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Background: Tumor-specific cytotoxic T lymphocytes (CTL) can be activated in vivo by vaccination with dendritic cells (DC). However, clinical responses to DC-based vaccination have only been observed in a minority of patients with solid cancer. Combination with other treatment modalities such as chemotherapy may overcome immunoresistance of cancer cells. We have previously shown that gemcitabine sensitizes human pancreatic carcinoma cells against CTL-mediated lysis. Here, we used a murine pancreatic carcinoma model to investigate whether combination with gemcitabine increases therapeutic efficacy of DCbased vaccination. Methods: Bone marrow-derived DC from C57BL/6 mice were loaded with UV-irradiated, syngeneic Panc02 carcinoma cells and were administered subcutaneously. For prophylactic vaccination, mice were vaccinated three times in weekly intervals prior to tumor challenge with Panc02 cells. Therapeutic vaccination was started when tumors formed a palpable nodule. Gemcitabine was administered intraperitoneally twice weekly. Results: Prophylactic DC-based vaccination completely prevented subcutaneous and orthotopic tumor development and induced immunological memory as well as tumor antigen-specific CTL. In the subcutaneous tumor model, therapeutic DC-based vaccination was equally effective as gemcitabine (14 % vs. 17 % survival at day 58 after tumor challenge; controls: 0 %). Combination of the two strategies significantly increased survival of tumor-bearing mice (50 % at day 58 after tumor challenge). DC-based vaccination also prevented death from pulmonary metastatization after i.v.-injection of Panc02 cells. Conclusion: DC-based immunotherapy may not only be successfully combined with gemcitabine for the treatment of advanced pancreatic carcinoma, but may also be effective in preventing local recurrence or metastatization in tumor-free patients.

Background/Aims: Restenosis after percutaneous transluminal angioplasty (PTA) of the internal mammary artery (IMA) grafts is much less pronounced than in other arteries and venous grafts. The aim of the study was to test whether various arteries respond differently to dilatation. Methods: PTA of the IMA, carotid, renal and circumflex coronary (RCx) arteries was performed in 9 pigs (balloon to artery ratio of 1:1.5). After 8 weeks, angiography was repeated and vessels prepared for histological analysis. Immunohistochemical staining was done to examine proliferative activity (Ki67) and to identify the vasa vasorum of the adventitia (F VIII-RA). Results: The intima-media ratio after PTA was lowest in the IMA (0.06), followed by the carotid (0.27) and renal arteries (0.49) and the RCx (0.69). Proliferation of the intima was seen at 287° of the vessel circumference in the RCx, at 286° in the renal and at 166° in the carotid artery. No proliferative activity was seen in the IMA. The intima-adventitia ratio was lower in the IMA than in the RCx and renal arteries (p < 0.05). Conclusion: Intima proliferation after PTA varies between the different vessels, with best results seen in the IMA. There are differences in remodeling after PTA between muscular, muscular/elastic and elastic arteries.

Background— We have recently demonstrated that activity of red blood cell glutathione peroxidase-1 is inversely associated with the risk of cardiovascular events in patients with coronary artery disease. The present study analyzed the effect of glutathione peroxidase-1 deficiency on atherogenesis in the apolipoprotein E-deficient mouse. Methods and Results— Female apolipoprotein E-deficient mice with and without glutathione peroxidase-1 deficiency were placed on a Western-type diet for another 6, 12, or 24 weeks. After 24 weeks on Western-type diet, double-knockout mice (GPx-1 −/− ApoE −/− ) developed significantly more atherosclerosis than control apolipoprotein E-deficient mice. Moreover, glutathione peroxidase-1 deficiency led to modified atherosclerotic lesions with increased cellularity. Functional experiments revealed that glutathione peroxidase-1 deficiency leads to increased reactive oxygen species concentration in the aortic wall as well as increased overall oxidative stress. Peritoneal macrophages from double-knockout mice showed increased in vitro proliferation in response to macrophage-colony-stimulating factor. Also, we found lower levels of bioactive nitric oxide as well as increased tyrosine nitration as a marker of peroxynitrite production. Conclusions— Deficiency of an antioxidative enzyme accelerates and modifies atherosclerotic lesion progression in apolipoprotein E-deficient mice.

