Anti-malignin antibody in serum and other tumor marker determinations in breast cancer.

In this study, 154 healthy volunteers and 76 patients were tested using the anti-malignin antibody in serum (AMAS) test. Of the 154 volunteers, three were AMAS positive. After further examination, two were positive for cancer and one had a history of ulcerative colitis. Tumor biopsies of 43 suspicious mammography patients revealed that 32 were cancerous and 11 were benign by pathology. For the cancer patients, 31/32 were positive for the AMAS test, while 4/11 of the pathological benign cases were AMAS positive. In comparison to cancer antigen tests, AMAS was more sensitive (97%) in detecting breast cancer than CEA (0%), CA 15-3 (10%), CA 19-5 (5%) or CA 125 (16%) in the same patients.

Inter-institutional reproducibility of flow cytometric DNA-analysis in breast carcinomas.

In order to study interinstitutional reproducibility of flow cytometric DNA-analysis (DNA-FCM), frozen pieces from 30 consecutive breast carcinomas were analysed by 5 laboratories. Different instruments, preparation and DNA staining methods were used. A concordance in DNA-ploidy status was obtained in 26 of the 30 tumours. The discrepancy can mainly be explained by intratumoural DNA-heterogeneity since a complete agreement in ploidy status was obtained when four of the laboratories analysed the same cell suspension, where solid bits showed differing results. The sampling method seems therefore to be a crucial step for the results and needs further studies. As far as the estimation of S-phase fraction was concerned, one laboratory obtained significantly higher values compared to the other four. The correlation between the other four laboratories varied between r = 0.66-0.92.

HIV-1 propagates in human neuroblastoma cells.

A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS.

Flow cytometric enumeration of colonies for in vitro chemotherapeutic drug evaluation.

The percentage of 50-100 micron colonies formed by LX-T cells in medium containing agarose was determined microscopically, and this value was compared with the percentage determined by a flow cytometric method based on the forward and 90 degree light scatter of the colonies. As assessed by both in vitro methods, LX-T cells exposed to chemotherapeutic agents formed fewer colonies as the drug concentration increased. However, flow cytometric analysis indicated that a change in the number of colonies formed was a consequence of changing chemotherapeutic drug concentration, whereas microscopic colony counting did not always detect the corresponding change in colony number. These experiments demonstrate that measurement of a drug's chemotherapeutic potential by flow cytometric counting of colonies is an alternative to the enumeration of colonies microscopically.

Effect of mitogens on the cell cycle progression and the quantification of T-lymphocyte surface markers in acquired immune deficiency syndrome.

The cell cycle progression and viability of stimulated and intact lymphocytes from 20 subjects with acquired immune deficiency syndrome (AIDS) was determined by flow cytometry. As compared to controls, 62% less AIDS lymphocytes, cultured for 72 hr in the presence of lectins (Con-A, PHA, PWM), had entered the proliferative phases of the cell cycle, while the respective value for periodic-acid (H5IO6)-stimulated cells was 34%. The helper-suppressor ratios and natural kill cell percentages of the unstimulated and PHA-activated AIDS lymphocytes increased approximately 3-fold after 72 hr in culture. The natural killer (NK) cell fraction of the PHA-stimulated and unstimulated AIDS cultures comprised approximately 20% as compared to 10% in controls. However, no changes in the percentages of T-lymphocytes were detected in the AIDS cell cultures. Throughout the culture period, viability of the unstimulated AIDS lymphocytes exceeded 90%, whereas in stimulated cultures it fluctuated within the 65-90% range. It is concluded that the liability of AIDS lymphocytes to mitogens is probably a direct consequence of the human immunodeficiency virus (HIV) infection.

Characteristics of monoclonal antibody measurements in human peripheral blood.

Three areas of monoclonal antibody measurements using flow cytometry have been presented. These include a description of a dual immunofluorescent method for measuring two antibodies simultaneously, the effects of blood storage on enumeration of helper (H) and suppressor (S) cells, and the relationship between absolute lymphocyte count and H/S ratio in both control and AIDS patients. These studies reveal that a dual immunofluorescent labeling method is useful for enumerating lymphocytes from peripheral blood which bear the helper, suppressor and/or thymus-derived (T) cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate helper T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for enumerating suppressor T-lymphocytes. Dual immunofluorescently stained lymphocytes, prepared from whole blood, were analyzed by flow cytometry. Two light scatter parameters, (forward and 90 degree scatter) were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper and suppressor distributions from 167 control patients were as follows: The average percentage +/- SD of the helper and suppressor cells were 42.8 +/- 7.5 and 21.6 +/- 6.4, respectively. The H/S ratio was 2.17 +/- .75. These studies show that the H/S ratio can be determined in a single preparative sample and analyzed by dual immunofluorescence in a single flow cytometric analysis even though the H/S ratio may vary from normal during a disease condition. The dual immunofluorescent assay enables one to correlate the activities of two antibodies against cell surface receptors and allows the measurement of a large number of samples in a minimal time. This study also compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor T-lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. Marked changes were observed in the percentages of helper cells at 4 degrees C, whereas the percentages of suppressor cells did not change appreciably regardless of the anticoagulant storage time or temperature. Finally, the relationship between ALC and the H/S ratio in control and AIDS patients was determined. The ALC varied considerably in both control and patient populations as a function of time. Conversely, the H/S ratio remained constant.(ABSTRACT TRUNCATED AT 400 WORDS)

