Expression of tumor-associated antigen (DF3) in atypical hyperplasias and in situ carcinomas of the human breast.

The monoclonal antibody (MoAb) DF3 prepared against a membrane-enriched fraction of human breast carcinoma has previously shown a differential reactivity to cytoplasmic antigen in carcinomas versus antigen concentrated on apical borders in benign lesions of the breast. In the present report the cytoplasmic reactivity of MoAb DF3 within a spectrum of benign and malignant breast lesions was studied to define whether the DF3 antigen is expressed in the cytoplasm of potentially premalignant lesions, i.e., atypical hyperplasias, or early malignant lesions, i.e., in situ carcinomas. Biopsy specimens of breast lesions from 108 women, including 28 patients with invasive carcinoma, 12 with in situ carcinoma, 17 with atypical hyperplasia, 25 with proliferative lesions without atypia, and 26 with nonproliferative lesions, were examined for DF3 antigen expression with the use of an indirect immunohistochemical method. Atypical hyperplasias were less reactive with MoAb DF3 than invasive carcinomas (P = .05 by Wilcoxon rank sum test). No significant statistical differences were observed, however, between invasive carcinomas and in situ carcinomas or between in situ carcinomas and atypical hyperplasias on the basis of cytoplasmic DF3 reactivity. Invasive carcinomas, in situ carcinomas, and atypical hyperplasias, however, demonstrated significantly higher reactivity with MoAb DF3 in the cytoplasm than proliferative lesions without atypia and nonproliferative lesions (P less than .01). These studies demonstrate that atypical hyperplasias express elevated levels of a given tumor-associated antigen and thus provide further immunologic evidence that these lesions are premalignant.

Evidence for a causal association between human papillomavirus and a subset of head and neck cancers.

BACKGROUND: High-risk human papillomaviruses (HPVs) are etiologic agents for anogenital tract cancers and have been detected in head and neck squamous cell carcinomas (HNSCCs). We investigated, retrospectively, an etiologic role for HPVs in a large series of patients with HNSCC. METHODS: Tumor tissues from 253 patients with newly diagnosed or recurrent HNSCC were tested for the presence of HPV genome by use of polymerase chain reaction (PCR)-based assays, Southern blot hybridization, and in situ hybridization. The viral E6 coding region was sequenced to confirm the presence of tumor-specific viral isolates. Exons 5-9 of the TP53 gene were sequenced from 166 specimens. The hazard of death from HNSCC in patients with and without HPV-positive tumors was determined by proportional hazards regression analysis. RESULTS: HPV was detected in 62 (25%) of 253 cases (95% confidence interval [CI] = 19%-30%). High-risk, tumorigenic type HPV16 was identified in 90% of the HPV-positive tumors. HPV16 was localized specifically by in situ hybridization within the nuclei of cancer cells in preinvasive, invasive, and lymph node disease. Southern blot hybridization patterns were consistent with viral integration. Poor tumor grade (odds ratio [OR] = 2.4; 95% CI = 1.2- 4.9) and oropharyngeal site (OR = 6.2; 95% CI = 3.1-12.1) independently increased the probability of HPV presence. As compared with HPV-negative oropharyngeal cancers, HPV-positive oropharyngeal cancers were less likely to occur among moderate to heavy drinkers (OR = 0.17; 95% CI = 0.05-0.61) and smokers (OR = 0.16; 95% CI = 0.02-1.4), had a characteristic basaloid morphology (OR = 18.7; 95% CI = 2.1-167), were less likely to have TP53 mutations (OR = 0.06; 95% CI = 0.01-0. 36), and had improved disease-specific survival (hazard ratio [HR] = 0.26; 95% CI = 0.07-0.98). After adjustment for the presence of lymph node disease (HR = 2.3; 95% CI = 1.4- 3.8), heavy alcohol consumption (HR = 2.6; 95% CI = 1.4-4.7), and age greater than 60 years old (HR = 1.4; 95% CI = 0.8-2.3), all patients with HPV-positive tumors had a 59% reduction in risk of death from cancer when compared with HPV-negative HNSCC patients (HR = 0.41; 95% CI = 0.20-0.88). CONCLUSIONS: These data extend recent molecular and epidemiologic studies and strongly suggest that HPV-positive oropharyngeal cancers comprise a distinct molecular, clinical, and pathologic disease entity that is likely causally associated with HPV infection and that has a markedly improved prognosis.

