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To advance preclinical testing of novel targeted drugs in colorectal cancer (CRC) we established a panel of 133 mouse xenograft models from fresh tumor specimens of 239 patients with CRC of all four UICC stages. A subgroup of 67 xenograft models was treated with cetuximab, bevacizumab and oxaliplatin as single agents. Mutation status of KRAS (G12, G13, A146T), BRAF (V600E) and PIK3CA (E542K, E545K, H1047R) was assessed in all xenografts by allelespecific real-time PCR. KRAS codon 61 was assessed by conventional sequencing. AREG and EREG expression levels were analyzed by real-time PCR expression assays. In the treatment experiment we observed response rates of 27% (18/67) for cetuximab, 3% (2/67) for bevacizumab, and 6% (4/67) for oxaliplatin. Classification based on KRAS, BRAF and PIK3CA mutation status identified 15 of the responders (sensitivity 83%, confidence interval at p = 0.05 (CI): 59% - 96%), and 38 nonresponders (specificity 78%, CI: 63% - 88%). If any mutation except in KRAS codon 13 were considered, the classifier reached sensitivity of 94% and specificity of 69%. We improved specificity of the classifiers to 90% and 86% respectively by adding AREG and EREG RNA expression thresholds retrospectively. In patient-derived xenograft models, we found a predictive classifier for response to cetuximab that is more accurate than established biomarkers. We confirmed its potential performance in primary human tumors. For patients, the classifier’s sensitivity promises increased response rates and its specificity limits unnecessary toxicity. Given the scope of our xenograft models across all UICC stages, this applies not only to mCRC but also to the adjuvant setting of earlier stages. The xenograft collection allows to mimic randomized phase II trials and to test novel drugs effectively as single agents or in combinations. It also enables the development of highly accurate companion diagnostics as demonstrated by us for cetuximab.

Summary A major reason for vein graft failure after coronary artery bypass grafting is neointimal hyperplasia and thrombosis. Elevated serum levels of homocysteine (Hcy) are associated with higher incidence of cardiovascular disease, but homocysteine levels also tend to increase during the first weeks or months after cardiac surgery. To investigate this further, C57BL/6J mice (WT) and cystathionine-beta-synthase heterozygous knockout mice (CBS+/−), a mouse model for hyperhomocysteinaemia, underwent interposition of the vena cava of donor mice into the carotid artery of recipient mice. Two experimental groups were examined: 20 mice of each group underwent bypass surgery (group 1: WT donor and WT recipient; group 2: CBS+/− donor and CBS+/− recipient). After 4 weeks, the veins were harvested, dehydrated, paraffin-embedded, stained and analysed by histomorphology and immunohistochemistry. Additionally, serum Hcy levels in CBS knockout animals and in WT animals before and after bypass surgery were measured. At 4 weeks postoperatively, group 2 mice showed a higher percentage of thrombosis compared to controls, a threefold increase in neointima formation, higher general vascularization, a lower percentage of elastic fibres with shortage and fragmentation in the neointima, a lower percentage of acid mucopolysaccharides in the neointima and a more intense fibrosis in the neointima and media. In conclusion, hyperhomocysteinaemic cystathionine-beta-synthase knockout mice can play an important role in the study of mechanisms of vein graft failure. But further in vitro and in vivo studies are necessary to answer the question whether or not homocysteine itself or a related metabolic factor is the key aetiologic agent for accelerated vein graft disease.

Aims Despite the lower patency of venous compared with arterial coronary artery bypass grafts, ∼50% of grafts used are saphenous vein conduits because of their easier accessibility. In a search for ways to increase venous graft patency, we applied the results of a previous pharmacological study screening for non-toxic compounds that inhibit intimal hyperplasia of saphenous vein conduits in organ cultures. Here we analyse the effects and mechanism of action of leoligin [(2 S ,3 R ,4 R )-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxyphenyl)tetrahydrofuran-3-yl]methyl (2 Z )-2-methylbut-2-enoat, the major lignan from Edelweiss ( Leontopodium alpinum Cass.). Methods and results We found that leoligin potently inhibits vascular smooth muscle cell (SMC) proliferation by inducing cell cycle arrest in the G1-phase. Leoligin induced cell death neither in SMCs nor, more importantly, in endothelial cells. In a human saphenous vein organ culture model for graft disease, leoligin potently inhibited intimal hyperplasia, and even reversed graft disease in pre-damaged vessels. Furthermore, in an in vivo mouse model for venous bypass graft disease, leoligin potently inhibited intimal hyperplasia. Conclusion Our data suggest that leoligin might represent a novel non-toxic, non-thrombogenic, endothelial integrity preserving candidate drug for the treatment of vein graft disease.

