text:
Chemotherapy Research and Practice
Volume 2012 (2012), Article ID 359041, 10 pages
CpG Island Methylation, Microsatellite Instability, and BRAF Mutations and Their Clinical Application in the Treatment of Colon Cancer
The Ohio State University Comprehensive Cancer Center, A454 Starling Loving Hall, 320 West 10th Avenue, Columbus, OH 43210, USA
Received 30 January 2012; Accepted 15 April 2012
Academic Editor: Vassilios A. Georgoulias
Copyright © 2012 Christina Wu and Tanios Bekaii-Saab. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
There have been significant developments in colon cancer research over the last few years, enabling us to better characterize tumors individually and classifying them according to certain molecular or genetic features. Currently, we are able to use KRAS mutational status as a guide to therapy with anti-epidermal growth factor receptor antibodies. Other molecular features under research include BRAF mutation, microsatellite instability, and CpG island methylation. These three molecular features are often associated with tumors that have overlapping phenotypes and can be present simultaneously in the same tumor. However, they carry different prognostic and predictive qualities, making analysis of their interaction relatively complex. Much research thus far has examined the clinical relevance of microsatellite instability in helping determine prognosis and the predictive value of adjuvant 5-fluorouracil chemotherapy in stages II and III colon cancers. BRAF mutation appears to be a biomarker for poor prognosis. CpG island methylation is tightly associated with microsatellite instable tumors and BRAF mutation, but its clinical utility remains uncertain. Hereby, we examine preclinical and clinical data that supports the utilization of all three phenotypes in future research applied to clinical practice.
1. Introduction
Colorectal cancer (CRC) is the third most common cancer in the USA and the second leading cause of cancer death, with 141,210 annual new cases and 49,380 annual deaths in 2011 alone [1]. Survival for patients with CRC has improved dramatically over the last decade with the availability of 5-fluorouracil (5FU-) based doublet chemotherapy and the addition of molecularly targeted agents, such as, bevacizumab, cetuximab, and panitumumab in the metastatic setting [2–8]. Significant progress has been made in genotyping tumors and extracting clinically relevant information that may help guide us in early cancer detection, predictive biomarkers, and new target discovery.
CpG island methylation phenotype (CIMP), microsatellite instability, and BRAF mutation may have clinical significance in colon cancer. These characteristics are due to genetic and epigenetic changes and have overlapping histopathological features that often occur in the same tumor. Their interaction is complex; for example, microsatellite instability-high (MSI) tumors carry a good prognosis whereas the presence of a BRAF mutation confers a poor outcome [9–15]. In this paper, we describe the clinical significance of CIMP, microsatellite instability, and BRAF mutation and their potential interactions.
2. Microsatellite Instability
2.1. Background
Approximately 15% of colon cancers carries the MSI characteristic, and those cancers are histopathologically and clinically distinct from microsatellite instability-low and microsatellite stable (MSS) tumors [16, 17]. Microsatellite instability low and MSS tumors behave similarly biologically and are often classified as MSS collectively [18]. Microsatellite instability is defined as a change in the length of short repeating nucleotide sequences called microsatellites, and this instability is detected in tumor cells when compared to normal tissue [19].
Microsatellite instability is due to defects in the DNA mismatch repair (MMR) system, resulting in the accumulation of nucleotide mutations and alteration in microsatellite length [20]. The MMR system is responsible for the surveillance and repair of errors that occur during DNA synthesis. It is composed of 4 MMR proteins: MLH1, MSH2, MSH6, and PMS2 [21]. Germline mutation of the MMR proteins results in hereditary nonpolyposis CRC (HNPCC) otherwise known as Lynch syndrome, an autosomal dominant hereditary disease in which patients are prone to early development of colon cancer [21]. The majority of patients with MSI tumors have defects in the MMR system, which is caused by epigenetic gene silencing of hMLH1 by sporadic hypermethylation [22]. Due to the causal relationship of loss of MMR and resultant MSI tumors, immunohistochemical analysis of the MMR proteins may be used instead of MSI analysis. Absence of any MMR protein is accepted as representing a MSI phenotype. Thus, tumors are identified as proficient in MMR (pMMR) in presence of all MMR proteins or deficient in MMR (dMMR) if 1 or more of the proteins are absent [23]. A limitation of this test is that the loss of protein may be due to gene silencing by hypermethylation or germ line mutation. Therefore, further tests need to be performed to delineate the etiology of MMR deficiency.
