Comparison of bone marrow, disseminated tumour cells and blood-circulating tumour cells in breast cancer patients after primary treatment.

The purpose of this study was to determine whether primary breast cancer patients showed evidence of circulating tumour cells (CTCs) during follow-up as an alternative to monitoring disseminated bone marrow tumour cells (DTCs) by immunocytochemistry and reverse transcriptase (RT)-PCR for the detection of micrometastases. We planned to compare CTC and DTC frequency in low-risk and high-risk patients. We identified two cohorts of primary breast cancer patients who were at low (group II, T(1)N(0), n=18) or high (group III, >3 nodes positive (with one exception, a patient with two positive nodes) n=33) risk of relapse who were being followed up after primary treatment. We tested each cohort for CTCs using the CellSearch system on 1-7 occasions and for DTCs by immunocytochemistry and RT-PCR on 1-2 occasions over a period of 2 years. We also examined patients with confirmed metastatic disease (group IV, n=12) and 21 control healthy volunteers for CTCs (group I). All group I samples were negative for CTCs. In contrast, 7 out of 18 (39%) group II primary patients and 23 out of 33 (70%) group III patients were positive for CTCs (P=0.042). If we count only samples with >1 cell as positive: 2 out of 18 (11%) group II patients were positive compared with 10 out of 33 (30%) in group III (P=0.174). In the case of DTCs, 1 out of 13 (8%) group II patients were positive compared with 19 out of 27 (70%) in group III (P<0.001). Only 10 out of 33 (30%) patients in group III showed no evidence of CTCs in all tests over the period of testing, compared with 11 out of 18 (61%) in group II (P=0.033). A significant proportion of poor prognosis primary breast cancer patients (group III) have evidence of CTCs on follow-up. Many also have evidence of DTCs, which are more often found in patients who were lymph node positive. As repeat sampling of peripheral blood is more acceptable to patients, the measurement of CTCs warrants further investigation because it enables blood samples to be taken more frequently, thus possibly enabling clinicians to have prior warning of impending overt metastatic disease.

Cardiac troponin I measurement with the ACCESS immunoassay system: analytical and clinical performance characteristics.

We evaluated the ACCESS cardiac troponin I (cTnI) immunoassay as a marker for myocardial infarction (MI). Total imprecision was 6.0% to 13.5%, the minimum detectable concentration was 0.007 microg/L, and the limit of quantitation was 0.046 microg/L. Comparison of cTnI measurement between the ACCESS and Stratus systems (n = 114) showed a proportional difference: ACCESS cTnI = 0.0996 Stratus cTnI + 0.049 microg/L (r = 0.811). Fifty-nine of 61 ambulatory patients without cardiac symptoms had no detectable cTnI (95% range, 0.00 to 0.025 microg/L). The optimum cutoff for discriminating MI (n = 289, 45 with MI) was 0.15 microg/L by receiver operator characteristic curve analysis; at this cutoff, the ACCESS cTnI assay showed a sensitivity of 88.9% (95% CI, 79.7-98.1%) and specificity of 91.8% (95% CI, 88.4-95.2%). The ACCESS cTnI assay results showed 89.4% and 93.0% concordance with the MB isoenzyme of creatine kinase (CK-MB) mass and Stratus cTnI results, respectively, for classification of patients with suspected MI. The ACCESS cTnI assay appears to show sensitivity and specificity comparable with those of both CK-MB mass and Stratus cTnI assays for the diagnosis of MI in patients presenting within 12 h of onset of symptoms.

Molecular forms of prostate-specific antigen and the human kallikrein gene family: a new era.

Without question, much has been learned about the glycoprotein PSA in recent years. By increasing our understanding of this tumor marker's biochemical and physiologic properties, we will be able to improve its clinical utility. The discovery of the various molecular forms of PSA represents a significant advancement. Knowing the concentration and ratio of these PSA forms will be valuable in deciding which patients require further evaluation with transrectal ultrasound and prostate biopsy and which men can be monitored safely without undergoing further invasive testing. This information will be most valuable in treating the patient with a mildly elevated serum PSA level. Although assays are not yet available to detect specifically hK2, the striking similarities of hK2 to PSA, including selective expression in the prostate, suggest that this marker may also prove useful in prostate cancer management. Indeed, a new era of PSA testing has been entered, and the entire field of prostate cancer will benefit.