Flow cytometry of blastogenesis and the concomitant viability assay of lymphocytes stained with 4', 6 diamidino-2-phenylindole.

The blastogenic response of lymphocytes induced by concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and by paraperiodic acid (H5IO6) was assayed by flow cytometry of 4',6 diamidino-2-phenylindole (DAPI) stained nuclei as well as by thymidine (3H-TdR) incorporation rates. The DNA content in DAPI-stained nuclei of viable and dead human, rat and mouse lymphocytes, was readily distinguishable from each other by flow cytometric methods (FCM).

Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum: a comparative study.

Several cytochemical studies of the DNA content and ploidy status of neuronal cell nuclei in the central nervous system have reported the occurrence of hyperdiploid amounts of DNA in Purkinje cells and suggest the existence of some type of 'extra' DNA, the biological significance of which is, as yet, unknown. To explore this phenomenon further, the DNA content of glial and Purkinje cell nuclei was determined in several vertebrate species, using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) to stain isolated cerebellar nuclei for analysis with a single parameter flow cytometer. The Feulgen reaction for DNA was used to stain liver and cerebellar tissue imprints for the measurement of individual nuclei with a Vickers M86 integrating microdensitometer. In both types of analyses, chicken erythrocyte nuclei served as an internal reference standard of 2.5 pg DNA per cell. The mean DNA content of Purkinje cells and glial or granule cells was essentially the same as that found for diploid (2C) non-neuronal cells, such as hepatocytes, in rainbow trout, Amazon molly fish, salamander (Plethodon), mouse, rat, rabbit, cat, dog, monkey and human. Although Purkinje cell nuclei with 4C DNA levels were found in all of these species, except salamander and rabbit, the frequency of such cells was low (1-7%) and varied with the species. There was a low incidence of Purkinje cell nuclei with interclass DNA amounts in all species examined. Our data show that most neuronal cell nuclei in the cerebellum contain 2C levels of DNA.

Prognostic indicators including DNA histogram type, receptor content, and staging related to human breast cancer patient survival.

Primary tumors from breast cancer patients were evaluated for the biochemical presence of three steroid cytosolic receptors and by DNA histogram analysis using flow cytometry. These parameters were compared with the histological and staging diagnoses and the patients' survival over a 36-month period. A total of 74 patients with primary breast tumors were evaluated. The breast samples invariably demonstrated a peak population of diploid G0/1 cells which contained 2C amounts of DNA, as determined by mixing experiments using normal human breast tissues or trout erythrocytes as fixed standards. The tumors were classified into five DNA histogram types based on their DNA index distributions established by flow cytometry. These results showed that 21% of the tumors were diploid and indistinguishable from the diploid population of normal breast cells, 8% were hypodiploid, 11% were hypertetraploid, 8% were multiploid, and the remaining 52% were hyperdiploid. The DNA index values varied from 0.78 (hypodiploid) to 2.60 (hypertetraploid). The percentages of S-phase cells were lowest in the diploid and hypertetraploid tumors and highest in the hypodiploid tumors. Among the 24 patients who died during the 36-month follow-up, 92% (22 of 24) were classified in one of the aneuploid groups. Three high-risk groups identified on the basis of survival after 36 months were distinguished: hypodiploid (50% survival); multiploid (43% survival); and hyperdiploid (50% survival). Rates of survival in the diploid and hyperdiploid groups were 87 and 71%, respectively. The hypodiploid group was distinguished by having the lowest mean estrogen cytosolic receptor value [26 +/- 13 (S.D.) fmol/mg], progesterone cytosolic receptor value (13 +/- 15 fmol/ mg), and androgen cytosolic receptor value (less than 1 +/- 1 fmol/mg). In contrast, the diploid tumors had some of the highest receptor values, with mean estrogen cytosolic receptor value equal to 102 +/- 114 fmol/mg, progesterone cytosolic receptor value equal to 74 +/- 110 fmol/mg, and androgen cytosolic receptor value equal to 65 +/- 80 fmol/mg. The lowest survival rates (17% after 36 months) occurred in patients over 67 years of age who had aneuploid tumors, compared to 100% survival in patients over 67 years of age with diploid tumors. Our results demonstrate the value of using flow cytometry and steroid receptor values as supplements to histopathology for the characterization of subgroups of mammary cancer patients. The ability to identify patients with a good prognosis compared to those at high risk of recurrence and death will be valuable in the design of future prospective treatment studies.

