Potential pathological application of immunocytochemical methods to the detection of micrometastases.

Immunocytochemical methods can be used to demonstrate tumor products and antigens at the cellular level. This approach facilitates the classification of tumors and the detection of small metastatic tumor foci in biopsy material from various sites including the bone marrow.

The study of apoptotic cells by flow cytometry.

Programmed cell death (PCD) is of fundamental importance in the normal development of an animal. It also plays a key role in tumour and radiation biology. PCD produces a sequence of changes occurring in cells called apoptosis. The main elements of this apoptotic cascade are rapidly being elucidated. Flow cytometry has been used to follow many of these changes. It has also been used to quantify the number of apoptotic cells in a culture and, more recently, in clinical samples. In this review, the properties of apoptotic cells and the main features of the apoptotic cascade are described. How flow cytometry can be used to follow changes during the apoptotic cascade is then discussed.

Prediction of response to neoadjuvant chemoendocrine therapy in primary breast carcinomas.

Our aim was to determine whether biological molecular markers can predict response to neoadjuvant chemoendocrine therapy in patients with early breast cancer. Ninety patients (median age 56 years; range, 28-69 years) with primary operable breast carcinoma were studied. They were treated with four 3-weekly cycles of chemotherapy with mitozantrone, methotrexate (+/- mitomycin C), and tamoxifen prior to surgery. Fine-needle aspiration was used to obtain samples from patients prior to therapy, and the following parameters were assessed: estrogen receptor (ER), progesterone receptor (PgR), p53, Ki67, Bcl-2, and c-erbB-2 measured by immunocytochemistry, and ploidy and S-phase fraction (SPF) by flow cytometry. The tumors of 78% of the subjects responded (complete response, 9%; partial response, 69%) and 22% did not (no change, 20%; progressive disease, 2%). Response rates according to disease stage and patient age were as follows: T1, 74%; T2, 79%; T3/T4, 78%; age 50, 79% (P = not significant). Response rates for other parameters were as follows: ER-positive, 82%, and -negative, 70%; PgR-positive, 86%, and -negative, 71%; p53-positive, 74%, and -negative, 81%; Bcl-2-positive, 85%, and -negative 61%; c-erbB-2-positive, 57%, and -negative, 93%; Ki67 high, 77%, and low, 81%; SPF high, 77%, and low, 77%; aneuploid, 71%; and diploid, 85%. Only the difference for c-erbB-2 was statistically significant (P = 0.007). A trend for higher response rates to neoadjuvant chemoendocrine therapy for tumors that were positive for ER, PgR, and Bcl-2 was observed but did not reach statistical significance. Tumors negative for c-erbB-2 had a higher response rate, which was statistically significant. In contrast, Ki67, ploidy, SPF, and p53 failed to predict for response.

Glycoprotein PAS-0 from the milk fat globule membrane carries antigenic determinants for epithelial membrane antigen.

Epithelial membrane antigen (EMA) can be found in human tissues using antisera raised against defatted human cream. PAS-0 is a glycoprotein which has been extracted from human milk fat globule membranes. Using a polyclonal antiserum and a series of monoclonal antibodies, we have shown that the major antigenic determinant for EMA is carried by PAS-0. A more detailed comparison of the two glycoproteins has been made by establishing a set of radioimmunoassays using the different antibodies.

A flow cytometric study of the effect of heat on the kinetics of cell proliferation of Chinese hamster V-79 cells.

Two methods involving labelling cells with bromodeoxyuridine (BrdUrd) have been used to study by flow cytometry the effect of hyperthermia (43 degrees C for up to 1 h) on Chinese hamster V79 cells. One method involved the use of an antibody to BrdUrd after pulse-labelling the cells either before or at time intervals after treatment. In the second method, the cells were incubated continuously in BrdUrd after heat treatment, and the components of the cell cycle were then visualized by staining with a combination of a bis-benzimidazole and ethidium bromide. All three methods showed that heating at 43 degrees C stopped DNA synthesis which, at 37 degrees C, subsequently recovered reaching the normal rate 8-12 h later. The cells in S phase at the time of treatment then progressed to G2 where they were further delayed. Cells heated in G1. after the recommencement of synthesis, progressed around the cycle, albeit slower than in unheated cells. The difference between the cells in G1 and S phases at the time of treatment may account for the greater sensitivity of S phase cells to hyperthermia.

