BRAF Colorectal Cancer Research Articles

The roles of protocadherin-7 in colorectal cancer cells on cell proliferation and its chemoresistance.

Despite the high mutation frequencies of KRAS, NRAS, and BRAF in colorectal cancer (CRC), there are no effective and reliable inhibitors for these biomarkers. Protocadherin-7 (PCDH7) is regarded as a potentially targetable surface molecule in cancer cells and plays an important role in their proliferation, metastasis, and drug resistance. However, the roles and underlying mechanisms of PCDH7 in CRC remain unclear. In the current study, we found that different colorectal cancer cells expressed PCDH7 over a wide range. The levels of PCDH7 expression were positively associated with cell proliferation and drug resistance in CRC cells but negatively correlated with the potential for cell migration and invasion. Our data indicated that PCDH7 mediated the resistance of CRC cells to ABT-263 (a small-molecule Bcl-2 inhibitor that induces apoptosis) by inhibiting cell apoptosis, which was supported by the downregulation of caspase-3, caspase-9, and PARP cleavage. We found that PCDH7 effectively promoted Mcl-1 expression at both mRNA and protein levels. Furthermore, PCDH7 activated the Wnt signaling pathway, which was confirmed by the increase in β-catenin and c-Myc expression. Finally, and notably, S63845, a novel Mcl-1 inhibitor, not only effectively attenuated the inhibitory effect of PCDH7 on cell apoptosis induced by ABT-263 in vitro but also sensitized PCDH7-overexpressed CRC cell-derived xenografts to ABT-263 in vivo. Taken together, although PCDH7 inhibited the migration and invasion of CRC cells, it could facilitate the development of drug resistance in colorectal cancer cells by positively modulating Mcl-1 expression. The application of the Mcl-1 inhibitor S63845 could be a potential strategy for CRC chemotherapy, especially in CRC with high levels of PCDH7.

Marcadores moleculares en los tumores digestivos. Implicaciones terapéuticas

Cancer is one of the main causes of morbidity and mortality in the world. For this reason, research in this area is ongoing and aims to develop new therapies that improve survival and patients’ quality of life. The identification of molecular alterations in each type of tumor allows for identifying prognostic and predictive factors of response to specific treatments. Along these lines, there are molecular markers that are used in healthcare practice (such as the determination of the mutational state of RAS and BRAF in colorectal cancer, HER2 in gastric cancer, or microsatellite instability in all), as well as others that are still being developed.

Loss of tumor intrinsic PD-L1 confers resistance to drug-induced apoptosis in human colon cancer.

Colorectal cancer (CRC) with BRAF (V600E) is associated with microsatellite instability (MSI) that predicts response to immune checkpoint inhibitors. We demonstrated the interrogation of TCGA RNA-seq human datasets revealed that BRAFV600E tumors had significantly higher Programmed Death Ligand 1 (PD-L1) mRNA compared to non-mutated BRAF CRCs. Also, MSI-H tumors were evaluated as higher PD-L1 than MSS CRCs. Inhibition of MEK/ERK by cobimetinib or CDK inhibitor dinaciclib was shown to attenuate mutant BRAF-induced PD-L1 coincident with reduced c-JUN and YAP expression whose combined knockdown reduced PD-L1. Using TCGA datasets, PD-L1 mRNA expression in human colon cancers was significantly associated with YAP expression. The deletion of PD-L1 can reduce tumor cell growth shown by clonogenic assay. Analysis of the role of PD-L1 as a mediator of chemosensitivity was then performed. Knockout of PD-L1 was shown to attenuate the induction of DNA double-strand breaks (pH2AX) and caspase-3 cleavage by 5-fluorouracil (5-FU) and paclitaxel compared to parental CRC cells. Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of wild-type PD-L1 promoted DNA damage and apoptosis. We also performed the clonogenic assay and flow cytometry to prove that loss of PD-L1 attenuated DNA damage and apoptosis induced by diverse anti-cancer drugs that could be reversed by restoration of wild-type PD-L1. Mechanistically, knockout of PD-L1 reduced chemosensitivity in association with reductions in p-AKT and in BH3-only proteins BIM and BIK, rather than STAT3 in CRC cells. However, STAT3 had a significant role in melanoma, which shows the heterogeneity of cancers. In summary, BRAFV600E can upregulate PD-L1 expression that was induced by c-jun and YAP to enhance chemotherapy-induced apoptosis. Together, we demonstrate a potential role for PD-L1 as a regulator of chemotherapy-induced apoptosis whose deletion or suppression confers chemoresistance. These findings expand the understanding of PD-L1 functions to include nonimmune mechanisms and suggest the potential use of PD-L1 as a biomarker of response to cytotoxic chemotherapy.

