Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Impact of mutant ß-catenin on ABCB1 expression and therapy response in colon cancer cells.

BACKGROUND: Colorectal cancers are often chemoresistant toward antitumour drugs that are substrates for ABCB1-mediated multidrug resistance (MDR). Activation of the Wnt/ß-catenin pathway is frequently observed in colorectal cancers. This study investigates the impact of activated, gain-of-function ß-catenin on the chemoresistant phenotype. METHODS: The effect of mutant (mut) ß-catenin on ABCB1 expression and promoter activity was examined using HCT116 human colon cancer cells and isogenic sublines harbouring gain-of-function or wild-type ß-catenin, and patients' tumours. Chemosensitivity towards 24 anticancer drugs was determined by high throughput screening. RESULTS: Cell lines with mut ß-catenin showed high ABCB1 promoter activity and expression. Transfection and siRNA studies demonstrated a dominant role for the mutant allele in activating ABCB1 expression. Patients' primary colon cancer tumours shown to express the same mut ß-catenin allele also expressed high ABCB1 levels. However, cell line chemosensitivities towards 24 MDR-related and non-related antitumour drugs did not differ despite different ß-catenin genotypes. CONCLUSION: Although ABCB1 is dominantly regulated by mut ß-catenin, this did not lead to drug resistance in the isogenic cell line model studied. In patient samples, the same ß-catenin mutation was detected. The functional significance of the mutation for predicting patients' therapy response or for individualisation of chemotherapy regimens remains to be established.

Suppression of tumorigenicity in breast cancer cells by the microfilament protein profilin 1.

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells.

Loss of the coxsackie and adenovirus receptor contributes to gastric cancer progression.

Loss of the coxsackie and adenovirus receptor (CAR) has previously been observed in gastric cancer. The role of CAR in gastric cancer pathobiology, however, is unclear. We therefore analysed CAR in 196 R(0)-resected gastric adenocarcinomas and non-cancerous gastric mucosa samples using immunohistochemistry and immunofluorescence. Coxsackie and adenovirus receptor was found at the surface and foveolar epithelium of all non-neoplastic gastric mucosa samples (n=175), whereas only 56% of gastric cancer specimens showed CAR positivity (P<0.0001). Loss of CAR correlated significantly with decreased differentiation, increased infiltrative depths, presence of distant metastases, and was also associated with reduced carcinoma-specific survival. To clarify whether CAR impacts the tumorbiologic properties of gastric cancer, we subsequently determined the role of CAR in proliferation, migration, and invasion of gastric cancer cell lines by application of specific CAR siRNA or ectopic expression of a human full-length CAR cDNA. These experiments showed that RNAi-mediated CAR knock down resulted in increased proliferation, migration, and invasion of gastric cancer cell lines, whereas enforced ectopic CAR expression led to opposite effects. We conclude that the association of reduced presence of CAR in more severe disease states, together with our findings in gastric cancer cell lines, suggests that CAR functionally contributes to gastric cancer pathogenesis, showing features of a tumour suppressor.

HIF-1alpha determines the metastatic potential of gastric cancer cells.

Gastric adenocarcinoma is characterised by rapid emergence of systemic metastases, resulting in poor prognosis due to vanished curative treatment options. Better understanding of the molecular basis of gastric cancer spread is needed to design innovative treatments. The transcription factor HIF-1alpha (hypoxia-inducible factor 1alpha) is frequently overexpressed in human gastric cancer, and inhibition of HIF-1alpha has proven antitumour efficacy in rodent models, whereas the relevance of HIF-1alpha for the metastatic phenotype of gastric adenocarcinoma remains elusive. Therefore, we have conducted a comprehensive analysis of the role of HIF-1alpha for pivotal metastasis-associated processes of human gastric cancer. Immunhistochemistry for HIF-1alpha showed specific staining at the invading tumour edge in 90% of human gastric cancer samples, whereas normal gastric tissue was negative and only a minority of early gastric cancers (T1 tumours) showed specific staining. Hypoxia-inducible factor 1alpha-deficient cells showed a significant reduction of migratory, invasive and adhesive properties in vitro. Furthermore, the HIF-1alpha-inhibitor 2-methoxy-estradiol significantly reduced metastatic properties of gastric cancer cells. The accentuated expression at the invading edge together with the in vitro requirement of HIF-1alpha for migration, invasion and adherence argues for a pivotal role of HIF-1alpha in local invasion and, ultimately, systemic tumour spread. These results warrant the exploration of HIF-1alpha-inhibiting substances in clinical treatment studies of advanced gastric cancer.

Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial.

PURPOSE: Metastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer. METHODS: Patients with histologically confirmed liver metastases from colorectal cancer were randomised to the vaccination or control group. After complete resection of liver metastases, patients randomised to the vaccination group received six doses of Newcastle disease virus (NDV) infected autologous tumour cell vaccine (ATV-NDV). The primary end-point was overall survival, secondary end-points were disease-free survival and metastases-free survival. RESULTS: Fifty-one patients were enrolled in the study with 50 patients available for analysis. The follow-up period was 116.1 +/- 23.8 month in the vaccination arm and 112.4 +/- 18.5 month in the control group. In the total patient group, no differences in the primary and secondary end-points were detected. Most interestingly, subgroup analysis revealed a significant advantage for vaccinated colon cancer patients with respect to overall survival [hazard ratio: 3.3; 95%, confidence interval (CI): 1.0-10.4; P = 0.042] and metastases-free survival (hazard ratio: 2.7; 95%, CI: 1.0-7.4; P = 0.047) in the intention-to-treat analysis. CONCLUSION: Active specific immunotherapy in unselected colorectal cancer patients was not effective for prevention of recurrent metastatic disease. However, in colon cancer patients, ASI with ATV-NDV appears to be beneficial prolonging overall and metastases-free survival.

Treatment of oesophageal anastomotic leaks by temporary stenting with self-expanding plastic stents.

BACKGROUND: Oesophageal anastomotic leakage is associated with considerable morbidity and mortality. The aim of the present study was to assess the feasibility of using temporary self-expanding plastic stents to treat postoperative oesophageal leaks. METHODS: Patients with anastomotic leakage after abdominothoracic oesophagectomy treated by endoscopic insertion of self-expanding plastic stents between 2001 and 2007 were studied. Clinical outcomes were analysed, including healing of the leak, morbidity and mortality. RESULTS: Stents were inserted successfully in all 22 patients without procedure-related complications. Ten patients also required computed tomography-guided drainage because surgical drains had been removed. Non-ventilated patients received oral nutrition a mean of 4 days after stent placement. Combined treatment with stenting and drainage resulted in resolution of the leak in 21 of 22 patients. The mean healing time (time to stent removal) was 23 days. Stent migration occurred in five of 22 patients, but endoscopic reintervention with placement of a new stent was successful in all patients. Repeat thoracotomy with intraoperative stent placement was necessary in one patient with an oesophagocolonic anastomosis. One patient died in hospital. CONCLUSION: In combination with effective drainage, self-expanding plastic stents are an option for the treatment of oesophageal anastomotic leaks, and may reduce leak-related morbidity and mortality.

Hepatic arterial Fotemustine chemotherapy in patients with liver metastases from cutaneous melanoma is as effective as in ocular melanoma.

AIM: Hepatic metastases from melanoma are associated with poor prognosis. Systemic chemotherapy and biological treatments remain unsatisfactory. This study investigated the impact of hepatic arterial chemotherapy in patients with ocular and cutaneous melanoma. METHODS: In a retrospectively analysed observational study, 36 consecutive patients with hepatic metastases from ocular or cutaneous melanoma were assigned for surgical hepatic port-catheter implantation. Fotemustine was delivered weekly for a 4-week period, followed by a 5-week rest and a maintenance period every 3 weeks until progression. Overall survival, response and toxicity were analysed and compared. RESULTS: After port-catheter implantation 30/36 patients were finally treated (18 with ocular and 12 with cutaneous melanoma). A median of 8 infusions per patient were delivered (range 3-24). 30% thrombocytopenia grade >or=3, 7% neutropenia grade >or=3 but no nausea or vomiting grade >or=3 were encountered. Nine out of 30 patients achieved partial remission, 10/30 stable disease; 11/30 patients were progressive. Median survival for all treated patients was 14 months. Partial remission and stable disease were associated with a survival advantage compared to progressive disease (19 vs. 5 months). No significant difference in survival was observed for ocular versus cutaneous melanoma. Serum LDH was a significant predictor of both response and survival. CONCLUSIONS: Hepatic arterial Fotemustine chemotherapy was well tolerated. Meaningful response and survival rates were achieved in ocular as well as cutaneous melanoma. Careful patient selection in consideration of extra-hepatic involvement is crucial for the effectiveness of this treatment. Independent from the primary melanoma site, it is debatable if patients with highly elevated serum-LDH may benefit from this approach.

