Types Of KRAS Mutations Research Articles

P-0206 - Kras Mutations in Metastatic Colorectal Cancer and the Effect of Bevacizumab

Background: Laboratory research of metastatic colorectal cancer subdivided the disease into a number of molecular sub-types mainly based on the status of KRAS gene. Recently different effect of certain monoclonal antibody (cetuximab) was shown with G13D KRAS mutation against previous data of drug resistance in KRAS mutant tumors. The question of different biological behavior of a tumor and treatment effect of bevacizumab was evaluated according to a KRAS sub-types status. Methods: Included were 397 colorectal cancer patients from the Israeli population-based Molecular Epidemiology of Colorectal Cancer study treated for their first metastatic event with irinotecan-based or 5-fluorouracil protocols, with or without bevacizumab, and no surgery. Patients were diagnosed between 1998 and 2010 and were followed up until October 2011; Mutations in KRAS codons 12, and 13 were identified by direct sequencing and SNP assay-by-design. Date of metastases and death and treatment details were extracted from the oncological follow-up records supported by computerized pharmacy records. Results: A total of 267 cases were treated with irinotecan-based chemotherapy, 96 (36%) of whom with combination with bevacizumab. A total of 130 cases were treated with 5-FU treatment, majority (98%) without bevacizumab. Mutation in codon 12 or codon 13 in KRAS was identified in 162 cases (41%), with 16.4%, 10.1% and 7.1% harboring mutations in the codon G12d, codon G12V and codon G13D, respectively. Combination 1st line treatment with IRINOTECAN-based chemotherapy and bevacizumab showed improved OS in cases with KRAS wild type tumors (HR = 0.69; p = 0.048), and KRAS codon 12 or codon 13 mutated tumors (HR = 0.68, p = 0.07). This effect was not different across types of KRAS mutation: G13D (HR = 0.46, p = 0.25), G12V (HR = 0.40; p = 0.049), G12D (HR = 0.76; p = 0.46), KRAS mutations (p for interaction =0.54) Assessment of prognostic effect in 5-FU only treated patients shows that relative to cases without any mutation in the codon 12 or codon 13, G13D (HR = 2.43) and G12D (HR = 1.75) mutations experienced a worse OS, while G12V (HR = 1.28) and all other codon 12 mutations combined (HR = 1.17) did not (p for difference = 0.075). Conclusion: The treatment effect of bevacizumab was not found to have a significant difference between KRAS sub-types of metastatic colorectal cancer. The data supports a prognostic effect of G13D and G12D KRAS mutations.

Outcomes of patients with advanced cancer and KRAS mutation in phase I clinical trials.

e22086 Background: The prognostic significance of KRAS mutations varies by mutation type. We compared the outcomes of patients with different KRASmutations treated in phase I clinical trials. Methods: Patients with advanced cancer and KRAS mutation who were treated in the phase I program from 9/08 to 12/12 were analyzed. The presence and type of KRAS mutation in the patients’ tumor samples were assessed in a CLIA-certified laboratory using PCR, sequenom analysis or next-generation sequencing. We compared clinical outcomes according to type of mutation, tumor, and therapy. Results: KRAS mutations were identified in 365 patients (codon 12, n=278; codon 13, n = 49; other codon, n = 38). The median age was 59 yrs (range, 18-87); and 46% men. Of 365 patients, 260 had KRAS mutation alone; 105 patients had 130 additional aberrations: PI3KCA, n=45 (39%); p53, n=29 (25.2%); PTEN, n=22 (19%); other, n=34 (29%). Of 260 patients, 159 were treated and evaluable for response. Outcomes in patients with KRAS mutation alone or with KRAS and additional molecular aberrations are shown in the Table. Conclusions: Keeping in mind that the number of patients was small, our results demonstrate heterogeneity in outcomes of patients with KRAS mutations. Combination studies of MEK inhibitors with other drugs may have improved antitumor activity. [Table: see text]

Comparison of the characteristics and clinical course of 677 patients with metastatic lung cancers with mutations in KRAS codons 12 and 13.