Dysregulated cytokine responsiveness by myeloid cells can be a trigger for the development of chronic inflammation as well as inflammatory bowel disease. Thus, mice with a myeloid-specific defect in signal transducer and activator of transcription (Stat) 3 develop spontaneous colitis secondary to the inability of myeloid cells to respond to the immunosuppressive cytokine interleukin-10. We now examined whether the inflammation caused by Stat3-deficient macrophages is cell autonomous or dependent on their interaction with lymphocytes. For this purpose, myeloid-specific Stat3-deficient mice (LysMcre/Stat3(flox) mice) were intercrossed with RAG-1 knockout mice to generate LysMcre/Stat3(flox) RAG(-/-) mice. In these mutants and LysMcre/Stat3(flox) littermate control mice we determined the onset and severity of spontaneous chronic enterocolitis, and the reaction to dextran sodium sulphate (DSS)-induced epithelial damage, as well as to lipopolysaccharide (LPS) challenge. In contrast to LysMcre/Stat3(flox) mice, LysMcre/Stat3(flox) RAG(-/-) animals are protected from chronic enterocolitis. Although they respond to oral dextran sulphate with acute colitis symptoms, the inflammation heals similarly to wild type mice whereas LysMcre/Stat3(flox) mice exhibit continued colitis pathology. In addition, the hyperreactivity of LysMcre/Stat3(flox) mice to LPS-challenge in vivo was less severe in the absence of lymphocytes. Despite clear differences in the strength of inflammatory responses, macrophages of both LysMcre/Stat3(flox) mice and LysMcre/Stat3(flox) RAG(-/-) animals exhibited increased costimulatory capacity. In conclusion, our findings demonstrate that Stat3-deficient myeloid cells alone are not capable of inducing the severe pathology seen in LysMcre/Stat3(flox) mice. Yet when these cells can interact with lymphocytes their increased costimulatory potential will trigger an overshooting inflammatory response.

Premalignant breast lesions are defined by their characteristic histologic features. Based on these criteria, an increased risk is assigned of eventually progressing towards a malignant lesion, but the most majority of these lesions will never progress to cancer. Hence, the clinical management depends on correct sampling and thorough histologic examination.

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.

Objective: The MRL mouse strain shows extraordinary wound healing capacities. Some years ago, Leferovich et al. (Proc Natl Acad Sci U. S. A. 2001;98:9830-35) have reported the absence of scar formation after cryogenically-induced right ventricular myocardial infarcts in adult MRL mice. An independent group (Oh et al., Cardiovasc Pathol 2004; 13:203-6) found that MRL mice repair left ventricular ischemic infarcts after coronary artery ligation with regular scar formation. Given the divergent outcomes in infarct healing in MRL mice reported by those two studies, we have investigated whether MRL mice heal myocardial infarcts, without scar both in the cryoinjury and in the coronary ligation model. Methods and Results: Four different protocols of cryogenically induced right and left ventricular injury, as well as permanent ligation of the left anterior descending coronary artery, were tested in adult MRL and control C57Bl/6 mice. At 60 days after experimental infarction, MRL mice showed pronounced scarring of the affected right and left ventricular areas, with no significant differences in infarct size and thickness between MRL and C57Bl/6 mice using any of the five experimental protocols. Analysis of cell proliferation by 5-bromo-2' -deoxyuridine (BrdU) incorporation into the DNA did not show any difference between the two strains of mice after infarction. Histological analysis of infarct areas using picrosirius red staining did not show differences in extent of collagen and distribution between the two mouse strains. Conclusions: NIRL mice heal myocardial infarcts with scar formation in response to ischemic as well as to cryogenic injuries. (c) 2008 Elsevier Inc. All rights reserved.

