A pilot study on potential plasma hypoxia markers in the radiotherapy of non-small cell lung cancer. Osteopontin, carbonic anhydrase IX and vascular endothelial growth factor.

BACKGROUND: Hypoxic radioresistance plays a critical role in the radiotherapy of cancer and adversely impacts prognosis and treatment response. This prospective study investigated the interrelationship and the prognostic significance of several hypoxia-related proteins in non-small cell lung cancer (NSCLC) patients treated by radiotherapy ± chemotherapy. MATERIAL AND METHODS: Pretreatment osteopontin (OPN), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CA IX) plasma levels were determined by ELISA in 55 NSCLC (M0) patients receiving 66 Gy curative-intent radiotherapy or chemoradiation. Marker correlation, association with clinicopathological parameters and the prognostic value of a biomarker combination was evaluated. RESULTS: All biomarkers were linearly correlated and linked to different clinical parameters including lung function, weight loss (OPN), gross tumor volume (VEGF) and T stage (CA IX). High OPN (p = 0.03), VEGF (p = 0.02) and CA IX (p = 0.04) values were significantly associated with poor survival. Double marker combination additively increased the risk of death by a factor of 2 and high plasma levels of the triple combination OPN/VEGF/CA IX yielded a 5.9-fold risk of death (p = 0.009). The combined assessment of OPN/VEGF/CA IX correlated independently with prognosis (p = 0.03) in a multivariate Cox regression model including N stage, T stage and GTV. CONCLUSION: This pilot study suggests that a co-detection augments the prognostic value of single markers and that the integration of OPN, VEGF and CA IX into a hypoxic biomarker profile for the identification of patients with largely hypoxic and radioresistant tumors should be further evaluated.

Co-detection of members of the urokinase plasminogen activator system in tumour tissue and serum correlates with a poor prognosis for soft-tissue sarcoma patients.

BACKGROUND: The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet. METHODS: We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA. RESULTS: A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients' prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination. CONCLUSION: As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

Are overexpressed alternative survivin transcripts in human bladder cancer suitable targets for siRNA-mediated in vitro inhibition?

In order to reduce side effects of survivin-inhibiting anticancer therapies, we determined the expression of the survivin transcripts survivin-wild-type (survivin-wt), survivin-DeltaEx3 (DeltaEx3) and survivin-2B (2B) in cryo-preserved tumor and non-malignant bladder tissues (18 tumor and 22 non-malignant samples, including 17 autologous tissue pairs) by quantitative PCR. Furthermore, we investigated the biological effects following specific inhibition of the alternative transcripts DeltaEx3 and 2B in bladder cancer (BCa) cells. In BCa and non-malignant bladder tissues survivin-wt was the quantitatively dominant transcript followed by DeltaEx3 and 2B. The mean mRNA expression of DeltaEx3 (0.37 vs. 0.06 zmol/amol GAPDH, respectively) and 2B (0.13 vs. 0.01 zmol/amol GAPDH, respectively) was significantly higher in BCa compared to non-malignant bladder tissues, indicating their accessibility for an expression inhibition in BCa cells. Effective and long-lasting small interfering RNA-mediated inhibition of one alternative survivin transcript caused lower cell growth reduction effects (apoptosis induction, cell cycle arrest, colony formation) compared to simultaneous inhibition of multiple survivin transcripts including survivin-wt. Inhibition of one alternative survivin transcript increased the apoptosis rate by 11% vs. 33-46% when reducing several survivin transcripts. We observed no G2/M arrest or reduction of cell colony formation after inhibiting one alternative survivin transcript. Reduction of cell viability by the chemotherapeutics cisplatin, mitomycin C or gemcitabine was stronger in combination with inhibition of several survivin transcripts than in combination with the reduction of one alternative survivin splice variant. Furthermore, reducing one alternative transcript caused chemosensitization to only one chemotherapeutic agent in contrast to inhibition of several survivin transcripts. Therefore, the alternative survivin transcripts DeltaEx3 and 2B do not represent reasonable targets for anticancer, at least BCa, treatment.

Constitutive tissue factor expression of human breast cancer cell line MCF-7 is modulated by growth factors.

Expression of tissue factor, the initiator of the extrinsic coagulation protease cascade, is a feature of certain malignant tumours. To study the modulation of tissue factor expression we incubated the breast cancer cell line MCF-7 with several growth factors. Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and interleukin-1 (IL-1) rapidly increased tissue factor expression of MCF-7 cells peaking at 6-8 h after starting point of incubation, as determined by clotting test, enzyme linked immunosorbent assay and flow cytometry. The data presented support the hypothesis that modulation of constitutive tissue factor expression in tumour cells by TGF alpha and IL-1 could also occur in vivo possibly resulting from interactions of stromal and cancer cells. The meaning for tumour biology, however, remains unclear.

