Perioperative FOLFOX4 chemotherapy and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC 40983): long-term results of a randomised, controlled, phase 3 trial.

BACKGROUND: Previous results of the EORTC intergroup trial 40983 showed that perioperative chemotherapy with FOLFOX4 (folinic acid, fluorouracil, and oxaliplatin) increases progression-free survival (PFS) compared with surgery alone for patients with initially resectable liver metastases from colorectal cancer. Here we present overall survival data after long-term follow-up. METHODS: This randomised, controlled, parallel-group, phase 3 study recruited patients from 78 hospitals across Europe, Australia, and Hong Kong. Eligible patients aged 18-80 years who had histologically proven colorectal cancer and up to four liver metastases were randomly assigned (1:1) to either perioperative FOLFOX4 or surgery alone. Perioperative FOLFOX4 consisted of six 14-day cycles of oxaliplatin 85mg/m(2), folinic acid 200 mg/m(2) (DL form) or 100 mg/m(2) (L form) on days 1-2 plus bolus, and fluorouracil 400 mg/m(2) (bolus) and 600 mg/m(2) (continuous 22 h infusion), before and after surgery. Patients were centrally randomised by minimisation, adjusting for centre and risk score and previous adjuvant chemotherapy to primary surgery for colorectal cancer, and the trial was open label. Analysis of overall survival was by intention to treat in all randomly assigned patients. FINDINGS: Between Oct 10, 2000, and July 5, 2004, 364 patients were randomly assigned to a treatment group (182 patients in each group, of which 171 per group were eligible and 152 per group underwent resection). At a median follow-up of 8·5 years (IQR 7·6-9·5), 107 (59%) patients in the perioperative chemotherapy group had died versus 114 (63%) in the surgery-only group (HR 0·88, 95% CI 0·68-1·14; p=0·34). In all randomly assigned patients, median overall survival was 61·3 months (95% CI 51·0-83·4) in the perioperative chemotherapy group and 54·3 months (41·9-79·4) in the surgery alone group. 5-year overall survival was 51·2% (95% CI 43·6-58·3) in the perioperative chemotherapy group versus 47·8% (40·3-55·0) in the surgery-only group. Two patients in the perioperative chemotherapy group and three in the surgery-only group died from complications of protocol surgery, and one patient in the perioperative chemotherapy group died possibly as a result of toxicity of protocol treatment. INTERPRETATION: We found no difference in overall survival with the addition of perioperative chemotherapy with FOLFOX4 compared with surgery alone for patients with resectable liver metastases from colorectal cancer. However, the previously observed benefit in PFS means that perioperative chemotherapy with FOLFOX4 should remain the reference treatment for this population of patients. FUNDING: Norwegian and Swedish Cancer Societies, Cancer Research UK, Ligue Nationale Contre Cancer, US National Cancer Institute, Sanofi-Aventis.

Activation of the CMV-IE promoter by hyperthermia in vitro and in vivo: biphasic heat induction of cytosine deaminase suicide gene expression.

The cytomegalovirus-immediate early (CMV-IE) promoter is widely used as a strong and constitutively active promoter. Although the CMV-IE promoter does not harbor heat-responsive sequences, we determined its heat inducibility. We analyzed in vitro and in vivo heat responsiveness and possible mechanisms of heat induction of the CMV-IE promoter. We used transfected SW480 human colon carcinoma cells (SW480/CMVCD), expressing CMV-IE promoter-driven bacterial cytosine deaminase (CD) gene. These cells were heated at 42 degrees C. The SW480/CMVCD cells were also used for in vivo studies, in which tumor-bearing animals were treated with hyperthermia at 41.5 degrees C. As controls, SW480 (SW480/HSPCD) cells were used, in which CD expression is driven by the HSP70-promoter. In vitro, we observed a biphasic, up to 25-fold heat induction of CMV-IE-driven CD expression after hyperthermia in SW480/CMVCD cells. In vivo, we found a 2.5-fold induction of CD expression after hyperthermia in SW480/CMVCD tumor-bearing animals. The analysis of the CMV-IE promoter sequence revealed several transcription factor-binding sites, which mediate stress responsiveness. YB-1 and C/EBP-beta might mediate heat responsiveness of the CMV-IE promoter. These data point to limitations in heat-induction gene therapy studies, in which the CMV-IE promoter is used as control system. In addition, the CMV-IE promoter itself could well be used for construction of heat-inducible vectors.