8025 Background: Patients (pts) with KRAS mutant lung cancers have a shorter survival compared to pts with KRAS/EGFR wild type tumors (Johnson et al, Cancer 2012). Whether outcomes for patients with KRAS mutant metastatic lung cancers differ by smoking status or specific amino acid substitution is unknown. In order to understand the impact of KRAS mutation subtype in the metastatic setting, we analyzed a large cohort of patients with KRAS mutant metastatic lung cancer. Methods: We identified all pts with KRAS mutant metastatic or recurrent lung cancers from Feb 2005 to Aug 2011. KRAS mutation type, clinical characteristics, and outcomes from diagnosis were obtained from the medical record. A multivariate cox proportion hazard model was used to identify factors associated with overall survival. Results: KRAS mutations were identified in 677 pts (53 at codon 13, 624 at codon 12). Median age: 66 (range 31-89), women: 62%, never smokers: 7%. Pts with transition mutations (n=157) were more likely to be never-smokers (p<0.0001). There was no difference in outcome for pts with KRAS transition versus transversion mutations (p=1) or when comparing current/former smokers to never smokers (p=0.33). There was no difference in overall survival (OS) when comparing specific amino acid substitutions (G12C=366, G12V=141, G12D=114, G12A=68, G13C=27, G13D=23, G12S=19, G12F=11)(p=0.20). Pts with KRAS codon 13 mutant tumors had inferior OS compared to pts with codon 12 mutant tumors, median 13 months (mo) (95% CI 13-17 mo) and 16 mo (95% CI 9-16 mo), respectively (p=0.009). There was no difference in frequency of receiving platinum-based chemotherapy or chemotherapy of any kind between pts with codon 12 and 13 mutant tumors. In a multivariate Cox model which included age, gender and smoking status, KRAS codon 13 mutation was associated with worse overall survival than KRAS codon 12 mutation (HR 1.52 95% CI 1.11-2.08 p=0.008). Conclusions: Among pts with KRAS mutant metastatic lung cancers, smoking history, and specific amino acid substitution do not affect outcome. Patients with KRAS codon 13 mutant metastatic lung cancer have shorter survival compared to pts with KRAS codon 12 mutant lung cancer.

Abstract 1199: NSCLC KRAS G12C and G12V mutations drive different pathways and display specific drug sensitivity patterns.

Abstract Background: Although a recent body of literature has described the molecular environment associated with KRAS G12D mutations (6%) in lung adenocarcinoma, the global mutational and gene expression environments of the most frequent types of KRAS mutations : G12C (45%) and G12V (25%) remain largely unknown. This study aimed: (i) to describe the differences in the mutational and gene expression environment in KRAS G12C and G12V genotypes; (ii) to correlate molecular models predictive of KRAS G12C and G12V genotypes with in vitro drug sensitivity in a large drug panel screen of cell lines (Cancer Cell Line Encyclopedia, CCLE). Materials and Methods: Data sets were extracted from public repositories (TCGA LUAD n= 249, BATTLE n= 124, CCLE Lung n= 82) or those pending release upon publication (CHEMORES n= 123). We used gene set enrichment analysis (GSEA) to identify pathways associated with mutational and gene expression changes, and penalized logistic regression (ElasticNet) to build models of KRAS G12C and G12V mutations. Correlations were computed between these models and drug sensitivity represented by the IC50 of 21 drugs on the 82 lung cancer cell lines of the CCLE. Results: The KRAS G12C phenotype, defined either by the mutational environment or by the associated gene expression signature, was enriched in pathways related to direct KRAS binding proteins such as GRB2, SOS1, SHC, FRS2 pathways (adjusted P values <.01). Conversely, the KRAS G12V phenotype was consistently enriched in pathways related to cell-cycle checkpoint gene signaling including P53, Rb, ATM and PML (adjusted P values <.05). Separate G12C and G12V gene expression models trained to predict the respective mutations in TCGA were robust by independent validation in BATTLE and CCLE with AUC = 0.78 & 0.61 and AUC= 0.60 & 0.76 respectively. In CCLE we computed cell-line specific scores for our G12C and G12V models and correlated that with IC50 for the 21 available drugs. Interestingly, G12C model scores positively correlated with sensitivity to AEW541 (IGF1R inhibitor) (P=.001), while G12V model scores were positively correlated with sensitivity to PD-0332991 (cyclin D kinase 4/6 inhibitor) (P=.02) and with resistance to TKI258 (multi-target VEGFR/PDGFR kinase inhibitor) (P=.02). Conclusion: Our analyses reveal singular differences between KRAS G12C and G12V mutations in lung cancer that are consistent when viewed either in terms of associated mutations, gene expression profiles, or sensitivity to drugs. The G12C phenotype exhibits a strong association with direct KRAS interactors and correlates with sensitivity to IGF1R inhibitors. The G12V phenotype demonstrates a strong association with cell-cycle checkpoint pathways and predicts sensitivity to the Cyclin D kinase inhibitor, PD-0332991. Citation Format: Charles Ferte, Justin Guinney, Jonathan Derry, erich H. huang, Benjamin Besse, Jean-Charles Soria. NSCLC KRAS G12C and G12V mutations drive different pathways and display specific drug sensitivity patterns. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1199. doi:10.1158/1538-7445.AM2013-1199

Phase II trial of combined chemotherapy with irinotecan, S-1, and bevacizumab (IRIS/Bev) in patients with metastatic colorectal cancer (mCRC): Hokkaido Gastrointestinal Cancer Study Group (HGCSG) trial—Comparison of the efficacy of KRAS status.

