Urokinase system expression in gastric carcinoma: prognostic impact in an independent patient series and first evidence of predictive value in preoperative biopsy and intestinal metaplasia specimens.

BACKGROUND: The prognostic relevance of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR), and plasminogen activator inhibitor 1 (PAI-1) in gastric carcinoma was demonstrated in an independent patient series. To the authors' knowledge,the roles of these activators as predictors of aggressive phenotypes in preoperative biopsies, Helicobacter pylori infection, and intestinal metaplasia have to date not been investigated simultaneously in resected tumors. The objectives of the current study were 1) to demonstrate the prognostic relevance of u-PA, u-PAR, and PAI-1 in an independent series; 2) to evaluate u-PA system expression in preoperative biopsy specimens compared with resected tumors; and 3) to evaluate u-PA system expression in intestinal metaplasias and samples with H. pylori infection. METHODS: In 104 patients with gastric carcinoma (median follow-up, 68 mos), u-PA, u-PAR, and PAI-1 in tumors and metaplasias were evaluated immunohistochemically. Preoperative biopsies were evaluated in a subset of patients. Patients were screened for H. pylori (urease) and tumor cells in bone marrow (u-PAR/CK18). RESULTS: u-PA and PAI-1 were confirmed as independent prognostic parameters, and u-PAR was associated with a trend toward a poor prognosis. u-PA system tumor expression was found to be correlated significantly with u-PAR in disseminated tumor cells and H. pylori-infected tumors, implicating a role of H. pylori in protease induction. There was a significant correlation noted between u-PA system staining between preoperative biopsies and the results in resected tumors. The expression of u-PAR and PAI-1 in intestinal metaplasias was found to be associated significantly with advanced tumor stage (depth of invasion; pathologic tumor status) and lymph node involvement (pathologic lymph node status) and was correlated significantly with u-PA system expression in tumors. CONCLUSIONS: To the author's know the current study is the first to date to demonstrate that u-PA system expression may serve as a predictor of risk in intestinal metaplasias and preoperative biopsies, implicating consequences for neoadjuvant therapy. The independent impact on recurrence and survival and a correlation with u-PAR-expression of minimal residual disease were identified in this independent series.

Immunotherapy of malignant ascites with trifunctional antibodies.

A new class of intact bispecific antibodies shows unmet effector qualities by activation of not only T cells but also simultaneous activation of Fcgamma receptor type I/III+ cells (macrophages, NK-cells and DC). These trifunctional antibodies (trAb) lead to efficient specific killing of targeted tumor cells without any pre- or co-stimulation. This concept was investigated in vivo in patients with malignant ascites in a clinical situation that allowed monitoring of tumor cell elimination and correlation with clinical effects. In a prospective study, 8 patients with malignant ascites due to peritoneal carcinomatosis were treated with intraperitoneal application of trAb, which bound either the EpCAM- or Her2/neu-antigen on tumor cells. Treatment consisted of 4-6 applications within 9-23 days with a total amount of 145-940 microg. Seven of eight patients required no further paracentesis during follow-up or until death with a mean paracentesis-free interval of 38 weeks (median = 21.5, range = 4-136). Tumor cell monitoring showed a complete elimination of tumor cells in ascites already at total doses as low as 40-140 microg. Clinical response with disappearance of ascites accumulation was seen in all patients, which was correlated with elimination of tumor cells (p = 0.0014). Severe adverse events were not observed. Clinically relevant side effects were fever, moderate abdominal pain and skin reactions. Intraperitoneal immunotherapy with trAb showed convincing efficacy in patients with malignant ascites. This treatment offers new therapeutic options for patients with peritoneal carcinomatosis.

Reduced survival of rectal cancer patients with increased tumor epidermal growth factor receptor levels.