Evaluation of higher-order time-domain perturbation theory of photon diffusion on breast-equivalent phantoms and optical mammograms.

Time-domain perturbation theory of photon diffusion up to third order was evaluated for its accuracy in deducing optical properties of breast tumors using simulated and physical phantoms and by analyzing 141 projection mammograms of 87 patients with histology-validated tumors that had been recorded by scanning time-domain optical mammography. The slightly compressed breast was modeled as (partially) homogeneous diffusely scattering infinite slab containing a scattering and absorbing spherical heterogeneity representing the tumor. Photon flux densities were calculated from densities of transmitted photons, assuming extended boundary conditions. Explicit formulas are provided for second-order changes in transmitted photon density due to the presence of absorbers or scatterers. The results on phantoms obtained by perturbation theory carried up to third order were compared with measured temporal point spread functions, with numerical finite-element method (FEM) simulations of transmitted photon flux density, with results obtained from the diffraction of diffuse photon density waves, and from Padé approximants. The breakdown of first-, second-, and third-order perturbation theory is discussed for absorbers and a general expression was derived for the convergence of the Born series in this case. Taking tumor optical properties derived by the diffraction model as reference we conclude that estimates of tumor absorption coefficients by perturbation theory agree with reference values within +/-25% in only 65% (first order), 66% (second order), and 77% (third order) of all mammograms analyzed. In the remaining cases tumor absorption is generally underestimated due to the breakdown of perturbation theory. On average the empirical Padé approximants yield tumor absorption coefficients similar to third-order perturbation theory, yet at noticeable lower computational efforts.

[R1 resection in the region of the lower gastrointestinal tract: relevance and therapeutic consequences]. / R1-Resektion im Bereich des unteren Gastrointestinaltrakts: Bedeutung und therapeutische Konsequenzen.

Incomplete resection (R1) and local recurrence of colorectal cancer continue to be a significant surgical problem. Radical resection of bowel and lymph node bassin are clearly necessary after incomplete endoscopic resection or local surgical excision. However, the situation is more difficult after previous conventional surgery. Anastomotic recurrence following resection and lymph nodal recurrence can often precede curative reresection. Locoregional lymph node metastases due to incomplete surgical clearance of the lymphatic drainage of colonic cancer may also be cured by radical reresection. Despite application of neoadjuvant therapy, integration of modern surgical concepts such as the circumferential resection margin and advances in surgical technique, R1 resection of rectal cancer remains a major problem. Although primary surgical therapy may be considered in selected cases, this situation will require multimodal therapy in most instances.

[Transcutaneous ultrasound]. / Transkutane Sonographie.

Preoperative transcutaneous ultrasound allows surgeons to assess the pathology directly, thus supplementing clinical examination of the patient. Technical advances including power doppler, three-dimensional ultrasound, and the advent of ultrasound contrast agents have increased the quality and broadened the diagnostic spectrum of ultrasound. This article reviews relevant new aspects of transcutaneous ultrasound in the surgical setting.

Uptake, biodistribution, and time course of naked plasmid DNA trafficking after intratumoral in vivo jet injection.

Nonviral jet injection is an applicable technology for in vivo gene transfer of naked DNA. However, little is known about the biodistribution and clearance of jet-injected DNA, or about its localization within tissue and cells. Therefore, in this study we analyzed the intratumoral and systemic biodistribution of jet-injected naked DNA in human colon carcinoma-bearing NCr-nu/nu mice, which were jet-injected with the pCMVbeta plasmid DNA. Intratumoral and systemic plasmid DNA biodistribution was analyzed 5, 10, 20, and 40 min and 3, 6, 24, 48, and 72 hr after jet injection, using quantitative real-time polymerase chain reaction. In the tumors, a rapid drop in naked DNA load within 24 hr of jet injection was shown. Detailed analysis of intratumoral distribution of rhodamine-labeled DNA revealed the presence of plasmid DNA within tumor cells 5 min after jet injection and further accumulation of significant DNA amounts in the cell nuclei 30 to 60 min after jet injection. In the blood, DNA amounts rapidly dropped within 10 to 40 min of jet injection to less than 0.001 pg of plasmid per 250 ng of tissue DNA and only minimal plasmid DNA dissemination was detected in liver, lung, spleen, kidney, and ovaries, which was cleared 3 to 6 hr after jet injection. By contrast, in heart, bone marrow, and brain almost no plasmid DNA was detectable.