Detection of circulating tissue factor and factor VII in a normal population.

The plasma tissue factor (TF) concentration was correlated to factor VII concentration (FVIIag) and factor VII activity (FVIIc) in 498 healthy volunteers ranging in age from 17 to 64 years. Immunoassays using monoclonal antibodies (mAbs) were developed for the determination of TF and FVIIag in plasma. The mAbs and the test systems were characterized. The mean value of the TF concentration was 172 +/- 135 pg/ml. TF showed no age- and gender-related differences. For the total population, FVIIc, determined by a clotting test, was 110 +/- 15% and the factor VIIag was 0.77 +/- 0.19 microgram/ml. FVII activity was significantly increased with age, whereas the concentration demonstrated no correlation to age in this population. FVII concentration is highly correlated with the activity as measured by clotting assay using rabbit thromboplastin. The ratio between FVIIc and FVIIag was not age-dependent, but demonstrated a significant difference between men and women. Between TF and FVII we could not detect a correlation.

A human lymphokine released by mononuclear blood cells upon contact with CEA.

As revealed by the macrophage electrophoretic mobility (MEM) technique mononuclear blood cells from certain cancer patients respond to carcinoembryonic antigen (CEA). This phenomenon appeared to be due to a specific lymphokine release. In this study, the lymphokine activity of supernatant pools was stepwise enriched by gel filtration on Sephadex. The mediator activity was recovered within a molecular mass region less than 47 kDA. The lymphokine was highly enriched during further gel filtration steps and showed a single activity peak in the molecular mass region of 23.5 kDa. Gel filtrations of appropriate control supernatants resulted in biologically inactive fractions. The lymphokine was heat-labile at 56 degrees C, showed a clear-cut, dose-dependent effect on macrophages, and could be blocked by fucose. Preparative gel electrophoresis of radiolabeled and unlabeled lymphokine resulted in two corresponding peaks of biological activity and radioactivity.

New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Interleukin-1beta-induced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in human articular chondrocytes.

The urokinase-type plasminogen activator receptor (uPAR) plays a critical role in cartilage degradation during osteoarthritis as it regulates pericellular proteolysis mediated by serine proteinases. Another important family of proteinases responsible for ECM destruction in arthritis are the matrix metalloproteinases (MMPs). MMPs are regulated by IL-1beta, a cytokine that plays a pivotal role in pathogenesis of osteoarthritis. This study was undertaken to address two questions: 1. Is uPAR-expression regulated by proinflammatory cytokines such as IL-1beta? 2. Does a functional co-localization exist between uPAR and MMPs? By immunohistochemical analysis we observed enhanced expression of uPAR on chondrocytes derived from osteoarthritic human cartilage compared to non-osteoarthritic controls. We found an IL-1beta-mediated expression of uPAR by immunoelectron microscopy. Western blot analysis demonstrated that IL-1beta-stimulated expression of uPAR on chondrocytes in vitro increased in a dose-dependent manner. Furthermore, we found a functional co-localization between uPAR and MMP-9 on IL-1beta-stimulated chondrocytes by means of a co-immunoprecipitation assay. Expression of uPAR in osteoarthritic cartilage but not in healthy cartilage suggests that uPAR plays a role in cartilage breakdown. We propose that uPAR-mediated effects e.g. pericellular proteolysis are one of other cytokine (IL-1beta)-mediated events that contribute to the pathogenesis of osteoarthritis. Furthermore, we found that MMPs and uPAR were part of the same cell surface complexes in chondrocytes. This finding underlines a functional interaction between MMPs and the serine proteinase system in the fine regulation of pericellular proteolysis. Interfering with uPAR signaling may present a novel target in arthritis therapy to prevent excessive proteolytic degradation.

Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides.