To evaluate the association of dynamic enhancement parameters of benign and malignant breast lesions at magnetic resonance (MR) imaging with microvessel distribution and histologic prognostic tumor characteristics.Regional review board approval and informed consent were obtained. Surgical resection specimens of breast lesions (32 benign, 86 malignant) in 118 patients (age range, 28-86 years; mean, 58 years) who had undergone dynamic T1-weighted MR imaging of both breasts were included in the study. Different MR enhancement parameters and microvessel density (MVD) distribution were determined. In malignant lesions, TNM stage, tumor grade, proliferative activity, and hormone receptor expression were determined. Spearman correlation coefficients; Wilcoxon, Fisher exact, Kruskal Wallis, and chi(2) tests; and logistic regression analysis were used for evaluation.Malignant lesions exhibited a higher ratio of microvessels in tumor periphery versus tumor center than did benign lesions (P.0005). High vessel ratios (P = .001) and low central vessel numbers (P = .007) were associated with high tumor grade. In malignant lesions, initial enhancement ratios of periphery to center of lesion correlated with the corresponding microvessel ratios (r = 0.61). Yet, a high peripheral MVD was not associated with strong peripheral enhancement (r = -0.09). High enhancement ratios, washout rates, and early enhancement peaks were associated with unfavorable, albeit not significant, prognostic indicators. Visible rim enhancement was the most accurate prognostic enhancement criterion for estrogen receptor status (P = .007), tumor grade (P = .06), and lymph node status (P = .046). Washout was the best discriminating criterion for proliferative activity.The different enhancement behaviors of malignant and benign breast lesions cannot be explained by MVD alone; however, a low MVD in the center of carcinoma is reflected quantitatively by a high enhancement ratio and qualitatively by rim enhancement, with an implication of adverse prognosis.

Medical progress depends on the availability of human tissue for teaching, quality control, and research. These tissue samples typically originate from resection specimen obtained during therapeutic and diagnostic interventions and it is hence the pathologist in charge of selecting and conserving tissue samples and eventually making them avaliable for the diverse academic purposes. These activities happen in a vaguely defined framework of medical, ethical, and legal constrictions, with important and partly conflicting interests of patient autonomy and data protection. This article introduces the reader to this complex subject, focusing on questions of informed individual versus general consent, and proposes practical solutions for dealing with these conflicting interest in the daily routine work.

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized on the X-chromosome may provide effective treatment for an extended number of patients.

PEComas, occasionally associated with the tuberous sclerosis complex, are defined by the presence of perivascular epithelioid cells that coexpress muscle and melanocytic markers. This family of tumors includes angiomyolipoma (AML), clear cell sugar tumor of the lung (CCST), lymphangioleiomyomatosis (LAM), and very rare tumors in other locations. Because non-AML/non-LAM PEComas are extremely rare and their natural history and prognostic features undefined, we present our experience with 26 PEComas of soft tissue and the gynecologic tract, the largest series to date. We also performed a detailed review of the literature, with special attention to features predictive of clinical behavior. All PEComas exclusive of AML and LAM were retrieved from our consultation files. Immunohistochemistry for pan-cytokeratin (CK), S-100 protein, smooth muscle actins (SMA), desmin, vimentin, HMB45, Melan-A, microphthalmia transcription factor (MiTF), TFE3, CD117, and CD34 was performed. Clinical follow-up information was obtained. Fisher's exact test was performed. The median patient age was 46 years (range, 15-97 years); there was a marked female predominance (22 females, 4 males). Sites of involvement included the omentum or mesentery (6 cases), uterus (4 cases), pelvic soft tissues (3 cases), abdominal wall (2 cases), uterine cervix (2 cases), and vagina, retroperitoneum, thigh, falciform ligament, scalp, broad ligament, forearm, shoulder, and neck (1 case each). The tumors ranged from 1.6 to 29 cm in size (median, 7.8 cm). Tumors were epithelioid (N = 9), spindled (N = 7), or mixed (N = 10). Multinucleated giant cells were present in 18 cases. High nuclear grade was noted in 10 cases, high cellularity in 7 cases, necrosis in 8 cases, and vascular invasion in 3 cases. Mitotic activity was 0 to 50 mitotic figures (MF)/50 high power fields (HPF) (median, 0 MF/50 HPF) with atypical MF in 6 cases. IHC results were: SMA (20/25), desmin (8/22), HMB45 (22/24), Melan-A (13/18), MITF (9/18), S-100 protein (8/24), CK (3/23), vimentin (12/14), TFE3 (5/17), c-kit (1/20), and CD34 (0/7). Clinical follow-up (24 of 26 patients, 92%; median, 30 months; range, 10-84 months) showed 3 local recurrences and 5 distant metastases. At last available clinical follow-up, 2 patients (8%) were dead of disease, 4 patients (17%) were alive with metastatic or unresectable local disease, and 18 patients (75%) were alive with no evidence of disease. No patient in our series had a history of tuberous sclerosis complex. Recurrence and/or metastasis was strongly associated tumor size > median size (8 cm), mitotic activity greater than 1/50 HPF, and necrosis. We conclude that PEComas of soft tissue and gynecologic origin may be classified as "benign," "of uncertain malignant potential," or "malignant." Small PEComas without any worrisome histologic features are most likely benign. PEComas with nuclear pleomorphism alone ("symplastic") and large PEComas without other worrisome features have uncertain malignant potential. PEComas with two or more worrisome histologic features should be considered malignant. Occasional PEComas express unusual markers, such as S-100 protein, desmin, and rarely CK. The role of TFE3 in PEComas should be further studied.

