KRAS Wild-type CRC Research Articles

Secondary resistance to anti-EGFR therapy by transcriptional reprogramming in patient-derived colorectal cancer models

BackgroundThe development of secondary resistance (SR) in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (anti-EGFR) antibodies is not fully understood at the molecular level. Here we tested in vivo selection of anti-EGFR SR tumors in CRC patient-derived xenograft (PDX) models as a strategy for a molecular dissection of SR mechanisms.MethodsWe analyzed 21 KRAS, NRAS, BRAF, and PI3K wildtype CRC patient-derived xenograft (PDX) models for their anti-EGFR sensitivity. Furthermore, 31 anti-EGFR SR tumors were generated via chronic in vivo treatment with cetuximab. A multi-omics approach was employed to address molecular primary and secondary resistance mechanisms. Gene set enrichment analyses were used to uncover SR pathways. Targeted therapy of SR PDX models was applied to validate selected SR pathways.ResultsIn vivo anti-EGFR SR could be established with high efficiency. Chronic anti-EGFR treatment of CRC PDX tumors induced parallel evolution of multiple resistant lesions with independent molecular SR mechanisms. Mutations in driver genes explained SR development in a subgroup of CRC PDX models, only. Transcriptional reprogramming inducing anti-EGFR SR was discovered as a common mechanism in CRC PDX models frequently leading to RAS signaling pathway activation. We identified cAMP and STAT3 signaling activation, as well as paracrine and autocrine signaling via growth factors as novel anti-EGFR secondary resistance mechanisms. Secondary resistant xenograft tumors could successfully be treated by addressing identified transcriptional changes by tailored targeted therapies.ConclusionsOur study demonstrates that SR PDX tumors provide a unique platform to study molecular SR mechanisms and allow testing of multiple treatments for efficient targeting of SR mechanisms, not possible in the patient. Importantly, it suggests that the development of anti-EGFR tolerant cells via transcriptional reprogramming as a cause of anti-EGFR SR in CRC is likely more prevalent than previously anticipated. It emphasizes the need for analyses of SR tumor tissues at a multi-omics level for a comprehensive molecular understanding of anti-EGFR SR in CRC.

Systems analysis of protein signatures predicting cetuximab responses in KRAS, NRAS, BRAF and PIK3CA wild-type patient-derived xenograft models of metastatic colorectal cancer.

Antibodies targeting the human epidermal growth factor receptor (EGFR) are used for the treatment of RAS wild-type metastatic colorectal cancer. A significant proportion of patients remains unresponsive to this therapy. Here, we performed a reverse-phase protein array-based (phospho)protein analysis of 63 KRAS, NRAS, BRAF and PIK3CA wild-type metastatic CRC tumours. Responses of tumours to anti-EGFR therapy with cetuximab were recorded in patient-derived xenograft (PDX) models. Unsupervised hierarchical clustering of pretreatment tumour tissue identified three clusters, of which Cluster C3 was exclusively composed of responders. Clusters C1 and C2 exhibited mixed responses. None of the three protein clusters exhibited a significant correlation with transcriptome-based subtypes. Analysis of protein signatures across all PDXs identified 14 markers that discriminated cetuximab-sensitive and cetuximab-resistant tumours: PDK1 (S241), caspase-8, Shc (Y317), Stat3 (Y705), p27, GSK-3β (S9), HER3, PKC-α (S657), EGFR (Y1068), Akt (S473), S6 ribosomal protein (S240/244), HER3 (Y1289), NF-κB-p65 (S536) and Gab-1 (Y627). Least absolute shrinkage and selection operator and binominal logistic regression analysis delivered refined protein signatures for predicting response to cetuximab. (Phospo-)protein analysis of matched pretreated and posttreated models furthermore showed significant reduction of Gab-1 (Y627) and GSK-3β (S9) exclusively in responding models, suggesting novel targets for treatment.

Association of KRAS and BRAF mutations with progression-free survival (PFS) with second-line FOLFIRI +/- regorafenib in metastatic colorectal cancer (mCRC).