Plasminogen activator inhibitor type-1 is a key regulatory protein of the fibrinolytic system that is involved in a variety of physiological and pathophysiological processes. A panel of 14 monoclonal antibodies directed against plasminogen activator inhibitor type-1 was analyzed regarding epitope specificity on plasminogen activator inhibitor type-1. For this purpose, chimera consisting of plasminogen activator inhibitor type-1 and another plasminogen activator inhibitor, plasminogen activator inhibitor type-2, with different portions of the respective wild-type proteins, were generated and plasminogen activator inhibitor type-1-derived 20-mer and 10-mer linear peptides were synthesized. Nine of the 14 monoclonal antibodies recognized an epitope located in the region between amino acid 76-188 of plasminogen activator inhibitor type-1, which encompasses the binding sites for vitronectin, heparin, and part of the fibrin binding region. Of these nine monoclonal antibodies, six reacted with a quadruple plasminogen activator inhibitor type-1 mutant (N152H, K156T, Q321L, M356I), and seven detected a plasminogen activator inhibitor type-1 deletion mutant (DeltaF111-H114). Two of the remaining five monoclonal antibodies recognized epitopes located between amino acid 209-227 and amino acid 352-371, respectively, while the other three antibodies reacted with wild-type plasminogen activator inhibitor type-1, only. Additional experiments revealed that two of the 14 mAbs neutralized and one monoclonal antibodies increased plasminogen activator inhibitor type-1 activity toward urokinase-type plasminogen activator, one of its target proteases.

Tissue factor antigen is elevated in patients with microvascular complications of diabetes mellitus.

Patients with late diabetic complications have increased levels of parameters indicating activation of coagulation (Takakashi et al., 1989; Ceriello, 1993; Murakami et al., 1993; Kario et al., 1995; Shimizu et al., 1995; Yokoyama et al., 1996), endothelial cell damage (Jensen, 1989; Iwashima et al., 1990; Sernau et al., 1995; Gabath et al., 1996). TF is believed to activate the coagulation mechanism in patients with late complications of diabetes. We studied the TF antigen plasma levels in 72 patients with diabetes mellitus (36 type I, 36 type II) with respect to its relevance as a marker of microvascular diabetic complications. TF levels did not correlate with macrovascular disease, diabetes type or age. Sixty patients with decreased renal function not due to diabetes were studied for evaluation of the contribution of renal failure to TF antigen plasma levels. We did not find a significant correlation of TF with s-creatinine in non diabetic patients (r = 0.27, p > 0.05). However, TF levels were elevated in diabetic patients with microvascular disease. Patients with retinopathy had higher TF levels than without (0.30 ng/ml vs 0.11 ng/ml, p < 0.007). When patients were divided into subgroups according to the urine albumin concentration, TF antigen of patients without albuminuria (0.019 ng/ml, n = 25) did not differ from patients with microalbuminuria (0.095 ng/ml, n = 19 p > 0.05). However, TF levels were significantly higher in patients with macroalbuminuria (n = 28; 0.215 ng/ml, p < 0.005). Thus activation of coagulation in patients with microvascular complications of diabetes may be triggered by tissue factor.

Cathepsin B, plasminogenactivator-inhibitor (PAI-1) and plasminogenactivator-receptor (uPAR) are prognostic factors for patients with non-small cell lung cancer.

To evaluate the possible role of cysteine proteases and serine proteases, as well as their respective inhibitors and receptors, as new prognostic factors in NSCLC, we examined, for the first time, 10 biological parameters related to three proteolytic systems within a homogeneous collective of 147 cases of NSCLC. Activities (cath B(AT), cath B(A7.5)) and protein levels of cath B(C), cath L(C), uPA, PAI-1, uPAR [measured by three different assays uPAR (ADI), uPAR (HD13), uPAR (IIIF10)] and TF were measured in homogenates of lung tumour tissue and corresponding non-malignant lung parenchyma. Total cath B activity (cath B(AT)) and enzymatic activity of the fraction of cath B, which is stable and active at pH 7.5 (cath B(A7.5)), were determined by a fluorogenic assay using synthetic substrate Z-Arg-Arg-AMC. The concentrations of cath B(C), cath L(C), uPA, PAI-1, uPAR and TF were determined by ELISAs. uPAR was determined using three different ELISA formats. The median levels of cath B(AT) (5.1-fold), cath B(A7.5) (2.5-fold), cath B(C), (8.5-fold), cath L(C) (6.6-fold), uPA (6.5-fold), PAI-1 (4.2-fold), uPAR (ADI) (2.2-fold), uPAR (HD13) (4.0-fold) and uPAR (IIIF10) (2.6-fold) were higher in tumour tissue compared to the lung parenchyma. Cath B(AT), cath B(A7.5) and cath B(C) in primary tumours correlated with lymph node metastases. Regarding histologies, the concentration of PAI-1 seems to be associated with the histological cell types of NSCLC. We found the highest values of PAI-1 in large cell carcinoma > SCC, AC > carcinoid and lowest values in metastases of primary tumours of other organs. Only PAI-1 was significantly increased in poorly-differentiated cells (G3) compared to well- and moderately- differentiated cells (G1/G2). PAI-1 significantly correlated with cath B(AT) and cath B(A7.5) with uPAR (ADI), uPAR (HD13), uPAR (IIIF10) with uPA, and only weakly with TF, but not with cath B(C) and cath L(C). Significant correlations with overall survival in the total population of NSCLC patients were observed in univariate analysis for cath B(AT), cath B(C), PAI-1, uPAR (ADI), uPAR (HD13), and uPAR (IIIF10). Cath L(C) was not significantly associated with poor prognosis. Regarding the histological tumour type, only in patients with squamous cell carcinomas did cath B(A7.5) and PAI-1 remain significant prognostic factors. In multivariate survival analysis only two proteolytic factors, PAI-1 and uPAR (III101F), stayed significant. In conclusion, among 10 biological parameters evaluated within the same cohort of patients, only PAI-1, uPAR (ADI), uPAR (HD13), uPAR (IIIF10), cath B(AT) and cath B(C) are prognostic factors for overall survival of NSCLC patients. Moreover, PAI-1 and uPAR (IIIF10) add independent prognostic information with regard to established clinical and histomorphological factors in NSCLC.

