Pleomorphism of the nuclear envelope in breast cancer: a new approach to an old problem

Abstract

In routine practice, nuclear pleomorphism of tumours is assessed by haematoxylin staining of the membrane-bound heterochromatin. However, decoration of the nuclear envelope (NE) through the immunofluorescence staining of NE proteins such as lamin B and emerin can provide a more objective appreciation of the nuclear shape. In breast cancer, nuclear pleomorphism is one of the least reproducible parameters to score histological grade, thus we sought to use NE proteins to improve the reproducibility of nuclear grading. First, immuno-fluorescence staining of NE as well as confocal microscopy and three-dimensional reconstruction of nuclei in cultured cells showed a smooth and uniform NE of normal breast epithelium in contrast to an irregular foldings of the membrane and the presence of deep invaginations leading to the formation of an intranuclear scaffold of NE-bound tubules in breast cancer cells. Following the above methods and criteria, we recorded the degree of NE pleomorphism (NEP) in a series of 273 invasive breast cancers tested by immunofluorescence. A uniform nuclear shape with few irregularities (low NEP) was observed in 135 cases or, alternatively, marked folds of the NE and an intranuclear tubular scaffold (high NEP cases) were observed in 138 cases. The latter features were significantly correlated (P-value <0.002) with lymph node metastases in 54 histological grade 1 and in 173 cancers with low mitotic count. Decoration of the NE might thus be regarded as a novel diagnostic parameter to define the grade of malignancy, which parallels and enhances that provided by routine histological procedures.

4

Haematoxylin and eosin staining of G3 invasive carcinoma: the nuclei meet the criteria for score 3, being vesicular, with prominent nucleoli and exhibiting marked variation in size (A). NE staining with anti-emerin antibody in IF shows instead a smooth profile (B), corresponding to low NEP.

1

SKBr3 breast cancer cell line, stained with (A) anti-lamin B antibodies (revealed with fluorescein) and (B) anti-emerin antibodies (revealed with rhodamin). The two proteins linked to the nuclear membrane share a similar distribution, not only at the nuclear surface, but in intranuclear deposits as well.

2

The arrangement of the nuclear membrane, as revealed by tagging membrane-associated lamin B, was observed by confocal microscopy, in cells from primary cultures of normal mammary epithelium (A) and in BT474 breast cancer cells (C). The 3D reconstruction of the serial sections allows to appreciate the smooth roundish shape on ‘normal’ nuclei (B) as compared to the deep foldings and intranuclear tubules in cancer cells (D).

3

IF staining with anti-emerin antibody outlined high NEP (A) in a histologically low-grade ductal carcinoma (G1) as shown by staining with haematoxylin and eosin of a parallel section (B). Anti-emerin antibody delineates the smooth profile of nuclei of normal residual epithelium (arrows) in a ductal high nuclear grade carcinoma in situ (C, D).