196 Background: LCCC1029 was a 2:1 randomized phase II trial of 2nd-line FOLFIRI plus either regorafenib or placebo in mCRC. The addition of regorafenib improved PFS (median PFS 6.1 vs 5.3 mo, HR 0.73, 95% CI 0.53-1.01). However, the effect of somatic mutations on regorafenib activity has not been tested. Methods: We performed whole exome sequencing on archival primary tumor tissue and paired normal tissue in 85 patients of LCCC1029. We compared PFS and OS using Kaplan-Meier method and log-rank tests, and hazard ratios (HR) were estimated using Cox proportional hazards method. Results: Among the 85 subjects, 54 (64%) had tumors wild-type (WT) for KRAS and BRAF, 26 (31%) had tumors with KRAS mutations in exons 2-4, and 5 (6%) had tumors with BRAF V600E. The addition of regorafenib to FOLFIRI improved PFS in the KRAS/ BRAF WT subgroup (median PFS 8.0 vs 4.9 mo, HR 0.68, 95% CI 0.48-0.97, log-rank p=0.028), but not in the KRAS mutant subgroup (median PFS 6.8 vs 5.5 mo, HR 0.90, 95% CI 0.61-1.35, log-rank p=0.617) or the BRAF mutant subgroup (log-rank p=0.156). In all of these subgroups, the addition of regorafenib was not associated with significant difference in OS. BRAF V600E was prognostic and associated with significantly worse OS (median OS 8.4 vs 18.0 mo, HR 2.59, 95% CI 1.01-6.66, log-rank p=0.04). Conclusions: The addition of regorafenib to FOLFIRI improves PFS among the subgroup of patients with KRAS and BRAF dual WT CRC, but not among the KRAS mutant subgroup. These results indicate that the addition of anti-angiogenic therapy to second-line chemotherapy backbones may be more effective in KRAS/ BRAF WT tumors in particular. More confirmatory studies are needed to corroborate this finding.

PO-325 Novel recurrent high-level amplifications in microsatellite stable colorectal cancer

IntroductionColorectal cancer (CRC) is a molecularly diverse disease with few targeted treatment options. Focal and high-amplitude DNA copy number aberrations are potential tumour drivers, and their identification may contribute to improved therapeutic outcomes in small patient subgroups, as recently illustrated by targeting the over-expression of HER2 protein resulting from high-level ERBB2 amplification in KRAS wild-type metastatic CRC. However, few recurrent amplifications have been detected in CRC.Material and methodsWe analysed focal high-level amplifications in 203 microsatellite stable (MSS) primary colorectal tumours using genome-wide high-resolution Affymetrix SNP6.0 arrays. The ASCAT algorithm was used to derive discrete allele specific copy number estimates.Results and discussionsThe overall copy number profiles confirmed the frequent gains and losses previously described in MSS CRC studies. Extreme focal amplifications (defined as >15 copies and <50 genes in peak) were found in 77 regions in tumours from 22 unique patients, distributed across 16 different chromosome arms. Recurrent amplifications included ERBB2, which was amplified to 97, 27 and 22 additional copies in tumours from three individual patients (amplification frequency 1.5%). The transcription factor TOX3 (16q) was also recurrently amplified in 1.5% of the patients, while MYC (8q), CCND2 (12 p) and a region on 10q22.3-q23.1, where ANXA11 was nominated as a likely target by GISTIC analysis, were recurrent in 1% of the patients. Regions with extreme and focal amplifications were also investigated for lower-amplitude aberrations (5–15 additional DNA copies), revealing a 3% amplification frequency of TOX3 in our cohort.ConclusionWe have identified several recurrent amplifications in cancer-critical genes in CRC MSS tumours, including the transcription factor TOX3, suggesting novel drug targets for preclinical studies.

