Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.

Abstract

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.Cyclins are proteins that vary in abundance and associate with and activate different cyclin-dependent kinases (cdk) at different stages of the cell cycle, a given cyclin-cdk complex being essential for passage through a specific stage in the cycle. The periodic appearance of the cyclins in distinct phases of the cell cycle suggests that they can be used as markers for proliferation of tissues. Because the three different cyclins mark different phases of the cell cycle-E for G1 and early S, A for S and G2, and B for late G2 (1-3)-the fraction of cells positive for a given cyclin should predict the fraction in a given phase of the cell cycle. Further, increased expression of various cyclins has been noted in extracts of human cancers, cyclin E in particular being markedly elevated in multiple tumors of different origins (4, 5), and an easy method for detecting such tumors will allow their biological behavior to be separately followed in clinical studies. Estimation of the proliferative behavior of a tumor is important for the management and prognosis of breast cancers. After local excision of node-negative tumors, two-thirds of the patients are expected to do well without further adjuvant therapy, and one-third are expected to relapse. Since chemotherapy is most effective before widespread metastases, it is useful to be able to predict which cancers are likely to recur in order to avoid undertreating these patients or overtreating patients whose tumors are not expected to recur (6, 7). One such prognostic factor is the S phase fraction (SPF) of the tumor estimated by fluorescence-activated cell sorting (FACS) analysis for DNA content of singly suspended tumor cells (8-10). Patients with a large fraction of tumor cells in S and G2 have a poorer prognosis and SPF has been used to guide the selection of patients for adjuvant therapy.Despite its value, flow cytometry of breast cancers is not performed widely, mostly because such analyses require fresh, relatively large tumors with enough tumor left over after tissueThe publication ...

Prognostic value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in patients with squamous cell lung carcinomas

“…Along with other researchers (Dutta et al, 1995;Paterlini et al, 1995), we found that the expression of cyclin A is closely correlated to the proportion of S-phase cells as measured by flow cytometry (data not shown). Univariate analysis indicates that cyclin A may also be a good prognostic indicator for the survival of patients with squamous cell lung carcinomas, but this was no longer seen in the multivariate analysis.…”

Lack of cyclin E immunoreactivity in non-malignant breast and association with proliferation in breast cancer

“…In addition, cyclin E expression was significantly associated with a high proliferation fraction, which had previously been demonstrated in the immunohistochemical study by Dutta et al (1995). Dutta et al identified a small fraction of tumours that overexpressed cyclin E relative to proliferation.…”

“…The following were used: mouse monoclonal antibody against cyclin E (HE12) generated against recombinant human cyclin E protein (Santa Cruz), as used by Dutta et al (1995). (In immunoblotting it recognizes the major human cyclin E-encoded proteins as three bands including a doublet at around 50 kDa and a single band at 42 kDa.)…”

“…It has been suggested that deregulation of cyclin E may be a factor contributing to the malignant phenotype (Keyomarsi and Pardee, 1993;Keyomarsi et al, 1994;Dutta et al, 1995). Recently, Western blotting of 114 breast tumour specimens showed that women with tumours with high cyclin E levels had a significantly increased risk of death and relapse from breast cancer (Nielsen et al, 1996).…”

“…Although this will provide information about different molecular weight isoforms, it restricts the spectrum of breast carcinomas that can be investigated as it requires large amounts of fresh tissue. Dutta et al (1995) used immunohistochemistry and showed in 48 breast cancers that there was a correlation between expression and tumour proliferation. Immunohistochemistry has the advantage of being applicable to a wide range of material, including benign, non-invasive and invasive carcinomas of all sizes.…”