In contrast to acute preparations such as the exteriorized mesentery or the cremaster muscle, chronically instrumented chamber models allow one to study the microcirculation under “physiological” conditions, i.e., in the absence of trauma-induced leukocyte rolling along the venular endothelium. To underscore the importance of studying the naive microcirculation, we implanted titanium dorsal skinfold chambers in hamsters and used intravital fluorescence microscopy to study venular leukocyte rolling in response to ischemia-reperfusion injury or extracorporeal blood circulation. The experiments were performed in chambers that fulfilled all well-established criteria for a physiological microcirculation as well as in chambers that showed various extents of leukocyte rolling due to trauma, hemorrhage, or inflammation. In ideal chambers with a physiological microcirculation (100 leukocytes/mm), we observed a paradoxical reduction of leukocyte rolling after ischemia-reperfusion injury or extracorporeal blood circulation. A similar effect was observed when we superfused leukotriene B4(LTB4) onto the chamber tissue. The initial increase in leukocyte rolling in response to an LTB4challenge was reversed by a second superfusion 90 min later. These observations underscore 1) the benefit of studying leukocyte-endothelial cell interaction in chronically instrumented chamber models and 2) the necessity to strictly adhere to well-established criteria of a physiological microcirculation.

Objective—Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied.Methods and Results—Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein–activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP expression, whereas rbCRP–positive males had significantly higher serum cholesterol levels than the rbCRP-negative counterparts. All mice exhibited extensive atherosclerotic lesions, as studied en face, and no differences were noted between rbCRP-negative and rbCRP-positive animals. Atherosclerotic luminal obstruction of aortic arch and first-order neck branches did not differ significantly between rbCRP-positive and rbCRP-negative mice. There was no correlation between rbCRP levels and atherosclerotic lesion formation.Conclusions—No marked effect of rbCRP on the formation of moderately advanced atherosclerotic lesions could be discerned in the apoE knockout mouse. Because of the oddities of the mouse complement system, however, this may not be a good model to investigate the role of CRP in human atherosclerosis.

The exact etiologic mechanisms leading to the formation of nasal polyps have remained largely obscure. A key phenomenon of this specific type of chronic inflammatory disease in nasal respiratory mucosa is remarkable edema. Vascular permeability/vascular endothelial growth factor (VPF/VEGF) plays an important role in inducing angiogenesis and modulating capillary permeability.To study the expression and localization of VPF/ VEGF as a putative key factor in nasal polyp development.Specimens of nasal polyps (n = 12) were harvested during endonasal sinus surgery in patients with polypous chronic rhinosinusitis. Specimens of healthy nasal respiratory mucosa (n = 12) served as controls and were obtained from inferior turbinates of patients undergoing surgery for nasal obstruction without signs and symptoms of inflammatory disease. Frozen sections were immunohistochemically stained for VPF/VEGF and quantitatively analyzed, using computer-based image analysis.The expression of VPF/VEGF in specimens of nasal polyps was significantly stronger than in specimens of healthy nasal mucosa of controls. VPF/VEGF in polypous tissue was mainly localized in vascular endothelial cells, in basal membranes and perivascular spaces, and in epithelial cells.The markedly increased expression in nasal polyps as opposed to healthy nasal mucosa suggests that VPF/VEGF may play a significant role in both the formation of nasal polyps and in the induction of heavy tissue edema. This finding is discussed with respect to the differential expression of cyclooxygenase (COX) isoenzymes-1 and -2 (COX-1 and COX-2) in nasal polyps was significantly stronger than in specimens of healthy nasal mucosa of controls. VPF/VEGF in polypous tissue was mainly localized in vascular endothelial cells, in basal membranes and perivascular spaces, and in epithelial cells. Conclusion: The markedly increased expression in nasal polyps as opposed to healthy nasal mucosa suggests that VPF/VEGF may play a significant role in both the formation of nasal polyps and in the induction of heavy tissue edema. This finding is discussed with respect to the differential expression of cyclooxygenase (COX) isoenzymes-1 and -2 (COX-1 and COX-2) in nasal polyps.

