Low-grade stromal sarcoma: DNA flow cytometric analysis and estrogen progesterone receptor data.

DNA flow cytometry (FCM) data and estrogen receptor (ER) and progesterone receptor (PR) status were studied in three cases of low-grade stromal sarcoma (LGSS). One case was a primary presentation and the remaining two were recurrent tumors. DNA FCM showed a DNA index (DI) equal to 1.00, consistent with a diploid cell population, for four of the six specimens studied. The other two showed near-diploid populations. Proliferation indices (PI) were low in two of the patients' tumors (8.0 and 12.7%). These findings are consistent with the clinical history of LGSS and its propensity for indolent growth, long intervals between recurrences, and generally favorable prognosis. In case 2, a patient with several recurrences, the PI was increased to 20.3% in a specimen from the first recurrence. She subsequently recurred within 1 year with a more aggressive tumor, characterized by a mitotic index of greater than 10 mitoses/10 high-power fields (HPF), absence of ER and PR, and an aneuploid population (DI = 1.19). Receptor data, obtained by dextran-coated charcoal assay, showed that all tumors except the aggressive recurrence in case 2 had high ER (average 316 fmole/mg protein) and high PR (average 753 fmole/mg protein) levels. These ER and PR data are similar to the two other reports in the literature and the usual clinical response to progestational therapy was demonstrated. Further studies will help define the possible role of FCM and ER and PR determinations in patients with LGSS. These preliminary data suggest that they may be of prognostic significance.

The role of the H-ras oncogene in radiation resistance and metastasis.

The sensitivity of tumor cells to the killing effects of ionizing radiation is thought to be one of the major determinants of curability of tumors in patients treated with radiation therapy. This paper reviews the evidence from our laboratory and other groups which supports a role for oncogenes in the induction of radioresistance in cultured mammalian cells. Primary rat embryo cells (REC) were chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offered several useful advantages. The cells prior to transformation are diploid and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of REC permitted the study of the effects of synergism between oncogenes on the induction of the radioresistant phenotype. The results show that the activated H-ras oncogene induces radiation resistance in primary rat cells after transformation, but that the effect of the oncogene itself is small. However, the myc oncogene, which has no effect on radiation resistance by itself, appears to have a synergistic effect on the induction of radiation resistance by H-ras. Radiation resistance induced by H-ras plus myc is characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. The AdenoE1A oncogene which will also act synergistically with ras in transformation assays plays a less clear-cut role in assays of radiation resistance. The H-ras oncogene is also known not only to transform cells but also to induce metastatic behavior in the tumors which form after these transformed cells are injected into syngeneic animals or nude mice. We have also shown in our primary rat embryo cell system that the induction of metastatic behavior in transformed cells, like the induction of radioresistance depends on a complex interaction between oncogenes and the cellular background. This evidence will be reviewed to demonstrate some of the analogies between radiation resistance and metastasis as examples of the complex alterations in cellular phenotype which occur after oncogene transfection.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of c-erbB-2 protein expression in conjunction with DNA content using multiparameter flow cytometry.

Breast cancer is a leading cause of cancer death among women. Factors useful for determining the prognosis of breast cancer include axillary lymph node involvement, tumor size, hormonal receptor status, nuclear grade, and relative DNA content. The c-erbB-2 protooncogene is amplified in 10-40% of primary breast tumors, as well as in breast cancer cell lines; where it is amplified there is increased expression of its product. We have investigated the DNA content and c-erbB-1 protein expression in tumor cell lines and in breast cancer patient specimens by multiparameter flow cytometry. The study was enabled by the discovery that both cellular integrity and c-erbB-2 antigen reactivity were preserved in cells and tissues following fixation in 70% ethanol. We demonstrate that flow cytometric analysis of c-erbB-2 expression in populations of ethanol-fixed tumor cells is a reliable and sensitive quantitative method that correlates well with previously documented semiquantitative techniques. This is a feasible method for analyzing archived clinical samples, and further allows correlations between c-erbB-2 levels and other cellular parameters. Additionally, this method detects abnormal populations not identified by DNA content analysis alone. Further studies utilizing this approach are necessary to evaluate the prognostic value of this oncoprotein in human breast cancer.

Cytologic smears of peritoneal surfaces as a sampling technique in epithelial ovarian carcinoma.

Ovarian carcinoma disseminates primarily through the shedding of cells into the peritoneal cavity and subsequent implantation onto peritoneal surfaces. Accurate evaluation of the extent of disease is important both at initial surgical evaluation for staging and at second-look operations to determine the necessity of further therapy. Techniques used for such assessment include node sampling, peritoneal washings, and random biopsies of peritoneal surfaces. Although random biopsies are used by some, others have found them to be of negligible value in the absence of gross disease. The use of cytologic smears of peritoneal surfaces offers a simple method by which cells from a large surface area may be evaluated, and uses equipment readily available to the gynecologist. We compared results of 125 cytologic smears with washings and biopsy specimens obtained during 33 laparotomies for ovarian carcinoma. Cytologic smears identified disease in 48 of 125 sites, whereas biopsy identified only 29 areas of disease. Thirteen of the positive Papanicolaou smears were obtained from clinically disease-free areas. Although the cytologic evaluation of the parietal peritoneal surfaces was more frequently positive than were biopsy specimens, each method identified disease in 16 patients when paired with standard techniques of examination and washing. We conclude that the peritoneal cytologic smear offers an alternative method of further evaluating the extent of disease, particularly when no gross evidence of extraovarian disease is detected.

DNA content of juvenile granulosa tumors determined by flow cytometry.