Micrometastases in breast cancer: long-term follow-up of the first patient cohort.

"Micrometastases" can be identified in the bone marrow of patients with apparently localised breast cancer using an immunocytochemical stain for epithelial membrane antigen (EMA). Of 39 women who had marrow samples examined at the time of initial presentation (37), or with locally recurrent disease (2), 13 (33%) had samples which contained small numbers of EMA positive cells. 10 out of 23 (44%) lymph-node positive patients were marrow positive, compared to 1 out of 14 (7%) lymph node negative cases (P = 0.03). Long-term follow-up (median 9.5 years) has shown that 11 out of 13 (85%) patients with micrometastases have developed metastatic disease compared to 8 out of 26 (31%) with negative bone marrow aspirates (P less than 0.05). The small number of EMA positive cells detected in bone marrow samples probably reflects the high metastatic potential of primary or recurrent cancers rather than established microscopic deposits; it is not yet clear whether the finding of such micrometastases will act as an independent variable compared to established prognostic factors.

Ploidy and proliferative index in medulloblastoma: useful prognostic factors?

Paraffin sections from 32 patients with primary medulloblastoma were analysed by flow cytometry for DNA ploidy and proliferative index to assess the value of these measurements in determining prognosis. Twenty-seven samples were informative. Of these 27 patients, 8 had had a total resection. The tumors were diploid in 13 patients and aneuploid in 14. Neither ploidy nor S-phase fraction were prognostic factors for survival, even when considered in conjunction with the type of surgery performed. This is in contrast to other published data, emphasising the need for large multicentre studies of biological prognostic factors in this rare tumour.

Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a 'sub-G1' peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

The distribution of effector cells for antibody-dependent cytotoxicity is species dependent.

Murine lymphoma cells were lysed by using either an allogeneic or a xenogeneic (rat) antiserum and antibody-dependent effectors from mice and rats. It was found that murine spleens were poorly antibody-dependent effectors against this target. The peritoneal cavities of mice were a good source of effectors. In rats, the spleen contained effectors, whereas adherent cells from the peritoneal cavity were comparatively ineffective. Cells from both rat and murine spleens lysed sheep erythrocytes in conjunction with an appropriate antiserum. The data emphasize that there is a difference in the antibody-dependent lysis of erythrocytes compared to nucleated mammalian cells. They also demonstrate that the distribution of antibody-dependent effectors is species dependent.

Destruction of allogeneic tumour cells by peritoneal macrophages.

The population of peritoneal macrophages from mice immunised with allogeneic tumor cells contains two types of cytotoxic cell. One lyses the specific target cell, the other initiates activation of macrophages and hence leads to inhibition of growth of the specific target cell. The yield of lytic effector macrophages was found to depend on the route and the nature of the cell used for immunisation and the condition of the mice. The yield correlated with the yield of complement-dependent cytotoxic antibodies. In contrast, production of specific "activator" macrophages did not depend critically on these factors. The results underline the difference between the two types of cell and suggest that they are produced independently of one another.

Dexamethasone and etoposide induce apoptosis in rat thymocytes from different phases of the cell cycle.

Dexamethasone and etoposide both induce apoptosis in immature rat thymocytes. We investigated the dependence of apoptosis on the phase of the cell cycle after incubation with these drugs. Cell cycle progression was followed by a combination of pulse labelling with 5-bromo-2'-deoxyuridine (BrdU), labelling fixed cells with an anti-BrdU antibody and flow cytometry. Dexamethasone had little effect on the cell cycle progression of proliferating thymocytes, while etoposide caused cell cycle arrest. Normal and apoptotic thymocytes were separated by centrifugation on discontinuous Percoll gradients into four fractions (F1-F4). It was found that both dexamethasone and etoposide induced apoptosis in cells in G0/G1 and G2/M of the cell cycle, whereas only etoposide induced apoptosis of cells in S phase. These results demonstrated that dexamethasone induced apoptosis in quiescent cells while only etoposide could induce apoptosis in cells from the proliferative compartment. Following treatment of thymocytes with etoposide, some of the proliferating thymocytes (F1) were converted to cells with intermediate size and density (F3). We have recently identified these cells as a population of preapoptotic thymocytes, at an early stage of apoptosis. These cells then further progressed to fully apoptotic cells (F4). These data support the hypothesis that normal thymocytes (F1) became apoptotic (F4) via an intermediate population (F3).

