Special considerations when treating breast cancer in the elderly.

The growing aging population of the U.S. will lead to an absolute and proportional increase in elderly women with breast cancer. While the underlying biologic characteristics of the disease will not likely change, the health status and life expectancy of the patients will improve. Our decisions regarding therapy must take into account not just the disease we are treating, but the characteristics of the host as well.

Human breast cancer cells contain an error-prone DNA replication apparatus.

The mechanisms responsible for creating genetic errors and genomic instability in cancer cells have not been fully defined. Recently, it has been shown that human cells contain a highly organized complex of proteins, termed the DNA synthesome, that is fully competent to carry out all phases of SV40 in vitro DNA replication (J. M. Coll et al, Oncol. Res., 8: 435-447, 1996; L. H. Malkas et al., Biochemistry, 29: 6362-6374, 1990; Y. Wu et al., J. Cell. Biochem., 54: 32-46, 1994; N. Applegren et al., J. Cell. Biochem., 54: 32-46, 1994). DNA replication fidelity analyses of the DNA synthesome derived from malignant and nonmalignant human breast cells demonstrate that the malignant cell synthesome is mutagenic. The decrease in tumor cell replication fidelity was not due to an increased proliferative capacity of the tumor cells or an increase in the synthetic activity of their DNA synthesome. The ratios of insertions, deletions, and mismatches created by the synthesome from malignant and nonmalignant breast cells were essentially identical, despite the greater overall number of mutations made by the breast cancer cell synthesome. These data define, for the first time, a mechanism unique to cancer cells that contributes to the observed increase in genetic mutation in cancer cells.

A unique form of proliferating cell nuclear antigen is present in malignant breast cells.

Despite extensive research efforts to identify unique molecular alterations in breast cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been shown that the multiprotein DNA replication complex (synthesome) from breast cancer cells has a significantly decreased replication fidelity compared to that of nonmalignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in DNA replication and DNA repair and is a component of the synthesome. Using two-dimensional PAGE analysis, we have identified a novel form of PCNA in malignant breast cells. This unique form is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and nonmalignant breast cells. This novel form is most likely the result of an alteration in the post-translational modification of PCNA in malignant breast cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The novel form of PCNA may prove to be a new signature for malignant breast cells.

Mapping specific protein-protein interactions within the core component of the breast cell DNA synthesome.

We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.

The human breast cell DNA synthesome: its purification from tumor tissue and cell culture.

In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.

Relationship of the size of the invasive component of the primary breast carcinoma to axillary lymph node metastasis.

BACKGROUND: Invasive ductal carcinomas of the breast frequently have an intraductal (in situ) component at the tumor periphery that, in some cases, is included in the measurement of the tumor and thereby increases the size of the tumor beyond that of the invasive component. METHODS: Thirty-seven ductal carcinomas containing intraductal and invasive components were analyzed. The total tumor size, the size of the invasive component, the percentage of intraductal component, and the estimated tumor volume were assessed for each tumor. RESULTS: The mean size of the invasive component was 6.5 mm in axillary lymph node negative patients and 14.3 mm in those with axillary lymph node metastasis (P = 0.0001). The mean total tumor size was 13.7 mm and 17.6 mm (P = 0.035) and the mean percent of intraductal component was 52% and 26% (P = 0.015) in patients with negative and positive axillary lymph nodes, respectively. Ninety-two and four tenths percent of the difference in mean estimated total tumor volume between patients with negative and positive axillary lymph nodes was attributable to the difference in the volume of the invasive component alone. CONCLUSIONS: In small ductal carcinomas of the breast, the size of only the invasive component, as determined by microscopic measurement, is a better predictor of axillary lymph node status than is the total tumor size. The well established prognostic value of total tumor size largely is due to its reflection of the size of the invasive component.

Mastopathy in insulin-requiring diabetes mellitus.