[Significance of palliative resection of gastrointestinal tumors]. / Hat die palliative Resektion bei gastrointestinalen Tumoren noch einen Stellenwert?

Before any palliative tumor resection, the morbidity and mortality risks must be carefully weighed against the continued prognosis (including quick and lasting relief of discomfort from the tumor) and alternative strategies such as bypass, chemotherapy, and radiotherapy. Multimodal concepts have seen considerable progress in recent years, and endoscopic and interventional methods have expanded the instrumentarium for palliative tumor therapy. Thus the value of palliative resection must be reassessed. The most important criteria and study results are described here, as they have resulted in increased interest in palliative tumor resection within a multimodal treatment for most gastrointestinal tumors. More studies are needed to learn how much can realistically be expected of these new approaches.

[Abdominal vacuum device with open abdomen]. / Abdomineller Vakuumsaugverband beim offenen Abdomen.

BACKGROUND: For the treatment of peritonitis or abdominal compartment syndrome, an open abdomen can be required. Because of the high complication rate associated with this method, different technical modifications were developed that are now being applied. Abdominal vacuum-assisted closure is increasingly favoured. We analyse our experience with this device in a distinct group of patients from gastrointestinal cancer surgery. PATIENTS AND METHOD: From June 2003 to December 2005, 36 patients were treated with 151 double-layer abdominal vacuum devices. Indications for applying this device were peritonitis (n = 22), abdominal compartment syndrome (n = 11), and necrotising fasciitis (n = 3). Thirty-four patients gave anamneses of malignoma. RESULTS: Overall, the vacuum therapy treatment lasted a median of 13 days (range 3-48). With it, four enteric fistulas (11%) and four abdominal wall bleedings (11%) occurred. In our patient group, no new intra-abdominal abscesses were observed. Four patients died during treatment with the vacuum-assisted device and four afterward because of multiple organ failure in acute sepsis (in-hospital mortality 22%). Twenty-six patients (72%) underwent direct fascial closure after a median treatment duration of 10 days. Six patients (17%) required synthetic mesh for fascial closure. After a median follow-up of 100 days, two patients developed ventral hernias and two others showed ossification of the scar. CONCLUSION: Compared with other methods of temporary abdominal closure, our experience with the vacuum-assisted device demonstrates its advantages concerning clinical feasibility and the relatively low complication rate. The high rate of direct fascial closure with an acceptable rate of ventral hernias following vacuum-assisted abdominal closure are further benefits of this technique.

[Need for psychosocial care in in-patients with tumour disease. Investigations conducted in a clinic specializing in tumour surgery]. / Psychosozialer Betreuungsbedarf bei stationären Tumorpatienten. Untersuchungen an einer chirurgisch-onkologischen Schwerpunktklinik.

BACKGROUND: Patients with tumour disease are in particularly stressful situation at all times. The aim of the present study was to find what proportion of patients on a surgical oncology ward would also benefit from psycho-oncological care. PATIENTS AND METHOD: Within a period of 6 months (IIIrd and IVth quarters of 2004) 406 of our tumour patients were questioned with the aid of a method (Po-Bado) developed specifically for use with such patients. RESULTS: According to this inquiry, it can be assumed that 41.4% of tumour patients are in need of professional psycho-oncological support. Patients who are in hospital for diagnostic procedures to confirm or exclude the suspicion of tumour disease have a greater need for such support (48.7%) than do patients who have been admitted for a scheduled operation (37.3%). Correlations were found between the need for this therapy and different disease situations. The prevalence of need was highest among patients with a second tumour, in whom it was 66.7%. The type of tumour disease also had an influence whether psycho-oncological care was indicated. The study revealed that patients with malignant soft-tissue tumours (49%) and patients with tumours of the upper digestive organs (48.7%) find the mental stress more difficult to cope with than patients who are in hospital for treatment of malignant skin tumours (31.8%) or malignant tumours of the mammary gland (38.7%). CONCLUSION: These results suggest that an adequate psycho-oncologic diagnostic at the start of a stationary stay are reasonable. This is a precondition for a well-directed psycho-oncologic intervention in order to enhance the disease accomplishment but at the same time the target-oriented supply of psycho-oncologic care in hospitals is a limited resource.