Enhanced immunogenicity of leucine enkephalin following coupling to anti-immunoglobulin and anti-CD3 antibodies.

Leucine enkephalin (Leu-enk) was coupled to both T and B cell antibodies in order to investigate the possibility of enhanced immunogenicity via targeted immunization. The two antibodies used were Hm x Mo CD3 and Gt x Mo Ig, respectively. The data indicate that while both antibody carriers enhanced the immunogenicity of Leu-enk, the use of the Hm x Mo CD3 antibody resulted in a greater number of mice with positive Leu-enk specific serum titers. 12 Leu-enk cell lines were produced and one, LE4H8, was chosen for characterization.

Production of IgG monoclonal antibodies to the tumor-associated antigen, CA-195.

The isotype of a monoclonal antibody is closely associated with its biologic activity. Certain immunoglobulin subclasses are more effective than others regarding their ability to execute complement-mediated lysis of cells, antibody-dependent cell-mediated cytotoxicity, and tumor localization. Many potential targets for cancer immunotherapy are tumor-associated antigens with high percentages of carbohydrate. Immunizations of mice with carbohydrate antigens usually produce IgM and IgG3 antibodies. The use of different adjuvants in immunization protocols has been associated with the induction of isotype-specific antibody responses. In experiments reported here, we compare the use of four different adjuvants on the generation of an IgG immune response to the carbohydrate-rich, tumor-associated antigen, CA-195. We report the production of IgG1 and IgG2a monoclonal antibodies (mAbs) to CA-195.

Decreased expression of the common acute lymphoblastic leukaemia antigen (CALLA/CD10) on neutrophils from patients with thermal injury.

The common acute lymphoblastic leukaemia antigen (CALLA/CD10) is a normal component of the circulating neutrophil cell surface membrane. In order to examine the potential functional significance of CALLA/CD10 we analysed the expression of this molecule on neutrophils isolated from thermal injury patients, since these patients have a well-documented constellation of neutrophil defects affecting their microbicidal functions. Expression of neutrophil CALLA/CD10 was monitored by indirect immunofluorescence and flow cytometry. We observed that CALLA/CD10 expression was quantitatively reduced on burn patient neutrophils, compared to healthy donors (P less than 0.001). In contrast, burn patient neutrophils expressed normal levels of class I HLA molecules and the C3bi receptor. Reduced expression of CALLA/CD10 was not associated with neutrophil activation or exposure to plasma 'factor(s)' in vivo. Analysis of normal bone marrow neutrophils by cell sorting indicated that expression of CALLA/CD10 occurs late in neutrophil maturation, since 25% of polymorphonucleated bone marrow neutrophils did not express cell surface CALLA/CD10. Attempts to examine the chemotactic responses of CALLA/CD10 positive and negative neutrophils from burn patients were hampered by previous exposure of these cells to chemoattractants in vivo. Collectively, our findings suggest that burn patient peripheral blood neutrophils may be deficient in CALLA/CD10 due to insufficient maturation time in the bone marrow following thermal injury.

Identification and characterization of a unique subpopulation (CALLA/CD10/negative) of human neutrophils manifesting a heightened chemotactic response to activated complement.

We have previously demonstrated that human neutrophils synthesize the common acute lymphoblastic leukemia antigen (CALLA/CD10). To determine whether CALLA/CD10-positive and -negative neutrophils have similar or distinct functional attributes, we sorted normal peripheral blood neutrophils for CALLA/CD10 expression and compared their chemotactic ability. Surprisingly, the low-frequency (approximately 5%), CALLA/CD10-negative neutrophils displayed a dramatically heightened chemotactic response to activated complement (C') that was (a) specific for C', (b) not observed with other minor subpopulations of neutrophils, (c) not due to previous activation in vivo or in vitro, and (d) apparently not due to an increase in C5a receptors. These results underscore the concept of neutrophil heterogeneity and prompt the hypothesis that CALLA/CD10-negative neutrophils may participate in an inflammatory response to trauma involving complement activation.