Preoperative short-course radiotherapy versus combined radiochemotherapy in locally advanced rectal cancer: a multi-centre prospectively randomised study of the Berlin Cancer Society.

BACKGROUND: The additional use of radiotherapy has changed the treatment of locally advanced rectal cancer (LARC) dramatically. But a major achievement has been the development of total mesorectal excision (TME) as a surgical standard and the recognition that the surgeon is the predominant prognostic factor. The benefit of preoperative hypofractionated radiotherapy (SCRT; five fractions each of 5 Gy), initially established by the Swedish Rectal Cancer Trial, has been demonstrated in conjunction with TME by the Dutch Colorectal Cancer Group. The concept of combined neoadjuvant radiochemotherapy (conventional radiation of about 50 Gy with chemotherapy) has not been compared over surgery alone with TME. However, the German Rectal Cancer Study Group recently demonstrated that preoperative radiochemotherapy (RCT) was better than postoperative radiochemotherapy in terms of local control. METHODS AND DESIGN: Patients with histological proven rectal cancer staged T2N+ or T3 are randomized to receive either SCRT (25 Gy in five fractions of 5 Gy) plus TME-surgery within 5 days or RCT (50.4 Gy in 28 fractions of 1.8 Gy, continuous infusion 5-fluorouracil) plus TME-surgery 4-6 weeks later. All patients receive adjuvant chemotherapy (12 weeks continuous infusional 5-FU) and are followed up for 5 years. TME-quality is independently documented by the surgeon and the pathologist. Hypothesis of the study is that RCT is superior to SCRT in terms of local recurrence after five years. Secondary endpoints are overall survival, disease-free survival, complete resection rate (R0 resection), rate of sphincter saving resection, acute and late toxicity (radiation related side effects), and quality of life (including long term bowel function). DISCUSSION: Similar long-term survival, local control and late morbidity have been reported for both concepts of preoperative therapy in non-comparative studies. In addition to other ongoing (and recently published) comparative trials we include a larger number of patients for adequate power, apply quality-controlled TME and try to avoid the adjuvant treatment bias by mandatory adjuvant chemotherapy in both groups. Further more, stratification of the initially planned surgical procedure and sphincter-preservation will generate valid evidence whether RCT will allow a less aggressive (sphincter saving) surgical approach.

Heat-inducible in vivo gene therapy of colon carcinoma by human mdr1 promoter-regulated tumor necrosis factor-alpha expression.

The promoter of the human multidrug resistance gene (mdr1) harbors defined heat-responsive elements, which could be exploited for construction of heat-inducible expression vectors. To analyze the hyperthermia inducibility of the mdr1 promoter in vitro and in vivo, we used the pcDNA3-mdrp-hTNF vector construct for heat-induced tumor necrosis factor alpha (TNF-alpha) expression in transfected HCT116 human colon carcinoma cells at mRNA level by quantitative real-time reverse transcription-PCR and at protein level by TNF-alpha ELISA. For the in vitro studies, the pcDNA3-mdrp-hTNF-transfected tumor cells were treated with hyperthermia at 43 degrees C for 2 h. In the animal studies, stably transfected or in vivo jet-injected tumor-bearing Ncr:nu/nu mice were treated for 60 min at 42 degrees C to induce TNF-alpha expression. Both the in vitro and in vivo experiments show that hyperthermia activates the mdr1 promoter in a temperature- and time-dependent manner, leading to an up to 4-fold increase in mdr1 promoter-driven TNF-alpha expression at mRNA and an up to 3-fold increase at protein level. The in vivo heat-induced TNF-alpha expression combined with Adriamycin (8 mg/kg) treatment leads to the inhibition of tumor growth in the animals. These experiments support the idea that heat-induced mdr1 promoter-driven expression of therapeutic genes is efficient and feasible for combined cancer gene therapy approaches.

