SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival.

MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I-III), and survival time was analyzed by Kaplan-Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC metastasis in mice. In patients, SPON2 serves as prognostic indicator for CRC metastasis and survival, and might represent a promising target for therapeutic approaches.

Local recurrence rates after radiofrequency ablation or resection of colorectal liver metastases. Analysis of the European Organisation for Research and Treatment of Cancer #40004 and #40983.

AIM: The aim of this study is to describe local tumour control after radiofrequency ablation (RFA) and surgical resection (RES) of colorectal liver metastases (CLM) in two independent European Organisations for Research and Treatment of Cancer (EORTC) studies. BACKGROUND: Only 10-20% of patients with newly diagnosed CLM are eligible for curative RES. RFA has found a place in daily practice for unresectable CLM. There are no prospective trials comparing RFA to RES for resectable CLM. METHODS: The CLOCC trial randomised 119 patients with unresectable CLM between RFA (±RES)+adjuvant FOLFOX (±bevacizumab) versus FOLFOX (±bevacizumab) alone. The EPOC trial randomised 364 patients with resectable CLM between RES±perioperative FOLFOX. We describe the local control of resected patients with lesions ≤4 cm in the perioperative chemotherapy arm of the EPOC trial (N=81) and the RFA arm of the CLOCC trial (N=55). RESULTS: Local recurrence (LR) rate for RES was 7.4% per patient and 5.5% per lesion. LR rate for RFA was 14.5% per patient and 6.0% per lesion. When lesion size was limited to 30 mm, LR rate for RFA lesions was 2.9% per lesion. Non-local hepatic recurrences were more often observed in RFA patients than in RES patients, 30.9% and 22.3% respectively. Patients receiving RFA had a more advanced disease. CONCLUSIONS: LR rate after RFA for lesions with a limited size is low. The local control per lesion does not appear to differ greatly between RFA and surgical resection. This study supports the local control of RFA in patients with limited liver metastases. Future studies should evaluate in which patients RFA could be an equal alternative to liver resection.

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.

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.

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.

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.

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.

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.

A new measure to assess the difficulty of liver resection.

BACKGROUND: There is no valid measure to assess surgical difficulty and feasibility of a planned liver resection. It is the objective of this study to evaluate a mathematical measure from a 3D graphical analysis. METHODS: Eleven different 3D models of hepatic tumours were evaluated by experts for resectability and analysed with Amira graphic software taking into consideration the portal and hepatic venous vascular relationships. Virtual resection volumes with increasing resection margins from 1 to 30 mm were determined separately for portal veins, hepatic veins, their intersections and volume unions. The integral of the increasing resection volumes was defined as risk coefficient. The risk coefficients from this volumetric analysis were compared with the expert opinion. RESULTS: The risk coefficient based on the integral of portal venous and hepatic venous volume unions reproduced the expert opinion highly significantly (correlation coefficient 0.9, p<0.05) and more accurately than volumetric analysis of the planned resection margin. CONCLUSION: With automated volumetric analysis, anatomically problematic situations in liver surgery can be reproduced and scaled. The risk coefficient obtained is a suitable objective measure for defining risk areas in liver surgery.

Lymphatic mapping and sentinel lymph node biopsy in epidermoid carcinoma of the anal canal.

AIM: Although 15-25% of patients with anal cancer present with superficial inguinal lymph node metastases but the routine application of groin irradiation is controversial because of serious side effects. Inguinal sentinel lymph node biopsy (SLNB) can be used to select patients appropriately for inguinal radiation. The study evaluates the efficiency and clinical impact of SLNB. METHODS: Forty patients with anal cancer underwent 1 ml Tc(99m)-Nanocolloid injection in four sites around the tumour. Patients with inguinal radio colloid enrichment were selected for sentinel lymph node biopsy (SLNB). Lymph node status was examined by haematoxylin and eosin (H&E) as well as immunohistochemistry-staining. All SLN-positive patients were scheduled for inguinal radiation; SLN-negative patients with T1 and early T2 tumours were not scheduled for inguinal radiation. RESULTS: SLN were detected in 36/40 patients. Three common patterns of lymphatic drainage were observed: mesenterial, iliacal and inguinal. Twenty patients with inguinal SLN underwent SLN-biopsy. 6/20 patients were SLN-positive. In 10/20 patients SLNB altered the therapy plan--four patients with T1-tumours and positive SLN had additional groin irradiation, whereas 6 patients with small T2-tumors and tumour-free inguinal SLN did not undergo inguinal irradiation. CONCLUSIONS: Inguinal sentinel node biopsy in anal cancer is efficient and could assist in the decision for inguinal radiation. The validity and safety of the proposed therapeutic algorithm has to be proven by a larger, prospective study.