Voltage-dependent ion channels in small-cell lung cancer cells.

Small-cell carcinoma of the lung is a highly lethal form of cancer associated with a wide variety of paraneoplastic syndromes. Using the patch-clamp technique, we have directly demonstrated the presence of voltage-gated K+, Na+, and Ca2+ channels in three cell lines of human small-cell carcinoma, NCI-H128, NCI-H69, and NCI-H146. Whole-cell currents were measured from the tumor cells held at -80 mV and depolarized to -60 to +120 mV. Outward K+ current (IK), which was found in every cell tested, reached 1.58 +/- 0.12 nA (mean +/- SE, n = 24 cells) for H128 cells and 2.14 +/- 0.18 nA (n = 41) for H69 cells in response to a test potential of +80 mV. Unlike H69 and H128 tumor cells, IK from H146 cells occasionally exhibited partial inactivation during the 60-ms pulse length and reached 0.94 +/- 0.15 nA (n = 18) in response to a +80 mV test potential. IK from each of the cell lines was significantly reduced by 4-aminopyridine and tetraethylammonium. The rapidly inactivating inward Na+ current (INa), recorded in H146 cells and about 30% of the H69 and H128 cells tested, demonstrated a peak amplitude of 58 +/- 6 pA (n = 11) at 0 mV and a reversal potential of 47 +/- 2 mV (n = 11). Externally applied tetrodotoxin quickly suppressed INa. For the H128 and H69 tumor cells, inward Ca2+ current (ICa), observed in about 25% of the cells exposed to 10 mM [Ca2+]o, peaked at 5.1 +/- 0.4 ms (n = 5) with an amplitude of 46 +/- 14 pA (n = 5) at +20 mV and partially inactivated over the 40-ms depolarization. In H128 cells exposed to isotonic Ba2+ (110 mM), inward currents with time courses similar to those of ICa were recorded. Nearly all H146 tumor cells demonstrated a significant inward Ca2+ current which peaked with an amplitude of 93 +/- 16 pA (n = 26) at +30 to +40 mV in the presence of 10 mM [Ca2+]o. Application of test potentials 2 s in duration revealed that H146 ICa inactivated in a voltage-dependent manner with a time constant on the order of seconds. Adjustment of the holding potential from -80 mV to -40 mV had no observable effect on the amplitude of the evoked current. These voltage-dependent ion channels may have integral roles in several small-cell carcinoma bioelectric phenomena, including secretion, resting membrane potential, and action potential generation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjunct to the diagnostic distinction between adenocarcinomas of the ovary and the colon utilizing a monoclonal antibody (COL-4) with restricted carcinoembryonic antigen reactivity.

Malignant ovarian tumors may represent either primary ovarian cancers or metastatic lesions (from patients with demonstrated primary cancers at other body sites) whose distinction may be difficult using clinical, surgical, and pathological criteria. Monoclonal antibody (MAb) COL-4, reactive with carcinoembryonic antigen, has previously been shown to react preferentially with adenocarcinomas of the colon versus a variety of normal tissues. We report here that MAb COL-4 is strongly reactive with primary colonic carcinomas (N = 50), as well as regional (N = 42), and distant (N = 20) metastases of colonic adenocarcinoma. In contrast, MAb COL-4 demonstrated little to no reactivity with primary (N = 53) and metastatic carcinomas of the ovary (N = 23) including serous, mucinous, and poorly differentiated adenocarcinomas using immunohistochemical techniques. This differential reactivity was statistically significant (P less than 0.001), suggesting the potential clinical utility of MAb COL-4 in the differentiation of ovarian from colonic adenocarcinoma. Solid-phase quantitative radioimmunoassays and Western blotting techniques confirmed these results. Data are also presented that the carcinoembryonic antigen molecules or epitopes recognized by a more classical broadly reactive anti-carcinoembryonic antigen MAb are distinct from those recognized by MAb COL-4. Other carcinomas which also metastasize to the ovary and may be confused clinically with a primary ovarian tumor such as adenocarcinomas of the stomach and breast were also evaluated for reactivity with MAb COL-4. COL-4 was also reactive with all gastric carcinomas evaluated, but failed to react with breast carcinomas. Hence, COL-4 can now be utilized as an immunohistochemical adjunct for the differentiation of ovarian from gastrointestinal adenocarcinoma which can be difficult to distinguish by clinical, surgical, and histological parameters.