Juvenile granulosa tumors (JGT) often exhibit worrisome morphologic features, yet usually behave in a benign fashion. Thirteen JGT were examined by flow cytometric analysis of paraffin material to determine if DNA content and cell kinetics are related to prognosis. The patients ranged in age from stillborn to 16 years. Unilateral salpingoophorectomy was the most common therapy. Eleven patients with follow-up were free of disease. Marked nuclear atypia was evident in three cases, and high mitotic counts were observed in six, but only marked atypia correlated with DNA content. Flow cytometry revealed that 46% of the JGT had abnormal DNA content and increased average growth fraction. The neoplasms with the highest DNA indices were found predominantly in postmenarchal girls. JGT may exhibit abnormal DNA content, nuclear atypia, and numerous mitoses, yet behave benignly. DNA flow cytometric studies of higher stage JGT are warranted.

Synergistic effect of the v-myc oncogene with H-ras on radioresistance.

Resistance of tumors to irradiation or chemotherapeutic agents is thought to be one of the reasons why patients who present with early malignancies may not be cured. Much is now known about the molecular mechanisms that underlie drug resistance, but until recently little was known about genetic contributions to radiation resistance. Some evidence now links oncogenes, particularly the ras family of oncogenes, to radiation resistance but heterogeneity between tumors and cell lines has complicated this analysis. Primary rat embryo cells have been chosen as a model system in which the effects on radiation resistance of the H-ras oncogene could be studied on a uniform genetic background. These cells offer several useful advantages. The cells prior to transformation are diploid, and because they have been in culture only for a few passages prior to transformation with the oncogene it is unlikely that any preexisting mutation affecting radiation response could be present. Additionally, the use of rat embryo cells permitted the study of the effects of a second oncogene on the appearance of the radioresistant phenotype. The results show that the activated H-ras oncogene is associated with radiation resistance in primary rat cells after transformation but that the effect of the oncogene by itself is small. However, the oncogene v-myc, which has no effect on radiation resistance by itself, has a synergistic effect on radiation resistance with H-ras. There appear to be differences in the phenotype of radiation resistance associated with these two forms of transfectants. Thus, radiation resistance seen with H-ras by itself is characterized by a change in the slope of the radiation survival curve at high radiation doses but little or no change within the should region of the radiation survival curve. Radiation resistance seen in H-ras plus v-myc transformants is also characterized by an increase in the slope of the curve at high doses but there is also a large effect within the shoulder region of the radiation survival curve. These studies led to the following conclusions: (a) the radioresistant phenotype is not due to preexisting genetic heterogeneity in the cells prior to transfection; (b) the radiation resistant phenotype of cells transformed by H-ras is seen to a greater degree in cells which also contain the v-myc oncogene; (c) the v-myc oncogene may play an important role in the phenotype of radiation resistance at low doses that is within the range most critical for clinical practice.

Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells.

The low (NF-L) and middle (NF-M) molecular weight (Mr) neurofilament (NF) subunits are expressed before the high (NF-H) Mr NF subunit in embryonic neurons. Thereafter, NF-M attains its mature state of phosphorylation more rapidly than does NF-H. However, little is known about NF subunit expression during cell division. A rapidly dividing medulloblastoma cell line (D283 MED), therefore, was examined using flow cytometry, immunochemistry, and a large panel of NF subunit-specific polyclonal and monoclonal antibodies. Many of the monoclonal antibodies (MAbs) distinguished NF-H and NF-M in different states of phosphorylation. By flow cytometry, more than 90% of the D283 cells expressed NF-H and NF-M in different states of phosphorylation, and an antiserum specific for the carboxy terminus of NF-L labeled more than 60% of these cells. Furthermore, the fluorescence intensity produced by MAbs that detected phosphorylated versus nonphosphorylated NF-H and/or NF-M epitopes, appropriately decreased or increased, respectively, by preincubating the D283 cells with alkaline phosphatase. In contrast, cell staining with antibodies specific for phosphate-independent NF protein epitopes did not change substantially as a result of enzymatic dephosphorylation. These results agreed closely with those obtained from studies of normal human spinal cord NF extracts. However, NF-H, NF-M, and NF-L were expressed throughout the cell cycle in dual parameter studies of D283 cells labeled with an antibody and propidium iodide. Nevertheless, reductions in the fluorescence intensity produced with most of these antibodies late in the cell cycle suggested that NF proteins may be subject to modifications in their structure or accessibility to antibody probes during different phases of the cell cycle. These data led to the conclusion that NF subunits are expressed throughout the cell cycle in cultured human medulloblastoma cells, but that subtle changes in the immunoreactivity of these proteins occur during cell division.

Monitoring hepatocellular carcinoma by using a monoclonal immunoenzymometric assay for alpha-fetoprotein.

A monoclonal immunoenzymometric assay for alpha-fetoprotein (M-AFP) was evaluated with respect to its utility in monitoring hepatocellular carcinoma (HCC) patients. Earlier (Clin Chem 1986;32:1318-22), we found this immunoassay to demonstrate abilities similar to polyclonal AFP assays, and we suggested that changes in M-AFP correlated with changes in intrahepatic tumor volume in most HCC patients. In the present study, 107 HCC patients were evaluated between 1978 and 1986. Patient demographics characterized this study population as being similar to those seen in regions with low incidence of HCC. Changes in serum M-AFP concentration correlated moderately (r = 0.55) with changes in intrahepatic tumor volume. The AFP concentration in serum was found to be a statistically significant independent predictor of survival; patients with above-normal M-AFP (AFP[+]) at presentation demonstrated a median survival time of 10 months, compared with 16 months for patients with "normal" values for M-AFP (AFP[-]) (P = 0.008). This prognostic pattern persisted when adjusted for serum bilirubin concentration (AFP[+] 12 months vs AFP[-] 29 months, P = 0.01).