Radiomics Texture Features in Advanced Colorectal Cancer: Correlation with BRAF Mutation and 5-year Overall Survival.

To explore the potential of radiomics texture features as potential biomarkers to enable detection of the presence of BRAF mutation and prediction of 5-year overall survival (OS) in stage IV colorectal cancer (CRC). In this retrospective study, a total of 145 patients (mean age, 61 years ± 14 [standard deviation {SD}]; 68 female patients and 77 male patients) with stage IV CRC who underwent molecular profiling and pretreatment contrast material-enhanced CT scans between 2004 and 2018 were included. Tumor radiomics texture features, including the mean, the SD, the mean value of positive pixels (MPP), skewness, kurtosis, and entropy, were extracted from regions of interest on CT images after applying three Laplacian-of-Gaussian filters known as spatial scaling factors (SSFs) (SSF = 2, fine; SSF = 4, medium; SSF = 6, coarse) by using specialized software; values of these parameters were also obtained without filtration (SSF = 0). The Wilcoxon rank sum test was used to assess differences between mutated versus wild-type BRAF tumors. Associations between radiomics texture features and 5-year OS were determined by using Kaplan-Meier estimators using the log-rank test and multivariate Cox proportional-hazards regression analysis. The SDs and MPPs of radiomic texture features were significantly lower in BRAF mutant tumors than in wild-type BRAF tumors at SSFs of 0, 4, and 6 (P = .006, P = .007, and P = .005, respectively). Patients with skewness less than or equal to -0.75 at an SSF of 0 and a mean of greater than or equal to 17.76 at an SSF of 2 showed better 5-year OS (hazard ratio [HR], 0.53 [95% confidence interval {CI}: 0.29, 0.94]; HR, 0.40 [95% CI: 0.22, 0.71]; log-rank P = .025 and P = .002, respectively). Tumor location (right colon vs left colon vs rectum) had no significant impact on the clinical outcome (log-rank P = .53). Radiomics texture features can serve as potential biomarkers for determining BRAF mutation status and as predictors of 5-year OS in patients with advanced-stage CRC.Keywords: Abdomen/GI, CT, Comparative Studies, Large BowelSupplemental material is available for this article.© RSNA, 2020.

Circulating tumor DNA to monitor treatment response and investigate resistance mechanism in BRAF inhibitor treated colorectal cancer patients.