PURPOSE: The epidermal growth factor receptor and its various ligands (epidermal growth factor, transforming growth factor-alpha, amphiregulin, heparin-binding epidermal growth factor, heregulin, and betacellulin) have been implicated in growth and regeneration of intestinal mucosa and might be related to the development and progression of gastrointestinal tumors. Although some studies have investigated levels of epidermal growth factor receptor by radioligand binding studies, none of them have further analyzed these levels in patients with rectal cancer and investigated their prognostic value. METHODS: We quantitatively determined tumor epidermal growth factor receptor levels in 38 patients with colorectal cancer compared with adjacent normal mucosa by iodine-125-labeled epidermal growth factor binding studies and Scatchard analysis. Patients were followed up for 49.5 +/- 32.2 (range, 2-120) months. RESULTS: Epidermal growth factor receptor capacity was increased in invasive colorectal carcinomas according to T classification (P < 0.001), tumors with lymph node infiltration (P = 0.038), and advanced International Union Against Cancer stage (P < 0.001). Survival of colorectal cancer was reduced in patients with advanced International Union Against Cancer stage (P < 0.001), tumors with positive lymph nodes (P < 0.001), and tumors with elevated epidermal growth factor receptor levels (P = 0.024). In rectal cancer patients, poor prognosis was associated with advanced International Union Against Cancer stage (P = 0.029), tumors with lymph node infiltration (P = 0.040), and increased epidermal growth factor receptor levels (P = 0.002). Multivariate Cox regression analysis indicated that elevated levels of epidermal growth factor receptor were an independent predictor of reduced survival in patients with rectal cancer (P = 0.005). CONCLUSION: The epidermal growth factor receptor/ligand system appears to be involved in tumor development and tumor progression of colorectal carcinomas, with prognostic implication especially in patients with invasive rectal carcinomas. These patients might take advantage of therapies that specifically block epidermal growth factor receptor-mediated signal transduction.

Detrimental immunologic effects of preoperative chemoradiotherapy in advanced rectal cancer.

PURPOSE: Preoperative chemoradiotherapy for advanced rectal cancer has been an important therapeutic tool to improve the long-term results of curative resection. It is not known whether preoperative chemoradiotherapy for advanced rectal cancer influences the perioperative course of immune parameters. METHODS: Thirty patients with rectal cancer underwent surgery with (study group, n = 15) or without (control group, n = 15) preoperative chemoradiotherapy (2 cycles of 5-fluorouracil, 45 Gy). Blood samples were taken before neoadjuvant therapy, preoperatively, and on Days 1, 2, and 5 after surgery. Cell numbers of lymphocyte subpopulations, granulocytes, monocytes, and natural killer cells were determined by flow cytometry; tumor necrosis factor-alpha and interleukin-6 serum levels were measured with enzyme-linked immunosorbent assay. RESULTS: Significant differences between study and control patients (P < 0.05) were detected regarding circulating interleukin-6 and tumor necrosis factor-alpha levels, with depression of the proinflammatory response to surgery in study patients. Similarly, granulocytosis and monocytosis after surgery were significantly lower in patients after neoadjuvant therapy. Furthermore, cell counts of total T lymphocytes, T helper cells, B lymphocytes, and natural killer cells were significantly reduced after preoperative chemoradiotherapy. This depression of cell-mediated immunity in study patients was even more pronounced after surgery. CONCLUSIONS: Preoperative chemoradiotherapy for advanced rectal cancer results in a significant preoperative and postoperative immune dysfunction as indicated by depression of lymphocyte subpopulations, monocytes, granulocytes, and proinflammatory cytokine release. These findings are of importance because increased perioperative morbidity and mortality rates have been observed after preoperative chemoradiotherapy.

Clinical implications of the EGF receptor/ligand system for tumor progression and survival in gastrointestinal carcinomas: evidence for new therapeutic options.

The epidermal growth factor (EGF) receptor and its various ligands (EGF, TGF-alpha, amphiregulin, heparin-binding (HB)-EGF, heregulin, betacellulin) seem to be involved in the growth regulation of intestinal mucosa and might be related to the development and progression of gastrointestinal tumors. However, few quantitative data investigating the impact of tumor-EGF receptor levels in gastrointestinal carcinomas on tumor stage and prognosis are available. Therefore, EGF receptors were quantitatively determined in colorectal carcinomas in comparison to adjacent normal mucosa by 125I[EGF]-binding studies. EGFR capacity was increased in advanced invasive colorectal carcinomas (T1/2 vs. T3/4 tumors, p<0.001) and advanced UICC stages (UICC I vs. UICC II/III, p<0.001). These findings were confirmed with quantitative 125[I]EGF autoradiography performed on frozen tissue slides and analyzed by laser densitometry (p=0.020). EGF receptor analysis with immunohistochemistry with EGFR antibodies directed against the extracellular domain of the receptor was not correlated with tumor invasion or prognosis. mRNA-expression of EGFR ligands was investigated using semiquantitative RT-PCR amplification using specific primers. RT-PCR transcripts of EGFR ligands (EGF, TGF-alpha, HB-EGF, and amphiregulin) were detected in both carcinomas and normal mucosa, indicating that autocrine growth stimulation of colorectal carcinomas is mediated by coexpression of EGF receptor ligands and upregulation of EGF receptors. Survival of colorectal cancer patients with increased tumor EGF receptor levels was significantly reduced in comparison to patients with low/unchanged tumor EGF receptor levels (mean survival+/-SD, 36.2+/-4.0 vs. 46.8+/-4.3 months; p=0.017). Further studies investigating EGF receptor levels in gastric cancer patients have shown that increased tumor EGF receptor levels were associated with poor prognosis in gastric cancer patients with tumors localized distal from the cardia. Several specific EGF receptor tyrosine kinase inhibitors have recently entered clinical phase I-III studies, with promising antitumor effects in several tumors, including gastrointestinal cancer. Therefore, patients with invasive gastric or colorectal carcinomas might benefit from therapies specifically blocking EGFR-mediated signal transduction.

