LETTERS to the EDITOR

p53 immunostaining in benign breast disease

This study attempts to clarify the role of c-erbB-2 overexpression in human non-small cell lung cancer (NSCLC) and relate it with the p53 alterations, DNA index (D.I.) and epidermal growth factor receptor (EGFR) content in sixty four patients with NSCLC. c-erbB-2 and EGFR quantification were carried out from tissue homogenates using quantitative ELISA procedures. p53 alterations were determined by immunohistochemical (IHC) detection with the monoclonal antibody DO-7 and analysis for p53 mutations on exons 4 to 8 by single strand conformation polymorphism (SSCP). The D.I. was performed by flow cytometry. c-erbB-2 hyperexpression was found in 13 of 58 LC (22%), and it was closely associated with hyperdiploid tumors (D.I.>1.3; P=0.00). The p53 abnormalities detected by SSCP were statistically more frequent in hyperdiploid tumors (16/25; P=0.015) than in diploid ones (8/30). No relationship between the results of IHC p53 and SSCP was found. The patients with c-erbB-2 hyperexpressing tumors were prone to have frequent relapses (P=0.03), although the patients with hyperdiploid NSCLC are the ones with the highest relapse rate (P=0.008). From the results obtained in this study the following conclusions can be drawn: (a) c-erbB-2 hyperexpressing NSCLC are associated with abnormalities in other biological markers and with a greater rate of relapses; (b) SSCP seemed to be more specific that IHC to detect p53 molecular abnormalities; and (c) the D.I. is the parameter more tightly related with relapse.

This chapter describes the biology of high risk benign breast lesions. The natural history of breast cancer indicates that it develops over years—even decades—and may progress through recognizable stages of proliferative breast disease. Although breast cancers are slow growing, they metastasize early. The ability to recognize and treat high-risk, precursor breast lesions is desirable. In the human breast, a spectrum of microscopic changes is termed as proliferative breast disease (PBD). The progression of histopathological features of PBD is correlated with increased risk for the development of invasive carcinoma, but the focal and microscopic lesions of PBD provide scant tissue for genetic or other biological analyses. Carcinoma in situ (CIS), with an associated tenfold increased risk for breast cancer, is accepted as a precursor lesion because subsequent development of invasive cancer is frequently in the same breast in which CIS was detected earlier. Patterns of loss of heterozygosity (LOH) and gene expression in cancer and in experimental cell models support the notion that breast cancer can develop from a number of pathways of interdependent genetic changes.

The development of mechanical supports for biochemically active compounds serving as immunochemical sensors has been the goal of many studies. A new compound in the form of gel fiberglass (GFG) membranes was recently developed as an example of such supports. These membranes prepared from glass fibers covered with oxysilanes to create a matrix `gel fiberglass' (R. Zusman, USA Patent #08/112,087, 1993) were used to prepare affinity chromatography columns for the isolation of soluble p53 protein from the serum of cancer patients. A thin layer of protein, trapped in gel glass during its preparation, was deposited on a lattice of glass fibers. Derivatization of the support eliminated nonspecific adsorption of proteins. Under such conditions, external agents percolating through a membrane may contact a maximum number of protein molecules trapped in the gel glass. The membranes are very stable, and can be stored in dry conditions for several months at room temperature. Affinity chromatography columns were prepared from the GFG membranes and used to isolate various proteins, including tumor-associated antigens (TAA). The capacity of the columns was calculated as the amount of protein (mg ml⁻¹) isolated from TAA-containing solution, and amounted to up to 9 mg ml⁻¹ of serum in colon cancer patients. The cytoplasmic p53 protein was one of the main components of TAA isolated in our experiments. Its concentration was determined by HPLC. This protein was isolated from the serum of cancer patients in the highest concentration yet reported, up to 5 mg ml⁻¹. The described method allows an easy and highly effective isolation of TAA and can be used for important goals including cancer diagnosis.

This study attempts to clarify the oncological significance of the p53 molecular abnormalities and p53 expression in lung cancer (LC) and their relationship with flow cytometry (FC) parameters and epidermal growth factor receptor (EGFR). The study includes 65 samples taken from both LC and normal lung (NL). The p53 molecular abnormalities of exons 4–8 were studied by single strand conformation polymorphisms (SSCP) and the loss of heterozygosity (LOH) of exon 4 by the Metzler method. P53 protein was detected by Western blot. EGFR was determined by a radioligand assay using [¹²⁵I]EGF. The FC parameters S phase fraction (SPF), DNA index (D.I.), G1G0 and growth rate (G2M+SPF) were evaluated from cellular monosuspensions. The LC with SSCP p53 molecular abnormalities have a significantly higher EGFR content (P<0.001), SPF (P<0.007), D.I. (P<0.017) and a lower proportion of G1G0 cells (P<0.04) than LC with no molecular abnormalities. No relationship between p53 molecular abnormalities and tumor TN or evolutive events was found. Neither the relationship between the molecular results and p53 expression detected by Western blot nor that of the p53 expression detected by Western with FC parameters or EGFR could be shown. In NL the growth fraction cells decrease significantly (P<0.05) with the intensity of p53 expression. The lack of biological functionality of p53 with molecular abnormalities seemed to relate to fast growing LC whereas p53 expression detected by Western seemed more related to the wild type of p53.