e16112 Background: BRAF mutations are found in about 10% of colorectal cancer (CRC) patients and is a poor prognosis factor in standard chemotherapy. Combination of BRAF and EGFR inhibition exerts a better therapeutic effective, whereas the resistance develops through undefined mechanisms. Circulating tumor DNA (ctDNA) is a non-invasive approach to assess the genetic evolution of tumors and prognosis in response to therapy, which would help better understanding the treatment response and resistance mechanism in BRAF inhibitor treated colorectal cancer patients. Methods: We performed panel next-generation sequencing of 425 cancer-related genes in 39 serial plasma samples collect from 19 patients who have BRAF V600E mutation CRC to track the resistance during the Vemurafenib treatment in combination with Irinotecan and Cetuximab (VIC) and evaluate the treatment response. Tumor responses were assessed radiologically every two months and are used to determine patients with innate or acquired resistance Results: A total of 19 patients were enrolled into the vemurafenib treatment with irinotecan and cetuximab. By January 20, 2020, treatment had been discontinued in 12 (63.2%) of the patients due to disease progression, while the other 7 cases were still under treatment. Among them, four patients with innate resistance (n = 4, 21.1%) were defined as those with PFS of less than 2 months, while patients (n = 15, 78.9%) with acquired resistance were defined as those with PFS of greater than 2 months. The VIC regimen demonstrated efficacy in patients with BRAF-mutant mCRC, with overall response rates was 63.2% (n = 12). Changes in levels of ctDNA at 4 weeks predicts therapeutic responses. CBLB, TP53 and APC baseline mutations were enriched in baseline samples of innate resistant patients. In contrast, MYC and Cell-cycle pathway alterations were enriched in baseline samples of patients with acquired resistance. Acquired RAS and MAPK mutations, known to confer resistance to BRAF inhibitors, were identified in 60% of acquired resistance patients. Moreover, acquired TGFBR2 and SMAD4 loss-of-function mutations were identified as novel resistant mechanism to the combination of BRAF and EGFR inhibition. Experimental validation is ongoing. Conclusions: Longitudinal analysis of ctDNA in BRAF CRC patients provides insights of molecular difference between innate and acquired resistance and gene alternation during resistance requiring. Serial ctDNA monitoring provides early indication of response to BRAF inhibitor in CRC patients.

Continued cetuximab in second-line treatment for patients with unresectable metastatic wild-type KRAS, NRAS, and BRAF colorectal cancer after disease progression during first-line cetuximab-based therapy.

127 Background: Cetuximab plus chemotherapy is a first-line treatment option for metastatic RAS wild type colorectal cancer patients. Currently, no data are available on continuing cetuximab or changing bevacizumab as second-line therapy beyond first-line cetuximab-based chemotherapy. Methods: Patients (aged ≥18 years) with metastatic, histologically and genetically confirmed wild-type KRAS, NRAS and BRAF colorectal cancer progressing after first-line cetuximab plus chemotherapy were randomly assigned (1:1 ratio) to second-line chemotherapy with cetuximab (arm A) or with bevacizumab (arm B) 2·5 mg/kg per week equivalently. The choice between oxaliplatin-based or irinotecan-based second-line chemotherapy depended on the first-line regimen (switch of chemotherapy). The primary endpoints were progression-free survival (PFS) and objective response rate (ORR). The second endpoint was overall survival (OS). Results: 77 Patients (from July 1, 2016 to Sept 20, 2019, 77) were randomized (41 in arm A and 36 in arm B). ORR was 29.3% and 19.4% in Arm A and Arm B ( p= 0.31). PFS was 7.2 months (95% CI 5.2–9.2) for Arm A and 5.9 months (95% CI 5.1–6.7) for Arm B ( p= 0.677). OS was 18.5 months (95% CI 15.1–21.8) for Arm A and 17.5 months (95% CI 15.4–19·7) for Arm B ( p= 0.444). Patients with ECOG PS 0 had significantly longer PFS and OS than ECOG PS 1 in second-line therapy whether cetuximab or bevacizumab combined with chemotherapy. ECOG 0 group vs ECOG 1 group, PFS was 8.7 months vs 4.6 months (p = 0.00) and OS was 21.2 months vs 12.3 months (p = 0.00). Moreover, ETS may predict efficacy of second-line continued cetuximab. The most frequently grade 3–4 adverse events in both arms were neutropenia (19.4% VS 16.7%), diarrhea (7.5% vs 11.1%), and nausea(10% vs 13.9%). Conclusions: Continuing cetuximab or changing bevacizumab plus standard second-line chemotherapy in patients with metastatic wild-type KRAS, NRAS and BRAF colorectal cancer after first-line cetuximab plus chemotherapy have similar clinical benefits. ECOG score is an independent predictor of prognosis and second-line treatment efficacy for colorectal cancer.