Targeting the RAS-dependent chemoresistance: The Warburg connection

RAS protein family members (KRAS4A, KRAS4B, HRAS and NRAS) function as GDP-GTP-regulated on-off switches, which regulate cytoplasmic-nuclear signaling networks ruling diverse normal cellular processes. Constitutive activating mutations in RAS genes are found in up to 30% of human cancers, and remarkably, the oncogenic Ras mutations and mutations in other components of Ras/MAPK signaling pathways seem to be mutually exclusive in most tumors, pointing out that deregulation of Ras-dependent signaling is an essential requirement for tumorigenesis. Up to 30% of solid tumors are known to have a mutated (abnormal) KRAS gene. Unfortunately, patients harboring mutated KRAS CRC are unlikely to benefit from anti-EGFR therapy. Moreover, it remains unclear that patients with KRAS wild-type CRC will definitely respond to such therapies. Although some clinically designed-strategies to modulate KRAS aberrant activation have been designed, all attempts to target KRAS have failed in the clinical assays and K-RAS has been assumed to be invulnerable to chemotherapeutic attack. Recently, different encouraging publications reported that ascorbate may have a selective antitumoral effect on KRAS mutant cancer cells. In this review we aim to describe the prevalence and importance of KRAS mutation in cancer and associated problems for the clinical handling of patients harboring these tumors. We highlight the role of mutated KRAS in boosting and keeping the tumor associated aberrant cell metabolism stating that further in-depth studies on the molecular mechanism of ascorbate to bypass mutated KRAS-related metabolic alterations may constitute a new pathway to design novel molecules in order handle tumor resistance to anti EGFR-therapies.

Targeting KRAS Mutant CMS3 Subtype by Metabolic Inhibitors.

Cancer cells rewire their metabolism in order to boost growth, survival, proliferation, and chemoresistance. The common event of this aberrant metabolism is the increased glucose uptake and fermentation of glucose to lactate. This phenomenon is observed even in the presence of O2 and completely functioning mitochondria. This is known as the "Warburg Effect" and it is a hallmark in cancer. Up to 40% of all CRC's are known to have a mutated (abnormal) KRAS gene, found at differing frequencies in all consensus molecular subtypes (CMS). CMS3 colon cancer molecular subtype contains the so-called 'metabolic tumours' which represents 13% of total CR cases. These tumours display remarkable metabolic deregulation, often showing KRAS mutations (68%). Unfortunately, patients harbouring mutated KRAS are unlikely to benefit from anti-EGFR therapies. Moreover, it remains unclear that patients with KRAS wild-type CRC will definitely respond to such therapies. Although some clinically designed-strategies to modulate KRAS aberrant activation have been designed, all attempts to target KRAS have failed in the clinical assays and KRAS has been assumed to be invulnerable to chemotherapeutic attack. Quest for metabolic inhibitors with anti-tumour activity may constitute a novel and hopeful approach in order to handle KRAS dependent chemoresistance in colon cancer.

Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer.

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for those patients whose tumors harbor a mutation in the KRAS oncogene (40-45% of all patients). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a double stranded (ds) RNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. Toll Like Receptor (TLR) 3, a member of the toll like receptor family of the host innate immune system is the pattern recognition motif for dsRNA pathogens. Using TLR3 expressing commercial HEK-Blue™-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for the detection of reovirus. Further, our investigation with KRAS mutated HCT116 cell line showed that effective expression of host TLR3 dampens the infection potential of reovirus by mounting a robust innate immune response. Down regulation of TLR3 expression with siRNA improves the anticancer activity of reovirus. In vivo experiments using human CRC cells derived xenografts in athymic mice further demonstrate the beneficial effects of TLR3 knock down by improving tumor response rates to reovirus. Strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved reovirus efficacy to specifically target the dissemination of KRAS mutated CRC.

Anti-KRAS siRNA nanoparticles for targeted colorectal cancer therapy.

636 Background: Standard treatment for metastatic colorectal cancer (mCRC) is systemic chemotherapy with anti-EGFR treatment, depending on KRAS mutational status. However, tumors harboring a KRAS mutation do not respond to existing targeted therapy. Moreover, targeting mutant KRAS has, to date, not been possible. Herein, we explore using a KRAS inhibitory nanoparticle (NP), to directly knock down mutant KRAS. Methods: Utilizing fluorescent-labeled small interfering RNA (siRNA) NPs, uptake was assessed via fluorescent microscopy. KRAS mutant CT26 and wild-type MC38 CRC cell lines were incubated with either scramble (Sc) sequence siRNA NP, KRAS siRNA NP, or FOLFOX (fluorouracil + oxaliplatin) chemotherapy ± KRAS siRNA NP. Cell viability was assessed via a luminescent viability assay. KRAS and cleaved caspase 3 protein expression were assessed using western blotting. Results: Fluorescent NP uptake was demonstrated in CT26 cells as early as 260 minutes post treatment, with increased uptake through 780 minutes. Decreased cellular viability was seen with KRAS siRNA NP treated CT26 cells, as compared to both Sc siRNA NP and non-treated CT26 cells (both p &lt; 0.0001). Cell viability was significantly diminished with FOLFOX combined with KRAS siRNA NP as compared to FOLFOX alone for CT26 cells ( p = 0.0003), but not MC38 cells (p = 0.2259). Western blot demonstrated decreased KRAS and increased cleaved caspase 3 expression in CT26 cells treated with KRAS siRNA NP. Conclusions: A KRAS siRNA tagged NP was internalized by the CRC cells in vitro, and induced cellular death via apoptosis in mutant type KRAS CRC. In addition, KRAS siRNA NP acted synergistically with FOLFOX chemotherapy to enhance cell death. We believe KRAS inhibition based NP treatment is a promising target for mutant type KRAS CRC. [Table: see text]