Effects of Bay K 8644 on spontaneous and evoked transmitter release at the mouse neuromuscular junction.

The dihydropyridine, Bay K 8644, was applied in vitro to mouse phrenic nerve-diaphragm muscle preparations. The drug increased both spontaneous and evoked release of acetylcholine from the motor nerve terminal in a concentration- and time-dependent manner. The rise in miniature endplate potential frequency, however, was the result of an increased intraterminal mobilization of free calcium, rather than well-established activation of voltage-dependent calcium channels. This view is supported by the following observations: (1) an increase in frequency was apparent in Ca2+-free medium; (2) Bay K 8644 is known to require a moderate depolarization to affect Ca2+ channels, but no membrane depolarization was detected; and (3) exposure to low Ca2+ and high Mg2+ medium did not diminish the effect on miniature endplate potential frequency. In a medium containing low Ca2+ and high Mg2+, Bay K 8644 increased quantal content of the evoked endplate potentials to a greater degree and with a faster time course than the frequency of miniature endplate potentials. This enhancement in evoked release did not appear to be caused solely by an increase in cytoplasmic Ca2+, but rather reflected at least in part the Bay K 8644-induced activation of voltage-gated Ca2+ channels, perhaps L-type, at the presynaptic nerve terminal. Thus, we propose that Bay K 8644 exerts dual effects on the motor nerve endings, characterized by a primary action on the presynaptic Ca2+ channels and a secondary action associated with the elevation of intracellular Ca2+ concentration.

A brachial plexopathy due to myositis ossificans. Case report and review of the literature.

Myositis ossificans (MO) is a disorder characterized by the intramuscular proliferation of fibroblasts and osteoblasts, with subsequent deposition of bone and cartilage. A typical clinical presentation involves traumatic injury to a young adult, usually localized to the thigh, buttock, or upper arm, with resultant MO and mildly restricted range of motion in adjacent joints. Rarely, MO is associated with peripheral neuropathies involving the radial, median, sciatic, and sural nerves. The authors present an unusual case of MO causing a brachial plexopathy. To their knowledge, this is the first description of such a presentation.

Prenatal counseling for cleft lip and palate.

Prenatal diagnoses of cleft lip and palate can occur during both routine screening obstetrical ultrasound and high resolution obstetrical ultrasound done for other reasons. The affected family or obstetrician may request prenatal consultation with the plastic surgeon. To define this population, a survey was done of all families who were referred to our cleft program with a prenatal diagnosis of cleft between 1990 and 1994. Of 80 newborn referrals, 13 had a prenatal diagnosis of cleft. These children had a higher incidence of bilateral cleft than our average population (53.8 percent versus 28.7 percent, p < 0.03, chi square test). No isolated cleft palates were identified. Nine families were available for follow-up. Only one-third of the families felt that they had been given adequate information about clefts from their obstetrician or ultrasonographer. All who had prenatal contact with the cleft team felt it was valuable. A review of prenatal diagnosis of cleft is given including limitations. Specific counseling information is discussed.

Comparative analysis of paracrine immunotherapy in experimental brain tumors.