An ELISA for tissue factor using monoclonal antibodies.

Whereas tissue factor, a high-affinity cell-surface receptor and essential cofactor for the serine protease factor VII is constitutively present in certain tissues such as epithelial tissue, brain and placenta, it is not normally expressed by cells within the vasculature. However, the stimulation of monocytes and endothelial cells by a variety of inflammatory and immunological reactions results in the induction of cell surface tissue factor (TF) expression. TF is also expressed on tumour cells, and may play a role in tumour growth and metastasis formation. To examine the role of TF in these processes we developed monoclonal antibodies to human tissue factor apoprotein. The antibodies were characterized by neutralization of the procoagulant activity and by immunoblotting. With two of these monoclonal antibodies a sandwich ELISA was developed for the rapid quantitation of TF. The sensitivity of the assay permits extensive studies involving the modulation of TF expression on small numbers of cells. The results are comparable to the functional clotting assay as evaluated with unpurified TF and with the tumour cell line MCF-7. For certain applications, monitoring of cellular TF expression by ELISA using anti-TF monoclonal antibodies is preferable because it is not influenced by other coagulation factors or by inhibitors of procoagulant activity on the cells.

Analysis of carcinoembryonic antigen determinants by monoclonal antibodies and lectins.

Two out of five determinants recognized by monoclonal antibodies (MoAbs) on carcinoembryonic antigen (CEA) were analyzed by blocking the CEA-MoAb interaction with 9 different lectins in a solid phase radioimmunoassay. Two batches of CEA were first studied for their binding to lectins at the solid phase. Lotus, soybean and wheat germ lectins were most active, but the reaction pattern was different with the two CEA batches. The corresponding sugars were capable of inhibiting the lectin-CEA binding. MoAbs were fixed to the solid phase, and their reaction with CEA was tried to be blocked by preincubating the antigen with lectins. Lotus lectin and wheat germ agglutinin were clearly blocking the MoAb-CEA interaction, but this was strikingly dependent on the CEA batch used and the particular CEA determinant investigated. These reactions could not be blocked by sugars. The data provide evidence for a role of sugars in the sterical configuration of CEA determinants. In view of the heterogeneity of the findings, however, the conclusion is drawn that the protein moiety plays the more important role in the configuration of the CEA determinants studied.

Studies on lymphokine activity in concanavalin A-stimulated lymphocyte supernatants using the macrophage-electrophoretic-mobility-test (MEM-test).

Blood lymphocytes from guinea pigs and human donors were incubated with varying amounts of soluble Con A or, in the human system, with Sepharose-bound Con A for 60 min and 24 h at 310 K. The cellfree lymphocyte supernatants were incubated with guinea pig peritoneal macrophages which were measured in a cell electrophoresis apparatus "Parmoquant II" to detect lymphokine activity. Differences were found in the slowing capacity of short-term (60 min) and long term (24 h) incubations. In the latter mobility reduction was higher at comparable Con A concentrations. Greatest macrophage slowing was found at Con A concentrations of 5--10 micrograms Con A/10(6) lymphocytes in the guinea pig system and of 40--50 micrograms Con A/10(6) lymphocytes in the human system. But there was no great difference in the heights of the macrophage slowing at the optimal Con A concentration indicating no species restriction for human lymphokine(s) in our experiments. Stimulation of lymphocytes seems to be possible with Sepharose-bound Con A during a long-term incubation as tested in the human system.