[Minimal residual tumor in gastrointestinal carcinoma. Relevance to prognosis and oncologic surgical consequences]. / Minimalresiduale Tumorerkrankung bei gastrointestinalen Karzinomen. Prognoserelevanz und onkologisch-chirurgische Konsequenzen.

Isolated tumor cells as a consequence of minimal residual disease are often not detectable by routine diagnostic procedures. However, before or after surgery, isolated tumor cells in lymph nodes, the peritoneal cavity, blood, or bone marrow can frequently be identified by immunohistochemical or molecular methods. Failure to reveal the presence of such cells results in under-staging of tumor patients and may constitute the source of unexpected tumor recurrence after radical surgery. These facts emphasize the importance of isolated tumor cells at least as a surrogate marker. The frequency of appearance of isolated tumor cells in different organ systems also depends on the type of primary tumor. Developments in modern detection methods have led to increasing sensitivity but at the expense of specificity. Isolated tumor cells demonstrate remarkable heterogeneity with respect to proliferative potential and tumorigenicity. This characteristic is also reflected by a striking variability in the expression of various genes conditioning the aforementioned biological behavior. Unfortunately there is also remarkable heterogeneity in methods used for sampling and processing patient material as well as for the enrichment and detection of isolated tumor cells. Despite the ongoing controversies concerning detection methods and biological significance of isolated tumor cells, several clinical trials providing data supporting the prognostic relevance of minimal residual disease should also be considered for gastrointestinal carcinoma. In future this finding should be integrated in the planning of trials in surgical oncology, and "minimal residual disease" should receive stronger attention as a stratification criterion in such clinical studies.

Prognostic significance of activated CD8(+) T cell infiltrations within esophageal carcinomas.

The purpose of this study was to explore whether there is a linkage between the infiltration of CD8(+) T cells and the risk of death from esophageal cancer. Cases of 70 consecutive patients in whom esophageal squamous cell carcinomas ( n = 33) or adenocarcinomas (n = 37) were R0-resected between 1993 and 1999 were reviewed. The presence of activated CD8(+) T cells was evaluated by quantitative real-time PCR and immunohistochemistry and compared to clinical and pathological stages. The primary end point analyzed was overall survival, and a multivariate analysis was performed to distinguish any factors conferring an improved survivorship. Intratumoral (i.t.) CD8(+) T cells accumulating within the epithelial complexes were detected in 11 of all (16%) cases: in 9 of 25 (36%) patients with Union Internationale Contrele Cancer stage I or II versus 2 of 45 (4%) patients with Union Internationale Contrele Cancer stage III or IV (P = 0.001). Intratumoral CD8(+) T cell infiltrations showed proliferative activity and also IFN-gamma secretion. The presence of i.t. CD8(+) T cell infiltration more than peritumoral infiltration was associated with a good prognosis in both squamous cell and adenocarcinomas. Multivariate analysis showed that i.t. CD8(+) T cell infiltration was an independent prognostic factor (hazard ratio, 0.5; P = 0.0004) indicating favorable outcome. In conclusion, the presence of CD8(+) T cell infiltration in esophageal carcinomas is a favorable prognostic factor that should have diagnostic and therapeutic implications.

GLI gene expression in bone and soft tissue sarcomas of adult patients correlates with tumor grade.

The GLI gene encodes a transcription factor harboring five zinc finger motifs that bind to DNA in a sequence-specific manner. The gene was originally identified because of its amplification in a human glioblastoma, and previous studies have shown it to be amplified in a significant proportion of mesenchymal tumors, such as childhood sarcomas. Here we evaluate GLI gene expression in bone and soft tissue sarcomas of adult patients. Samples from 40 patients (37 sarcomas and 3 benign mesenchymal tumors) and samples of 15 normal mesenchymal tissues were examined for GLI gene amplification and expression by Southern hybridization, reverse transcription-PCR of tissue RNA, and immunohistochemistry, using a new polyclonal GLI antibody developed against an epitope outside of the zinc finger region. In contrast to childhood sarcomas, amplification of the GLI gene was not observed in sarcomas of adult patients. Although GLI gene expression in sarcomas was significantly higher than that in normal mesenchymal tissues (P < 0.0001), the levels were very variable. Attempts to correlate the expression data with different pathophysiological parameters only showed a significant relationship to tumor grade. Based on these data, increased levels of GLI gene expression may be indicative of the aggressiveness of the tumor.