The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer.

BACKGROUND & AIMS: Activation of the Wnt/beta-catenin pathway is frequently observed in colorectal cancers. Our aim was to elucidate the impact of gain-of-function beta-catenin on the metastasis-associated gene S100A4 in human colon cancer cell lines and tumors. METHODS: We analyzed cell lines heterozygous for gain-of-function and wild-type beta-catenin, and variants homozygous for gain- or loss-of-function mutation in beta-catenin, for S100A4 expression, cell motility, and in vivo metastasis. beta-catenin-mediated S100A4 promoter activation was tested by reporter assays. For human colon carcinomas, S100A4 expression, beta-catenin genotype, and metachronous metastasis were correlated. RESULTS: We identified S100A4 as the most regulated gene by gain-of-function beta-catenin using a 10K microarray. Cell lines with gain-of-function beta-catenin expressed up to 60-fold elevated S100A4 levels, displayed strongly increased migration and invasion in vitro, and induced metastasis in mice. S100A4 small interfering RNA, beta-catenin small interfering RNA, or dominant negative T-cell factor (TCF) knocked down S100A4 and blocked biological effects. S100A4 complementary DNA transfection increased migration and invasion. We identified a TCF binding site within the S100A4 promoter and demonstrated the direct binding of heterodimeric beta-catenin/TCF complexes. Reporter assays confirmed the beta-catenin-induced S100A4 promoter activity. Furthermore, S100A4 mRNA expression was increased in primary colon cancers, which later developed distant metastases, compared to non-metastasizing tumors. Colon tumors heterozygous for gain-of-function beta-catenin showed concomitant nuclear beta-catenin localization, high S100A4 expression, and metastases. CONCLUSIONS: S100A4 is a direct beta-catenin/TCF target, induces migration and invasion in vitro and metastasis in vivo, and has value for prognosis of metastasis formation in colon cancer patients.

Genetic dissection of apoptosis and cell cycle control in response of colorectal cancer treated with preoperative radiochemotherapy.

BACKGROUND: In previous analyses we identified therapy-induced upregulation of the CDK inhibitor p21CIP/WAF-1 and consequently decreased tumor cell proliferation or loss of Bax as adverse factors for survival in rectal cancer treated with radiochemotherapy. Here, we address the individual role of p53 and its transcriptional targets, p21CIP/WAF-1 and Bax, on apoptosis induced by individual components of multimodal anticancer therapy, i.e. 5-fluorouracil (5-FU), ionising gamma-radiation (IR) and heat shock/hyperthermia. METHODS: We analysed tumor samples 66 patients with rectal carcinoma treated by a neoadjuvant approach with radiochemotherapy +/- heat shock/hyperthermia for the expression and mutation of p53 and the expression of p21CIP/WAF-1 and Bax. These data were correlated with the tumor response. The functional relevance of p53, p21CIP/WAF-1 and Bax was investigated in isogeneic HCT116 cell mutants treated with 5-FU, IR and heat shock. RESULTS: Rectal carcinoma patients who received an optimal heat shock treatment showed a response that correlated well with Bax expression (p = 0.018). Local tumor response in the whole cohort was linked to expression of p21CIP/WAF-1 (p < 0.05), but not p53 expression or mutation. This dichotomy of p53 pathway components regulating response to therapy was confirmed in vitro. In isogeneic HCT116 cell mutants, loss of Bax but not p53 or p21CIP/WAF-1 resulted in resistance against heat shock. In contrast, loss of p21CIP/WAF-1 or, to a lesser extent, p53 sensitized predominantly for 5-FU and IR. CONCLUSION: These data establish a different impact of p53 pathway components on treatment responses. While chemotherapy and IR depend primarily on cell cycle control and p21, heat shock depends primarily on Bax. In contrast, p53 status poorly correlates with response. These analyses therefore provide a rational approach for dissecting the mode of action of single treatment modalities that may be employed to circumvent clinically relevant resistance mechanisms in rectal cancer.

