Characterization of breast precancerous lesions and myoepithelial hyperplasia in sclerosing adenosis with apocrine metaplasia

2D gel Western blotting of whole breast tumour extracts reacted with various antibodies. (A) 15-PGDH (dilution 1:1000; IEF). (B) p53 (dilution 1:1000; IEF). (C) MRP14 (dilution 1:500; IEF). (D) Psoriasin (dilution 1:1000; IEF). (E) CK14 (dilution 1:2000; IEF). (F) CK15 (dilution 1:500; IEF). (G) CK5 (dilution 1:4000; NEPHGE). (H) SMA (dilution 1:2000; IEF). (I) CK7 (dilution 1:500; IEF). (J) CK8 (dilution 1:500; IEF). (K) MAGE-4 (dilution 1:12; IEF).

Identification of the various structures observed in sclerosing adenosis with apocrine metaplasia in a tissue section of patient 11 stained with the 15-PGDH antibody. (A) Apocrine adenosis (arrow indicates nuclear staining). (B) Apocrine hyperplasia. (C) Apocrine microcysts. Arrows indicate type I (apocrine; black arrow) and type II (non-apocrine; blue arrow) microcysts. (D) Intraductal papilloma and columnar cell changes. The antibody was used at a dilution 1:4000.

Analyses of the various structures observed in sclerosing adenosis. Serial paraffin-embedded tissue sections were reacted with antibodies against components of the benign apocrine phenotype. Adenosis with apocrine atypia (patient 14): (A) p53. (B) 15-PGDH. (C) MRP14. (D) Psoriasin. Hyperplasia with apocrine atypia (patient 11): (E) p53. (F) 15-PGDH. (G) MRP14. (H) Psoriasin. Blue arrows indicate cells that are p53, 15-PGDH, MRP14, and psoriasin positive. Yellow arrows indicate cells that are positive for p53, 15-PGDH, and MRP14, but negative for psoriasin. Red arrows indicate cells that are negative for p53 and psoriasin, but are 15-PGDH and MRP14 positive. Microcysts with apocrine atypia (patient 14): (I) p53. (J) 15-PGDH. (K) MRP14. (L) Ki67 (arrow indicates a mitotic cell). The following antibody dilutions were used: p53 (1:250), 15-PGDH (1:4000), MRP14 (1:1000), psoriasin (undiluted culture supernatant), and Ki67 (1:200).

IHC analysis of paraffin-embedded sections of tissue biopsies from patient 23 stained with various specific antibodies. (A, D, G, J and M) Lesion I stained with antibodies against (A) 15-PGDH, (D) p53, (G) AR, (J) MRP14, and (M) MAGE-4. (B, E, H, K and N) Lesion II stained with antibodies against (B) 15-PGDH, (E) p53, (H) AR, (K) MRP14, and (N) MAGE-4. (C, F, I, L and O) Tumour biopsy stained with antibodies against (C) 15-PGDH, (F) p53, (I) AR, (L) MRP14, and (O) MAGE-4. The MAGE-4 and AR antibodies were both used at a dilution of 1:100. All other antibodies were used at the dilutions given in the legend of Figure 3.

IEF 2D gels of whole protein extracts stained with silver nitrate. (A) Lesion II and tumour biopsy from patient 23. (B) Apocrine macrocyst with ME hyperplasia. (C) Non-malignant mammary cell line MCF-10A. Exponentially growing MCF-10A cells were resuspended in lysis solution as previously described Celis et al., 2006d. The identity of the proteins indicated with arrows was determined by mass spectrometry (see also Table 3).

p53 positive non-apocrine lesions found in the SA biopsy of patient 14. (A and B) Serial sections stained with the p53 (A) and the 15-PGDH (B) antibodies. (C and D) Other areas of the above preparations stained with the 15-PGDH (C) and the p53 (D) antibodies. (E) Section stained with the Ki67 antibody. (F) Area from one of the p53 stained sections showing necrosis (indicated with an arrow). The antibody dilutions are given in the legend of Figure 3.

Confocal laser scanning analysis of indirect triple-label immunofluorescence of paraffin-embedded sclerosing adenosis tissue sections from patients 18 (A and B) and 10 (C and D) reacted with specific antibodies. (A) Tissue section reacted with CK15 (Alexa Fluor^® 594; red channel) and CK14 (Alexa Fluor^® 488; green channel) antibodies, and counterstained with the nuclear stain TO-PRO-3 (white channel). In the merged image, cells expressing both antigens are yellow and are indicated with a white arrow. Cells expressing only CK14 are green and are indicated with a yellow arrow. (B) Tissue section reacted with p63 (Alexa Fluor^® 594; red channel) and CK15 (Alexa Fluor^® 488; green channel) antibodies, and counterstained with the nuclear stain TO-PRO-3 (white channel). In the merged image, cells expressing both antigens are indicated with white arrows. Cells expressing only CK15 are indicated with a yellow arrow. (C) Tissue section reacted with CK15 (Alexa Fluor^® 633, blue channel), CK19 (Alexa Fluor^® 594, red channel), and CK14 (Alexa Fluor^® 488, green channel) antibodies. In the merged image, cells expressing CKs 15 and 19 and that are CK14 negative are indicated with white arrows. Cells that are CK19+, CK15− and CK14− are indicated with a yellow arrow. (D) Tissue section reacted with CK15 (Alexa Fluor^® 633; blue channel), CK8 (Alexa Fluor^® 594, red channel), and CK14 (Alexa Fluor^® 488, green channel) antibodies. In the merged image, cells expressing all three antigens are white.

Section from an invasive apocrine carcinoma reacted with the antibody against 15-PGDH. (A) Apocrine CIS. (B) Apocrine adenosis. (C) Apocrine hyperplasia. (D) Invasive area. The antibody was used at a dilution of 1:4000.

IHC staining of an invasive apocrine carcinoma using antibodies against components of the benign apocrine signature. (A, E, and I) Area with AA stained with antibodies against (A) MRP14, (E) psoriasin and (I) p53. (B, F, and J) Area with apocrine hyperplasia stained with antibodies against (B) MRP14, (F) psoriasin, and (J) p53. Area with apocrine CIS (cribriform) stained with antibodies against (C) MRP14, (G) psoriasin, and (K) p53. (D, H, and L) Area with invasive disease stained with antibodies against (D) MRP14, (H) psoriasin and (L) p53. The antibodies were used at the dilutions given in the legend of Figure 3.