536 Background: Mutations of the KRAS gene were identified as a predictive marker in mCRC for anti-EGFR antibody. Previously reported data suggest that the longer overall survival (OS) observed with bevacizumab (BV) treatment in mCRC is independent of alterations in the KRAS status. We analized efficacy of BV combined irinotecan and S-1 (IRIS/Bev) in mCRC relative to KRAS status. Methods: In the retrospective analysis (n=53) of patients who participated in the Phase II trial of IRIS/Bev, additional statistical analyses were done with data from KRAS mutational analyses. In this trial, eligible patients had to have mCRC with a confirmed diagnosis of adenocarcinoma, an age of >20 years, and no history of prior chemotherapy. S-1 40-60 mg twice daily p.o. was given on days 1-14 and irinotecan 100 mg/m2 and bevacizumab 5 mg/kg i.v. were given on days 1 and 15 of a 28-day cycle. The Response Evaluation in Solid Tumors (RECIST) criteria version 1.0 was used to assess tumor response. The Kaplan–Meier method was used to determine Progression-free survival (PFS) and OS. Log-rank test was used to compare with mutant or wild-type KRASin terms of PFS and OS. All statistical tests were performed using SPSS. Results: The target number of 53 patients was enrolled as of March 2009. KRAS status was assessed in 43 patients (wild = 27, mutant = 16). Response rate was 63.0% with wild-type and 68.8% with mutant-type KRAS, that was not significant (p=0.752). The median Progression-free survival was 17.1 months with wild-type and 22.7 months with mutant-type KRAS, that was not significant (p=0.531). And median OS was 49.0 months with wild-type and 38.0 months with mutant-type KRAS, that was not significant(p=0.906) as well. Conclusions: IRIS/Bev provides clinical benefit in patients with mCRC expressing either mutant or wild-type KRAS. Randomized control trial comparing this regimen with oxaliplatin containing regimen(XELOX or mFOLFOX6 plus bevacizumab: TRICOLORE study) is already started. Comparison of the efficacy of KRAS status is also planned in this study. Clinical trial information: NCT00569790.

The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non–small cell lung cancer

The role of KRAS mutations in molecular targeted therapy by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) has not been fully understood. The present investigation is aimed at an elucidation of the role of specific KRAS mutation types in predicting outcomes of patients with advanced NSCLC receiving EGFR-TKI therapy. Initially, 448 NSCLC patients were tested for the presence of KRAS mutations, to obtain frequencies of specific KRAS mutation types. Subsequently, the clinical outcome of treatment was evaluated in a subgroup of 38 KRAS-positive patients receiving EGFR-TKI therapy. KRAS mutations were detected in 69 of 448 patients (15.4%), mostly in smokers (17.86% vs. 5.8%, P = 0.0048), and appeared more frequently in adenocarcinomas than in squamous cell NSCLC or NSCLC that is not otherwise specified (21% vs. 6.99% vs. 4.4%, P = 0.0004). The most frequent type of KRAS mutation was G12C. The progression-free survival (PFS) was doubled in a group of non-G12C patients compared with that of the G12C group (9.0 wk vs. 4.3 wk, P = 0.009). The overall survival (OS) was not significantly different between non-G12C and G12C groups (12.1 wk vs. 9.3 wk, P = 0.068). The G12C KRAS mutation is a strong negative predictor for EGFR-TKI treatment, whereas other KRAS mutation types have not negatively predicted treatment efficacy compared with that for the wild-type KRAS genotype.

KRAS and BRAF mutations in 203 esophageal squamous cell carcinomas: pyrosequencing technology and literature review.

BackgroundEpidermal growth factor receptor (EGFR) signaling is one of the most promising targets for molecular-targeted therapies in esophageal squamous cell carcinoma (ESCC). Thus, the molecular diagnosis of KRAS and BRAF mutations is clinically important in therapeutic decision making. However, the frequency of KRAS and BRAF mutations in ESCCs remains inconclusive because of the limited sample sizes of previous studies (all N ≤ 80). Pyrosequencing is a nonelectrophoretic nucleotide extension sequencing technology that can be used for mutation testing.MethodsThe frequency of KRAS and BRAF mutations was examined using a nonbiased database of 203 resected ESCCs and a high-throughput pyrosequencing assay.ResultsThe validity of the KRAS pyrosequencing method was initially demonstrated by detection of all 4 types of KRAS mutations [c.35G>T (codon 12 GGT>GTT), c.35G>A (codon 12 GGT>GAT), c.34G>T (codon 12 GGT>TGT), c.38G>A mutation (codon 13 GGC>GAC)], which had been previously diagnosed using Scorpion-ARMS technology, in 9 colon cancer tissues (9 of 9; 100 %). Similar results were demonstrated for BRAF mutational status in 3 colon cancer cell lines (HCT116, Colo201, and HT29), which were validated by Sanger dideoxy sequencing. Subsequently, the KRAS mutation was found to be extremely rare (1 of 203; 0.5 %), and the BRAF mutation was absent (0 of 203; 0 %), in the dataset of 203 ESCCs.ConclusionsThese results suggest that KRAS and BRAF mutations play a limited role in the development of ESCC and that mutation analysis is not useful as a screening test for sensitivity to anti-EGFR therapy in ESCC.

Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: higher susceptibility of women to smoking-related KRAS-mutant cancers.

The molecular epidemiology of most EGFR and KRAS mutations in lung cancer remains unclear. We genotyped 3,026 lung adenocarcinomas for the major EGFR (exon 19 deletions and L858R) and KRAS (G12, G13) mutations and examined correlations with demographic, clinical, and smoking history data. EGFR mutations were found in 43% of never smokers and in 11% of smokers. KRAS mutations occurred in 34% of smokers and in 6% of never smokers. In patients with smoking histories up to 10 pack-years, EGFR predominated over KRAS. Among former smokers with lung cancer, multivariate analysis showed that, independent of pack-years, increasing smoking-free years raise the likelihood of EGFR mutation. Never smokers were more likely than smokers to have KRAS G > A transition mutation (mostly G12D; 58% vs. 20%, P = 0.0001). KRAS G12C, the most common G > T transversion mutation in smokers, was more frequent in women (P = 0.007) and these women were younger than men with the same mutation (median 65 vs. 69, P = 0.0008) and had smoked less. The distinct types of KRAS mutations in smokers versus never smokers suggest that most KRAS-mutant lung cancers in never smokers are not due to second-hand smoke exposure. The higher frequency of KRAS G12C in women, their younger age, and lesser smoking history together support a heightened susceptibility to tobacco carcinogens.

The Usability of Allele-Specific PCR and Reverse-Hybridization Assays for KRAS Genotyping in Serbian Colorectal Cancer Patients

Colorectal cancers (CRCs) with wild-type KRAS respond to EGFR-targeted antibody treatment. Analysis of the hotspot clustered mutations in codons 12 and 13 is compulsory before therapy and no standardized methodology for that purpose has been established so far. Since these mutations may have different biological effects and clinical outcome, reliable frequency and types of KRAS mutations need to be determined for individual therapy. The purpose of this study was to describe the KRAS mutation spectrum in a group of 481 Serbian mCRC patients and to compare the general performances of allele-specific PCR and reverse-hybridization assays. KRAS testing was performed with two diagnostic analyses, DxS TheraScreen K-RAS PCR Kit and KRAS StripAssay™. KRAS mutations in codons 12 and 13 were present in 37.6 % of analyzed formalin-fixed paraffin-embedded (FFPE) DNA samples. The seven most frequent mutation types were observed with both assays: p.G12D 34.6 %, p.G12V 24.9 %, p.G12A 10.3 %, p.G12C 8.1 %, p.G12S 5.4 %, p.G12R 1.6 %, and p.G13D 15.1 %. Regarding double mutants, 0.8 % of them were present among all tested samples and 2.2 % among KRAS mutated ones. Two screening approaches that were used in this study have been shown as suitable tests for detecting KRAS mutations in diagnostic settings. In addition, they appear to be good alternatives to methods presently in use. In our experience, both methods showed capacity to detect and identify double mutations which may be important for potential further subgrouping of CRC patients.

1302P - Clinical Impact of Presence and Type of Kras Mutation in a Population of EGFR Wild Type (WT) Advanced Non-Small Cell Lung Cancer (NSCLC) Patients (PTS) Treated with Platinum-Based Chemotherapy: A Retrospective Analysis