Overexpression of sialyltransferase CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-Sialyltransferase is related to poor patient survival in human colorectal carcinomas.

Thomsen-Friedenreich (TF)-related blood group antigens, such as TF, Tn, and their sialylated variants, belong to a family of tumor-associated carbohydrates. The aim of the present study was to examine tumor-associated alterations of glycosyltransferases involved in the biosynthesis of the TF glycotope in colorectal carcinomas. To this end, glycosyltransferase expression was examined in 40 cases of colorectal carcinoma specimens classified according to the WHO/Union International Contre Cancer guidelines and in "normal" mucosa of the same patients. Occurrence of TF glycotope was examined by immunohistochemistry with the monoclonal antibody A78-G/A7. Expression of sialyltransferases CMP-sialic acid:Galbeta1,3GalNAc-R alpha3-sialyltransferase I and II (ST3Gal-I and ST3Gal-II) and CMP-sialic acid:Galbeta1,3GalNAc-R alpha6-sialyltransferase (ST6GalNAc-II) and of core 2 beta1,6-N-acetylglucosaminyltransferase was determined by reverse transcription-PCR in the same cryostat sections used for immunohistochemistry. Additionally, alpha2,3-sialyltransferase enzyme activity was studied in each of these tissues. The TF glycotope was detected in 7% of the normal mucosa, but in 57% of the carcinoma samples. Expression of alpha2,3-sialyltransferases ST3Gal-I, ST3Gal-II, and enzyme activity of alpha2,3-sialyltransferase was significantly increased (P < 0.001) in carcinoma specimens compared with normal mucosa. ST3Gal-I mRNA expression was significantly increased (P = 0.05) in cases showing invasion of lymph vessels. Expression of ST6GalNAc-II was significantly increased (P = 0.04) in cases with metastases to lymph nodes along the vascular trunk. Moreover, ST6GalNAc-II expression provides an prognostic factor for patient survival (log rank, P = 0.02). In an attempt to study the functional relevance of the glycosyltransferases for TF biosynthesis, SW480 colorectal cells were transfected with each of the enzymes, and cell surface expression of the TF glycotope was examined by flow cytometry. The presence of TF was not altered by transfection of the cells with either sialyltransferase ST3Gal-I or ST3Gal-II. However, successful transfection with core 2 beta1,6-N-acetylglucosaminyltransferase led to reduced expression of TF. In contrast, increased cell surface expression of TF was found after ST6GalNAc-II transfection. Thus, expression of TF on the cell surface of SW480 colorectal carcinoma cells depends on the ratio of core 2 beta1,6-N-acetylglucosaminyltransferase and ST6GalNAc-II. Earlier immunohistological studies demonstrated that TF is a prognostic factor for patient survival. Our results suggest that sialyltransferase ST6GalNAc-II is of crucial relevance for the prognostic significance of TF.

Protoporphyrin IX occurs naturally in colorectal cancers and their metastases.

Colorectal cancers exhibit a red fluorescence. The nature of the responsible fluorophore and its eventual diagnostic potential were investigated. Thirty-three consecutive colorectal resection specimen, 32 of which with histologically confirmed cancer, and a total of 1053 palpable mesenteric nodes were fluorimetrically characterized ex vivo. Furthermore, frozen material from 28 patients was analyzed, selected for the availability of primary tumor material and metastatic tissue, e.g., lymphatic and liver metastases from the same patient. Biochemical characterization was carried out through chemical extraction and reversed phase high-performance liquid chromatography. The fluorescence spectra of tissues, tissue extracts, and standard solutions of porphyrins were determined using a pulsed solid-state laser system for excitation and an imaging polychromator, together with an intensified CCD camera for time-delayed observation. Protoporphyrin IX (PpIX) was identified as the predominant fluorophore in primary tumors and their metastases. The fluorophore occurred in the absence of necrosis and in sterile locations. In untreated cases (n = 24), PpIX fluorescence discriminates metastatically involved lymph nodes from all other palpable nodes with a sensitivity of 62% at a specificity of 78% (P < 0.0001). After neoadjuvant treatment of rectal cancer, the PpIX fluorescence level of the primary tumors was reduced and a discrimination of lymph nodes based on PpIX-fluorescence was impossible. We conclude that colorectal cancer metastases accumulate diagnostic levels of endogenous PpIX as a result of a tumor-specific metabolic alteration.