Human skin is exposed to a number of harmful agents of which the ultraviolet (UV) component of solar radiation is most important. UV-induced damages include direct DNA lesions as well as oxidative damage in DNA, proteins and lipids caused by reactive oxygen species (ROS). Being the main site of ROS generation in the cell, mitochondria are particularly affected by photostress. The resulting mitochondrial dysfunction may have negative effects on many essential cellular processes. To counteract these effects, coenzyme Q10 (CoQ10 ) is used as a potent therapeutic in a number of diseases. We analyzed the mitochondrial respiration profile, the mitochondrial membrane potential and cellular ATP level in skin fibroblasts after irradiation. We observed an accelerated regeneration of cellular ATP level, a decrease in mitochondrial dysfunction as well as a preservation of the mitochondrial membrane potential after irradiation in human skin fibroblasts by treatment with CoQ10 . We conclude that the faster regeneration of the ATP level was achieved by a preservation of mitochondrial function by the addition of CoQ10 and that the protective effect of CoQ10 is primarily mediated via its antioxidative function. We suggest also that it might be further dependent on a stimulation of DNA repair enzymes by CoQ10 .

517 Background: wt KRAS and wt BRAF are established clinical markers to predict response to CE in patients (pts) with mCRC. Some pts with KRAS mut in codon 13 respond to CE, while the majority of them do not. Recently, it was shown that pts with CRC of Duke C and wt KRAS and wt BRAF do not benefit from CE added to FOLFOX (ASCO 2011 abstr 3607). In addition, data indicated that AREG and EREG RNA expression is also correlated with CE response. We established a panel of 133 xenograft models from primary tumor tissue of pts with CRC of all four Dukes stages and conducted a therapy experiment with CE (AACR '11, LB9086). Methods: mut status of KRAS (G12, G13, A146T); BRAF (V600E) and PIK3CA (E542K, E545K, H1047R) was assessed in all xenografts by allele-specific RT-PCR. KRAS codon 61 was sequenced. AREG and EREG expression levels were analyzed by RT-PCR expression assays. Results: We observed response to CE (treated to control ratio < 20%) in 18/67 (27%) of the treated xenografts. The distribution of responder (R) to non-responder (NR) is: Duke A: n=8; 2R / 6NR; B: n= 22; 8R / 14NR; C: n=28; 4R / 24 NR; D: n= 9; 4R / 5NR. Retrospective classification based on KRAS mut status identified R with a sensitivity (S+) of 83%, and NR with a specificity (S-) of 61%. If mut status in BRAF and PIK3CA were added the classifier showed S+ of 83% / S- of 78%. If any mut except in KRAS codon 13 were considered, the classifier reached S+ of 94% / S- of 69%. We improved S- of the classifiers to 86%; 90%, and 86%, respectively by adding AREG and EREG RNA expression data. The most accurate classifier combined wt KRAS, wt BRAF, wt PIK3CA with mut in codon 13 and RNA expression of AREG and EREG and reached S+ of 94% / S- of 86%. Classification in corresponding primary tumors showed similar accuracy. Conclusions: We developed a powerful classifier for all Dukes stages to predict response to CE with S+ of 94% and S- of 86%. In adjuvant setting the mut based classifier reaches S+ of 100% and S- of 68%; S- can be improved to 79% by adding AREG/EREG. This novel classifier has the potential to be used in the adjuvant and metastatic setting to improve pts selection for CE therapy.

393 Background: We previously reported on the discovery and prospective validation of a blood based test (Detector C) for early detection of colorectal cancer (CRC). Detector C measures 202 RNA markers in white blood cells as a response of the host to tumor formation and growth. Detector C was validated using a prospective, multicenter case-control study with 343 patients (pts), 210 cases with confirmed CRC and 133 controls undergoing a complete screening colonoscopy. Detector C has a validated sensitivity (S+) of 90% (95% CI 0.851-0.937) and specificity (S-) of 88% (95% CI 0.812-0.930) (Rosental A. et al, J Clin Oncol 28:7s, 2010 (suppl; abstr 3580)). We now present the discovery of Detector C 2.0 based on 445 samples representing most of the pts previously used in the discovery and validation sets of Detector C. Methods: We used Affymetrix U133 plus 2.0 expression data of 291 CRC cases and 154 controls for discovery of Detector C 2.0. Random forest was used for feature (gene) selection and the support vector machine algorithm was employed as classifier in 600 repetitions of double-nested bootstraps to discriminate between cases and controls. Within each repetition, randomly chosen 23 controls and 160 CRC cases served as prospective validation set. The most frequent chosen genes for discrimination between cases and controls formed the consensus signature, namely Detector C 2.0. Results: Choosing a signature length of 1000 genes resulted in the following second order unbiased prospective performance estimates: S+=0.916 (95% CI 0.863-0.954) and S-= 0.948 (95% CI 0.773-0.994). S+ for UICC stage I and stage II cases were 0.93 and 0.94. S+ for high-grade intraepithelial neoplasia was ∼ 0.67 and S+ for adenoma ≥ 10 mm was ∼ 0.45. Conclusions: Using a three times larger discovery set for Detector C 2.0 than for Detector C we improved S+ (cancer detection rate) by 1.6% to 91.6%. The most important enhancement is the high S- of 94.8% of Detector C 2.0 which is six percent higher than the S- of Detector C. Detector C 2.0 is based on a much larger pts set and should be even more robust than Detector C. We will prospectively validate this test in the largest case-control study ever performed in early detection of CRC.