ABSTRACT Background In this retrospective analysis we assessed whether KRAS mutation would affect the clinical outcome of EGFR WT advanced NSCLC pts treated with a first-line platinum-based chemotherapy. Moreover, the effect of specific mutant KRAS was evaluated. Methods One hundred and ninety-five EGFR WT, advanced NSCLC pts were included in the analysis. Study pts were treated at the Medical Oncology of the Perugia Hospital from Jan 2005 to March 2012. EGFR (exons 18 to 21) and KRAS (codons 12, 13 and 61) genes were amplified by nested PCR and sequenced in both sense and antisense directions. Results Median age was 60 years (29-81); 181 pts (92.8%) were PS 0 or 1; 155 pts (79.4%) belonged to the non-squamous subtype and 39 pts (20.0%) were never-smokers. Treatment was as follows: platinum + a third generation agent in 103 pts (52.8%); platinum + pemetrexed in 81 pts (41.6%); platinum-based doublet + bevacizumab in 11 pts (5.6%). Seventy-five pts (38.4%) were KRAS mutant (MUT), of which 60 pts at codon 12 (COD 12 MUT), 12 pts at codon 13 (COD 13 MUT) and 3 pts at codon 61 (COD 61 MUT). The most common amino acid changes found were: Gly12Cys (29 pts), Gly12Val (11 pts) and Gly13Cys (10 pts). In the whole study population, 69 pts (35.3%) responded to treatment and 62 pts (31.7%) achieved stable disease, for a disease control rate of 67.0%. At a median follow-up of 14 months (2-102), median progression-free survival (PFS) and overall survival (OS) were 6.2 and 21.6 months, respectively. When analyzed according to KRAS mutation status, a significantly shorter PFS was noted for the EGFR WT/KRAS MUT subgroup (n = 75) compared with the EGFR WT/KRAS WT population (n = 120) [5.1 vs 6.6 months, respectively, P = 0.02; HR = 1.43 (95% CI, 1.05 to 1.95)]. Similarly, a significant difference was observed between the two groups in terms of OS [13.7 vs 26.1 months, respectively, P = 0.02; HR = 1.56 (95% CI, 1.06 to 2.30)]. In the EGFR WT/KRAS MUT subrgoup, OS for COD 12 MUT, COD 13 MUT and COD 61 MUT was 17.2, 10.2 and 8.0 months, respectively, P = 0.17. Multivariate analysis for PFS and OS confirmed that KRAS mutation was an independent predictor of poorer oucome. Conclusions EGFR WT/KRAS MUT pts appear to experience a less favorable prognosis compared with the EGFR WT/KRAS WT genotype, with a significant difference in clinical outcome according to the mutant codon of KRAS. Disclosure All authors have declared no conflicts of interest.

Effect of KRAS codon13 mutations in patients with advanced colorectal cancer (advanced CRC) under oxaliplatin containing chemotherapy. Results from a translational study of the AIO colorectal study group

BackgroundTo evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines.MethodsTumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS).Results201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens.ConclusionsOur data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important.Trial registration number2002-04-017

Involvement of K-RAS mutations and amino acid substitutions in the survival of metastatic colorectal cancer patients

The efficacy of epidermal growth factor-targeting therapies has been found to be limited in tumors with the wild-type K-RAS gene, suggesting a predictive value of K-RAS gene analysis in tumoral response. However, the prognostic value of K-RAS is controversial. This study included patients diagnosed with metastatic colorectal cancer. The presence of K-RAS mutations was analyzed, and the tumors positive for a K-RAS mutation were further analyzed to identify the mutation type. Similarly, the following clinical and pathological variables were also collected. The study was composed of 53.3 % of patients with wild-type K-RAS and 46.7 % of patients with mutated K-RAS (mutated codon 12 was the most frequent). With a mean follow-up of 15 months (range, 1-45), the median survival of patients with wild-type K-RAS was 31.6 months. The median survival was 24.8 months for patients with K-RAS mutated in codon 12 and 17.8 months for patients with mutated codon 13 (p = 0.37). In a univariate analysis, K-RAS was associated with stage IV at diagnosis (p < 0.005). When K-RAS was mutated, a lower overall survival was observed in cases of G → A transition compared with G → T transversion (19.5 vs. 24.2 months, respectively; p = 0.47). When the amino acid change resulted in an acidic substitution, survival was lower, but it increased when the substitution resulted in a polar or nonpolar amino acid (19.5 vs. 23.2 vs. 24.4 months, p = 0.79). The type of K-RAS mutation or amino acid changes may have prognostic implications in metastatic colon cancer patients. Further research is needed in patients treated in prospective controlled trials.

O-0021 Randomised Phase III Study of Bevacizumab + Chemotherapy Beyond Progression in Bevacizumab-Treated Patients with Metastatic Colorectal Cancer: TML Study Kras Subgroup Findings