Abstract 3170: BRAF expression is associated with poor survival in colorectal cancers independent of BRAF (V600E) mutation

Abstract The RAS pathway is known to be commonly activated and a key determinant of colorectal cancer (CRC) biology, with mutational activation of RAS/RAF genes observed in ~50% of CRC patients. The relationship between gene mutation and expression is, however, poorly understood. We analyzed how the expression of RAS/RAF genes vs. their respective mutations impacted outcomes. The relationship to other rarely mutated RAS/RAF genes (HRAS, ARAF, RAF1 (i.e. c-RAF)) was also examined in these tumors. While KRAS expression was positively associated with KRAS mutations (r=0.26, p<0.0001), BRAF expression was, unexpectedly, negativelycorrelated with BRAF (V600E) mutation (r= -0.18, p=0.0001). Thus, a substantial number of patients express relatively high levels of BRAF absent of BRAF mutations. Additional analysis indicates that lower BRAF expression was significantly correlated with MSI or the CMS1 subtype that is strongly associated with BRAF (V600E). Intriguingly, despite negative association with mutant BRAF (V600E) that is well-established to be associated with poor prognosis, higher BRAF expression was also associated with poor overall survival, as indicated by Kaplan-Meier survival analysis of BRAF expression by quartiles in either 458 CRCs (Logrank test for trend p=0.0262) in 406 WT BRAF CRCs (p=0.0102). Note that the expression of all other five RAS/RAF genes (HRAS, KRAS, NRAS, ARAF and RAF1) was not associated with survival. Further analysis with the gene expression signatures measuring RAS pathway activation reveals that unlike BRAF (V600E) (or KRAS mutations or KRASexpression), BRAF expression appeared to be negatively correlated with RAS pathway activation signature scores. Taken together, these data suggest that higher BRAF expression in CRC patients may activate an “alternative” pathway to the canonical pathway directed by mutant BRAF (V600E) as yet another means to deliver tumor progression. Citation Format: Mingli Yang, Michael J. Schell, Timothy J. Yeatman. BRAF expression is associated with poor survival in colorectal cancers independent of BRAF (V600E) mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3170.

Long term response on Regorafenib in non-V600E BRAF mutated colon cancer: a case report

BackgroundNon-V600E BRAF mutated colorectal cancer (CRC) is a rare disease entity with specific clinical features. These tumors are less likely to have microsatellite instability than CRC with a V600E BRAF mutation and often harbor a KRAS or NRAS mutation. Notably, median overall survival is longer than in wild-type BRAF CRC. Little is known about treatment possibilities in these patients.Case presentationWe present the case of a 59 year old patient with a rare mutation in BRAF codon 594, who progressed rapidly on all classical therapies but experienced a clear and long lasting response on treatment with Regorafenib.ConclusionLittle is known about therapies that can be effective in the rare non-V600E BRAF mutated CRCs. We present a patient who had a definite response to treatment with Regorafenib. There are no predictive markers that define a subset of CRC patients who benefit most from Regorafenib. The specific features of this non-V600E BRAF mutated CRC may be relevant in the exploration of predictive biomarkers for the efficacy of Regorafenib.

5-Arylidene(chromenyl-methylene)-thiazolidinediones: Potential New Agents against Mutant Oncoproteins K-Ras, N-Ras and B-Raf in Colorectal Cancer and Melanoma.

