BACKGROUND:This phase I dose-escalation study was designed to determine the maximum tolerated dose (MTD) and recommended dose of cetuximab administered on an every-second-week schedule to patients with metastatic colorectal cancer, on the basis of safety, pharmacokinetic and pharmacodynamic evaluation.PATIENTS AND METHODS:The study comprised two parts: a 6-week cetuximab monotherapy dose-escalation phase and a subsequent combination therapy phase, during which patients received cetuximab, at the same dose/schedule as in the monotherapy phase, followed by irinotecan plus infusional 5-fluorouracil/folinic acid (FOLFIRI). Patients in the control group received cetuximab as a 400 mg/m(2) initial dose, then 250 mg/m(2)/week and in the dose-escalation group, at 400-700 mg/m(2), every second week.RESULTS:Sixty-two patients were included in the study. The MTD of cetuximab administered on an every-second-week schedule was not reached. The safety profiles were similar across all groups. Response rates in the cetuximab monotherapy and combination therapy phases were 15% and 42%, respectively. Trough levels for the 500, 600 mg/m(2) and standard weekly regimens were comparable.CONCLUSION:Cetuximab can be safely administered once every second week at doses between 400 and 700 mg/m(2), with 500 mg/m(2) being the most convenient and feasible dose for future studies.

4000 Background: Efficacy analyses of the randomized phase II OPUS trial have previously failed to show significant improvements in progression-free survival time (PFS) or overall response, although a significantly higher response rate was achieved in patients with good performance status (ECOG 0/1) and a significantly higher curative surgery rate for cetuximab added to FOLFOX versus FOLFOX alone in the first-line treatment of mCRC was observed. KRAS mutation status has been related to the efficacy of anti-epidermal growth factor receptor (anti-EGFR) targeted therapies in different cancer models. Efficacy analyses have been repeated to evaluate the influence of KRAS mutation status in first-line patients treated with standard therapy, with or without cetuximab, under controlled study conditions. Methods: Genomic DNA was isolated from archived tumor material. The KRAS mutation status of codons 12/13 was determined using a sensitive, quantitative PCR-based assay. Best overall response and PFS time (IRC evaluation) are presented by KRAS mutation status. Results: In general, the population with tissue available for KRAS analysis (n=233) was representative of the overall intention- to-treat population (n=337) in terms of demographic and efficacy parameters. KRAS mutations were detected in 42% (99/233) of evaluable samples. Conclusions: These data suggest that the benefit from addition of cetuximab to standard treatment is higher for the population with wild-type KRAS. For patients with KRAS mutations, no benefit could be shown of adding cetuximab to FOLFOX in this study. PFS and overall response rate (RR) by KRAS mutation status KRAS status Median PFS (mo) Cetuximab + FOLFOX Median PFS (mo) FOLFOX Overall RR (%) Cetuximab + FOLFOX Overall RR (%) FOLFOX Wild-type 7.7 (n=61) 7.2 (n=73) HR:0.57 p=0.02 61 (n=61) 37 (n=73) p=0.01 Mutation 5.5 (n=52) 8.6 (n=47) HR:1.83 p=0.02 33 (n=52) 49 (n=47) p=0.11 HR, hazard ratio. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Merck Serono Merck Merck, Merck Serono

The formation of new bloodvessels, called angiogenesis, is critical for a tumour to grow beyond a few mm3 in size. A provisional matrix promotes endothelial cell adhesion, migration, proliferation and survival. Synthesis and degradation of this matrix closely resemble processes that occur during coagulation and fibrinolysis. Degradation of the matrix and fibrinolysis are tightly controlled and balanced by stimulators and inhibitors of the plasminogen activation system. Here we give an overview of these processes during tumour progression. We postulate a novel way to inhibit angiogenesis by removal of the matrix through specific and localised overstimulation of the plasminogen activation system.

