Axillary lymph node dissection in breast cancer patients after sentinel node biopsy^*

Axillary lymph node dissection in breast cancer patients after sentinel node biopsy<a href="#FN0001" target="_blank">^*</a

Strain elastogram of a 15 mm BI-RADS 5 tumour.

<p>The strain elastogram is displayed left of the B-mode image and shows a stiff tumour appearing mainly blue with small green areas (horizontal arrow). The tumour appears larger in the strain elastogram than in the B-mode image, indicating malignancy. The green curve on the strain quality indicator (lower right corner of the elastogram) displays the applied compression and decompression and should ideally be between the two dotted lines (red/white). The colour scale (upper left corner of the elastogram) shows the range of colours used to designate soft (red), intermediate (yellow and green) and stiff (blue) tissue. The B-mode image shows an irregular, spiculated, hypoechoic mass with posterior shadowing (horizontal arrow). Histology showed carcinoma.</p

Strain histograms are equal to strain ratios in predicting malignancy in breast tumours - Fig 3

<p><b>(A)</b> Strain histogram analysis of an 11 mm BI-RADS 3 tumour. The strain elastogram shows a predominantly blue tumour. Strain histogram analysis was performed from a single ROI within the tumour (ROI <b>A</b>). The B-mode image shows an oval, circumscribed, hypoechoic tumour, which is wider than tall and with slight posterior enhancement. Histology showed carcinoma. <b>(B)</b> The strain histogram is displayed as a bar chart, with the pixel colour values on the x-axis, and the number of pixels of a certain colour pr. 1000 pixels within the ROI on the y-axis. The mean colour value of the tumour is then calculated. In this case the mean colour value was 190, signifying a relatively stiff tumour, consistent with carcinoma.</p

Table of the histological classifications of the included tumours.

<p>Table of the histological classifications of the included tumours.</p

Table of the distribution of malignant and benign tumours for BI-RADS 1–5.

<p>Table of the distribution of malignant and benign tumours for BI-RADS 1–5.</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

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

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