[Studies on the immunologic specificity of the macrophage-electrophoresis mobility test in mice with syngeneic and allogeneic transplants of mammary carcinoma]. / Untersuchungen zur immunologischen Spezifität des Makrophagen-Elektrophorese-Mobilitätstests bei Mäusen mit syngenen und allogenen Mammakarzinom-Transplantaten

The study was performed to answer the question as to whether the macrophage electrophoretic mobility test (MEM-test) is suitable to detect allogeneic and syngeneic lymphocyte sensitization in the murine mammary tumour host system. Lymph node cells of both CBAf and XVII/Bln mice injected with allogeneic or syngeneic mammary tumour cells reacted specifically to hypertonic KCl extracts from allogeneic or syngeneic mammary tumours. Lymph node cells of nontreated control mice did not react at all. The MEM results reflect lymphocyte sensitizations which are specific with respect to the claim of transplantation and tumour immunology in the system employed.

[Discrimination of nonspecific effects in the macrophage-electrophoretic-mobility test using dimethyl sulfoxide]. / Untersuchungen zur Ausschaltung unspezifischer Effekte im Makrophagen-Elektrophorese-Mobilitätstest durch Dimethylsulfoxid.

Upon incubation with human encephalitogenic protein (HEP) blood lymphocytes from patients with malignant tumours release mediators (lymphokines) leading to a decreased electrophoretic mobility of guinea pig peritoneal macrophages. The lymphocyte supernatants used for incubating the macrophages contain HEP, nonspecific lymphocyte-derived proteins, and in case of sensitized lymphocytes also specific mediators. Whereas HEP or nonspecific lymphocyte products do not themselves exert any effect on macrophages, they produce a nonspecific mobility reduction when acting simultaneously. In the presence of 2.4% (v/v) dimethylsulfoxide (DMSO) this nonspecific effect is prevented. The specific lymphokine action, however, remains stable in the presence of DMSO. It cannot be decided whether DMSO exerts its effect via the membrane of macrophages or/and by influencing the interactions of proteins in the soluble phase.

Human lymphocyte response to CEA: titration of different CEA samples and neutralization experiments with monoclonal anti-CEA antibodies.

By means of the macrophage electrophoretic mobility technique a striking digestive system cancer-associated human lymphocyte response to CEA has been found during a large-scale study including tests in 499 individuals. The question to be answered by this study was whether this response is really CEA-specific. Titration experiments with 3 different CEA preparations in lymphocytes from 5 colorectal cancer patients showed that the threshold dose of CEA necessary to induce lymphocyte responses amounts to 50-100 ng CEA per ml and 10(6) lymphocytes, regardless of the CEA origin and its state of purity. The CEA specificity of the responses was proved by neutralization experiments with 3 CEA-specific monoclonal antibodies. When allowed to react with CEA before lymphocyte incubation, the MABs prevented CEA from inducing lymphocyte responses. Appropriate murine control myeloma protein did not influence these responses. The reactivity of these lymphocyte samples to a teratocarcinoma extract could not be prevented by treating this material with CEA-specific MABs before incubation. Preliminary attempts to enrich the lymphokine(s) released after CEA stimulation resulted in recovery of the activity within 2 arbitrarily cut Sephadex G-100 fractions comprising the mol. wt range of 3000-47,000.

Sensitization of human lymphocytes to carcinoembryonic antigen (CEA): neutralization experiments with anti-CEA sera.

By means of the macrophage electrophoretic mobility technique we could show lymphocytes of patients suffering from cancers of the digestive system to be sensitized to carcinoembryonic antigen (CEA). These findings conflict with the common view that CEA is not immunogenic in humans. The aim of the present study was to look as to whether conventional anti-CEA sera can neutralize the activity of a CEA preparation which is responsible for the human lymphocyte response. When 60 ng CEA were preincubated with highly diluted anti-CEA serum and the resulting immune complexes were thereafter co-precipitated by protein A-sepharose, positive lymphocyte responses could no longer be obtained. This effect was observed with 3 anti-CEA sera in 3 cancer patients (colon cancer, stomach cancer, teratocarcinoma), who's lymphocytes responded to CEA by lymphokine release. Normal serum had no neutralizing effect. The anti-CEA sera did not influence the activity of another tumour-relevant extract (teratocarcinoma-derived), to which cancer patients' lymphocytes reacted regardless of the tumour site. The lymphocytes from an oesophagus carcinoma patient, though reacting to the teratocarcinoma preparation, did not respond to CEA, thus, logically, all other tests with normal serum and anti-CEA sera were negative, too. The results show that the digest system cancer-associated lymphocyte reactivity to CEA can be abrogated by conventional anti-CEA sera, which finding indicates that there exist closely CEA-associated "tumour-specific" antigenicities.