Background and objectives: Cancer represents the miscommunication between and within the body cells. The mutations of the oncogenes encoding the MAPK pathways play an important role in the development of tumoral diseases. The mutations of KRAS and BRAF oncogenes are involved in colorectal cancer and melanoma, while the NRAS mutations are associated with melanoma. Thiazolidine-2,4-dione is a versatile scaffold in medicinal chemistry and a useful tool in the development of new antitumoral compounds. The aim of our study was to predict the pharmacokinetic/pharmacodynamic properties, the drug-likeness and lead-likeness of two series of synthetic 5-arylidene(chromenyl-methylene)-thiazolidinediones, the molecular docking on the oncoproteins K-Ras, N-Ras and B-Raf, and to investigate the cytotoxicity of the compounds, in order to select the best structural profile for potential anticancer agents. Materials and Methods: In our paper we studied the cytotoxicity of two series of thiazolidine-2,4-dione derivatives, their ADME-Tox properties and the molecular docking on a mutant protein of K-Ras, two isoforms of N-Ras and an isoform of B-Raf with 16 mutations. Results: The heterocyclic compounds strongly interact with K-Ras and N-Ras right after their posttranslational processing and/or compete with GDP for the nucleotide-binding site of the two GTPases. They are less active against the GDP-bound states of the two targets. All derivatives have a similar binding pattern in the active site of B-Raf. Conclusions: The data obtained encourage the further investigation of the 5-arylidene(chromenyl-methylene)-thiazolidinediones as potential new agents against the oncoproteins K-Ras, N-Ras and B-Raf.

The atypical cyclin CNTD2 promotes colon cancer cell proliferation and migration

Colorectal cancer (CRC) is one of the most common cancers worldwide, with 8–10% of these tumours presenting a BRAF (V600E) mutation. Cyclins are known oncogenes deregulated in many cancers, but the role of the new subfamily of atypical cyclins remains elusive. Here we have performed a systematic analysis of the protein expression levels of eight atypical cyclins in human CRC tumours and several cell lines, and found that CNTD2 is significantly upregulated in CRC tissue compared to the adjacent normal one. CNTD2 overexpression in CRC cell lines increases their proliferation capacity and migration, as well as spheroid formation capacity and anchorage-independent growth. Moreover, CNTD2 increases tumour growth in vivo on xenograft models of CRC with wild-type BRAF. Accordingly, CNTD2 downregulation significantly diminished the proliferation of wild-type BRAF CRC cells, suggesting that CNTD2 may represent a new prognostic factor and a promising drug target in the management of CRC.

Abstract 1665: AZ304, a novel dual BRAF inhibitor, exerts antitumor activity in colorectal cancers independent of BRAF status

Abstract Background: BRAF mutation is associated with poor clinical outcome of malignant tumors and mediates resistance to chemotherapy and targeted therapy. We aimed to determine whether wild type and V600E mutant BRAF colorectal cancers exhibit distinct sensitivities to dual BRAF inhibitor, AZ304. Methods: We measured kinase activity by AlphaScreen assay. MTT assays, colony-formation assays, and Western blot were performed to evaluate the in vitro anti-tumor effects of AZ304. In vivo efficacy was investigated by xenograft study and Immunohistochemistry. Results: AZ304 exhibited potent inhibitory activity against both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF with enzyme IC50values of 79nM and 38nM, respectively. By suppressing the ERK phosphorylation, AZ304 led to effective cell proliferation inhibition against a panel of human cancer cell lines with different BRAF and RAS generic status. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. Additionally, combination with the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independent of BRAF mutational status. Conclusions: The BRAF inhibitor, AZ304, had a broad spectrum of antitumor activities and the combination with Cetuximab significantly enhanced AZ304 potency against colorectal cancers in vitro and in vivo. Citation Format: Rui Ma, Ling Xu, Xiaofang Che, Xiujuan Qu, Lisa Drew, Minhui Shen, Tony Cheung, Yunpeng Liu. AZ304, a novel dual BRAF inhibitor, exerts antitumor activity in colorectal cancers independent of BRAF status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1665.

AZ304, a novel dual BRAF inhibitor, exerts anti-tumour effects in colorectal cancer independently of BRAF genetic status.