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.

Cytokine signaling pathways involving transcription factors of the signal transducers and activators of transcription (STAT) family play a key role in the pathogenesis of inflammatory bowel diseases (IBD). STAT proteins are latent cytoplasmic transcription factors that induce transcription upon phosphorylation, dimerization, and nuclear translocation. However, their activation pattern in IBD is poorly understood. The aim of our study was to characterize STAT-expression in IBD.Mononuclear cells were isolated from 36 colonic specimens of Crohn's disease, ulcerative colitis, or from control patients. Cells were stimulated overnight with antibodies against human CD2 and CD28 and mononuclear cells were analyzed by flow cytometry. Alternatively, CD4(+) T cells were immunomagnetically separated and then assessed by flow cytometry. Intracellular stainings of the following transcription factors were performed: STAT-1, STAT-2, STAT-3, STAT-4, and STAT-6. In addition, immunofluorescence staining on cryosections for phosphorylated STAT-1 and STAT-3 was performed.Average expression of the IFN-gamma inducible transcription factor STAT-1 was increased in Crohn's disease as compared to patients with ulcerative colitis and control patients. However, levels of phospho-STAT-1 were surprisingly not markedly upregulated in IBD as compared to controls. In contrast, STAT-3 and phospho-STAT-3 levels were significantly increased in IBD patients as compared to controls (p0.01). No differences could be detected in STAT-6 levels. Finally, average expression of STAT-2, which is involved in type I interferon signalling, was downregulated in IBD as compared to control patients.The analysis of STAT activation patterns could serve as a helpful tool to characterize intestinal inflammation. Furthermore, the IL-6/STAT-3 rather than the IFN-gamma/STAT-1 signaling pathway emerges as a key target for the development of future therapeutic concepts in IBD.

The transcription factor c-Maf controls IL-4 gene expression in CD4(+) T cells, and its expression is up-regulated in human asthmatic airways after allergen challenge. In the present study, we addressed the role of c-Maf in asthma by studying transgenic (Tg) mice overexpressing c-Maf in CD4(+) T cells under the control of the CD2 promoter. As shown, lung CD4(+) T cells of c-maf-Tg mice produced more IL-5 at the early stage (day 2) of culture in the presence of IL-4 than wild-type control cells. Consistently, c-maf-Tg mice spontaneously showed increased IL-5 expression and eosinophils in the bronchial alveolar lavage fluid (BALF) and activated IL-5 signal transduction via Raf-1 and Ras in lung eosinophils. Finally, IL-13 was suppressed in the BALF of c-maf-Tg mice and in supernatants of Tg lung CD4(+) T cells cultured in the presence of IL-2. Consistently, retroviral overexpression of c-Maf suppressed IL-13 production in developing lung Th2 cells. In summary, c-Maf induces IL-5 production in lung CD4(+) T cells at an early stage, but along with IL-2 suppresses IL-13 production in differentiating lung Th2 cells, thereby explaining the finding that overexpression of c-Maf does not cause airway hyperresponsiveness, a hallmark feature of asthma.