OBJECT: Local delivery of cytokines has been shown to have a potent antitumor activity against a wide range of malignant brain tumors. In this study, the authors examined the efficacy of treating central nervous system (CNS) tumors by transfecting poorly immunogenic B16/F10 melanoma cells with interleukin (IL)-2, IL-4, or granulocytemacrophage-colony stimulating factor (GM-CSF) gene, and using these cells to deliver the cytokine locally at the site of the CNS tumor. The object was to determine which cytokine would possess the greatest antitumor activity and to further elucidate its mechanism of action. METHODS: The transfected B16/F10 cells were irradiated to prevent replication and injected intracranially into C57BL/6 mice (10 mice per group) along with nonirradiated, nontransfected B16/F10 (wild-type) melanoma cells. Sixty percent of mice treated with IL-2 (p < 0.001 compared with control) and 10% treated with IL-4 (median survival = 31 days, p < 0.001 compared with control) were long term survivors (> 120 days). The median survival for animals treated with GM-CSF was 22 days with no long term survivors (p = 0.01 compared with control). Control animals that received only wild-type cells had a median survival of 18 days (range 15-20 days). Histopathological examination of brains from animals killed at different times showed minimal infiltration of tumor cells in the IL-2 group, moderate infiltration of tumor cells in the IL-4 group, and gross tumor invasion and tissue necrosis in the GM-CSF group. Animals treated with IL-2 showed a strong CD8 T cell-mediated response, whereas IL-4 evoked a prominent eosinophilic infiltrate in the area of the tumor. CONCLUSIONS: High levels of locally expressed IL-2 rather than IL-4 or GM-CSF stimulate a strong immunological cytotoxic antitumor response that leads to significant prolongation of survival in mice challenged with B16/F10 intracranial melanoma tumor cells. Consequently, IL-2 may be a superior candidate for use in paracrine immunotherapy.

Lambert-Eaton syndrome: antigen-antibody interaction and calcium current inhibition in chromaffin cells.

Plasma and IgG obtained from 10 Lambert-Eaton myasthenic syndrome (LES) patients (5 with carcinoma, 5 without associated cancer), 6 healthy subjects, and 1 patient with small-cell lung cancer (SCLC) were examined in their ability to recognize chromaffin cell antigens on Western blots. The pattern of antigen recognition was compared with the magnitude of inhibition of voltage-dependent calcium and sodium currents recorded with the patch-clamp technique from chromaffin cells. Eight of the 11 patients with LES and/or SCLC recognized plasma membrane proteins and 9 of the patients' IgG interacted with cytoplasmic antigens with no apparent pattern of antigen recognition between patients. Also, there was no obvious band pattern distinguishing patients with LES from those with LES and concurrent SCLC. Eighty percent of the LES patients' antibodies were capable of reducing the calcium current (ICa) in chromaffin cells. One of the novel findings of this study is that 30% of the patients had produced antibodies which were able to inhibit both calcium and sodium currents (INa). The heterogeneous response of the IgG on the Western blots does not appear to correlate with the efficacy of reducing the inward currents.

Expression and antibody inhibition of P-type calcium channels in human small-cell lung carcinoma cells.

P-type channels, a recently described form of voltage-gated calcium channels, are found in many central and peripheral neurons. In the present study, a partial cDNA clone sharing extensive nucleotide identity with a putative P-type voltage-gated calcium channel alpha 1 subunit was isolated from a small-cell lung carcinoma (SCLC) cell line. Anti-peptide antibodies generated to a unique acidic stretch in the IVS5-S6 linker region of the putative SCLC P-type channel reacted specifically with a SCLC fusion protein produced in bacteria and with a cell surface molecule in SCLC cells. Calcium currents in SCLC cells, measured by whole-cell patch clamp, were inhibited by these antibodies and by the P-type channel-specific toxin omega-agatoxin IVA. The inhibitory effects of the antibody and the toxin were not additive, consistent with their proposed action on the same type of channel. These results provide evidence for the expression of P-type calcium channels by SCLC cells. The expression of neuron-related molecules by these cells is of particular interest because small-cell lung carcinoma is frequently associated with paraneoplastic disorders affecting the nervous system.

Inhibition of calcium currents and exocytosis by Lambert-Eaton syndrome antibodies in human lung cancer cells.