Changes in nuclear chromatin precede internucleosomal DNA cleavage in the induction of apoptosis by etoposide.

Etoposide, a DNA topoisomerase II inhibitor, caused a concentration-dependent induction of apoptosis in immature thymocytes. Using a flow cytometric method to separate and quantify normal and apoptotic cells, etoposide-induced apoptosis was inhibited by cycloheximide and actinomycin D but not by zinc. Etoposide induced a marked cleavage of DNA into nucleosomal length fragments or multiples thereof, which was completely inhibited if the thymocytes were also incubated in the presence of zinc. Etoposide, alone, induced the classical ultrastructural features of apoptosis, but in the presence of zinc, the morphological pattern was markedly different and dominated by discrete clumps of condensed chromatin abutting the nuclear membrane. These latter changes resemble those described as the earliest changes in apoptosis. These results support the hypothesis that, in the induction of apoptosis, critical alterations in nuclear chromatin occur prior to endonuclease cleavage of DNA into nucleosomal fragments.

Comparative studies of total cross-linking, cell survival and cell cycle perturbations in Chinese hamster cells treated with alkylating agents in vitro.

The toxicities of 4-hydroperoxycyclophosphamide (4-OOH CY), phosphoramide mustard (PM), melphalan (MEL) and busulphan (BU) have been compared in Chinese hamster cells, V-79-753B. The initial total amount of cross-linking was a determining factor for the clonogenic survival of cells treated with MEL or PM. Although 4-OOH CY generated cross-links in this cell line, this damage did not account for the toxicity of the compound. There was no evidence for cross-link formation in cells treated with BU, even at a dose of the drug (1000 micrograms/ml) that was too toxic to measure clonogenic survival. Comparison for the four compounds at equitoxic doses showed that both PM and MEL caused the arrest of the cell cycle at G2 which persisted after drug removal. This was accompanied by a decline in the population growth rate and a decrease in total cell count. In contrast, both BU and 4-OOH CY caused a temporary arrest of the cell cycle G2, 24 hr after drug removal. However, the cell cycle distribution returned the control values within 3-4 days after treatment. Both BU and 4-OOH CY showed little effect on the initial growth rate of the cells. It is concluded that the initial amount of cross-links contributes to the toxicity of PM and MEL. However, it is unlikely that the generation of cross-links is of major importance for the toxicity of either 4-OOH CY or BU.

Flow cytometry used to distinguish between complete and partial hydatidiform moles.

Cells prepared from fresh tissue from a normal conception, and three complete hydatidiform moles (CHMs), were shown to be diploid by flow cytometry, while triploid populations were identified in tissue from two partial hydatidiform moles (PHMs). Similar results were found in cells from formalin-fixed, paraffin-embedded material. In nine cases where cytogenetic analysis was carried out this correlated with the results from flow cytometry. Using flow cytometry we were able to show that two cases which were pathologically complete were in fact diploid, despite the finding of trisomy at one locus in the molar tissue using biochemical markers. The most likely explanation of apparent trisomy of the enzymes in the molar tissue was contamination by maternal tissue.

Is DNA ploidy and proliferative activity of prognostic value in advanced gastric carcinoma?

We analyzed the DNA content of 116 gastric carcinomas and lymph node metastasis in 53 of these using flow cytometry on archival material. DNA index and proliferative activity (% S-phase) values were obtained in 111 and 76 tumors, respectively, and survival rates were obtained for 108. Tumor classification was based on histologic type, grade, and growth pattern and stage. All were advanced gastric carcinomas. Fifty four tumors (47%) were aneuploid. In single variable analysis, survival was related to stage (muscularis propria versus serosa P = .002) and lymph node metastasis (P = .04). Infiltrative tumor growth had a slightly worse prognosis (P = .06). In multifactorial analysis, tumor stage was the only independent prognostic factor. Neither DNA ploidy nor % S-phase values had any effect on survival, although, patients with tumors with higher DNA indexes (greater than 1.7) showed a tendency toward worse survival rates. We failed to prove the prognostic value of DNA ploidy in gastric carcinoma. Before its effect is ruled out, some factors have to be considered: (1) all tumors were in advanced stage and the true effect of ploidy on survival may have been diluted, (2) quality of fixation--near diploid aneuploid populations may have been overlooked, and (3) intratumoral DNA ploidy heterogeneity can cause sample discrepancy (20% in our series).