Dual immunofluorescent analysis of human peripheral blood lymphocytes.

These studies reveal that a dual immunofluorescent labeling method is useful for enumerating cells from human peripheral blood that bear the helper, suppressor, and/or T-cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate Helper (H) T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for quantitating suppressor (S) T-lymphocytes. FL-conjugated Leu-4 antibodies were used to measure the T-lymphocyte activity. Dual immunofluorescent stained lymphocytes, prepared either from whole blood or by Ficoll-Hypaque, gradient cell separation, were analyzed by flow cytometry. Two light scatter parameters, forward and 90 degree, were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper, suppressor, and T-lymphocyte distributions from 100 controls were as follows: The average percentages +/- SD of the helper and suppressor cells were 41.2 +/- 7.2 and 23.0 +/- 7.2, respectively. The H/S ratio was 1.99 +/- 0.77, while the T-cell distribution on 28 patients was 71.4 +/- 7.7. The Ficoll-Hypaque purified lymphocytes and lysed whole blood lymphocytes compared favorably in their H/S ratios. A comparison was made between the percentages of helper and suppressor cells enumerated by fluorescent microscopy and flow cytometry in which correlation coefficients of 0.80 and 0.86 were determined, respectively. These studies show that helper and suppressor T-lymphocytes can be quantitated simultaneously by flow cytometry, which enables one to correlate the phenotypic activities of two antibodies against cell surface receptors and permits the measurement of a large number of samples in a minimal time.

Picogram per cell determination of DNA by flow cytofluorometry.

Using nuclei isolated from less than 0.2 g tissue or 10(7) cells, a method is presented for the quantitative determination of amounts of DNA per cell at the picogram level. This technique is based on the enhanced fluorescence of 4',6-diamidino-2-phenylindole (DAPI) when it binds to DNA. A rapid, one-step nuclear isolation and DNA staining procedure is used to prepare tissue samples for flow cytometric analysis. Frozen tissues give results comparable to those for fresh tissue. Both chicken and trout erythrocyte nuclei were used as reference standards in the determination of amounts of DNA per diploid cell for several mammals and Amazon molly fish. The consistent values obtained for different tissues from the same organism show the accuracy of this method for DNA measurement.

The effects of anticoagulant and temperature on the measurements of helper and suppressor cells.

This study compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor Thymus-derived (T) lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. A dual immunofluorescent labeling method, using fluoresceinated-helper (LEU 3a + b) and B-phycoerythrinated-suppressor (LEU 2a) antibodies, was used to simultaneously determine the percentages of the lymphocyte types in whole blood preparations by flow cytometry. Light scatter distributions were stable for ACD or heparinized blood at room temperature, whereas EDTA or 4 degrees C caused changes in the granulocyte distributions. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. However, heparinized and EDTA blood showed marked decreases in the percentages of helper cells at 4 degrees C, and EDTA blood stored at room temperature showed an increase in helper cells. The percentages of suppressor cells did not change appreciably regardless of the anticoagulant, storage time, or temperature.

Cytologically malignant squamous-cell carcinoma arising in a verrucous carcinoma of the penis.

A case of verrucous carcinoma with a focus of cytologically malignant squamous-cell carcinoma is presented. This usually occurs following radiation therapy of the verrucous carcinoma, but may rarely occur de novo, as in this case. The potential usefulness of fine-needle aspiration in detecting focal anaplasia in verrucous carcinoma is discussed. This technique may be especially useful if the lesion is to be destroyed cryosurgically.

DNA flow cytometry of pleural effusions. Comparison with pathology for the diagnosis of malignancy.