Presence of mature DC-Lamp+ dendritic cells in sentinel and non-sentinel lymph nodes of breast cancer patients.

AIM: Our study examined differences in the presence of mature, DC-Lamp+ DC in the SLN and non-SLN according to the extent of metastatic involvement. PATIENTS AND METHODS: Paraffin blocks of the SLN and non-SLN from patients with primary breast cancer who had undergone SLN biopsy and axillary dissection were separated into three groups: (Group A) no tumor cell involvement in the SLN and non-SLN; (Group B) isolated tumor cells or micrometastases in the SLN, and tumor cell-free non-SLN; and (Group C) macrometastases in the SLN. One section of all the SLN and non-SLN was examined with immunohistochemistry using an anti-DC-Lamp-antibody. The densest area occupied by the DC-Lamp+ cells on each slide was quantified and recorded by an electronic imaging system. In this regard, the SLN and non-SLN were compared within the patients of each group using the Wilcoxon signed rank-test (p<0.05). RESULTS: One hundred and fourteen SLN and 1258 non-SLN from 79 patients were examined. A significantly larger area was occupied by the DC-Lamp(+) cells in the SLN compared to the non-SLN in Groups A (p=0.024) and B (p=0.009), whereas no significant difference was found within Group C (p=0.107). CONCLUSIONS: This study suggests that the DC-dependent immune response is altered during the process of metastasis formation and is primarily activated before and during formation of micrometastasis.

Markers of tumour angiogenesis and tumour cells in bone marrow in gastric cancer patients.

AIMS: Vascular endothelial growth factors VEGF-A, VEGF-C and VEGF-D are considered to be potentially angiogenetic and lymphangiogenetic. "Minimal residual disease" is responsible for cancer progression and recurrence. In this study, we investigated the relation between expressions of VEGF-A, VEGF-C and VEGF-D in gastric cancer tissue and the presence of tumour cells in bone marrow. METHODS: A total of 50 resected primary gastric adenocarcinomas, 44 non-cancerous gastric mucosa and 36 lymph node metastases were analyzed by immunohistochemistry for VEGF-A, VEGF-C and VEGF-D. The specimens used were drawn from a previous study cohort, where the presence of ITC in bone marrow was confirmed with immunohistochemical assay with cytokeratin (CK)-18. RESULTS: The levels of expression of VEGF-A, VEGF-C and VEGF-D were highest in tumour (p < 0.001), and the level in lymph node metastases was significantly higher (p < 0.01) than in mucosa. The expression of VEGF-A was correlated significantly with venous tumour invasion (p < 0.05) and the presence of tumour cells in bone marrow (p < 0.05). Tumours expressing high levels of VEGF-D showed significantly advanced stages of tumour infiltration (p < 0.05) and lymph node metastasis (p < 0.01). CONCLUSIONS: VEGF-A is a significant marker for the presence of tumour cells in the bone marrow of gastric cancer patients. Our results confirm VEGF-D as a predictor for the lymphatic spread of tumour cells. Therefore, the route of metastatic spread of gastric cancer could be determined, at least in part, by the profile of VEGF family members expressed in the primary tumour of gastric cancer patients.

The location of small tumor deposits in the SLN predicts Non-SLN macrometastases in breast cancer patients.