INTRODUCTION: The prognostic significance of HER-2/neu in breast cancer is a matter of controversy. We have performed a study in 101 node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting, and analysed the prognostic significance of immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH), both separately and in combination, in comparison with traditional prognostic factors. METHODS: Overexpression was classified semiquantitatively according to a score (0 to 3+) (HER-2_SCO). FISH was used to analyse HER2/neu amplification (HER-2_AMP). Patients classified 2+ by IHC were examined with FISH for amplification (HER-2_ALG). Patients with 3+ overexpression as well as amplification of HER-2/neu were positive for the combined variable HER2_COM. These variables were compared with tumour size, histological grade and hormone receptor status. RESULTS: HER-2_SCO was 3+ in 20% of all tumours. HER-2_ALG was positive in 22% and amplification (HER-2_AMP) was found in 17% of all tumours. Eleven percent of the tumours showed simultaneous 3+ overexpression and amplification. Only histological grade (relative risk [RR] 3.22, 95% confidence interval [CI] 1.73-5.99, P = 0.0002) and HER-2_AMP (RR 2.47, 95% CI 1.12-5.48, P = 0.026) were significant for disease-free survival in multivariate analysis. For overall survival, both histological grade (RR 3.89, 95% CI 1.77-8.55, P = 0.0007) and HER-2_AMP (RR 3.08, 95% CI 1.24-7.66, P = 0.016) retained their independent significance. CONCLUSION: The prognostic significance of HER-2/neu in node-negative breast cancer depends on the method of testing: only the amplification of HER-2/neu is an independent prognostic factor for the long-term prognosis of untreated node-negative breast cancer

The growing class of cancer/germ-line genes is characterized by a unique expression pattern with transcription restricted to germ cells and cancer cells. It is not known which fraction of germ-line genes is ectopically activated in tumor cells and whether this fraction displays common features as compared with strictly germ-line genes remaining silent in cancer. Using an unbiased genome-wide scanning approach, representative samples of both cancer/germ-line genes as well as strictly germ-line-specific genes were determined. Comparative analysis disclosed highly significant diametric characteristics for these two categories of genes with regard to sex specificity, developmental stage of physiological expression during gametogenesis, chromosomal localization, and epigenetic regulation of expression. Our findings provide class predictors for germ cell-specific gene activation in cancer. The identification of highly congruent expression patterns in cancer and in DNA methyltransferase-deficient cells suggests an underlying common epigenetic mechanism for activation of germ-line genes in cancer.

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5x10(8) IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I-restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

Because of its amplification and/or overexpression in many human tumors, the HER-2/neu proto-oncogene represents an attractive target for T-cell-mediated vaccination strategies. However, overexpression of oncogenes is often associated with defective expression of components of the MHC class I antigen-processing machinery (APM), thereby resulting in an immune escape phenotype of oncogene-transformed cells. To determine whether HER-2/neu influences the MHC class I antigen-processing pathway, the expression pattern of different APM components was examined in murine in vitro models of constitutive and tetracycline-controlled HER-2/neu expression. In comparison with HER-2/neu− control cells, HER-2/neu+ fibroblasts exhibit reduced levels of MHC class I surface antigens that were associated with impaired expression and/or function of the peptide transporter associated with antigen processing, the proteasome subunits low molecular weight protein 2 and low molecular weight protein 10, the proteasome activators PA28α and PA28β, and tapasin. These APM abnormalities resulted in reduced sensitivity to lysis by CTLs. The HER-2/neu-mediated immune escape phenotype could be corrected by IFN-γ treatment. The clinical relevance of this finding was supported by an inverse correlation between HER-2/neu and the peptide transporter associated with antigen-processing protein expression as determined by immunhistochemical analysis of a series of HER-2/neu− and HER-2/neu+ breast cancer specimens. Thus, a functional link between deficient APM component expression and HER-2/neu overexpression is proposed that might influence the design of HER-2/neu-targeted T-cell-based immunotherapeutic strategies.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.

Object. Acoustic neuroma is the most frequent benign tumor of the cerebellopontine angle, and surgery is still the most common form of treatment. To gain better insight into the dysregulated mechanisms causing growth of acoustic neuroma, the authors studied the proliferative activity of 34 consecutive samples by analyzing immunohistochemical staining with Ki-67 and proliferating cell nuclear antigen (PCNA), and apoptosis based on the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling. Data from these analyses were correlated with clinical parameters (that is, tumor size, duration of symptoms, and patient age). Methods. Apoptotic cells were found in none of the tumors. Proliferation measured on staining with Ki-67 and PCNA correlated with tumor size, but not with patient age or duration of symptoms. The authors demonstrated that tumors 18 mm or smaller in diameter have lower proliferation indices and growth rates, compared with tumors larger than 18 mm with high proliferative indices and growth rates. Additionally, they observed that these more aggressive, larger tumors occur mostly in patients younger than 50 years of age. Conclusions. Patients with tumors larger than 18 mm in diameter and who are younger than 50 years of age sustain an enhanced risk for fast-growing tumors because of these lesions' enhanced proliferative activity. For these patients the authors recommend active therapy.