Diabetic mastopathy is a recently described collection of histopathological features found in dense fibrous breast masses in insulin-requiring diabetics. Fifty-seven breast biopsy specimens showing nonspecific benign disease were examined in a blinded fashion from 21 diabetics (seven insulin-requiring, 14 non-insulin-requiring), 30 age-matched controls and six patients with thyroid disease. Five diabetics had the constellation of extensive keloidal fibrosis, mononuclear perivasculitis, and mononuclear ductitis and/or lobulitis, whereas none of the controls or patients with thyroid disease had all of these features. All five patients with diabetic mastopathy were insulin-requiring (two type I, three type II). Epithelioid fibroblasts in the stroma, a previously described component of this constellation, were present in three of the five cases but do not appear to be essential in making the diagnosis. Four of the five diabetics were hypertensive, and three had secondary diabetic complications. The mean duration of diabetes in the five patients was greater than 13 years. Based on a previous report and the current study, this constellation of histological features appears to be relatively specific for insulin-requiring diabetes mellitus. The single clinical factor common to all patients with diabetic mastopathy in this article and in a previous study was exogenous insulin use.

Expression of the retinoic Acid nuclear receptors (rars) and retinoid x-receptor (rxr) genes in estrogen-receptor positive and negative breast-cancer.

Retinoid inhibition of breast carcinoma growth correlates with the estrogen receptor (ER) positivity and elevated retinoic acid nuclear receptor (RARalpha) mRNA levels. We therefore examined retinoid nuclear receptor mRNA levels in patient breast carcinoma biopsy specimens to determine if such a correlation exists between ER positivity and RARalpha mRNA levels. We have now shown that RARalpha mRNA levels are significantly higher in ER positive samples. RARgamma mRNA is expressed at relatively high levels in a majority of the tumor samples independent of the ER-status while RARbeta mRNA was expressed at low levels in only one tumor sample. We also found high RXRalpha mRNA levels in all of the tumor samples examined while RXRgamma mRNA could not be detected. Our study demonstrates that RARalpha mRNA levels are either low or absent in ER-negative patient samples and that RARalpha levels may serve as a potential marker to determine patient responsiveness to RA therapy.

IGFBP-3 gene expression and estrogen receptor status in human breast carcinoma.

Insulin-like growth factors (IGF) I and II are potent mitogens for breast carcinoma proliferation. IGF-mediated proliferative activity can be markedly enhanced by the presence of specific IGF-binding proteins (IGFBPs). IGFBP-3 has been shown to enhance IGF-mediated growth in a number of systems. Studies have demonstrated IGFBP-3 secretion only in estrogen receptor (ER)-negative breast carcinoma cell lines while IGFBP-3 could not be detected in media conditioned by ER-positive cell lines. We investigated whether a relationship exists between ER status and IGFBP-3 mRNA expression in human breast carcinoma biopsy specimens. We have detected IGFBP-3 mRNA in breast carcinoma tissue obtained from patients utilizing in situ hybridization. Quantitation of IGFBP-3 mRNA levels was performed utilizing image cytometry. There was a significantly higher expression of IGFBP-3 mRNA in ER-negative breast carcinoma specimens when compared to the ER-positive specimens. Whether this higher expression of IGFBP-3 mRNA and presumed secretion of IGFBP-3 by ER-negative tumors play a role in the rapid proliferation and poor prognosis of these tumors remains to be determined.

A prospective randomized study of sutured versus stapled bowel anastomoses in patients with cancer.

Eighty-eight cancer patients with the presence of one or more adverse factors for healing (carcinomatosis, adhesions, prior chemotherapy and radiation therapy, bowel obstruction, anemia, and low leukocyte count or albumin value) were prospectively randomized to undergo conventional two-layer hand suturing (45 patients) or mechanical stapling with a GIA/TA instrument (U.S. Surgical Corp., Norwalk, CT) (43 patients) of the large or small bowel anastomosis. Age, sex, complete blood count findings, and all biochemical plasma values were comparable in both groups. The anastomosis took an average of 19 minutes for the sutured and 9 minutes for the stapled technique (P = 0.0001), but the average length of operation, postoperative return of bowel function, and hospital stay were comparable in both groups. Bowel fistula was seen in one case of stapled anastomosis (P = not significant). The pulmonary and wound complication rates were the same in both groups. Of the four deaths (4.5%) due to causes unrelated to bowel anastomosis, three occurred in the stapled and one in the sutured group. It was concluded that a stapled anastomosis is as safe as a sutured one in patients with advanced-stage cancer. It saves time in anastomosis, but does not save time in postoperative return of the bowel function and hospital stay.

ELISA tests for antibodies against mycobacterial glycolipids.

ELISA tests with purified mycobacterial glycolipids and bovine heart cardiolipin are described. The possible clinical use of ELISA tests with mycobacterial glycolipids for the diagnosis of tuberculosis and other mycobacterioses is discussed.