Estrogen receptor expression profile of disseminated epithelial tumor cells in bone marrow of breast cancer patients.

The estrogen receptor (ER) status in primary breast cancer represents an important prognostic factor and has a profound impact on therapeutic decisions. However, ER expression profile on disseminated breast cancer cells is largely unknown, although these cells are one of the main target structures in adjuvant therapy after local curative resection (R0) achieved in most breast cancer patients. Thus, the present pilot study was designed to evaluate the ER expression profile on disseminated epithelial cells in bone marrow, one of the preferential organs for manifestation of distant metastases in breast cancer. Using the alkaline phosphatase anti-alkaline phosphatase-immunogold double staining procedure, in a panel of 17 breast cancer patients, epithelial cells (mab CK2) detected in bone marrow were analyzed for ER expression (mab 1D5) and compared with ER expression in the corresponding primary tumors. Whereas eleven of the 17 patients (64.7%) were ER-positive in primary carcinomas, only two patients (11.8%) revealed ER-positive epithelial cells in bone marrow. In addition, one of these two patients demonstrated a heterogeneous ER expression pattern, with both ER-positive and ER-negative epithelial cells in bone marrow. Although in both of these cases the ER-positive epithelial cells in bone marrow derived from ER-positive primary tumors, in this small patient cohort none of the prognostic relevant clinical and pathological factors tested, i.e., TNM-classification, grading, and ER status in primary breast cancer, correlated with the ER status in bone marrow. The striking discrepancy between ER expression in primary breast cancers and the corresponding disseminated epithelial cells in bone marrow suggests either the selective dissemination of ER-negative tumor cells into the bone marrow or a negative impact of the bone marrow microenvironment on epithelial ER expression. This phenomenon might influence therapeutic effects of antihormonal treatment.

Different immune responses to abdominal surgery in men and women.

BACKGROUND: Animal experiments reveal significant gender differences in the immunological response to surgical trauma. This raises the possibility that gender differences may also exist in patients after major abdominal surgery. PATIENTS AND METHODS: This prospective study included 40 patients (20 men, 20 women) with colorectal diseases requiring surgical intervention. To evaluate the immune response to surgery circulating lymphocyte populations and natural killer cells were determined by flow-cytometry, and IL-6 serum levels were measured by enzyme-linked immunosorbent assay. Blood samples were taken before and on days 1, 2, and 5 after surgery. RESULTS: Despite comparable preoperative cell counts we detected significant postoperative gender differences regarding B-lymphocyte, T-lymphocyte, T-helper cell counts, and NK cell counts. While only a short, insignificant depression of these immune competent cells was detected in women, men suffered long-lasting (5 days) depression of these cells. Furthermore, women showed a more pronounced immediate (day 1) proinflammatory response (circulating IL-6) after abdominal surgery. CONCLUSIONS: Significant immunological gender differences following major abdominal surgery were observed in this prospective clinical study. Our findings support the experimental observations of better posttraumatic immune competence in women than in men. These gender differences may be of relevance for short- and long-term results after surgery for colorectal diseases. Future studies will address the use of sex-steroids and/or their antagonists as a therapeutic option for the improvement in perioperative immune dysfunction in patients with major surgery.

Induction of cytotoxicity against autologous tumour cells by interleukin-12: evidence for intrinsic anti-tumor immune capacity in curatively resected gastrointestinal tumour patients.