Detection of microsatellite instability is by DNA extraction from either paraffin embedded or frozen tissue, followed by PCR amplification of specifically defined microsatellites [24]. PCR products are then analyzed by gel or capillary zone electrophoresis. The National Cancer Institute Workshop on Microsatellite Instability for Cancer Detection and Familial Predisposition compiled an accepted reference panel of 5 loci markers for identifying MSI: BAT25, BAT26, D5S346, D2S123, and D17S250 [25]. However, investigators have used >5 loci markers, with additional loci to the reference panel, for improved sensitivity. MSI is identified when >2 of the 5 loci are unstable, or when ≥30% of >5 loci are unstable [25].
2.2. Clinical Significance
Tumors with MSI are generally right sided and typically diagnosed at an earlier stage. Pathological features of MSI colon cancers include a Crohn's-like host response, mucinous phenotype, and high histological grade. Detection of MSI tumors in colon cancer is clinically significant, and retrospective studies of large randomized control trials in Stage II/III colon cancers have analyzed the prognostic and predictive value of MSI (Table 1) [9–12, 25, 26]. When examining the treatment arm in which no adjuvant therapy was given, the presence of MSI conferred a better prognosis than MSS. Interestingly, this positive prognostic effect seems more significant in stage III colon cancers, although more analysis is needed to validate these findings given the low incidence of MSI in colon cancers and the lower frequency of MSI tumors in stage III than stage II [26]. Tejpar et al. reported that in patients who did receive adjuvant chemotherapy, those with MSI tumors had superior overall survival (OS) and recurrence-free survival (RFS) compared to those with MSS tumors [12]. Moreover, patients with MSS tumors (especially Stage III cancers) seem to benefit the most from adjuvant 5-fluorouracil/Leucovorin (5FU) chemotherapy [9, 10]. Conversely, patients with MSI tumors and more specifically those with stage II cancer, do not seem to benefit from adjuvant chemotherapy [9, 10]. Overall, the absolute risk reduction with adjuvant 5FU therapy in stage II colon cancer is low. Practicing oncologists typically refer to clinicopathologic high-risk features (e.g., T4 lesion, bowel obstruction, lymphovascular invasion, perineural invasion, perforation, positive margins, or <12 lymph nodes sampled at surgery) to determine whether adjuvant therapy should be offered. We believe that based on the published data, tumor MSI status should be integrated into clinical decisions about therapy for stage II colon cancer [9–12, 25, 26].
Patients with stage III colon cancer did not have differences in benefit from adjuvant 5FU treatment when comparing tumors with MSI versus MSS [11]. However, results from a recent study suggest that patients with stage III MSI tumors, and more specifically those with germ line mutation, seem to benefit from adjuvant 5FU chemotherapy [26]. It is unknown how these results apply to the current standard adjuvant therapy for stage III colon cancer which consists of adding oxaliplatin to 5FU, suggesting the need for further study.
One of the major clinical implications of finding MSI tumors is that it is a cost-effective method to screen for HNPCC. Once dMMR tumors are detected, they can then be tested for the germ-line mutation of the missing MMR protein. Identifying HNPCC can lead to a great impact on survival outcomes for patients and their families through early introduction of genetic counseling and cancer screening practices. In the CAPP2 study, 861 HNPCC patients were randomized to receive aspirin or placebo for 2 years, and the treatment arm was found to have a reduced incidence of cancer with a HR of 0.41 (0.19–0.86, ) [41]. As such, identifying those at risk may help with primary prevention for colon cancer in this patient population.
