Prognostic value of morphometry in patients with normal breast tissue or usual ductal hyperplasia of the breast.

Women with usual ductal hyperplasia have a relative risk of 1.6-1.9 of subsequent breast cancer development. This slightly increased risk is generally not considered sufficiently high to justify (chemo)preventive therapy. It is therefore important to identify high-risk ductal hyperplastic lesions that would benefit from such a treatment. Nuclear morphometric features have been shown in previous work to be useful for objectively describing morphologic features associated with high risk in (pre)invasive breast lesions. The aim of this study was to evaluate whether such morphometric features can also predict subsequent invasive cancer development in patients with the common pattern of usual ductal hyperplasia or a normal breast biopsy. The present case-control study included 423 women with normal breast biopsies (n = 89) or biopsies containing usual ductal hyperplasia (n = 334). Of these 423 women, 132 developed invasive breast cancer during follow-up (mean 16.7 +/- 7.0 years). On the original hematoxylin and eosin-stained sections, nuclear morphometry was performed with a digitizing video overlay system, and mitotic and apoptotic indices were assessed. Patients with mean nuclear feature values for area, perimeter, diameter or longest axis above the 75th percentile had 1.6-1.7 times the breast cancer risk of women with mean nuclear feature values below this value. Pairwise combinations of these features yielded slightly higher cancer risks for the fourth quartile patients, with the highest risk (1.9) for patients with SD of nuclear area and perimeter values above the 75th percentile. The number of apoptotic or mitotic cells had no prognostic value for patients with apparently normal tissue or usual ductal hyperplasia. Our results give a first indication that normal breast tissue or usual ductal hyperplasia harbor nuclear morphologic changes that, when assessed by morphometry, may be used to predict breast cancer development. It is worthwhile studying this further in independent groups of patients with long-term follow-up.

Similarity in expression of cell cycle proteins between in situ and invasive ductal breast lesions of same differentiation grade.

There is increasing evidence that there are different progression routes leading to invasive breast cancer, depending on histology and differentiation grade. The aim of this study was to determine alterations in the expression of proteins involved in proliferation and apoptosis in non-invasive and invasive ductal breast lesions. Immunohistochemistry was performed on 106 usual ductal hyperplasias (UDH), 61 DCIS lesions and 53 invasive ductal breast carcinomas. Increased proliferation (Ki67), overexpression of cyclin D1, HER-2/neu, p21 and p53, and decreased expression of bcl-2 and p27 could already be found in UDH. Significant differences between UDH and DCIS lesions were found for only one protein when UDH was compared with well-differentiated DCIS (p27), for three proteins when compared with intermediately differentiated DCIS (p21, cyclin D1, Ki-67), and for all proteins when compared with poorly-differentiated DCIS. Comparing DCIS with invasive lesions of same differentiation grade, proliferation was elevated in the invasive lesions. Altered expression of the other proteins was in general only slightly increased in the invasive lesion compared with DCIS. The number of proteins with altered expression per lesion was highest in poorly-differentiated lesions and was comparable between DCIS and invasive cancer of the same differentiation grade. In conclusion, the biggest changes in expression of these proliferation and apoptosis related proteins appear to occur during the transition from hyperplasia to DCIS; they probably play a minor role in the transition from DCIS to invasive breast lesion of same differentiation grade. Well-differentiated in situ and invasive breast lesions share many of the aberrations in expression of these proteins, as do poorly-differentiated in situ and invasive lesions. However, there are many differences between the well and poorly-differentiated lesions. This further supports the existence of different progression routes leading to breast cancer.

Aberrant expression of MUC1 mucin in ductal hyperplasia and ductal carcinoma In situ of the breast.

MUC1 mucin is a high molecular weight transmembrane glycoprotein expressed on the apical cell surface of normal glandular epithelia. In many human adenocarcinomas, this protein is up-regulated and/or underglycosylated, and its expression changes from apical to the entire cell membrane. It is thought that entire cell membrane expression of MUC1 reduces cell-cell and cell-extracellular matrix interactions and therefore may facilitate invasive growth and development of metastases. In this study, we determined immunohistochemically the expression of normal and underglycosylated MUC1 in normal breast tissue (n = 8) and in a spectrum of breast lesions, including usual ductal hyperplasia (n = 23), atypical ductal hyperplasia (n = 7), and ductal carcinoma in situ (DCIS) (n = 22). We used 4 monoclonal antibodies; 115D8 is directed to a glycopeptide, the other 3 to the peptide core of the molecule, of which 139H2 is not affected by the degree of glycosylation of MUC1, whereas SM3 and VU-4-H5 stain only underglycosylated forms. All cases showed apical positivity for 115D8 and 139H2. Entire cell membrane expression of fully (normal) glycosylated MUC1 was mainly found in DCIS lesions. Apical staining of SM3 was found in 38% of normal cases and 60% of the ductal lesions with no difference between the different subgroups. Apical staining of VU-4-H5 was found more often in DCIS (27%) than in normal tissue or ductal hyperplasia (3%). Membrane expression of underglycosylated MUC1 was found only in poorly differentiated DCIS. In conclusion, aberrant expression of MUC1, i.e., on the entire cell membrane and/or underglycosylated forms, can be found in ductal hyperplasia with atypia and especially in DCIS of the breast. This finding implies that these lesions with aberrant expression are at higher risk for developing subsequent invasive breast carcinoma.

