Helene S. Smith

Loss of Heterozygosity in Normal Tissue Adjacent to Breast Carcinomas

Loss of heterozygosity (LOH) was detected in morphologically normal lobules adjacent to breast cancers. The most frequent aberration was at chromosome 3p22-25; of ten cases with this LOH in the carcinoma, six displayed the same LOH in adjacent normal lobules. This suggests that in a subset of sporadic breast cancers, a tumor suppresser gene at 3p22-25 may be important in initiation or early progression of tumorigenesis. Among sixteen breast cancers with LOH at 17p13.1 and five breast cancers with LOH at 11p15.5, one case each displayed the same LOH in adjacent normal lobules. Thus the molecular heterogeneity that characterizes invasive breast cancers may occur at the earliest detectable stages of progression.

Induction of cell division in medium lacking serum growth factor by SV40

Abstract Infection with SV40 induces many cells to synthesize DNA and divide in a medium lacking serum protein growth factor(s) essential for division of uninfected cells. Virus infection can induce the cells to go through several rounds of cell division since colonies containing more than 100 cells are formed. A functioning virus genome is necessary to induce cell division since UV-irradiation of the virus destroys this capacity. The total number of cell divisions induced is proportional to the input multiplicity. Most of the colonies induced to divide show no evidence of permanent functioning virus information: they no longer have cells with T-antigen, and most do not grow to high saturation density when shifted to complete medium. The ability of a cell to grow in factor-free medium is not the same property as its loss of contact inhibition; a transformed clone has been selected which still is strongly contact inhibited in spite of producing SV40 T-antigen and having rescuable virus information. This transformant would not have been recognized in the standard transformation assay. It has gained one of the growth properties associated with virus-transformation, the ability to grow in factor-free medium.

Growth of diploid cells from breast cancers

Cell cultures were derived from normal and cancerous breast tissues and from metastases by methods that selected for relatively adherent epithelial aggregates. Karyotypic analyses of first or second passage cultures yielded predominantly normal diploid cells. Nonclonal aberrations were more common in tumor-derived than in normal cultures. Three of the cultures that originated from metastases were characterized by abnormal clones. These results support observations based on DNA content, which indicate that a considerable fraction of breast cancers are composed predominantly of diploid cells. They differ greatly from chromosomal findings in long-term cultures of tumor effusions and thus emphasize the karyotypic diversity that can be found in tumors from a single tissue of origin--the breast.

The biology of breast cancer at the cellular level

Two properties seem fundamental to cancer; heterogeneity and progression (Foulds (1975) Academic Press, New York; Heppner et al. (1979) Commentaries on Research in Breast Disease, Vol. 1 (Bulbrook, R. and Taylor, D.J., eds.), pp. 177-191, Plenum Press, New York). Relatively little is understood about the premalignant stages of human breast disease in vivo. When the disease manifests as invasive carcinoma, its behavior exhibits great diversity, sometimes metastasizing rapidly, while in other cases 10-30 years pass before metastases proliferate. Here we review various aspects of breast cancer in vivo and consider how they predict properties of breast cancer found in culture. All of the experiments are consistent with the hypothesis proposed by Nowell (1976) Science 194, 23-28, that a fundamental aspect of malignancy is an increased genetic instability and that many of the cells within tumors are nonviable results of genetic instability. We suggest that most of the viable cells within primary breast carcinomas are diploid and are not yet capable of aspects of metastatic spread. What these cells have attained is an increased propensity for genetic instability which enables them to generate randomly aneuploid but frequently lethal genetic configurations. Occasionally one of these altered genomes is associated with the ability to proliferate at a metastatic site. This hypothesis implies that metastases from various patients may have arisen by divergent pathways and may also be divergent in many other aspects of their physiology, unrelated to malignancy. Such extreme heterogeneity may hamper attempts to understand fundamental aspects of malignancy. Hence we suggest that the less anaplastic and less divergent diploid cells within the primary carcinomas might be an important resource to gain insights into the critical alterations that are responsible for initiating frankly malignant behavior.

Detection of retinoblastoma gene copy number in metaphase chromosomes and interphase nuclei by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was applied to detect the copy number of the retinoblastoma (RB1) tumor suppressor gene in metaphase chromosomes and interphase nuclei. We used 14 lambda phage clones spanning the whole RB1 gene region as a probe and obtained a specific hybridization signal in normal metaphase chromosomes at 13q14. Normal interphase nuclei showed two RB1 signals in about 90% of cases, whereas two cell lines with cytogenetically defined deletions involving the RB1 gene showed only one hybridization signal in about 80% of the nuclei. Analogous changes were detected in metaphase chromosomes. Multicolor FISH with subsets of the phage clones allowed visualization of subregions within the 200-kb gene in interphase nuclei. Analysis of clinical breast cancer samples showed that most of the cells contained two copies of the RB1 gene, even when restriction fragment length polymorphism analysis showed loss of heterozygosity (LOH) at the RB1 locus. This indicates that LOH at the RB1 locus in breast cancer cells probably involves mechanisms other than physical deletion.