Response prediction by FDG-PET after neoadjuvant radiochemotherapy and combined regional hyperthermia of rectal cancer: correlation with endorectal ultrasound and histopathology.

Accurate response assessment after neoadjuvant therapy is essential in patients with rectal cancer. The aim of this study was to assess the value of fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) in predicting response of locally advanced rectal cancer to preoperative multimodal treatment. Twenty-two consecutive patients with locally advanced (uT3/4) primary rectal cancer were entered in this prospective pilot study. FDG-PET was performed before and after neoadjuvant radiochemotherapy (RCT) with combined regional hyperthermia (RHT). Treatment consisted of external-beam radiotherapy (45 Gy), chemotherapy (folinic acid and 5-fluorouracil) and regional pelvic hyperthermia followed by curative tumour resection 6-8 weeks later. Semi-quantitative measurements (SUV) of tumour FDG uptake were made before and 2-4 weeks after completion of neoadjuvant treatment. Two patients who did not receive post-therapeutic restaging by FDG-PET were excluded from the analysis. Results were correlated with findings on endorectal ultrasound (EUS, n=17 patients) and histopathology. Histopathological evaluation of the resected tumour revealed complete response in one patient, partial response in 12 and stable disease in seven. SUV reduction in tumours was significantly greater in responders than in non-responders [60% (+/-15%) vs 30% (+/-18%), P=0.003, CI=95%). Using a minimum post-therapeutic SUV reduction of 36% to define response, FDG-PET revealed a sensitivity of 100% (EUS: 33%) and a specificity of 86% (EUS: 80%) in response prediction; the corresponding positive and negative predictive values were 93% (EUS: 80%) and 100% (EUS: 33%), respectively. FDG-PET results were statistically significant (P<0.001, CI=95%). FDG-PET has great potential in the assessment of tumour response to neoadjuvant RCT in combination with RHT and is superior to EUS for this purpose.

[Diagnosis of tumors of the small intestine with the aid of CT contrast enema. Sellink CT technique evaluated in 63 patients]. / Diagnostik von Dünndarmtumoren mit Hilfe des CT-Enteroklysma. Die CT--Sellink ausgewertet bei 63 Patienten.

PURPOSE: Aim of the study was to compare the visualisation of small bowel tumours particularly lymphoma of the small bowel by enteroclysma, computed tomography (CT) and computed tomography following enteroclysma. METHODS: We examined 97 examinations in 63 patients. Non Hodgkin's Lymphoma was the primary malignancy in 44 patients, metastasis of different malignancies in 8 patients, primary malignancies of the small intestine in 5 patients, mesenteric tumours in 4 patients and postoperative stricture in another 2 patients. CT following enteroclysma (CT Sellink) was performed as helical CT in 55 patients and as incremental CT in 42 patients. Examinations were evaluated by two radiologists. Evaluation criteria were small bowel distension, perceptibility of details and topographic correlation. RESULTS: Manifestations of lymphoma were found in 32 patients, infiltration of bowel wall in 12 patients. In three patients metastases of melanoma were found. In three patients the suspicion for small bowel tumours was not verified in CT Sellink. The perceptibility of details was evaluated as ameliorated in 45.5% of examinations over all. CONCLUSIONS: CT Sellink offers remarkable advantages in the diagnosis of small bowel tumours compared with enteroclysma and "conventional" computed tomography under intra-venous and oral contrast media. CT Sellink was feasible over a time of 7 years now in clinical praxis. This examination represents an optimized standard in small intestine examination.