Filiform serrated adenoma (SA) is an uncommon type of polyp that shows morphological features similar to traditional serrated adenoma (TSA). Unlike TSA, filiform SA is composed predominantly of prominent, thin, elongated filiform projections lined by neoplastic epithelium with a serrated contour. However, the molecular pathogenesis underlying filiform SA is unclear and its relationship with TSA has not been explored yet. The purpose of this study was to determine the clinicopathological and molecular characteristics of filiform SA in a cohort of Korean patients.Thirteen filiform SAs were evaluated for mutations of BRAFand KRAS genes, microsatellite instability (MSI), and promoter hypermethylation of hMLH1, MGMT, p16, MINT1, MINT2, MINT31 and the APC genes. The clinicopathological and molecular results were compared to results from previously published studies of left-sided TSAs among Koreans.All but one filiform SAs were located in the left colon and showed low grade dysplasia. BRAF and KRAS mutations were observed in six (46.2%) and four (30.3%) filiform SAs, respectively. Hypermethylation of hMLH1 (using both Herman et al. and Park et al.), MGMT, p16, MINT1, MINT2, MINT31 and the APC gene was found in 30.3% and 7.7%, 38.5%, 15.4%, 53.8%, 46.2%, 38.5% and 15.4% of cases, respectively. Thirteen filiform SAs were MS stable and classified with a CpG island methylator phenotype (CIMP) of high in five, CIMP low in five and CIMP negative in three cases. Compared to TSAs in the left colon, methylation of hMLH1, APC, and MGMT was less frequent in cases of filiform SA, but the filiform SA sizes were larger.Our findings suggest that filiform SA may grow larger without acquisition of additional genetic alterations and can be categorised as a rare, less aggressive variant of TSA with unique morphology.

Clinical trials in irritable bowel syndrome are associated with high placebo response rates. We aimed to identify the magnitude of the placebo response and the contributing factors to this occurrence.We did a systematic review and meta-analysis with a search of MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials between April 1, 1959, and April 30, 2020. We included all randomised controlled trials that compared an active pharmacotherapeutic agent with placebo and had a dichotomous outcome of response to therapy (in terms of global improvement or improvement in abdominal pain) in adults (aged ≥18 years) with irritable bowel syndrome. Exclusion criteria were trials reporting on treatment satisfaction as a dichotomous outcome of response to therapy or clinician-reported outcomes and a treatment duration of less than 4 weeks. Our main outcome was identification of the magnitude of the pooled placebo response rate for the following endpoints: global improvement, abdominal pain, and US Food and Drug Administration (FDA) endpoints. We extracted information from published reports and pooled proportions through meta-analysis with random effects. The study was registered with PROSPERO, CRD42020170908.Of the 6863 publications identified, 70 articles describing 73 randomised controlled trials were included in our analysis. The pooled placebo response rate was 27·3% (95% CI 24·3–30·9) using the global improvement endpoint, 34·4% (31·2–37·8) using the abdominal pain endpoint, and 17·9% (15·2–21·0) using the composite FDA endpoint responder definition, all with substantial heterogeneity between the trials. Studies published before 2006, and those done in Europe, with a parallel design, a run-in period of 2 weeks or less, a dose schedule of three times a day or more, or a smaller sample size of the control group were significantly associated with an increased pooled placebo response rate.More than a quarter of patients with irritable bowel syndrome had a placebo response in terms of global improvement, with multiple associated moderators. We recommend future trials apply a run-in period of at least 2 weeks and dose once or twice a day to minimise the placebo response rate.None.