Structure/function studies of the common acute lymphoblastic leukemia antigen (CALLA/CD10) expressed on human neutrophils.

The common acute lymphoblastic leukemia antigen (CALLA/CD10) is a nonintegral membrane glycoprotein expressed on normal and neoplastic cells of hematopoietic and nonhematopoietic origin. We have undertaken a series of experiments to examine 1) the structural homology between leukemia cell and neutrophil CALLA/CD10 and 2) the putative function CALLA/CD10 subserves to human neutrophils. Biosynthetic labeling, peptide mapping, and two-dimensional gel electrophoresis indicate that neutrophils synthesize and express a CALLA/CD10 molecule that is similar, but not identical, to leukemic cell CALLA/CD10. The level of CALLA/CD10 expression is similar on the two cell populations, and neutrophil CALLA/CD10 (like its leukemic cell counterpart) undergoes antigenic modulation. Finally, we report that neutrophil cell surface-bound anti-CALLA/CD10 monoclonal antibodies inhibit the chemotactic response to both N-Formyl-methionyl-leucyl-phenylalanine (F-mlp) and zymosan-activated sera (ZAS), but had no inhibitory effect on random migration, degranulation, or aggregation. The anti-class I monoclonal antibody W6/32 exerted a similar effect on chemotaxis. We conclude that CALLA/CD10 has no clearly defined role in neutrophil function but may play a role in some distal event in chemotaxis.

p24 and p26, structurally related cell surface molecules identified by monoclonal antibody BA-2.

This paper describes additional structural analyses of the p24 cell surface molecule recognized by monoclonal antibody BA-2. Since BA-2 is broadly reactive with a variety of normal and malignant lymphohematopoietic and nonlymphohematopoietic cells, we examined the structure of p24 expressed on different cell types. Tryptic peptide mapping and 2-dimensional gel electrophoresis of p24 isolated from colon carcinoma cells, fresh leukemic cells, leukemic cell lines, and activated T-cells indicated that p24 exhibits no structural polymorphism within the cells examined. As has recently been demonstrated with several other cell surface molecules, p24 is shown to possess a covalently-attached fatty acid, based on the incorporation of [3H]palmitate. We have also identified an additional protein, designated p26, that is coprecipitated with p24. The p26 molecule is not disulfide-linked to p24, and can be immunoprecipitated from a variety of 125I- or [35S]methionine-labeled cells. V8 protease peptide mapping indicated that p24 and p26 are structurally homologous. Pulse-chase analysis using [35S]methionine and digestion with endoglycosidase-F indicated that p24 and p26 are probably derived from a p23 precursor, but no precursor-product relationship exists between p24 and p26. Based on this data we propose that p24 and p26 are most likely differentially-processed protein products of the same gene.

Two mechanisms of inhibition of human lymphocyte proliferation by soluble yeast mannan polysaccharide.

The literature on chronic mucocutaneous candidiasis contains multiple reports which suggest that loss of cell-mediated immunity in this disease may be related in part to the presence of an inhibitory factor(s) present in patient plasma. One such inhibitory factor has been suggested to be mannan polysaccharide released from the cell wall of the pathogen. The present report describes results of experiments to consider mechanisms by which yeast mannan influences proliferative responses of human lymphocytes. Mannan for these experiments was isolated from Saccharomyces cerevisiae. We observed that mannan-mediated inhibition of proliferative responses to a battery of stimuli (phytohemagglutinin, pokeweed mitogen, and Candida, mumps, streptococcus, cytomegalovirus, and herpes simplex virus antigens) was related in part to an effect of copper associated with the mannan and possibly to the superoxide dismutase activity of the mannan-copper complex. Mannan made deficient in copper by use of a copper-chelating resin appeared to inhibit only lymphoproliferation stimulated by the Candida antigen. These results suggest that inhibitory effects of yeast mannans on lymphoproliferative responses may involve at least two mechanisms, one related to hydrogen peroxide production augmented by mannan-copper complexes and another related to still unknown effects independent of the metal ligand. We propose that our results represent a significant novel observation which may be useful in understanding mechanisms of immunoinhibitory effects of C. albicans mannan.