Abstract 3788: Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer

Abstract Introduction New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for patients whose tumors harbor a mutation in the KRAS oncogene (40-45%). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a dsRNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. We hypothesize that effective expression of host Toll Like Receptors 3 (TLR3) will dampen the infection potential of reovirus propagation by mounting an innate immune response. Development of strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved virus productivity to specifically target the KRAS mutated cancer cells. Methodology: TLR3 expressing HEK293 cells were treated with reovirus to confirm that TLR 3 is the host pattern recognition motif of the host cellular machinery that is responsible for the detection of dsRNA harboring reovirus. TLR 3 was next down regulated by shRNA technology in KRAS mutated HCT116 CRC cell line and treated with reovirus at dose of 5 MOI (multiplicity of infection) for 48 hours. The cell proliferation profile under this treatment condition was measured by MTT assay and corelated with the proliferation pattern of TLR3 expressing HCT116 cells undergoing similar treatment. Cytokine ELISA was performed for interferon (INF) alpha (α)and beta (β) to determine the downstream consequences of the gene silencing. Finally xenograft tumor models of athymic nude mice (Foxn1nu) were developed with TLR3 silenced HCT116 cells and treated with reovirus at a daily intra tumoral (IT) dose of 1×107 TCID (tissue culture infective dose) and compared to tumors generated by HCT116 cells receiving identical treatment. Results ¬ TLR 3 is confirmed to be the host pattern recognition motif for dsRNA containing reovirus ¬ TLR3 which is constitutively expressed by colon epithelium is also a mediator of virus recognition in CRC cell line HCT116. ¬ Down regulation of TLR3 by shRNA technology enhances viral propagation as measured by MTT assay. ¬ Cytokine ELISA assay of INF α β distinctly reveals that downstream activation post reovirus infection is compromised. ¬ In xenograft models, those of TLR3 down regulated HCT116 cells, showed improved control of tumor growth with reovirus treatment as compared to those with TLR 3 expressing HCT 116 cells (p = 0.04) Conclusion TLR3 plays an important role in recognition of reovirus and mounting an innate immune response by inducing the secretion of type I INF. Mitigation of the TLR3 receptor mediated pattern recognition by shRNA down regulates host immune response and thus improves virus mediated cell cytotoxicity. The findings can be therapeutically utilized towards improved and beneficial killing of cancer cells by reovirus. Citation Format: Radhashree Maitra, Titto Augustine, Matt Coffey, Sanjay Goel. Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3788.

C6-ceramide's effect on KRAS-defined colorectal cancer cells treated with oxaliplatin, 5-fluoruracil with and without cetuximab.