ABSTRACT Introduction Bevacizumab in combination with fluoropyrimidine-based chemotherapy is a standard treatment for patients with metastatic colorectal cancer (mCRC) in the first-line and bevacizumab-naive second-line settings. This randomised study evaluated the benefit of continuing bevacizumab with standard chemotherapy as second-line treatment for patients with mCRC who progressed after receiving a standard bevacizumab-containing regimen in the first-line setting. The study included a broad biomarker collection as an exploratory objective. We present here analysis of outcomes according to tumour KRAS status. Methods Patients with unresectable, histologically confirmed mCRC who progressed within 3 months after discontinuation of first-line bevacizumab + chemotherapy were randomised to second-line fluoropyrimidine-based chemotherapy ± bevacizumab (2.5 mg/kg/wk equivalent). The choice of oxaliplatin- or irinotecan-based second-line chemotherapy was based on the regimen used in the first-line setting (crossover) and was included in the stratification variables. The primary endpoint was overall survival (OS); secondary endpoints included progression-free survival (PFS), response rate and safety. Subgroup analysis according to KRAS status was performed for both OS and PFS; no adjustment was made for multiplicity. Results Conclusive tumour KRAS mutation data were available for 616 of the 820 patients (75%). Baseline characteristics were similar in the biomarker and randomised populations. Overall, 300 (49%) patients had mutant-type (MT) KRAS tumours and 316 (51%) had wild-type (WT) KRAS tumours. Median OS in the KRAS WT group was 15.4 months for bevacizumab + chemotherapy vs 11.1 months for chemotherapy alone (p = 0.0052; HR = 0.69) and 10.4 months for bevacizumab + chemotherapy vs 10.0 months for chemotherapy alone in the KRAS MT group (p = 0.4969; HR = 0.91). Median PFS in the KRAS WT group was 6.4 months for bevacizumab + chemotherapy vs 4.5 months for chemotherapy alone (p Conclusion This is the first randomized study evaluating the benefit of continuing bevacizumab in combination with standard chemotherapy as second-line treatment for patients with mCRC who progressed after receiving a standard bevacizumab-containing regimen in the first-line setting. The biomarker findings from this study suggest that patients with both WT and MT KRAS tumours are likely to benefit from bevacizumab treatment.

Involvement of K-RAS mutations and amino acid substitutions in the survival of metastatic colorectal cancer patients.

e14116 Background: The efficacy against of epidermal growth factor therapy was found to be limited to those wild-type K-RAS tumours, suggesting a predictive value of response. However, the prognostic value of K-RAS status is controversial. The aim is to determine the relationship between K-RAS mutations and types of amino acid changes with the survival (OS) of metastatic colorectal patients (mCRC). Methods: The retrospective analysis included 120 patients diagnosed of mCRC. The presence and type of K-RAS mutations were analysed. Similarly, the clinical and pathological variables were also collected including OS. Results: The sample characteristics were summarized in Table 1. 53.3% of tumours were wild-type and 46.7 mutated KRAS. In mutated patients, codon 12 was affected in 76.79% and codon 13 in 23.21%. The median OS for wild-type was 31.6 months. In mutated patients (codon 12 or 13), median OS was 24.8 and 17.8 months (p=0.37), respectively. In the univariate analysis, mutated K-RAS was associated with stage IV at diagnosis (p&lt;0.005). The relationship between OS and amino acid mutation are summarized in table 1. Conclusions: Our study suggest that the types of mutation or amino acid changes of KRAS in mCRC provides prognostic information. These data need to be confirmed in prospective or controlled trials. [Table: see text]

Molecular and clinicopathologic evidence of heterogeneity in KRAS-mutant colon cancers.

3575 Background: The activation of the MEK/ERK signaling cascade by KRAS or BRAF mutations has a high incidence in colorectal cancer (CRC). While these mutations are mutually exclusive, we have identified a highly conserved gene expression pattern, characteristic to BRAF mutants, that can also be observed in KRAS mutants (AACR 2011, JCO 2012), and which defines a distinct subpopulation. Its characterization is important as it may shed a light on the observed survival and treatment response variability of this group of CRCs. Methods: A set of 257 stage II and III KRAS mutant CRCs from the PETACC3 clinical trial and a subset of 150 CRCs from TCGA project were used for this study. Gene expression data was available for all samples and, additionally, CNV and methylation data was available for some of the samples. Due to the limited number of MSI samples, the analyses were focused on MSS subpopulation. Results: The cluster analysis on the genes from the BRAF signature clearly indicated a major split of the KRAS mutants into two subgroups, that were further compared in terms of clinico-pathological and survival parameters. The BRAF-mutant-like (BML) subgroup was clearly enriched in MSI-H (p&lt;0.001), right-sided (p=0.038) and mucinous histology (p&lt;0.001) tumors. The enrichment in mucinous tumors remained significant also in stratified analysis by MSI status. In MSS, the BML subpopulation of KRAS mutants had the worst prognosis for overall survival (p=0.05, HR=1.6) and survival after relapse (p=0.004, HR=2.1). Analysis of differentially expressed genes between BML and the rest of KRAS mutants identified genes responsible for colon crypt differentiation, Wnt pathway activation and, more intriguingly, 50 other genes, all located on chromosome 20q, that had significantly different activation levels in BML tumors. Among these genes, there are a number of important tumor suppressors, like PLAGL2, TP53RK and POFUT1. No differences in type of KRAS mutation nor in PIK3CA mutations were found between BML and the rest of KRAS mutants. Conclusions: We identified a subgroup of KRAS mutants formed of more aggressive, poorly differentiated tumors. All these results prove that KRAS mutant CRCs form a heterogeneous group tumors.