Introduction: Tumour growth is dependent upon the balance of positive and negative regulators of angiogenesis. Antiangiogenic compounds such as antiangiogenic Antithrombin III (aaAT III) inhibit endothelial cell biology in vitro and angiogenesis in vivo. Until now it is unknown whether coagulation active Antithrombin (AT III) is effective as an antiangiogenic inhibitor. The aim of the study was to determine the effect of coagulation active AT III (Kybernin © ) on the growth of human pancreatic cancer in a mouse model (SCID). Methods: Human pancreatic cancer cell lines (BxPC3 and AsPC-1) were injected subcutaneously (s. c.) into the dorsa of male, immunodeficient (SCID) mice. When tumour volume was 100 mm 3 , mice were randomized into each five groups (n = 8/group) receiving Kybernin © (s. c.) (10, 50, 100, 200 mg/kg /day with n = 8 /group), latent AT III (antiangiogenic AT III) (100 mg/kg/day with n = 4/group) injections or Buffer (n = 5/group) as control for 19 days. Tumours were measured every 3–5 days and the tumour inhibition rate was determined for the last time point. Inhibitory effects of Kybernin © versus native (purified) AT III (0.1–20 mg/ml) on proliferation and migration was measured using micro- and macrovascular endothelial cell assays (BCE and HUVEC). Statistical analysis was performed. Results: Tumour inhibition rate (%) for BxPC-3 (slow growing tumour) was 69 % for 10 mg/kg/day, 90 % for 50 mg/kg/day 81 % for 100 mg/kg/day Kybernin © and 80 % for latent AT III (100 mg/kg/day). The inhibition of tumour growth was statistically significant in all treatment groups compared to placebo. For the fast growing tumour (AsPC-1) the inhibition rate was 44 % for 50 mg/kg/day, 46 % for 100 mg/kg/day, 56 % for 200 mg/kg/day Kybernin © and 65 % for latent AT III (100 mg/kg/day). The most effective dose with 90 % inhibition in the BxPC-3 model was 50 mg/kg/day. The same dosage in the AsPC-1 model leads to only 44 % inhibition. All mice gained weight throughout the study. A significant and dose dependent inhibition was observed in vitro for Kybernin © compared to native (purified) AT III in the migration and proliferation assay. Discussion: Coagulation active AT III (Kybernin © ) revealed significant inhibitory effects in vivo and in vitro. Depending on the angiogenic phenotype and the ability of different tumours to cleave native AT III by secreted enzymes to antiangiogenic AT III an effective tumour therapy with AT III (Kybernin © ) in combination with chemo- and/or radiation therapy or with other antiangiogenic

3554 Background: Response to the epidermal growth factor receptor (EGFR) inhibitor, cetuximab appears to correlate with the intensity of the associated skin reaction. This randomized multicenter study investigated the effect of cetuximab dose escalation on EGFR activation and downstream signaling in skin and tumor biopsies, on activity and skin reactions in patients (pts) with EGFR-expressing mCRC, failing prior irinotecan (I). Methods: Pts with ≥1 measurable lesion suitable for biopsy were eligible and were randomized 22 days after starting treatment with cetuximab (initial dose 400mg/m2 then 250mg/m2/week(w)) plus I (2-weekly regimen 180 mg/m2) if they had not experienced >grade 1 skin reaction, any other >grade 2 cetuximab-related toxicity and were not intolerant to I. Pts were randomized to standard cetuximab dose (arm A; 250 mg/m2/w) or dose-escalation (arm B; cetuximab dose increased by 50 mg/m2 q 2 w up to 500 mg/m2, until >grade 2 toxicity or tumor response). Pts not randomized (arm C) continued with the standard regimen after day 22. Skin and tumor biopsies were taken pre- and during treatment. Results: Of 273 screenedpts, 221 were EGFR-expressing and 166 were recruited. 45 pts were randomized to arm A, 44 to arm B and 76 were in arm C. By August 2005, 18 pts had reached the highest dose (500mg/m2/w), with no concerns over safety. All baseline and second biopsies are available. Safety results are presented for the first 36 pts in arm B. M/F 23/13, median age 60 y (34–79), median Karnofsky performance score 90 (80–100). All pts reported here had received cetuximab for at least 6 weeks. Maximum dose reached at w 6: 350mg/m2/w (300mg/m2 in 36 pts at w 4; 350mg/m2 in 31 pts at w 6). Relevant grade 3/4 toxicities were diarrhea (2.8%) and fatigue (5.6%). No grade 3/4 skin reactions occurred. Conclusions: Cetuximab at a dose of 350 mg/m2 is well tolerated in pts who have had no or mild skin reactions in response to standard dose treatment. Further dose escalation is possible. No early increase of skin toxicity is observed. Analysis of efficacy and pharmacokinetics/dynamics is ongoing. [Table: see text]