Preclinical studies have shown increased sensitivity of MSI cancer cell lines to irinotecan, which should be explored in the clinical setting [42]. Although irinotecan has not shown to be effective in the adjuvant setting in stage III cancers, it may prove to be effective in MSI tumors specifically [43]. A recent study reported that patients with stage III colon cancer who had MSI tumors and were treated with 5FU and irinotecan (IFL) had an improved 5-year disease-free survival (DFS) when compared to MSS tumors with HR of 0.76 (95% CI, 0.64 to 0.88) versus 0.59 (95% CI, 0.53 to 0.64), respectively () [11]. In contrast, another study of 1,327 patients with stages II and III cancers, infusional 5FU, and irinotecan did not provide additional benefit in OS or RFS over 5FU treatment alone for MSS or MSI tumors [12]. More work, however, needs to be done to explore the role of irinotecan in the adjuvant setting. Finally, preclinical data suggests a role for poly (ADP ribose) polymerase (PARP) inhibitors as candidates to target MSI tumors via synthetic lethality [44]. PARP inhibitors block base excision repair and cause double-stranded DNA breaks, which MSI cells are unable to repair thus causing tumor cell death [44]. This strategy has already been employed in the treatment of BRCA mutant breast cancer [45]. Currently clinical trials are ongoing examining the efficacy of PARP inhibitors in MSI metastatic colon cancers.
3. CpG Island Methylation Phenotype (CIMP)
3.1. Background
CIMP is detected in approximately 30–40% colon cancers [46, 47]. CpG island methylation is DNA methylation at the cytosine base of CpG dinucleotide islands, by DNA methyltransferase enzymes [48]. The normal genome contains about 70–80% CpGs that are usually methylated, but CpG islands located proximal to the promoter region of genes are usually left unmethylated [49]. Conversely, in cancer cells it has been observed that there is genome-wide hypomethylation and gene promoter hypermethylation. Hypermethylation contributes to gene silencing and genomic instability and affects tumor-suppressor genes, DNA repair, and cell-cycle control.
Methylation analysis is performed by either methylation-specific polymerase chain reaction (PCR) or real-time PCR (Methylight) [50, 51]. One of the challenges in studying CIMP tumors is that there is no general consensus of which specific methylated loci to use to define CIMP. The majority of studies have typically included the classic panel: hMLH1, p16, MINT1, MINT2, and MINT31 [27]. However, in addition to these 5 loci, CIMP marker panel may be extended to include: CACNA1G, CRABP1, IGF2, NEUROG1, RUNX3, SOCS1, HIC1, IGFBP3, and WRN, with no consensus on how many markers are required to be positive to define CIMP [27–36, 38–40]. When analyzing clinical outcome results across studies, caution should be taken because the loci marker panel and criteria for CIMP vary.
Toyota et al. first described CIMP in CRC, identifying cancer-specific methylation and distinguishing it from age-specific methylation [48]. Subsequently, Weisenberger et al. performed unsupervised two-dimensional cluster analysis of DNA methylation and classified CRC into CIMP-negative or CIMP-positive cancers [52]. They observed a strong relationship of CIMP cancers with BRAF mutations. In addition, there is now evidence of different subgroups of CIMP (high, low, and negative) [53]. In an analysis of 97 colon cancer tumors, whereby genetic and epigenetic alterations were studied, unsupervised hierarchical clustering of DNA methylation identified 3 distinct groups of CIMP, described as CIMP1, CIMP2, and CIMP-negative [54]. CIMP1 tumors were highly associated with MSI (80%) and BRAF mutation (53%). CIMP2 tumors had a high incidence of KRAS mutations (92%), and CIMP-negative correlated with p53 mutations (71%). A group from Singapore performed comprehensive methylation profiling of 1,505 CpG sites and identified 3 distinct CIMP groups (CIMP-H, CIMP-M, and CIMP-L), with different features to those described in the previous study, for example, CIMP-H cluster has a high incidence of KRAS and BRAF mutation and thus now suggests 4 CIMP categories [55]. CIMP classification continues to be refined as technology improves.