Sensitive three-parameter flow-cytometric detection of abnormal cells in human cervical cancers: a pilot study.

The cell volume, the DNA, and the carcino-embryonic (CEA) or epithelial-membrane (EMA) antigen of formaldehyde-fixed human cervical cells from 21 malignant cervix tumors and 11 normal patients were measured simultaneously with a Fluvo-Metricell flow cytometer. The simultaneous cell volume and DNA measurement provided the distinction of morphologically intact cells from cell debris, the determination of the cell cycle phase combined with the detection of aneuploid cells, and the distinct of inflammatory cells from parenchymal and tumor cells. Malignant samples were recognized because they contained more than 0.5% CEA positive cells which were of intermediate size. CEA and EMA expression in the malignant samples was not linked. The false positive rate in a total of 32 samples was 6.3% when the sum of CEA and EMA positive cells of each cell sample was calculated. No false negative malignant sample was observed.

Epithelial Membrane Antigen. Its use in the cytodiagnosis of malignancy in serous effusions.

The cytological distinction between reactive mesothelial and malignant cells frequently causes problems for the diagnostic cytologist. In order to determine whether an immunocytochemical method might help resolve these difficult cases, we have stained smears from 309 serous effusions from 246 patients for the Epithelial Membrane Antigen (EMA). The EMA staining was classified as strong, weak or negative. Carcinoma cells (as diagnosed by conventional cytology) stained strongly for EMA in 63 of the 116 positive smears (54%). Five out of 15 (33%) of cytologically suspicious smears from patients with known carcinomas gave a strong EMA stain. Of particular interest were three effusions in which malignant cells were not identified in conventionally stained smears and in which a small number of EMA positive cells were identified. The EMA positive cells were subsequently restained by the Papanicolaou method and identified as malignant on retrospective morphological examination.

A new antigen on the epithelial membrane: its immunoperoxidase localisation in normal and neoplastic tissue.

Using antisera raised against defatted human cream we have demonstrated a new antigen on the epithelial membrane. An indirect immunoperoxidase technique on routinely processed histological sections was used, and we describe results which show that this antigen has a widespread but highly selective distribution, being apparently localised to membranes that have a secretory function. The antigen is carried by a variety of adenocarcinomas, suggesting that its localisation may have a role in the diagnosis and differential diagnosis of malignant disease.

Epithelial membrane antigen in cells from the uterine cervix: immunocytochemical staining of cervical smears.

Smears made from cervical scrapes have been stained immunocytochemically for epithelial membrane antigen using a polyclonal antiserum and two monoclonal antibodies. With the polyclonal antiserum malignant cells and those showing dysplasia consistently expressed the antigen. Normal cells were generally negative, with the exception of some metaplastic cells. The monoclonal antibodies, although they stained the abnormal cells less consistently, gave the same pattern of staining. All three antibodies showed considerable heterogeneity in the intensity of stain. This appears to be a general feature of the expression of this type of epitope in epithelial cells. While the results confirm that an immunohistochemical stain might have potential application for improved diagnostic methods, the staining of metaplastic cells with the presently available antibodies limits the usefulness of an antiserum to epithelial membrane antigen.

Use of antisera to epithelial membrane antigen for the cytodiagnosis of malignancy in serous effusions.

A new human antigen, designated epithelial membrane antigen (EMA), has recently been described on surface membranes of a wide variety of normal epithelium but not on connective tissue cells. The antigen is only weakly expressed on normal or reactive mesothelium. Increased expression of the antigen has been observed in most neoplasms of epithelial origin and in malignant mesothelioma. We have investigated the possibility of using this difference in the expression of the antigen to distinguish between mesothelial cells and malignant cells in cytological smears of serous effusions. This distinction cannot always be made on morphological grounds alone and problems of differential diagnosis are encountered in about 15% of all specimens of serous effusions sent for cytological examination. Using antisera to EMA we have applied an indirect immunoalkaline phosphatase technique to alcohol-fixed smears prepared from serous effusions and have found that intense staining of the antigen is confined to effusions from patients in whom there is either clinical or cytological evidence of malignancy. The technique proved to be especially useful in cytologically equivocal cases, where there were problems of differential diagnosis.