Based on previous findings in SCC of the human uterine cervix and of the head and neck, metastasis- and survival-related metabolic classification was performed in human rectal adenocarcinomas. Such a characterization is suggested as basis of a novel clinical guideline for an appropriate therapy of epithelium-derived malignancies.Tissue concentrations of ATP, glucose, and lactate in viable tumor regions of 33 pretherapeutically taken cryobiopsies from 24 patients were measured at a microscopic level using the technique of imaging bioluminescence.In metastatic carcinomas, lactate levels were significantly higher (mean+/-SD: 13.4+/-3.8 vs 6.9+/-2.5 micro mol/g, P = 0.0046) and glucose levels significantly lower (0.3+/-0.2 vs 1.0+/-0.4 micro mol/g, P = 0.0020) than in non-metastatic carcinomas. No patients had distant metastasis with tumor lactate levels below the population median of 8.0 micro mol/g or glucose levels above 0.9 micro mol/g.These results support the hypothesis that lactate and glucose levels measured in human primary carcinomas may serve as an early prognostic tool in the clinic with regard to formation of metastasis and patient survival. The present data encourage further studies on possible strategies for a metabolic classification of tumors as a prognostic tool.

BACKGROUND The overexpression of glucose transporters, especially of Glut-1, is a common characteristic of human malignancies, including head and neck carcinoma. Recently, the assessment of glucose metabolism in the tumor with [18F]-2-fluoro-2 deoxy-D-glucose (FDG) and positron emission tomography (FDG-PET) has been used to identify particularly aggressive tumors. The authors tested the hypothesis that both glucose transport and its metabolism play a key role in the progression of oral squamous cell carcinoma (OSCC). METHODS Retrospective analysis of Glut-1 expression was performed by immunohistology in 118 patients with OSCC, and a Glut-1 labeling index (LI) was established for each. A separate group of 44 patients with primary OSCC was evaluated prospectively by FDG-PET prior to surgery. To link the expression of Glut-1 with glucose metabolism, both FDG-PET and immunohistology were determined in a subgroup of 31 patients, and the results were correlated with overall survival. RESULTS The patients who had OSCC with a low LI for Glut-1 survived significantly longer compared with patients who had OSCC with a high LI (138 months vs. 60 months; P = 0.0034). It was found that Glut-1 expression was an independent marker of prognosis in patients with OSCC. In patients who were evaluated by FDG-PET, the standardized uptake value (SUV) below the median split value of 5.6 was predictive of a longer survival (P 5.6 was associated with an increased hazard of death. In combination, a high Glut-1 level and a high SUV predicted shorter survival (P < 0.005) for patients with OSCC. Patients who achieved a complete response to preoperative radiation tended to have tumors with low glucose metabolism, as defined by both the Glut-1 LI and the SUV. CONCLUSIONS Both glucose transport and glucose metabolism determine the glycolytic tumor phenotype, which is a significant negative biomarker of prognosis and overall survival in patients with OSCC. Cancer 2003;97:1015–24. © 2003 American Cancer Society. DOI 10.1002/cncr.11159

Tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of Crohn's disease (CD). However, little is known about the role of TNF receptors (TNF-R) in this disease. Here, we found that TNF-R2 (in contrast to TNF-R1) was significantly up-regulated on lamina propria and peripheral blood T cells in CD compared to control patients. To directly test the functional role of TNF-R2 in Th1-mediated experimental colitis in vivo, we took advantage of transgenic animals overexpressing TNF-R2 in T cells. Reconstitution of SCID mice with CD4+ CD62L+ T cells from TNF-R2 transgenic mice led to an earlier wasting syndrome, a more severe colitis and augmented Th1 cytokine production than reconstitution with cells from wild-type littermates. In addition, TUNEL staining revealed a significantly decreased apoptosis rate of lamina propria mononuclear cells in mice reconstituted with TNF-R2 transgenic T cells compared to mice reconstituted with wild-type T cells. In summary, our data suggest a critical regulatory role of TNF-R2 signaling for disease exacerbation in Th1-mediated chronic colitis. Taken together with the increased expression of TNF-R2 in CD, selective targeting of TNF-R2 signaling thus emerges as a potentially novel approach to the treatment of CD.