3.2. Clinical Significance
Pathological features of CIMP tumors are the high rate of mutations (KRAS or BRAF), wild-type p53, proximal colon location, and higher occurrence in women and older patients. Characterizing CIMP alone is not routinely performed to guide treatment decisions. It is currently being explored as a marker for genetic and environmental factors that affect colon carcinogenesis and as a possible prognostic or predictive marker along with MSI and BRAF mutation. Interestingly, there has been an association reported between smoking and CIMP. In smokers, there is increased CpG methylation at the bronchial epithelium [56]. In colon cancer, cigarette smoking is associated with CIMP tumors and has a significant relationship to the number of cigarettes smoked [57]. Also, the increased risk for colon cancer in inflammatory bowel disease (IBD) is hypothesized to be due to repetitive mucosal inflammation and possibly age-related CIMP [58]. However, studies have not showed an increased incidence of CIMP in IBD-associated CRC as compared to sporadic cancers [59, 60]. A study from Cleveland Clinic matched 19 patients who had CRC and ulcerative colitis to 54 patients with sporadic CRC, and their tissue was examined for BRAF mutation, CIMP, KRAS mutation, and p53. The colitis-associated CRC did not have an increased number of patients with CIMP (5% versus 22%) but was more likely to have a p53 mutation (95% versus 53%, ) [54]. Similarly, a methylation microarray analysis of IBD CRC, sporadic CRC, and normal colonic tissue demonstrated that CIMP was less common in IBD-associated CRC than sporadic CRC [55]. Thus, it is unlikely that IBD-related CRC is via the CIMP pathway.
The predictive role of CIMP is controversial. It has been hypothesized that CIMP tumors have aberrant folate metabolism in cancer cells and thus could be more sensitive to antifolate therapies, such as 5FU [61, 62]. Conversely, adjuvant 5FU-based therapy is not beneficial in stage II colon cancers that are identified as MSI, and there is overlap between MSI and CIMP tumors, thus CIMP presence was also postulated to be a negative predictive marker for 5FU therapy. There are studies in which the presence of CIMP has predicted benefit of 5FU-based treatment in stages II/III colon cancer; however, there is also evidence showing a trend for resistance to chemotherapy in CIMP tumors (Table 2) [27, 28, 30, 31, 38, 39]. In the majority of the studies that have analyzed patients with all stages of CRC who did not receive chemotherapy treatment, tumors identified as MSS, and CIMP have a worse survival outcome [27, 29–34, 36]. However, two studies report better outcomes with CIMP tumors [35, 39]. The conflicting data may be due to the different criteria used across the studies to define CIMP status, or that CIMP tumors are heterogenous and need to be further classified. Based on the data available, CIMP cannot be used clinically as a prognostic or predictive marker.
DNA hypermethylation is under investigation both as a tool in colon cancer screening and a target for cancer therapy. One method of utilizing DNA methylation in CRC screening is by detecting abnormal DNA methylation from tumor cells shed in stool samples. In one study, stool samples were collected from patients who had undergone endoscopic examination and biopsy and identified as healthy control patients and patients diagnosed with adenomas or CRC [63]. DNA methylation was analyzed by Methylight. For diagnosing CRC, sensitivity was 90% (CI 56–100%) and specificity was 77% (CI 46–95%). Another proposed method to detect aberrant DNA methylation is by testing for cell-free DNA that is released by cancer cells undergoing apoptosis or necrosis in the serum or plasma [64]. However, a number of obstacles to this method exist including extracting sufficient DNA from the sample and determining which platform assays to use.
Hypermethylated CpG islands are also targeted in drug development. Preclinical studies suggest that azacytidine and decitabine, both DNA methyltransferase inhibitors, are able to demethylate CpG islands in colon cancer cell lines in a nonrandom and reproducible fashion [65]. There is currently an ongoing clinical trial with azacytidine in combination with entinostat, a histone deacetylase inhibitor in the treatment of advanced colon cancer. Another phase I/II trial studies the combination of azacitidine, capecitabine, and oxaliplatin in advanced CRC, with specific selection for patients with CIMP tumors.