The aim of this study was to investigate the cell-mediated immune response in 14 patients undergoing curative resection for a gastrointestinal tumor by the induction of peripheral blood mononuclear cell (PBMC)-mediated immune activity against autologous tumour cells. PBMC were stimulated by interleukin-12 (IL-12; 100 IU/ml) and IL-2 (1,000 IU/ml) without contact with tumour cells for 36 h. Specific cytotoxic activity against autologous tumour cells (auTu), natural killer (NK)-sensitive cells (K562) and allogeneic tumour cells (RF48/HT29) was determined by fluorescence cytotoxicity assay. Additionally, inhibition experiments using the mononuclear antibodies (mAb) FMC16 and W6/32 against major histocompatibility complex I (MHC I) on autologous tumour cells were performed in order to determine the involvement of specific T lymphocytes. The cytotoxic activity of unstimulated PBMC did not differ between the three target cells. IL-12 caused a 3.2-fold increase in activity against auTu ( P=0.002). In contrast, after stimulation with IL-2, only a slight increase in activity was observed. After IL-12 stimulation, cytotoxic activity against auTu was 2.5- to 2.7-fold higher than the corresponding activity against K562/allogeneic tumour cells ( P=0.002/ P=0.006). After blocking of the MHC I complex on auTu by FMC 16 or W6/32 mAb, a 62.9%/74.4% reduction in the specific cytotoxicity of IL-12-stimulated PBMC was found. In summary, IL-12 induced an effective immune response against auTu, which was partly mediated by specific cytotoxic T lymphocytes (CTL). It was considered that de novo generation of this activity during 36 h incubation without antigen contact was hardly possible, but that the observed induction of effective anti-tumor cytotoxicity was rather based on the re-activation of a pre-existing immune potential from the tumour-host interaction. These findings indicate the existence of an autologous anti-tumor immune response following curative resection in patients undergoing surgery for solid tumours, which might influence the development of tumour recurrence from disseminated tumour cells. Making use of this capacity could constitute an attractive immunotherapeutical approach for curatively operated tumour patients.

Chlamydia pneumoniae in atherosclerotic lesions of patients undergoing vascular surgery.

The pathophysiological implications of Chlamydia pneumoniae in atherosclerotic lesions and its contribution to atherosclerotic complications remain unclear. Therefore, the purpose of the present study was to evaluate whether or not there is an association between the presence of Chlamydia pneumoniae in atherosclerotic lesions and the clinical manifestations as well as risk factors of atherosclerotic disease in patients undergoing vascular surgery. Specimens from atherosclerotic arteries were collected during endarterectomy of the carotid artery (n = 15), endarterectomy of the femoral artery (n = 19), or repair of an abdominal aortic aneurysm (n = 28). Detection of Chlamydia pneumoniae was performed by using immunohistochemical staining (IHC) with specific antibodies. Clinical manifestations of atherosclerotic disease were defined by the presence of cardiovascular risk factors, coronary heart disease, and previous vascular surgery. Inflammatory serum markers were determined in all patients prior to surgery. The specimens of all 62 patients revealed severe atherosclerosis in histological examination and a positive IHC was observed in 41 samples (66%). There were no differences regarding cardiovascular risk factors, coronary heart disease, events of previous vascular surgery, or inflammatory serum markers when comparing patients with positive and negative IHC. In conclusion, our findings showed no correlation between clinical or laboratory parameters of atherosclerosis and the presence of Chlamydia pneumoniae in atherosclerotic lesions. Therefore, Chlamydia pneumoniae appears to be a concomitant phenomenon rather than a causative principle in atherosclerosis.

Results of long-term follow-up after curative resection of Dukes A colorectal cancer.

Patients with Dukes A (UICC I) colorectal cancer have a good prognosis after curative resection. It is not known, however, if the outcome is significantly different for UICC Ia and Ib patients or if patients with reduced risks of recurrences can be identified early after surgery. This is of interest, as it would permit a more cost-effective, patient-oriented, and tumor stage-oriented follow-up program. To study these questions, a prospective follow-up database, including 1375 patients after curative resection of colorectal cancer, was analyzed. A total of 296 patients with Dukes A colorectal cancer with a median follow-up of 44 months were studied. Perioperative and follow-up mortality rates were 3% and 14%, respectively. Recurrent disease developed in 10% of Dukes A patients after a disease-free interval of 16 months. Significantly more patients suffering from pT2 (UICC Ib) cancer had recurrent disease than patients with pT1 (UICC Ia) cancer (13% vs. 4%; p <0.05). Preoperative CEA levels in patients with recurrent disease were significantly higher than in long-term disease-free patients (5.3 +/- 1.8 vs. 3.5 +/- 0.6 ng/ml; p <0.05). Curative resection of recurrent disease was achieved in 38% of the patients with recurrences (4% of all patients). Survival analysis showed significantly better survival in patients with Dukes A cancer than in those at higher tumor stages (log rank, <0.0001), and only 39% of all Dukes A patients who died during follow-up had recurrent disease. Dukes A (UICC Ia and Ib) colorectal cancer was diagnosed in 22% of our patients treated for cure, and long-term survival was 86%. There were significantly fewer cases of recurrent disease after curative resection of UICC Ia (pT1N0M0) cancer, so we propose a novel, less intensive follow-up regimen for these patients, leading to a more cost-effective, patient-oriented, and tumor stage-oriented follow-up program.