619 Background: Cetuximab (Cet) is beneficial for patients with metastatic KRAS Wild type (WT) colorectal cancer (mCRC) only. C6-Ceramide (C6-Cer) can act synergistically with chemotherapy to induce cancer cell death. The aim of this study was to compare growth inhibition percentage (GIP) of cytostatics 5-fluoruracil (5-FU), oxaliplatin (Ox) and Cet with or without C6-Cer in KRAS WT and KRAS mutant (KRAS Mut) CRC cell lines (SW48 and SW480, respectively). Methods: Both cell lines were incubated with IC50 concentrations of test drugs. Drug concentrations included 0.8µM for 5-FU, 0.04µM for Ox, 25 µg/mL for Cet, and C6-Cer concentrations ranged from 5 to 10 µM. Cell survival was assessed 72h after using 0.4% Trypan Blue. Results: With above mentioned concentrations, C6-Cer’s GIP was 78.3% for SW-480 (vs. 33.33% for SW-48). It was also noted that the addition of C6-Cer to a combination of steady concentrations of Ox, Cet and 5-FU increased GIP with an especially significant effect on SW-480. Addition of 5 and 7.5µM resulted in doubling of GIP (75% and 86.25%, respectively, vs 32.5% of 5-FU + Ox + Cet). The greatest effect was seen when 10µM of C6-Cer was added to the IC50 concentrations of chemotherapeutic agents (using the 25µg/mL concentration of Cet), where GIP increased from 32.5% to 92.5% in SW-480. Same concentration of drugs increased GIP for SW-48 to a similar 93.5%. Conclusions: C6-Cer appears to have direct inhibitory properties, especially on KRAS Mut cells. Additionally when added to Ox, 5-FU and Cet, C6-Cer reversed the apparent insensitivity of KRAS Mut to Cet. Also, the study showed C6-Cer can provide additional synergism to their cytostatic properties in KRAS WT CRC cell lines. The true mechanism of these observations is still undergoing analysis, however the effect of isolated C6-Cer on KRAS Mut raises possibility of a different pathway that could bypass EFGR pathway. We believe the results of this study provide a starting point for clinical studies with C6-Ceramide in patients with relapsing or metastatic KRAS Mut CRC in combination with standard chemotherapy plus molecular target agents hoping they will translate into clinical benefit for this difficult to treat patient population.

Abstract B35: Acquired KRAS mutations after anti-EGFR therapy partially derive from low-frequency clones detectable in the primary tumor.

Abstract Background: The detection of acquired KRAS and EGFR extracellular domain mutations have been reported and correlated with acquired resistance to anti-EGFR monoclonal antibodies (MAb). The two original reports of KRAS mutations after EGFR therapy provided different interpretations on whether these mutations were pre-existing in a minor clone and expanded under selective pressure, or were not present in the original tumor and developed through acquisition of new mutations. To address such question, using high-sensitivity assay we re-analyzed twelve archival tissues from KRAS wild type patients that showed the presence of acquired KRAS mutation in plasma circulating free DNA(cfDNA) after progressing on EGFR therapy. Methods: Plasma and tissue samples were collected from metastatic CRC pts as part of the ATTACC program. Eligible pts had a source documentation of pre-treatment KRAS wild type tumor. KRAS and EGFR ectodomain mutations were analyzed by BEAMing technology (sensitivity of 0.02% mutant to wild type alleles) in DNA extracted from patient tumor tissue collected at the time of diagnosis and from plasma cfDNA collected after progression to anti-EGFR mAb treatment. Acquired mutations were defined as mutations not identified by standard-of-care sequencing of the primary tumor. Results: Plasma from 77 patients was analyzed for EGFR and KRAS mutation. The S492R EGFR mutation was detected in 4 patients, while 24 patients previously classified as KRAS wild type by standard of care sequencing of the primary tumor had an acquired KRAS mutation in the cfDNA after BEAMing analysis. For 12 of these patients we re-analyzed the untreated primary tumor using BEAMing technology to screen for low-frequency (LF) KRAS mutant clones. LF KRAS mutations were detected in 5 samples (42%), while the other 7 (58%) identified only KRAS wild type alleles. In addition, the analysis identified atypical KRAS mutation, such as codon 61 and 146 (present respectively in 33 and 10% of samples with acquired mutations), and more than one concurrent KRAS codon mutations (29% of samples with acquired mutations) were common features of acquired KRAS mutations compared with primary mutations. Conclusions: Acquired KRAS mutations are common in clinically KRAS wild type CRC after anti-EGFR therapy, and appear to derive from pre-existing clones in a significant proportion of cases. This data confirm the limit of current technique used for detection of clinically relevant mutations and suggests that the use of more sensitive sequencing technologies may be able to identify clinically relevant low-frequency KRAS mutations. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B35. Citation Format: Maria Pia Morelli, Michael Overman, Arvind Dasari, Ali Kazmi, Edoardo Vilar-Sanchez, Cathy Eng, Bryan Kee, Laurel Deaton, Chris Garrett, Frank Diehl, Dipen Maru, Philipp Angenendt, Scott Kopetz. Acquired KRAS mutations after anti-EGFR therapy partially derive from low-frequency clones detectable in the primary tumor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B35.