3006 Background: C and G are two anti-EGFR agents with different mechanisms of action. We had previously shown a synergistic effect combining the two agents in preclinical models (Matar et al, Clin Cancer Res 2004). This study aims to explore safety, PK and PD changes in tumor and skin at different doses of C/G to define the recommended dose (RD) for further development. Methods: pts were treated at the RD of weekly iv C (400 mg/m 2 initial dose, 250 mg/m 2 weekly) and oral daily G (250 mg/d) as single agents (5 pts each) and in successive cohorts of combined C/G (3–6 pts each): C (320/200) / G (100), C (400/250) / G (100), C (400/250) / G (250), C (320/200) / G (500) & C (400/250) / G (500) (ongoing). Dose escalation depended on dose limiting toxicity (DLT) rate during the first 28 d. Pre- & on-treatment steady-state (14 d) tumor & skin biopsies were obtained and have been evaluated by IHC for total (t) and phospho (p)-EGFR, p-MAPK, p-Akt, proliferation (Ki67), p27 expression and apoptosis by TUNEL. Gene profiling analysis is ongoing. Results: 35 pts have been treated so far: 20 CRC, 13 HNC & 2 NSCLC; median KI 90 % (70–100); median age 60 years (38–80); 24 males, 11 females. DLTs occurred in 3 pts: 1 pt with G alone with reversible ILD; 1 pt with G (250) / C (400/250) with reversible deafness & 1 pt with G (500) / C (320/200) with grade 3 anorexia and nausea. There were 1 CR (HNC) and 5 PRs (CRC) in the C/G cohorts, and 1 PR (CRC) in the C cohort. Overall, 5 out of 9 (56%) pts with CRC treated in the C/G cohorts presented a PR. PD studies show superior inhibition of p-EGFR, p-MAPK and p-Akt, reduction of proliferation and increased apoptosis (all p values <0.05) in the tumors of pts treated with C/G compared with the single agent cohorts. PK evaluation shows no PK interactions with the 2 drugs. Conclusions: This combination of an anti-EGFR MAb (C) and a TKI (G) is feasible at the RD of both agents. Our findings show encouraging clinical activity, especially in CRC, and superior PD signaling inhibition with the combination without any significant PK interaction. Combined anti-EGFR therapy deserves further evaluation. [Table: see text]

We show here that recombinant endostatin protein has a biphasic effect on the inhibition of endothelial cell migration in vitro. In tumor-bearing animals, there is a similar biphasic effect on the inhibition of tumor growth and on circulating endothelial cells after once-daily s.c. injections. This biphasic effect is revealed as a U-shaped curve in which efficacy is optimal between very low and very high doses depending on the tumor type. This result may be applicable to other inhibitors of endothelial growth and to angiogenesis. Furthermore, these results have important implications for clinicians who administer angiogenesis inhibitors for cancer or other angiogenesis-dependent diseases. When these results are taken together with two previous reports of angiogenesis inhibitors with a U-shaped dose-response, they suggest that other regulators of endothelial growth may display a similar pattern.

3580 Background: The EPIC trial is a randomized phase III study comparing cetuximab plus irinotecan to irinotecan in 2nd-line metastatic, EGFR-expressing colorectal cancer (CRC) patients (pts). A pre-planned safety assessment of the first 400 randomized patients was performed. Methods: Patients with EGFR-expressing mCRC who had failed 1st-line oxaliplatin in combination with a fluoropyrimidine therapy with an ECOG performance status ≤ 2 were randomized to either Arm A (cetuximab 400 mg/m2 followed by 250 mg/m2 weekly dose and irinotecan 350mg/m2 q 3 weeks) or to Arm B (irinotecan 350mg/m2 q 3 weeks). A pooled analysis of safety is presented. Results: Four hundred pts were randomized from May 2003 to July 2004 in the US, Europe and Australia, 162 women and 238 men, with a median age of 61 years. Eleven patients did not receive study therapy and are excluded from the analysis. A total of 1493 cycles were administered to the 389 pts with a median of 3 cycles per pt (range 1–18). Four (1%) severe infusion reactions (IR) occurred. All were grade 3 and none were fatal. In this pooled analysis, 24 deaths occurred within 30 days of last study therapy: 17 were disease-related, 2 were study drug- related (both due to neutropenia), 4 were classified as “other causes” and 1 was “unknown”. A summary table of adverse events (AE) is below. Conclusions: In this pooled analysis the characteristic toxicities of cetuximab and irinotecan do not appear to be increased. The incidence and severity of these events appear comparable to previous reports of cetuximab with chemotherapy or of irinotecan. The study is continuing to accrue to its target of 1300 patients. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bristol-Myers Squibb Bristol-Myers Squibb Aventis, Merck, Pfizer

We have isolated a selectively deglycosylated form of vitamin D binding protein (DBP-maf) generated from systemically available DBP by a human pancreatic cancer cell line. DBP-maf is antiproliferative for endothelial cells and antiangiogenic in the chorioallantoic membrane assay. DBP-maf administered daily was able to potently inhibit the growth of human pancreatic cancer in immune compromised mice (T/C=0.09). At higher doses, DBP-maf caused tumor regression. Histological examination revealed that treated tumors had a higher number of infiltrating macrophages as well as reduced microvessel density, and increased levels of apoptosis relative to untreated tumors. Taken together, these data suggest that DBP-maf is an antiangiogenic molecule that can act directly on endothelium as well as stimulate macrophages to attack both the endothelial and tumor cell compartment of a growing malignancy.