AIMS: The extent to which the location of micrometastases (MIC) or isolated tumor cells (ITC) in sentinel lymph nodes (SLNs) is correlated with the risk of downstream metastases is still unknown. This study examined this issue and compared the impact of MIC/ITC location with other established risk factors. METHODS: Paraffin slides of SLNs with MIC/ITC-involvement obtained from 68 breast cancer patients were evaluated for MIC/ITC location, lesion size, and various SLN morphologic features. These parameters, together with demographic data and primary tumor characteristics, were analyzed using univariate and multivariate analysis to determine their association with the presence of downstream macrometastases in Non-SLN. RESULTS: Eighteen of 68 patients with MIC (n=37) or ITC (n=31) had Non-SLN metastases. After multivariate analysis, the location of MIC/ITC in the SLN (parenchyma vs. sinus/vessel) had the strongest association with the presence of Non-SLN macrometastases (p<0.0001), followed by the pT-category (p=0.008). Sixteen of 18 patients with parenchymal involvement but only 2 of 31 without parenchymal involvement had Non-SLN macrometastases. The metric size of the primary tumor and the estrogen receptor status were significantly associated only on univariate analysis (p=0.041, 0.034), whereas the correlation to the size classification for tumor cell deposits (MIC vs. ITC) was not significant (p=0.077). CONCLUSIONS: The results indicate that lesion location is an important predictor of Non-SLN-macrometastases. This finding may simplify the decision for axillary treatment in patients with small tumor deposits in the SLN.

Development and validation of a three dimensional ultrasound based navigation system for tumor resection.

BACKGROUND: Intraoperative navigation is a rapidly emerging procedure in orthopaedic surgery and neurosurgery. For abdominal tumors (e.g. liver metastasis) and soft tissue tumors there is only limited experience with navigation techniques due to problems of organ shift and tissue deformation. We have developed a navigation system for tumor resection in soft tissue based on 3D ultrasound imaging and optical tracking. METHODS: Two different modes of navigation were evaluated and compared with conventional surgery in an experimental soft tissue model. Both techniques were based on 3D ultrasound and an optical tracking system for intraoperative real time registration of surgical instruments. These two techniques were used: a) Indirect navigation with ultrasound guided insertion of a tracked hook needle into the tumor; and b) Direct navigation using a 3D image which was obtained with an optically tracked 3D ultrasound probe. It was the aim of both techniques to achieve a circumferential resection margin of 2cm around the tumor. RESULTS: A total of 23 resections were performed consisting of indirect (n=7) and direct (n=10) navigation and conventional surgery (n=6) as gold standard. For indirect navigation a median deviation from the ideal resection margin (accuracy) of 0.32cm was measured. Direct navigation showed an accuracy of 0.16cm compared to 0.42cm with conventional surgery. Navigated surgery showed for both techniques a significant increase of resection accuracy compared to conventional resection (p<0.05). CONCLUSION: 3D ultrasound based indirect and direct optoelectronic navigation for resection of soft tissue tumors is feasible and may improve intraoperative orientation with increased surgical precision.

Sentinel node biopsy in synovial sarcoma.

AIMS: To examine the relevance of sentinel node biopsy in patients with synovial sarcoma. METHODS: Between July 2004 and February 2007 11 consecutive patients with synovial sarcoma treated in our clinic underwent sentinel node biopsy after a preoperative lymphoscintigraphy. A handheld gamma-probe was used during the procedure to identify the sentinel nodes, which were then resected and submitted for histopathologic evaluation. RESULTS: At least one sentinel node was identified in every patient. Of a total of 15 sentinels, one was positive and 14 negative. The patient with the positive sentinel underwent a regional lymph node dissection and remains disease-free 17 months later. One patient developed regional nodal metastases despite negative sentinel node biopsy and died 12 months after the procedure. No biopsy-associated complications were observed. CONCLUSIONS: Sentinel node biopsy can be successfully and safely applied to patients with synovial sarcoma. Further prospective studies are required to determine the optimal treatment approach, the false negative rate and the prognostic significance of a positive sentinel node biopsy.

Sentinel lymph node biopsy for gastrointestinal cancers.

Sentinel lymph node biopsy (SLNB) in gastrointestinal-(GI)-tract cancer is not yet of clinical relevance. Nevertheless, the results in the upper GI-tract promise to be helpful to individualize the indication for surgical therapy. SLNB in colon cancer still fails to show high validity to predict the nodal status, but may be helpful to clarify the prognostic role of micrometastases/isolated tumor cells. In anal cancer SLNB is able to guide the indication for groin irradiation.

[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.