Immunolocalization of a human basal epithelium specific keratin in benign and malignant breast disease

SummaryThis report describes the immunocytochemical localization of a human basal- or myoepithelial-specific antikeratin antibody in benign and malignant breast disease. Reactivity patterns with this antibody have demonstrated the lack of myoepithelial or basal epithelial participation in most benign breast specimens examined including those displaying cystic disease, fibrosis, or hyperplasia. However, in specimens of sclerosing adenosis, strong reactivity with the majority of cells in most ducts suggests a major participation of the myoepithelial cell type. Analysis of 118 breast carcinoma specimens has demonstrated strong, homogeneous reactivity in 4% of the specimens, suggesting a role for the basal epithelial cell in malignancy of the human mammary gland and implications for the prognosis of such tumors. Antigenic characterization of the malignant and benign mammary specimens which are uniformly reactive with the antibody has demonstrated the presence of a 51 kd keratin polypeptide not found in the non-reactive specimens.

The prognostic value of proliferation indices: a study with in vivo bromodeoxyuridine and Ki-67

Proliferation indices are intended to help patients and clinicians make treatment decisions. We have previously demonstrated that a proliferation index based on in vivo labeling of S-phase cells with bromodeoxyuridine (BrdUrd) correlates with Ki-67 labeling index (LI). We now compare the prognostic value of these indices.With written consent, we gave 129 women with biopsy confirmed breast cancer 200 mg/M2 BrdUrd during 30 min immediately preceding surgery. We used IU-4 anti BrdUrd antibody to count the immunohistochemical labeling index (LI) of DNA-incorporated BrdUrd in 2,000 cells and MIB-1 to count Ki-67 (118 cases). Patients received standard surgical and adjuvant treatment. No patients were lost to follow-up and patients were followed a minimum of 2 (median 5.1) years. We compared survival and recurrence in tumors with high vs low labeling indices. We found that women in the low BrdUrd LI group had better disease free survival (92% vs 67% 5-yr DFS p = 0.001) and overall survival (94% vs 70% 5-yr OS, p = 0.0001) than those with a high LI. In comparison, a low Ki-67 index predicted better OS (87% vs 80% 5-yr OS, p = 0.020) and a trend for better DFS (84% vs 72% DFS p = 0.055). The apparent superiority of BrdUrd LI over Ki-67 LI is likely due to chance (p = 0.18). In multivariate survival analyses we found that BrdUrd LI proliferative index significantly improves prediction of DFS or OS even when node status, age or tumor size is in the model. We conclude that markers of proliferation are useful adjuncts in predicting patient prognosis.

Biological properties of human melanoma cells in culture

SummaryThree human melanoma cell lines derived from one primary and two metastatic tumors from three different patients were characterized for growth properties usually associated with malignant transformation; these include cell morphology, growth rate, saturation density, growth in semisolid media, colony-forming ability on contact-inhibited monolayers of normal fibroblasts and epithelial cells, and tumorigenicity in immunosuppressed mice. Variations in expression of aberrant properties were evident among the lines. One of the metastatic lines satisfied all the parameters of malignancy tested and the other showed a number of these properties, whereas the primary essentially fulfilled only one. These results suggest that cultured melanoma cells reflect the clinical variability often observed among melanoma patients and the metastatic melanoma seems to display a higher degree of malignant transformation than the primary.

Correlation of bromodeoxyuridine (BRDU) labeling of breast carcinoma cells with mitotic figure content and tumor grade.

Tumor proliferation is inversely associated with survival in patients with breast carcinoma. Labeling of tumor cells with bromodeoxyuridine (BRDU) correlates highly with that seen with [3H]thymidine, the current “gold standard” for measuring tumor S-phase. However, the relationship of BRDU labeling to mitotic figure content and tumor grade remains incompletely defined. To determine this, we labeled 55 breast carcinomas with BRDU in vivo and correlated the results with mitotic figure content. The BRDU labeling index was the number of BRDU-positive cells/2, 000 tumor cells, the mitotic figure index was the number of mitotic figures per 1, 000 tumor cells, and the mitotic figure count was the number of mitotic figures per 10 high-powered fields. BRDU labeling was also correlated with tumor grade (ScarffBloomRichardson). The BRDU labeling index correlated highly with the mitotic figure index (r = 0.814, p = 7.0 ± 10−14), mitotic figure count (r = 0.725, p = 6.0 ± 10−10), and tumor grade (r = 0.68, p = 1.1 ± 10−8). The correlation of BRDU labeling with mitotic figure content was strong enough to suggest that a very carefully measured mitotic figure index provides an estimate of tumor growth fraction equivalent to the BRDU labeling index. Also, analysis of variance showed that the mitotic figure index was twice as precise as the mitotic figure count in estimating BRDU labeling, and thus was a more accurate measure of tumor proliferation. Moreover, measurements made by the mitotic figure index were as precise as those made by BRDU labeling. However, which method is optimal for estimating tumor proliferation rate remains unclear. Further studies are indicated.

Early expression of vimenti in human mammary cultures

SummaryThe intermediate filaments of most epithelial cells in vivo consist solely of cytokeratins. Using monoclonal antibodies to vimentin or keratin, we have examined the expression of vimentin in homologous specimens of frozen tissue sections and primary cultures of normal human mammary epithelium. In frozen sections, only epithelial cells reacted with the antikeratin antibody, whereas antivimentin reactivity was associated with stromal cells. All epithelial cultures were positive for cytokeratin and in addition coexpressed vimentin as strongly as cultured fibroblasts and as early as the 4th d after initiation of the culture. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis of cytoskeletal preparations of secondary cultures of normal mammary epithelium have also demonstrated the appearance of a moiety identical to the vimentin found in cultured fibroblasts. Our observations are consistent with the hypothesis that vimentin expression is induced, possibly as a result of changes in cell shape or growth rate, when cells are freed from three-dimensional restirctions imposed by the tissue of origin