HLA Class II Histocompatibility Antigen γ Chain (CD74) Expression Is Associated with Immune Cell Infiltration and Favorable Outcome in Breast Cancer

The triple-negative breast cancer (TNBC) subtype, defined as negative for ER, PgR, and HER2, is biologically more aggressive and with a poorer prognosis than the other subtypes, in part due to the lack of suitable targeted therapies. Consequently, identification of any potential novel therapeutic option, predictive and/or prognostic biomarker, or any other relevant information that may impact the clinical management of this group of patients is valuable. The HLA class II histocompatibility antigen γ chain, or cluster of differentiation 74 (CD74), has been associated with TNBCs, and poorer survival. However, discordant results have been reported for immunohistochemical studies of CD74 expression in breast cancer. Here we report validation studies for use of a novel CD74 antibody, UMAb231. We used this antibody to stain a TMA including 640 human breast cancer samples, and found no association with the TNBC subtype, but did find a positive correlation with outcome. We also found associations between CD74 expression and immune cell infiltration, and expression of programmed death ligand 1 (PD-L1). Given that CD74 may play a role in innate immune system responses and the potential of immunotherapy as a viable treatment strategy for TNBCs, CD74 expression may have predictive value for immune checkpoint therapies

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

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

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

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