BackgroundBRAF mutation is associated with poor clinical outcome of patients with malignant tumours, and mediates resistance to chemotherapy and targeted therapy. This study aimed to determine whether V600E mutant and wild type BRAF colorectal cancers exhibit distinct sensitivities to the dual BRAF inhibitor AZ304.MethodsKinase activity was assessed by the AlphaScreen assay. Then, MTT assay, EdU assay, colony-formation assay and Western blot were performed to evaluate the anti-tumour effects of AZ304 in vitro. In vivo efficacy was investigated by xenograft analysis and immunohistochemistry.ResultsAZ304 exerted potent inhibitory effects on both wild type and V600E mutant forms of the serine/threonine-protein kinase BRAF, with IC50 values of 79 nM and 38 nM, respectively. By suppressing ERK phosphorylation, AZ304 effectively inhibited a panel of human cancer cell lines with different BRAF and RAS genetic statuses. In selected colorectal cancer cell lines, AZ304 significantly inhibited cell growth in vitro and in vivo, regardless of BRAF genetic status. In addition, the EGFR inhibitor Cetuximab enhanced the potency of AZ304 independently of BRAF mutational status.ConclusionsThe BRAF inhibitor AZ304 has broad spectrum antitumour activity, which is significantly enhanced by combination with Cetuximab in colorectal cancers in vitro and in vivo.

Frequency of NRAS Gene Mutation in Wild Type KRAS and BRAF Colorectal Cancers; a Single Center Study.

BACKGROUND Incidence of colorectal cancer is increasing in countries such as Iran. Molecular biomarkers play very important role in the diagnosis, treatment, and prognosis of this cancer. Mutation in the RAS family (including KRAS and NRAS) is one of these important molecular biomarkers, which should be tested before starting treatment with anti-EGRF (Epidermal growth factor) drugs. Objectives: There has been very few reports about the frequency of NRAS mutation from Iran and no study from south of the country. In this article we will describe our experience about the frequency of NRAS mutation in colorectal cancers from the largest referral center in the south of Iran. METHODS During 5 years (2011-2015), we had 52 cases of colorectal cancers with wild type KRAS and BRAF in the hospitals affiliated to Shiraz University of Medical Sciences with enough tissue for molecular studies. NRAS mutation analysis was performed on paraffin embedded formalin fixed tissue of these cases by polymerase chain reaction (PCR)-sequencing method. RESULTS Among these 52 cases of colorectal cancer with wild type KRAS and BRAF, there has been 3 (5.7%) cases with mutant NRAS. One of the mutations has been in codon 12 and two in codon 61. No mutation in codon 13 was found. All the three cases were women with stage IV and well differentiated histomorphology. CONCLUSION Our results showed that frequency of NRAS mutation in colorectal cancer is rare, which is very close to other studies from different geographic areas of the world.

Probing a 3,4'-bis-guanidinium diaryl derivative as an allosteric inhibitor of the Ras pathway.

Mutations in the Ras-pathway occur in 40-45% of colorectal cancer patients and these are refractory to treatment with anti-EGFR-targeted therapies. With this in mind, we have studied novel guanidinium-based compounds with demonstrated ability to inhibit protein kinases. We have performed docking studies with several proteins involved in the Ras-pathway and evaluated 3,4'-bis-guanidinium derivatives as inhibitors of B-Raf. Compound 3, the most potent in this series, demonstrated strong cytotoxicity in (WT)B-Raf colorectal cancer cells and also cells with (V600E)B-Raf mutations. Cell death was induced by apoptosis, detected by cleavage of PARP. Compound 3 also potently inhibited ERK1/2 signalling, inhibited EGFR activation, as well as Src, STAT3 and AKT phosphorylation. Mechanistically, compound 3 did not inhibit ATP binding to B-Raf, but direct assay of B-Raf activity was inhibited in vitro. Summarizing, we have identified a novel B-Raf type-III inhibitor that exhibits potent cellular cytotoxicity.

Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients

AimsTo evaluate the accuracy, consumable cost and time around testing (TAT) of a next-generation sequencing (NGS) assay, the Ion Torrent AmpliSeq Colon and Lung Cancer Panel, as an alternative to...

3'-Deoxy-3'-[18F]-Fluorothymidine PET imaging reflects PI3K-mTOR-mediated pro-survival response to targeted therapy in colorectal cancer.

Biomarkers that predict response to targeted therapy in oncology are an essential component of personalized medicine. In preclinical treatment response studies that featured models of wild-type KRAS or mutant BRAF colorectal cancer treated with either cetuximab or vemurafenib, respectively, we illustrate that [18F]-FLT PET, a non-invasive molecular imaging readout of thymidine salvage, closely reflects pro-survival responses to targeted therapy that are mediated by PI3K-mTOR activity. Activation of pro-survival mechanisms forms the basis of numerous modes of resistance. Therefore, we conclude that [18F]-FLT PET may serve a novel and potentially critical role to predict tumors that exhibit molecular features that tend to reflect recalcitrance to MAPK-targeted therapy. Though these studies focused on colorectal cancer, we envision that the results may be applicable to other solid tumors as well.

Molecular profiling of patients (pts) with advanced colorectal cancer (CRC): Princess Margaret Cancer Centre experience.

459 Background: The prevalence of somatic mutations other than KRAS, PIK3CA and BRAF in CRC has not been well described. This study reports molecular profiling of pts with advanced CRC in clinical practice. Methods: Pts with advanced CRC are enrolled in an ongoing institution-wide screening program. Molecular profiling is performed on FFPE archival tissues using a customized Sequenom panel (23 genes, 279 mutations) or the Illumina MiSeq TruSeq Amplicon Cancer Panel (48 genes, 212 amplicons, ≥ 500x coverage) in our CLIA-certified laboratory. PTEN negative tumor is defined by IHC with H score <1. Demographic and clinical data are collected. Data was analyzed by chi-square test. Results: From March 2012 to August 2013, 132 pts were enrolled. One or more mutations were found in 50% (58/117) and 93%(14/15) of pts using Sequenom and MiSeq platforms respectively. Mutation rate was 40% for KRAS, 9% for PIK3CA, 2% for BRAF, 4% for NRAS, and 1% for each of CTNNB1, ERBB2, FGFR3, and EGFR. Mutations outside of codons 12/13 were found in 8% (4/53) of tumors with KRAS mutations. MiSeq showed additional mutations including ERBB4 (1/15), SMAD4 (1/15), FRXW7 (1/5), APC (5/15), and TP53 (12/15) in CRC. Co-mutation with KRAS was found in 58% (7/12) of pts with PIK3CA mutation and 80% (4/5) of pts with APC mutation. PTEN negative tumor was present in 20% (11/55) of pts. Presence of any mutation was significantly more common for pts with right-sided CRC (73%) compared to those with left-sided CRC (47%) and rectal CRC (38%) (p < 0.01). There was a trend toward increased proportion of KRAS mutation in right-sided CRC (p=0.06). There was no difference in age, ethnicity, gender, smoking status, stage at presentation, pathology grade, and response to first-line chemotherapy between right and left-sided CRC. Conclusions: Somatic alterations are common in advanced CRC. Targeted next-generation DNA sequencing identifies additional clinically relevant alterations than multiplex genotyping. Differences in the mutation profile of right and left-sided CRC tumors should be further investigated. Updated results will be presented at the meeting.

Associations between patient (pt) colorectal cancer (CRC) tumor KRAS and BRAF mutation (mut) status and overall survival (OS).