Chemotactic deactivation of human neutrophils: role of stimulation of hexose monophosphate shunt activity in nonspecific deactivation.

We have investigated the mechanism of the nonspecific component of chemotactic deactivation of human neutrophils. This component of deactivation occurs on exposure of neutrophils to larger doses of cytotaxin and is expressed as depressed subsequent spontaneous migration and chemotaxis toward all cytotaxins tested. We demonstrate a possible mechanistic association between stimulation of hexose-monophosphate shunt activity and the nonspecific component of deactivation. This association is evidenced by failure to effect deactivation of 0 degrees C, by the ability of non-chemotactic stimulatory agents to mimic cytotaxin-induced deactivation, by the ability of agents which block shunt activity to protect against deactivation, and by the ability of methylene blue to deactivate neutrophils from individuals with chronic granulomatous disease. Thus, excessive stimulation of shunt activity, possibly through generation of products of oxygen metabolism, appears to have an inhibitory influence on nonspecific neutrophil migratory functions.

Neutrophil function in lymphoreticular malignancy.

Neutrophil chemotaxis, phagocytosis, and metabolic stimulation were studied in 25 patients with untreated lymphoreticular malignancies, 8 patients with untreated carcinomas, and 8 patients undergoing therapy for lymphoreticular tumors. Phagocytic activity and metabolic stimulation during phagocytosis were comparable to controls in all patient groups. Slight, but significant, differences were found in chemotactic responsiveness of untreated patient groups when compared to controls. In almost all instances, chemotactic responsiveness reverted to normal in most patients studied after therapy for the malignant process. In patients with intact immunologic function these minor differences in chemotactic responsiveness are probably not sufficient to predispose to infection. However, this defect could be additive, and when present in combination with impaired immunity may contribute to compromised host defense.

Chemotactic deactivation of human neutrophils: possible relationship to stimulation of oxidative metabolism.

Neutrophils preexposed to high concentrations of activated complement or synthetic N-formyl methionyl peptides are inhibited in their subsequent spontaneous and chemotactic migratory responses. We have considered the possibility that a part of this nonspecific loss of migratory function may be attributable to the interaction of the leukocytes with reactive forms of oxygen deriving from the cytotaxin-induced burst of oxidative metabolic activity. For these studies we have assessed the effect of preexposure of neutrophils from patients with chronic granulomatous disease to cytotaxins on their subsequent migratory responses. We find that these responses are not altered by preexposure to either cytotaxin. Thus, there appears to be a functional relationship between deactivation and the ability of the normal neutrophil to undergo a cytotaxin-induced respiratory burst.

Chemotactic deactivation of human neutrophils: evidence for nonspecific and specific components.

Human polymorphonuclear neutrophils have been preexposed to activated complement as zymosan-activated serum (ZAS) or to the chemotactic oligopeptide N-formyl methionylphenylalanine (F-Met-Phe). Spontaneous migration and chemotactic responses toward the deactivating and other cytotaxins were monitored after washing and resuspension of cells in cytotaxin-free medium. Two patterns of deactivation were observed. Preexposure of the leukocytes to high doses of ZAS or F-Met-Phe decreased all subsequent migratory responses. Preexposure of the leukocytes to lower doses of ZAS or F-Met-Phe decreased only a subsequent chemotactic response to the deactivating cytotaxin. These results suggest two mechanisms, or components, of chemotactic deactivation.