Use and findings of K-ras in colorectal cancer (CRC) testing in administrative and electronic medical records (EMR) data from 2005 through 2010.

e14146 Background: The use of K-ras testing in clinical decision-making has grown over the past few years. The objective of this study was to evaluate, in a real world context, the trends and diagnostic findings of K-ras testing using managed care and EMR data. Methods: The Georgia Cancer Specialists Database EMR (2005-2010) and administrative data from the MarketScan and IMPACT database(s) was used to select patients with newly diagnosed colorectal cancer (CRC). We looked for trends in use of K-ras in relation to timing of chemotherapy administration. The EMR data provided information on k-ras mutation type. Results: In MarketScan, of the 23,548 patients with a diagnosis of CRC, 1,730 (7.3%) patients had a test ordered for K-ras between 2005 and 2010. The number of patients receiving K-ras increased with line of therapy: first line 336 patients (8.2%) of 4,098 treated, second line 455 patients (15.2%) of 2,984 treated, and third line 529 patients (33%) of 1,603 treated. We found similar results using the IMPACT database: 2,256 (7.8%) CRC patients had a test ordered for K-ras between 2005 and 2010. K-ras testing increased with line of therapy: first line 244 patients (7.8%), second line 510 (14.6%), and third line 650 patients (33.1%). EMR lab results from stage IV disease (n=349) consisted of 15% mutated type, 60% unknown and 25% wild type. For confirmed test results wild type represented 62.5% and mutated type 37.5%. Conclusions: Over the last six years, use of K-ras testing has increased in use in patients with CRC. The increase has occurred in later lines of therapy. The timing occurring late in therapy may limit the use of agents specific for this test.

KRAS, BRAF and PIK3CA Mutations and the Loss of PTEN Expression in Chinese Patients with Colorectal Cancer

BackgroundTo investigate the frequency and relationship of the KRAS, BRAF and PIK3CA mutations and the loss of PTEN expression in Chinese patients with colorectal cancer (CRC).Methodology/Principal FindingsGenomic DNA was extracted from the formalin-fixed paraffin-embedded (FFPE) tissues of 69 patients with histologically confirmed CRC. Automated sequencing analysis was conducted to detect mutations in the KRAS (codons 12, 13, and 14), BRAF (codon 600) and PIK3CA (codons 542, 545 and 1047). PTEN protein expression was evaluated by immunohistochemistry on 3 mm FFPE tissue sections. Statistical analysis was carried out using SPSS 16.0 software. The frequency of KRAS, BRAF and PIK3CA mutations and loss of PTEN expression was 43.9% (25/57), 25.4% (15/59), 8.2% (5/61) and 47.8% (33/69), respectively. The most frequent mutation in KRAS, BRAF and PIK3CA was V14G (26.7% of all mutations), V600E (40.0% of all mutations) and V600L (40.0% of all mutations), and H1047L (80.0% of all mutations), respectivly. Six KRAS mutatant patients (24.0%) harbored BRAF mutations. BRAF and PIK3CA mutations were mutually exclusive. No significant correlation was observed between the four biomarkers and patients' characteristics.Conclusions/Significance BRAF mutation rate is much higher in this study than in other studies, and overlap a lot with KRAS mutations. Besides, the specific types of KRAS and PIK3CA mutations in Chinese patients could be quite different from that of patients in other countries. Further studies are warranted to examine their impact on prognosis and response to targeted treatment.

Epidermal growth factor receptor and KRAS mutations in Brazilian lung cancer patients

OBJECTIVE:Epidermal growth factor receptor is involved in the pathogenesis of non-small cell lung cancer and has recently emerged as an important target for molecular therapeutics. The KRAS oncogene also plays an important role in the development of lung cancer. The aim of this study was to evaluate the frequency of epidermal growth factor receptor and KRAS mutations in a population of Brazilian patients with non-small cell lung cancer.METHODS:A total of 207 specimens from Brazilian patients with non-small cell lung cancer were analyzed for activating epidermal growth factor receptor and KRAS somatic mutations, and their associations with clinicopathological characteristics (including age, gender, ethnicity, smoking habits, and histological subtype) were examined.RESULTS:We identified 63 cases (30.4%) with epidermal growth factor receptor mutations and 30 cases (14.6%) with KRAS mutations. The most frequent epidermal growth factor receptor mutation we detected was a deletion in exon 19 (60.3%, 38 patients), followed by an L858R amino acid substitution in exon 21 (27%, 17 patients). The most common types of KRAS mutations were found in codon 12. There were no significant differences in epidermal growth factor receptor or KRAS mutations by gender or primary versus metastatic lung cancer. There was a higher prevalence of KRAS mutations in the non-Asian patients. Epidermal growth factor receptor mutations were more prevalent in adenocarcinomas than in non-adenocarcinoma histological types. Being a non-smoker was significantly associated with the prevalence of epidermal growth factor receptor mutations, but the prevalence of KRAS mutations was significantly associated with smoking.CONCLUSIONS:This study is the first to examine the prevalence of epidermal growth factor receptor and KRAS mutations in a Brazilian population sample with non-small cell lung cancer.