1. Human small-cell lung cancer (SCLC) cells are believed to express the antigens responsible for the production of pathological antibodies in the Lambert-Eaton syndrome (LES), a Ca2+ channel disorder in which quantal transmitter release from the motor nerve terminal is impaired. Whole-cell patch-clamp techniques were used to study the voltage-dependent Ca2+ channels expressed by H146 SCLC cells and the effects of LES antibodies on these channels. The types of Ca2+ channels were determined using biophysical properties and pharmacological sensitivity to several antagonists. 2. Whole-cell Ca2+ currents (ICa) in SCLC cells are sensitive to the dihydropyridine (DHP) nicardipine, omega-conotoxin GVIA (omega-CgTX GVIA) and omega-agatoxin IVA (omega-AgTX IVA). Nicardipine at 100 nM and 10 microM reduced ICa by 35 and 45% (n = 38 cells), respectively, while omega-CgTX GVIA (1 microM) inhibited ICa by 32% (n = 31). Application of omega-AgTX IVA at 50 and 100 nM to the cancer cells decreased ICa by 41 and 42%, respectively (n = 22). 3. Measurement of cell membrane capacitance (Cm) revealed that Ca(2+)-dependent exocytosis underlies the secretory activity of SCLC cells. Exocytosis, when induced by step depolarizing pulses and measured by increases in Cm, was markedly inhibited by nicardipine (10 microM) and omega-AgTX IVA (100 nM). In contrast, omega-CgTX GVIA (1 microM) was not as effective in altering increases in Cm. 4. From negative (-80 mV) and depolarized (-40 mV) holding potentials, both peak and plateau ICa were inhibited by the presence of LES antibodies (1 mg ml-1 IgG). LES serum also reduced depolarization-induced increases in Cm by 48% (n = 15). 5. To determine whether the LES antibodies are downregulating a specific type(s) of Ca2+ channel, nicardipine (10 microM), omega-CgTX GVIA (1 microM) or omega-AgTX IVA (100 nM) was applied to tumour cells that had been previously exposed to LES serum for 24 h. The most pronounced change was that omega-AgTX IVA was 38-84% less effective at reducing ICa after the IgG treatment. The effectiveness of nicardipine was diminished by 18% after incubation with the LES antibodies, whereas the omega-CgTX GVIA was seen to be more effective. These results suggest that LES IgG downregulates P-type Ca2+ channels and, possibly, to a lesser extent L-type channels. 6. In view of recent evidence that P-type Ca2+ channels mediate cholinergic transmitter release at the mammalian neuromuscular junction (NMJ), the expression of P-type Ca2+ channels in the SCLC cells and the reactivity of LES IgG with these channels support the hypothesis that P-type Ca2+ channels in these cancer cells may trigger the autoantibody production in this disorder. The antibodies so produced are implicated in the functional impairment of the Ca2+ channels characteristic of LES.

Verapamil-induced blockade of voltage-activated K+ current in small-cell lung cancer cells.

Verapamil, Ca++ channel antagonist, has proven clinically useful in the reversal of multiple drug resistance, which is a major detriment to chemotherapy. Recently, verapamil alone has been shown to diminish proliferation in a variety of neoplastic cell lines. Using the patch-clamp technique, the action of verapamil on voltage-gated K+ channels in two cell lines of human small-cell carcinoma of the lung, NCI-H146 and NCI-H82, was investigated. With inward Na+ current suppressed, virtually all control cells exhibited a slowly inactivating outward current that was insensitive to alterations in the external Ca++ concentration. Externally applied verapamil enhanced the rate and extent of outward K+ current (IK) inactivation. Verapamil at a concentration of 20 microM diminished peak IK, evoked by a test pulse to +60 mV from a holding potential of -80 mV, from 1.38 +/- 0.11 nA (mean +/- S.E.M., n = 29 cells) to 0.56 +/- 0.13 nA (n = 11) and caused IK to decay to less than 20% of the peak current within 60 msec. After blocking IK and Na+ current, Ca++ current (ICa) was measured in the presence of 10 mM Ca++. The addition of 100 microM verapamil to the external bath resulted in a 53% reduction of H146 ICa. Peak ICa fell from 81 +/- 9 pA (n = 22) to 38 +/- 8 pA (n = 12). Examination of the whole-cell K+ current on single cells before and immediately after the addition of 100 microM verapamil clearly revealed that the drug had no effect on the initial activation phase of IK, suggesting that K+ channels first open before interacting with the drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibody B72.3 reactivity with human endometrium: a study of normal and malignant tissues.