Mitochondria are the powerhouse of the cell and their disruption leads to cell death. We have used a peptide trivalent arsenical, 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO), to inactivate the adenine nucleotide translocator (ANT) that exchanges matrix ATP for cytosolic ADP across the inner mitochondrial membrane and is the key component of the mitochondrial permeability transition pore (MPTP). GSAO triggered Ca(2+)-dependent MPTP opening by crosslinking Cys(160) and Cys(257) of ANT. GSAO treatment caused a concentration-dependent increase in superoxide levels, ATP depletion, mitochondrial depolarization, and apoptosis in proliferating, but not growth-quiescent, endothelial cells. Endothelial cell proliferation drives new blood vessel formation, or angiogenesis. GSAO inhibited angiogenesis in the chick chorioallantoic membrane and in solid tumors in mice. Consequently, GSAO inhibited tumor growth in mice with no apparent toxicity at efficacious doses.

Angiostatin is a potent inhibitor of angiogenesis generated in cancer-bearing hosts by tumor-derived proteases. Because the naturally occurring bone and prostate cancers of pet dogs provide unique model systems to study factors that regulate cancer progression and tumor dormancy, we investigated the capacity of these tumors to generate angiostatin. We determined that angiostatin fragments are present in urine of dogs with bone cancer. The identity of these fragments was confirmed by comparison of the experimentally determined protein sequence to that of a clone of canine angiostatin. Importantly, these fragments were absent in urine collected from the same dogs after complete surgical removal of the primary tumor. We also demonstrate that canine prostate cancer cells are capable of processing plasminogen to angiostatin in vitro. These findings provide rationale for using spontaneous canine tumor models to isolate endogenous angiogenesis inhibitors and to investigate their therapeutic use against cancer.

Background. Because pancreatic adenocarcinoma is poorly responsive to chemotherapy and radiation therapy, novel treatments such as antiangiogenic gene therapy may have use in the adjuvant treatment of this malignancy. We evaluated the antitumor effects of the in vivo administration of an adenovirus vector encoding a soluble form of Flk1 (Flk1-Fc), a receptor for vascular endothelial growth factor, in 3 murine models of pancreatic adenocarcinoma. Methods. In a first model, immunocompetent C57Bl/6 mice were injected subcutaneously with Panc02 murine pancreatic adenocarcinoma cells before treatment. In a second model, immunodeficient severe combined immunodeficiency mice were injected subcutaneously with BxPc-3 human pancreatic adenocarcinoma cells before treatment. In a third model, C57Bl/6 mice were injected with Panc02 cells through an intrasplenic route before treatment, in an effort to model metastatic disease. In each model, half the tumor-bearing mice were injected intravenously with 109 Flk1-Fc adenovirus particles and half with control adenovirus. Results. In subcutaneous tumor models, Ad Flk1-Fc-treated animals were found to have 75% smaller murine and 78% smaller human pancreatic tumor volumes, relative to tumor volumes of Ad Fc-treated animals, 6 weeks after vector administration. In animals injected with tumor through the intrasplenic route, pathologic and histologic analyses made 10 days after injection of tumor revealed hepatic, pancreatic, and splenic tumors, together with a desmoplastic response consistent with pathologic findings in human pancreatic cancer. Cohorts of these tumor-bearing mice treated with Ad Flk1-Fc demonstrated significantly longer survival and decreased liver replacement with tumor at the time of death, relative to animals treated with Ad Fc. Conclusion. A recombinant adenovirus encoding soluble Flk-1 inhibited pancreatic tumor growth in mice. These studies suggest that the delivery of gene products such as Flk1-Fc through in vivo gene transfer may be useful in the future treatment of patients with pancreatic cancer. (Surgery 2002;132:857-65.)