative breast cancer. Magnification: x10. Representative areas are shown in higher magnification (x20) The cut-off values for FABP7 and HMGCS2 are specified in Material and methods. 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