Abstract C263: mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

Abstract Colorectal cancers (CRCs) harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that combines targeting of the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not wild-type CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C263. Citation Format: Erin M. Coffee, Anthony C. Faber, Carlotta Costa, Anahita Dastur, Hiromichi Ebi, Aaron N. Hata, Alan T. Yeo, Elena J, Youngchul Song, Ah Ting Tam, Jessica L. Boisvert, Randy J. Milano, Jatin Roper, David P. Kodack, Rakesh K. Jain, Ryan B. Corcoran, Miguel N. Rivera, Sridhar Ramaswamy, Kenneth E. Hung, Cyril H. Benes, Jeffrey A. Engelman. mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C263.

Hsa-mir-31-3p effects on gene expression in colorectal cancer cell lines and patients.

e14600 Background: Recently, hsa-miR-31-3p was found to be up-regulated in some tumors of metastatic colorectal cancer (mCRC) patients and was proposed as a prognostic marker for anti-EGFR therapy in KRAS wild-type (WT) patients. The aim of this study was to identify the target genes of hsa-miR-31-3p in a CRC context. Methods: Three different KRAS WT CRC cell lines, Caco-2, Colo205 and Colo320HSR were transfected with hsa-miR-31-3p mimic or control mimic. After 24h, cells were harvested and total RNA was extracted for an expression array analysis. Linear models for micro-arrays analysis from R package was used to analyze the results. A miRNA predictive target database including 6 available databases (microRNA.org, MicroCosm, TargetScan, PicTar, PITA, miRDB) was created. Validation of the identified target genes was performed on tumors sample of 39 patients treated with panitumumab and irinotecan as third line. Results: After transfection of the 3 cell lines with hsa-miR-31-3p mimic, an expression profile analysis was done. Results obtained in each cell lines were first compared in a pair wise analysis and secondly tested with eBayes procedure without matching. Fifty –four genes were down-regulated (fold change: fc≤0.77, p&lt;0.05). Using the predictive target database, hsa-miR-31-3p was the second most common miRNA as it targeted putatively 25 out the 54 genes, among which STAT3. To identify indirect target genes, we then studied the genes up-regulated by hsa-miR-31-3p over-expression in the 3 different cell lines and identified 27 genes. As expected all but one were not predicted to be direct target genes of hsa-miR-31-3p using the predictive target database. Interestingly, the proto-oncogene MET was up regulated by the over expression of hsa-miR-31-3p. Validation of the 25 putative targets and of the 27 genes up-regulated genes was done with RT qPCR on tumor samples of patients treated with anti-EGFR and some of the direct and undirected target genes were confirmed. Conclusions: This study shows that hsa-miR-31-3p, which expression level was shown to be correlated with prognosis of mCRC patients treated with anti-EGFR, plays an important role in CRC by regulating genes involved in cancer like STAT 3 and MET.

Phase III randomized trial of cetuximab (CET) plus either brivanib alaninate (BRIV) or placebo in patients (pts) with metastatic (MET) chemotherapy refractory K-RAS wild-type (WT) colorectal carcinoma (CRC): The NCIC Clinical Trials Group and AGITG CO.20 trial.