473 Background: Evolving information is emerging regarding patient tumor molecular data and associations with patient clinical characteristics and survival outcomes; we sought to evaluate pt clinical data with CRC tumor mut status. Methods: Since January 2008 tumor pts with stage IV CRC (principally primary tumors) were analyzed for KRAS (codon 12, 13, and 61) mut status as standard-of-care. A subset of pts tumors underwent BRAF and KRAS codon 164 mut testing. Results: 1,891 pts with CRC whose tumors underwent molecular mut analysis were identified from the clinical record. There was no statistically significant difference in pt overall survival from time of diagnosis for stages I/II (data not shown) and III; for stage IV pts there was an inferior OS associated with BRAF mut tumors (see Table). Tumor BRAF mut was significantly detected among ever smokers (10.2%) as compared to never smokers (5.4%), p < 0.001; meanwhile, tumor KRAS mut and wild type distribution were similar between ever smokers and never smokers. KRAS mut was significantly observed in CRC patients with distant lymph node, hepatic and lung metastasis, p < 0.001. Type 2 diabetes, intake of metformin or ACE inhibitors, were not significantly correlated with tumor KRAS or BRAF mut status. Conclusions: Pts with stage IV CRC BRAF mut tumors have a higher likelihood of prior or current smoking history in addition to having an adverse survival prognosis; pts with stage IV KRAS mut tumors have an increased association with distant lymph node, hepatic, and lung metastases as compared to KRAS wild type and BRAF mut tumors. Studies are ongoing to determine how tumor molecular characteristics may help to predict clinical outcomes and provide a useful to guide pt care. [Table: see text]

Off-label use of cetuximab plus sorafenib and panitumumab plus regorafenib to personalize therapy for a patient with V600E BRAF-mutant metastatic colon cancer

Sorafenib, the first agent developed to target BRAF mutant melanoma, is a multi-kinase inhibitor that was approved by the FDA for therapy of kidney and subsequently liver cancer, and is currently in clinical trials for thyroid, lung and brain cancer. Colorectal cancer with V600E BRAF mutation has shown relative resistance to standard chemotherapy regimens, as well as lack of efficacy to vemurafenib in clinical trials. New treatments are needed for BRAF-mutant colorectal cancer. We report a case of a patient with BRAF-mutant metastatic colon cancer whose disease had progressed on FOLFOX plus bevacizumab and subsequent FOLFIRI plus cetuximab. Based on preclinical data published in Nature in 2012 suggesting that successful therapeutic targeting of BRAF in colorectal cancer may require concomitant targeting of the EGFR, we offered this patient without other attractive options the combination of sorafenib plus cetuximab, in off-label use with informed consent. Sorafenib and cetuximab therapy led to a mixed radiographic response with some areas showing dramatic improvement and other areas showing stable disease over a 7-month period which is a notably long period of progression-free survival for V600E BRAF mutated colon cancer. The cetuximab plus sorafenib therapy was very well-tolerated by the patient who remained on it long enough until another therapy option, regorafenib, was approved in September 2012. The patient was offered single agent regorafenib at the time of progression. At the time of progression on single agent regorafenib, panitumumab was combined with regorafenib and this was also well-tolerated and appeared to slow disease progression. Further study of these approaches in the clinic as personalized treatment of BRAF-mutant advanced colorectal cancer is warranted.

Targeting mutant BRAF in colorectal cancer

Background Mutations in BRAF V600E oncogene (BRAFMT) occurs in 8-15% of colorectal cancer (CRC) patients1. This mutation constitutively activates MAPK signalling, resulting in a proliferative and survival advantage for the tumour cells and oncogenic BRAF status has been linked with poor prognosis2. Despite introduction of the BRAFMT specific inhibitor Vemurafenib in metastatic melanoma3, there is no effective treatment strategy for BRAFMT CRC patients. This study aimed to assess the effectiveness of Ganetespib (HSP90 inhibitor), the multi-kinase inhibitor (CRAF/ VEGFR/PDGFR) Sorafenib and the BRAFMT inhibitor Vemurafenib in BRAFMT CRC cell line models.