A study was undertaken to determine the potential value of DNA flow cytometry (FCM) for the diagnosis of malignancy in pleural effusions and to compare its results with those of traditional cytomorphology. Forty-one pleural fluids from 37 patients were evaluated by DNA FCM and routine cytologic techniques. Of the 41 pleural fluids, 29 (70.7%) demonstrated an abnormal DNA content by FCM. Two of the 29 pleural fluids had been originally diagnosed as benign by cytology. Cytologic review of these two cases showed no abnormal cells; therefore, there were no false-negative cases based on cytology. In 12 (29.3%) of the 41 fluids, DNA FCM and cytologic evaluation both indicated benign processes. Our preliminary observations indicate that FCM is an accurate and reliable technique that may be of aid in the diagnosis of malignant effusions. The technique may prove to be of special value in the differential diagnosis of reactive mesothelial cells versus malignant mesothelioma as well as for following patients who receive chemotherapy for malignant pleural effusions. DNA FCM may also complement cytomorphologic diagnoses in other serous, exfoliative or aspiration material.

Modulation of glucocorticoid hormone receptor levels in chicken lymphoid tissue following treatment with androgens in vivo.

Lymphatic tissues are highly sensitive to androgens and androgens are thought to contribute to sex differences in the immune response. In this study we have examined the effects of androgens on cytosolic glucocorticoid receptor levels in lymphoid tissues. The immature chick was chosen for our experimental model because it allows the separate evaluation of the bursa of Fabricius (primarily B-cells) compared to the thymus (primary T-cells). Treatment with dihydrotestosterone (a potent androgen in chicks) for 3-12 days in vivo reduced the cytosolic glucocorticoid (triamcinolone acetonide-[3H]) receptors in bursa tissue to approximately 42% of control levels after 5 days and less than or equal to 5% of control levels after 7 days of treatment. The chick thymus tissues were still approximately 92% of control triamcinolone acetonide receptor levels after 5 days of androgen treatments. However the thymus levels had dropped to less than or equal to 5% of control values after 12 treatment days. Thus a difference in the rate of decrease in the bursa of Fabricius compared to the thymus was indicated. The blastogenesis index (BI), a measurement of the percentage of cells progressing through the cell cycle, was figured using fluorescent DNA staining with diamidino phenylindole followed by flow cytometry analysis. After 3, 5, or 7 days of androgen treatment, the bursa of Fabricius from dihydrotestosterone treated chicks (2 mg/day/chick) had a mean BI = 11.17 (+/- 3.07 SD) which was significantly lower than the bursa of Fabricius from control chicks which showed a mean BI = 27.33 (+/- 3.42 SD). The thymus from dihydrotestosterone treated chicks had a mean BI = 19.57 (+/- 2.19 SD) which was slightly but not significantly higher than the control thymus CI = 17.38 (+/- 0.89 SD). In summary, androgen treatment in vivo induced a decrease in the cytosolic glucocorticoid hormone receptor levels in both the chick thymus and bursa of Fabricius tissues while decreasing the blastogenesis index in the bursa cells but not in the thymus cells.

Studies on the pharmacology and cytokinetics of 2,3-dihydro-1H-imadazo[1,2-b]pyrazole (NSC 51143) with P815 mastocytoma cells.

A study has been made of the biochemical, cytokinetic, and pharmacological effects of pyrazole-imidazole (NSC 51143) (IMPY) on P815 mastocytoma ascites cells maintained in mice and of cells maintained in culture. The distribution phase of IMPY equivalents from the peritoneal fluid of the mouse was found to be two hr, with an elimination phase of 69 hr. No consistent alteration in the ribonucleotide pools of the ascites tumor cells in vivo was observed by high-pressure liquid chromatography using i.p. doses of IMPY up to 1000 mg/kg (25% increase in survival). Correspondingly, no significant alteration occurred in the proportion of cells in G0, G1, S, or G2 + M in vivo by flow cytometric analysis. This is in contrast to the in vitro data which showed a signifcant blockage in S phase (50% effective dose, 1.6 x 10(-4) M). Using Dowex 1 chromatography of extracts from ascites tumor cells treated with IMPY in vivo, several intracellular drug metabolites were detected, and their proportion was noted to change with time. No such metabolism was detected in vitro. Some radiolabeled drug was detected in RNA and DNA from the cold, acid-insoluble fraction of ascites tumor cells. Analysis of alkaline sucrose sedimentation indicated that part of the radiolabeled IMPY was in the heavy-sedimenting DNA fraction.

Preparation of tissues for DNA flow cytometric analysis.

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.

The effect of the glutamine analog, AT-125, on the cell cycle of MCF-7 and BT-20 human breast carcinoma cells using DNA flow cytometry.

It was found by DNA flow cytometry that AT-125 preferentially inhibited the cell cycle progression in G1-phase of BT-20 more so than MCF-7 breast carcinoma cells in vitro and that cells washed free of the drug now possessed S-phase DNA content. It was also found that gamma-glutamyl transpeptidase levels were more than 2 times higher in BT-20 than in MCF-7 cells.