386 Background: The anti-EGFR monoclonal antibody CET has improved survival in pts with MET, chemotherapy refractory, K-RAS wild type (WT) CRC. The addition of BRIV, a tyrosine kinase inhibitor targeting vascular endothelial and fibroblast growth factor receptors (VEGFR/FGFR), to CET has shown encouraging activity in an early phase clinical trial. Methods: Pts with MET CRC previously treated with combination chemotherapy were randomized 1:1 to receive CET 400 mg/m2 IV loading dose followed by weekly maintenance of 250 mg/m2 plus either BRIV 800 mg PO daily (Arm A) or placebo (Arm B). Pts may have had 1 prior anti-VEGF, but no prior anti-EGFR therapy. The trial was amended shortly after opening to enrol K-RAS WT pts. Primary endpoint was overall survival (OS). Results: From 02/2008 to 02/2011, 750 pts were randomized (376 in Arm A and 374 in Arm B). Demographics: median age=64 (range 27-88); male=64%; ECOG 0:1:2 (%)=32:58:10; &gt;3 prior chemotherapy regimens=92%; prior anti-VEGF therapy=41%; K-RAS WT=97%. Median OS in the intent-to-treat population was 8.8 months in Arm A and 8.1 months in Arm B, hazard ratio (HR)=0.88; 95% CI=0.74 to 1.03; p=0.12. Median progression-free survival (PFS) was 5.0 months in Arm A and 3.4 months in Arm B, HR=0.72; 95% CI=0.62 to 0.84; p&lt;0.0001. Both partial responses (13.6% vs 7.2%, p=0.004) and stable disease (50% vs 44%) were higher in Arm A. Incidence of any ≥grade 3 adverse event (AE) was 78% in Arm A and 53% in Arm B. Most frequent ≥grade 3 AEs were fatigue (25%), hypertension (11%) and rash (10%) in Arm A, vs fatigue (11%), rash (5%) and dyspnea (5%) in Arm B. Time to deterioration of physical function was shorter and global quality of life scores were lower in Arm A vs Arm B. Pts received ≥90% dose intensity of CET=57% in Arm A vs 83% in Arm B; of BRIV/placebo=48% in Arm A vs 87% in Arm B. Conclusions: Despite positive effects on PFS and objective response, the combination of CET+BRIV did not significantly improve OS in pts with MET, chemotherapy refractory, K-RAS WT CRC. AEs were consistent with those reported for each drug given as monotherapy.

Abstract C35: Response of colorectal cancer cells to combination of EGFR mAbs with oxaliplatin is determined by the sequence of administration.

Abstract Monoclonal antibodies (mAb) targeting EGFR (epidermal growth factor receptor), cetuximab (Erbitux) and panitumumab (Vectibix), have been approved for the therapy of metastatic colorectal cancer (mCRC). However, the majority of the patients are primarily resistant to EGFR mAbs or become resistant during the treatment. While activating mutations in KRAS have been reported to confer resistance to EGFR mAbs, some recent randomised controlled trials with EGFR mAbs combined to oxaliplatin-based regimens have failed to demonstrate clinical effect even in patients with KRAS wild-type mCRC. We have addressed the effects of EGFR mAbs on the growth of KRAS wild-type and KRAS mutant CRC cell lines in the absence and presence of oxaliplatin. Our observations indicate that while the KRAS mutant genotype is predictive for resistance to single agent EGFR mAbs, the combination of EGFR mAb plus oxaliplatin is not superior to oxaliplatin alone regardless of the KRAS status. However, sequential administration of oxaliplatin prior to EGFR mAb results in a clear additive effect whereas a sequence of EGFR mAb prior to oxaliplatin antagonizes the effect of oxaliplatin. The opposing effects of the two different sequences seem to be independent on both KRAS and BRAF mutation status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C35.

Abstract A156: GA201 (RG7160) pretreatments and combination therapies improve efficacy without negatively affecting antitumoral ADCC.

Abstract GA201 is a humanized and glycoengineered IgG1 antibody which targets EGFR with boosted antibody dependent cell cytotoxicity (ADCC) in phase 2 clinical trials. Pretreatment with corticosteroids and/or anti-histaminics are of normal practice for monoclonal antibody therapy to prevent potential infusion reactions. GA201 follows a premedication regimen of anti-histaminics and hydrocortisone. Due to the dual GA201 mode of action, evaluation on the impact of these drugs on ADCC is critical and was previously assessed preclinically for potential negative impacts on immune effector cells and their capacity to mediate tumor cell killing. Combination phase II studies with GA201 + chemotherapy regimens are currently under investigation in NSCLC as first-line treatment (chemotherapy selection based on histology Cisplatin + gemcitabine for squamous and cisplatin + pemetrexed for non squamous) and in CRC as second-line treatment (FOLFIRI for KRAS-mutant CRC or cetuximab + FOLFIRI for KRAS-wildtype CRC). These combinations and others where tested prior use in the clinics to evaluate preclinical efficacy and their impact on the immune effectors in vitro, and in vivo. Chemotherapy combos showed additive or synergistc effect in anti-tumor efficacy as assessed in orthotopic xenograft models and all pre-medications and chemotherapies tested showed no negative impact on ADCC supporting their combination with GA201 in the clinics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A156.