Balance of cell proliferation and apoptosis in breast carcinogenesis.

We determined the mitotic and apoptotic index through the spectrum of pre-invasive ductal breast lesions to invasive carcinoma in search of disturbances in the proliferation/cell death balance in breast carcinogenesis. Seventy-two pure pre-invasive ductal breast lesions (without invasive carcinoma) and 103 invasive breast carcinomas were used. The numbers of mitotic and apoptotic cells were microscopically counted in hematoxylin and eosin stained sections (MI and AI, respectively), and the ratio of the values of MI and AI was calculated for each individual case (M/A index). A distinction was made between well differentiated and poorly differentiated breast lesions, based on histological type and nuclear grade, to arrive at two plausible progression models for breast carcinogenesis. For the well differentiated breast lesions, the MI was rather equal for hyperplasias and well differentiated DCIS, but increased 6-fold from DCIS to well differentiated invasive carcinoma. The AI remained in the same range, resulting in a 4-fold increase of the M/A index. For the poorly differentiated breast lesions, a significant increase in MI and AI was found from hyperplasia to poorly differentiated DCIS. From DCIS to poorly differentiated invasive carcinoma, the MI increased significantly and the AI decreased 2-fold (n.s.), resulting in a 2.5-fold significant increase of the M/A index. In conclusion, the net increase of the number of cells in the transition from well differentiated pre-invasive to well differentiated invasive carcinoma is accompanied by an increase of cell proliferation rather than decrease in apoptosis, suggesting that in these lesions, proliferation related mechanisms are most important in carcinogenesis and progression. In contrast, in poorly differentiated breast lesions, decreased apoptosis seems to be also important in carcinogenesis and progression. At present, we are gathering patients with invasive breast cancer who had a previous biopsy with a pre-invasive lesion to obtain further more direct evidence for this hypothesis.

Expression of proliferation and apoptosis-related proteins in usual ductal hyperplasia of the breast.

Expression of proliferation- and apoptosis-related proteins was studied by immunohistochemistry in 130 usual ductal hyperplasias of the breast, of which 39 cases (30%) had adjacent invasive cancer. Overexpression of cyclin D1 and Ki-67 was found in 6% and 29% of the cases, respectively. Only two mild ductal hyperplasias were Her-2/neu positive. Overexpression of p21 and reduced expression of p27, both cdk-inhibitors, was seen in 16% and 27% of the lesions, respectively. Reduced expression of bcl-2 was found in 16% of the cases, and p53 accumulation was present in 8%. Expression of six of the seven studied proteins showed no significant difference between mild, moderate, or florid ductal hyperplasias, indicating that there are no important cell biological differences with regard to the studied proteins between the lesions within this morphologically continuous spectrum. In addition, there were no differences between lesions with and without an invasive component. Cyclin D1 positivity was exclusively seen in lesions with 75% or more p27-positive nuclei. No significant correlations were found between other proteins. Twenty-three of 91 lesions (25%) had multiple events, of which five showed altered expressions of three or four proteins. In conclusion, altered protein expression of several proliferation- and apoptosis-related genes that are known to be involved in invasive breast cancer also may be found in usual ductal hyperplastic lesions, including several lesions with multiple events. This implies that usual ductal hyperplastic lesions may be among the earliest lesions within the breast oncogenetic spectrum.

Lack of granzyme expression in T lymphocytes indicates poor cytotoxic T lymphocyte activation in human papillomavirus-associated cervical carcinomas.

Changes in major histocompatibility complex (MHC) class I expression in neoplastic cells are frequently observed in human papillomavirus type 16 (HPV16)-associated cervical carcinomas. In order to investigate whether this affects cytotoxic T-cell (CTL) activation, 20 HPV16-positive cervical carcinomas with variable MHC expression were analyzed immunohistochemically for the expression of granzyme B and interleukin-2 receptor (IL-2R). The results revealed that most carcinomas show strong CD3+ T-lymphocyte infiltrates. In nine of these cases CD8+ cells outnumbered the CD4+ cells, whereas in the remaining cases equal amounts of CD4+ and CD8+ cells were found. Double staining revealed that CD3+ granzyme B+ cells were not detected in 19 out of 20 cervical lesions, whereas in one carcinoma an occasional cluster of granzyme B+ T cells was observed. Positive controls, including genital warts and renal allograft rejections, showed granzyme B+ T cells. In agreement with this observation was the extremely low frequency of IL-2R+ T cells in the carcinomas tested, while warts contained IL-2R+ lymphocytes. The data indicate that cytotoxic (CD8+) T cells in cervical carcinomas are not activated, as demonstrated by the lack of granzyme B and IL-2R expression in the T cells.