Long-term results after pulmonary resection of renal cell carcinoma metastases.

BACKGROUND: Until now no conclusive data exist regarding the factors influencing long-term survival after pulmonary resection of renal cell carcinoma metastases. The aim of the present study, therefore, was to discover definitive prognostic factors for survival using a large and homogeneous single center patient cohort. METHODS: Between 1980 and 2000, 105 patients, after curative resection of lung metastases from renal cell carcinoma, were followed in this long-term study. These patients underwent a total of 150 surgical procedures. Survival analysis was done using the Kaplan-Meier method and the log-rank test. Multivariate analysis of prognostic factors was performed using the Cox multivariate proportional hazard model. RESULTS: Median survival after curative resection reached 43 months (range, 1 to 218 months). Survival at 3, 5, and 10 years was 54%, 40%, and 33%, respectively. Univariate analysis revealed that a complete resection, a less than 4-cm diameter of the metastases and tumor-free lymph nodes at the time of primary operation, were highly significant dependent prognostic factors (p < 0.001). These factors were also shown to be independent prognostic factors as suggested by multivariate analysis (p < 0.05). CONCLUSIONS: The size of the metastatic nodule, the completeness of pulmonary resection, and the lymph node status at the time of nephrectomy are the most important prognostic factors that influence survival after resection of pulmonary metastases. Recurrence of resectable pulmonary metastases does not impair survival, thus favoring repeated resection in patients with recurrent disease.

Minimal residual disease in gastric cancer: evidence of an independent prognostic relevance of urokinase receptor expression by disseminated tumor cells in the bone marrow.

PURPOSE: To study the invasion-related molecule urokinase-type plasminogen activator receptor (u-PAR) expressed by disseminated tumor cells as a biologic predictor of poor survival in a large prospective series of patients with gastric cancer. PATIENTS AND METHODS: In 156 gastric cancer patients (prospective series), disseminated tumor cells in the bone marrow and the u-PAR expressed by these tumor cells were determined by cytokeratin (CK) 18 immunocytochemistry and u-PAR/CK18 double immunocytochemistry. RESULTS: In contrast to the mere detection of disseminated tumor cells at primary surgery, the additional evidence of u-PAR on these cells correlated significantly with pathologic T stage (P =.0474) and the expression of u-PAR (P =.0093) and plasminogen-activator inhibitor 1 (P =.0145) in the primary tumor (immunohistochemistry, chi(2)). Kaplan-Meier analysis revealed no association with prognosis for the mere detection of disseminated tumor cells. In contrast, a significant association was seen between detection of u-PAR on these cells and shorter disease-free (P <.0001) and overall survival (P <.0001). Multivariate analysis revealed that u-PAR on disseminated tumor cells at the time of primary surgery is an independent prognostic factor for disease-free (95% confidence interval [CI], 1.72 to 3.21; P =.024) and overall survival (P =.0049; relative risk, 2.89; 95% CI, 1.92 to 4.30). CONCLUSION: This is the first large study to show that u-PAR, detected on disseminated tumor cells in the bone marrow, is an independent prognostic parameter in gastric cancer, in contrast to the mere detection of minimal residual disease (MRD). u-PAR may be a promising marker to define a critical subpopulation of disseminated tumor cells and a target to eliminate MRD. Molecular phenotyping of MRD is critical for defining its individual clinical relevance.

[New ways in hepatocyte cultures: Cell immobilisation technique]

Hepatocyte cultures are used for a variety of investigations in the field of toxicology, pharmacology or hepatology. Above all rodent hepatocytes are used for these experiments. But many results from animal hepatocytes do not correspond to human metabolism. Therefore in vitro studies on human hepatocytes would be extremely useful to reduce animal experiments. One of the major obstacles to the ultimate success of approaches in the past was the inability of standard culture conditions to maintain hepatocyte viability and differentiated function over long term periods under standardised conditions. The technique of cell immobilisation mimicks the in vivo architecture of the liver in vitro. This culture technique not only supports liver specific functions over a period of 50 days but also preserves hepatocyte morphology. We conclude that the immobilisation technique is of considerable advantage as compared to other cell culture systems, especially in order to reduce animal experiments. Another advantage of cell immobilisation is the possibility of cryoconservation for repeated investigations on batch-controlled hepatocyte cultures. In combination with new techniques of cell isolation, it is also possible to culture porcine hepatocytes from slaughterhouse organs. This could also reduce animal experiments.