e14049 Background: Patients (pts) with advanced, metastatic colorectal cancer (CRC) and wild-type KRAS mutation status benefit from anti EGFR antibodies cetuximab (CE) and panitumumab. Pts with CRC...

e14055 Background: miRNAs have a length of 21-23 bp. They are more stable than mRNAs, especially in formalin-fixed paraffin embedded (FFPE) tumor tissue. The increased stability of miRNA makes them...

3558 Background: Adjuvant chemotherapy in stage II colorectal cancer (CRC) is generally not recommended. Patients (pts) with T4 tumors, performations, < than 12 lymph nodes assessed, or positive ve...

Accurate prediction of response to therapy is a prerequisite for individualized approaches to colorectal cancer treatment. Testing of various cancer drugs on the individual patient tumor can support selection of effective therapies. To this end, we have established 149 stably passageable NOD/SCID mouse tumor xenografts from patients of all four Dukes stages within the framework of the prospective multicenter clinical study. All patients gave written informed consent prior to surgery. In a pharmacological study, 75 xenograft tumor models underwent single-agent treatment with oxaliplatin (OX), cetuximab (CE), or bevacizumab (BE). Each of the 75 tumors was transplanted onto 20 mice (5 controls and 5 for each drug). Models with a treated-to-control tumor growth ratio of 20% or lower were defined as responders. This resulted in response rates of 7% for OX, 24% for CE, and 4% for BE. Mutation analysis was conducted on 71 xenografts by allele-specific RT-PCR for the following hotspots: KRAS: 34G>A, 34G>T, 34G>C, 35G>A, 35G>T, 35G>C, 38 G>A and 436 G>A; BRAF: 1799 T>A, and PIK3CA: 1624G>A, 1633G>A, 3140A>G. KRAS mutations were found in 46%, BRAF in 10%, and PIK3CA in 15% of the xenografts. 59% of the xenografts carry at least one mutation. KRAS mutations were never observed together with BRAF-mutations while it combined with PIK3CA in 8 cases (11%). The corresponding primary tumors showed the same mutation profile. We examined mutation status with response to therapy to CE and OX. 3/5 OX responders were KRAS wildtype (wt) (Sensitivity (S+) 66%) while 31/66 OX non-responders were KRAS mutated (Specificity (S-) 46%). If also mutations in BRAF and PIK3CA are considered 40/60 OX non-responders were identified (S-66%). Of the 18 CE responders, all are wt for BRAF and PIK3CA. Three show mutations of KRAS, two of them in codon 13, which seems to confirm a recently published clinical observation by De Roock et al. (JAMA 2010, 304:1812) that this mutation may be related to an improved response under CE treatment. Wt status in all three genes was observed in 83% of the responders. KRAS mutations were seen in 57% of the non-responders. If mutations in BRAF and PIK3CA are also considered, the fraction of recognized non-responders increases to 73%. We also investigated how well response can be predicted from the mutation data. A machine learning approach was used to estimate unbiased prospective prediction rates for response to CE based on the mutation profile. In each of a large number of bootstrap loops, a new predictor was trained on a new randomly selected subset of the tumors and applied to the remaining tumors. KRAS, BRAF and PIK3CAmutations achieve a S+ of 84% (95% CI: 59% and 96%) and a S- of 68% (95% CI: 54% and 80%). KRAS, BRAF, and PIK3CA mutation status combined has potential clinical utility higher than KRAS alone to individualize CE treatment, also in the adjuvant setting, for patients with Dukes B and Dukes C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2011-LB-317

3612 Background: We discovered a prognostic gene signature (Predictor-C) containing 32 Affymetrix probesets in a cohort of 55 pts with colorectal cancer (CRC) UICC stage I or stage II. From all pts...

A study at a replicated logistics workplace examines whether the Respiratory Exchange Ratio (RER) can be used to analyze stress during lifting operations by using a mobile cardiopulmonary exercise testing (CPET)-system. Based on previously conducted task analyses in the field of manual handling, an appropriate experimental design was developed where both the package weight (0kg, 6.5kg, 13kg) and the type of movement (bending-to-stretching, stretching-to-bending, normal-to-normal) are varied. To test the plausibility of the RER, relative oxygen consumption (V’O2/kg), the heart rate (HR) as well as subjective data (Borg RPE scale) are used.