Monoclonal antibody (MAb) B72.3, reactive with a high molecular weight mucin-like glycoprotein [termed tumor-associated glycoprotein (TAG)-72], has been shown previously to react preferentially with adenocarcinomas of the ovary, breast, and colon versus a variety of normal human tissues using immunohistochemical and radioimmunoassay (RIA) techniques. We report here B72.3 reactivity with normal postovulatory endometria, in contrast to proliferative phase epithelia that were nonreactive. Both immunohistochemical and RIA techniques were used to evaluate this phenomenon. TAG-72 expression was also detected in 100% of endometrial adenocarcinomas (n = 32) examined. No MAb B72.3 reactivity was noted in resting, postmenopausal endometria; however, it was present in hyperplastic lesions and appeared to correlate with the severity of histologic abnormality. The utility of MAb B72.3 for screening, radiolocalization, or perhaps therapy of endometrial adenocarcinoma is also discussed.

Voltage-dependent sodium channels in human small-cell lung cancer cells: role in action potentials and inhibition by Lambert-Eaton syndrome IgG.

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na+ channels are only weakly tetrodotoxin (TTX)-sensitive (Kd = 215 nM). With the membrane potential maintained at -60 to -80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (> 20 pA). Similar all-or-nothing spikes were also observed with anodal break excitation. Removal of external Ca2+ did not affect the action potential production, whereas 5 microM TTX or substitution of Na+ with choline abolished it. Action potentials elicited in the Ca(2+)-free condition were reversibly blocked by 4 mM MnCl2 due to the Mn(2+)-induced inhibition of voltage-dependent sodium currents (INa). Therefore, Na+ channels, not Ca2+ channels, underlie the excitability of SCLC cells. Whole-cell INa was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na(+)-selective channel. INa evoked by depolarizing test potentials (-60 to +40 mV) exhibited a transient time course and activation/inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m infinity and h infinity, also revealed biophysical similarity between SCLC and neuronal Na+ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at -20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit ICa and INa in bovine adrenal chromaffin cells, also significantly inhibited INa in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na+ channel conductance; (ii) fundamental characteristics of SCLC Na+ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na+ channels are an additional target of the pathological IgG present in the patients' sera.

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.

Comparison of monoclonal antibodies for the detection of occult breast carcinoma metastases in bone marrow.

Twenty percent (n = 6) of Stage III or IV breast cancer patients (n = 30) had bone marrow metastases detected in bilateral bone marrow biopsy/aspiration preparations using standard histologic preparations. Each metastasis was also detected by four separate monoclonal antibodies (MAbs) which recognize breast carcinoma associated antigens (DF3, anti-EMA, HMFG-2, and CAM5.2). These MAbs were then utilized to stain other bone marrow preparations (n = 81) to determine their utility for the detection of micrometastatic breast carcinoma. MAbs HMFG-2, anti-EMA, and DF3 were each strongly reactive with bone marrows containing histologically-evident metastatic breast carcinoma (18/18). These anti-epithelial membrane antigen MAbs, however, were also reactive with rare plasma cells and immature cells (as well as cell clusters) in some of the control bone marrow samples tested, including those from normal patients and patients with hematologic disorders. They also reacted with some of the preparations from patients with leukemia and lymphoma, and with uninvolved marrows from patients with non-epithelial malignancies. The anti-keratin MAb CAM5.2, in contrast, reacted with 83% (15/18) breast cancer metastases and failed to stain any cells in the various categories of control marrow preparations. These data suggested that MAb CAM5.2 might be utilized to immunohistochemically differentiate micrometastatic breast carcinoma from immature myeloid or erythroid elements. Each MAb was then reacted with histologically uninvolved marrow preparations from the remaining 24 of 30 breast cancer patients in an attempt to identify occult breast carcinoma metastases. While MAbs HMFG-2, DF3, and anti-EMA demonstrated reactive cells in some of these marrows, this reactivity was similar to that seen with control preparations. MAb CAM5.2, in contrast, was negative with all specimens. These data suggest that MAb CAM5.2 may be a useful immunologic probe for the detection and confirmation of metastatic breast carcinoma in bone marrow, while more caution must be employed in the interpretation of results obtained using MAbs anti-EMA, DF3, and HMFG-2.

Lambert-Eaton syndrome antibodies inhibit acetylcholine release and P/Q-type Ca2+ channels in electric ray nerve endings.

1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.