Association of KRAS G12D mutation with outcome in patients with pancreatic cancer after surgery.

221 Background: The frequency of KRAS gene mutations in pancreatic adenocarcinoma is high and most mutations occur in codon 12 and 13. However, the association of KRAS mutation status with clinical outcome of pancreatic cancer patients after surgery is not clearly understood. The aim of present study was to clarify whether presence of mutation in KRAS is of prognostic significance, and to determine whether the different type of KRAS mutation have different impact on clinical outcome in pancreatic cancer. Methods: A total of 62 pancreatic adenocarcinoma tissues from patients underwent curative surgery were analyzed for KRAS mutation. DNA was extracted from the tissue microarray. The domain containing the KRAS gene codons 12 and 13 was amplified by PCR, and KRAS mutation was detected by loop hybrid mobility shift assay (LH-MSA). Results: KRAS mutation was detected in 41 of 62 tumors (66.1%). Mutations of KRAS codon 12 and codon 13 were detected in 37 of 62 tumors (59.7%) and in 4 of 62 tumors (6.5%), respectively. G12D mutation were detected in 35.5% (22/62) and G12V mutation in 19.4% (12/62) of all. The patients with KRAS mutations had a significantly shorter overall survival (OS) (log rank, p=0.0375) and disease free survival (DFS) (p=0.0175). When the type of mutation were evaluated, G12D mutation was significantly associated with both shorter OS (p=0.0410) and DFS (p=0.0214), whereas G12V mutation was associated with neither OS (p=0.6727) nor DFS (p=0.9854). Conclusions: The presence of KRAS mutation was significantly associated with poor outcome of pancreatic cancer patients after surgery. A specific mutation G12D demonstrated significant association with shorter OS and DFS in pancreatic cancer. The different type of KRAS mutation might have different impact on clinical outcome in pancreatic cancer.

A novel and simple KRAS mutation detection method, FRET-Based Preferential Homoduplex Formation Assay (f-PHFA): Results from a multicenter, clinical evaluation trial.

476 Background: KRAS gene mutation in colorectal tumors is predictive of nonresponse to anti-epidermal growth factor receptor (EGFR) antibodies in patients with metastatic colorectal cancer (CRC). The F-PHFA has been developed as a simple and accurate mutation detection assay especially for somatic mutations. In this study, we evaluated KRAS mutation detection by F-PHFA which detects 7 common mutations in codon 12 and 13. This study was a multicenter, clinical evaluation trial designed to evaluate the efficacy of the F-PHFA method forKRAS testing in patients with CRC. Primary endpoint was the concordance rate of KRAS mutation between F-PHFA and direct sequencing with manual micro-dissection (MMD) to enrich the tumor concentration. Secondary endpoints included the concordance rate between F-PHFA and direct sequencing without MMD. Methods: The key eligibility criteria included histologically confirmed colorectal adenocarcinoma with adequate tumor samples. The materials were formalin-fixed paraffin-embedded (FFPE) 8 unstained slides at 10 um and 2 matching hematoxylin-eosin (HE)-stained slide, which were sent to the central pathology review to confirm the tumor content and to mark the tumor area. Genomic DNA was extracted using QIAamp DNA FFPE Tissue from both whole tissue of slide and tissue with MMD. F-PHFA and direct sequencing were conducted independently in two laboratories. Results: FFPE specimens from 165 patients with colorectal adenocaricinoma were registered. Success rates on KRAS tests of F-PHFA both of with or without MMD were 100% (165/165). However, those of direct sequencing were 99.4% (164/165). The concordance rate between F-PHFA and direct sequencing were 99.4% (163/164) for both of with MMD and without MMD. Only one discordant sample had a rare mutation other than the 7 common mutations. Total assay time was about 3.5 hrs. Also 20 ng of DNA was enough to examine 7 types of KRAS mutations. Conclusions: We confirmed that F-PHFA and direct sequencing shows high concordance rate regardless of MMD. F-PHFA for KRAS mutation detection method is very suitable for clinical use in terms of easiness and accuracy.