4.1. Background
The incidence of BRAF mutation V600E in colorectal cancer is 8–10% [66]. BRAF mutation is detected by DNA extraction from paraffin-embedded or frozen tissue and then analyzed by PCR amplification and either pyrosequencing or Sanger sequencing. The RAS/RAF/MAPK pathway is important in tumor cell proliferation, invasion, and inhibition of apoptosis [67, 68]. Activation of the MAPK pathway is initiated by ligand binding to a receptor tyrosine kinase, such as, epidermal growth factor receptor (EGFR) that has been shown to be critical in the development of colorectal cancer. The RAS proteins are small GTPases that are farnesylated and inserted into the cell membrane. They are activated downstream of EGFR and propagate signaling by recruiting BRAF to the plasma membrane. BRAF in turn phosphorylates MEK1/2. Once activated, MEK1/2 phosphorylates ERK1/2 which subsequently triggers downstream signaling by phosphorylating cytoplasmic and nuclear transcription factors and proteins. Both KRAS and BRAF mutations are mutually exclusive and may indicate that they individually play an important role in the MAPK pathway. BRAF mutation is commonly present in cancers, especially melanoma and colon cancers [69]. Eighty percent of the mutations is attributed to the V600E substitution in the kinase domain. Mutant BRAF results in increased serine threonine kinase activity, and activation of the RAS/RAF/MAPK pathway [70]. In vitro studies have shown that BRAF mutation correlates with ERK 1/2 activation in CRC lines [71]. BRAF mutants elicit tumorigenic properties, as demonstrated when transfection of BRAF mutant proteins transformed NIH3T3 cells and BRAF mutant expressing NCM640 cells underwent malignant transformation by gaining the ability to grow on soft agar [70, 72]. BRAF mutation also affects the mitotic spindle and spindle assembly [73, 74].
4.2. Clinical Significance
It is recognized that KRAS mutations in codon 12 and perhaps 13 are predictive of lack of efficacy of anti-EGFR monoclonal antibody therapy in advanced CRC [7, 8, 75, 76]. Studies have examined whether mutations in BRAF, which is downstream of KRAS, may also have an impact on the efficacy of anti-EGFR agents such as; cetuximab or panitumumab. One retrospective study suggested that the presence of BRAF mutation may be an adverse predictive biomarker for the activity of cetuximab in MCRC [60]. Another retrospective analysis of 113 patients who received panitumumab or cetuximab showed that none of the patients with a BRAF mutation responded to treatment [77]. However, retrospective analysis of the large phase III CRYSTAL trial in which patients with metastatic CRC were randomized to 5FU and irinotecan with or without cetuximab showed that patients with BRAF mutant tumors did poorly, regardless of the therapy they received [13]. This finding was also confirmed in the MRC FOCUS phase III trial [78]. Additionally, evaluation of BRAF in stages II and III colon cancers showed that BRAF mutation was a negative prognostic factor for overall survival in patients [15, 37, 40]. Compilation of all this data indicates that the presence of a BRAF mutation is prognostic rather than predictive.
BRAF mutation analysis is not yet routinely tested in the clinical setting. Nonetheless, BRAF mutation is being studied as a target for various therapeutic agents. In BRAF-mutated CRC, a number of novel agents are being developed to target the RAS/RAF/MAPK pathway, including BRAF inhibitors and MEK inhibitors. The selective BRAF inhibitor, PLX4720, has been shown to inhibit MAPK phosphorylation in BRAF mutated cancer cell lines [79]. PLX4720 causes cell-cycle arrest, apoptosis, and tumor growth delays in BRAF mutant tumor xenograft models. There is an ongoing trial of the BRAF inhibitor, PLX3603, in BRAF mutant cancers. Vemurafenib, a FDA-approved BRAF inhibitor for the treatment of BRAF-mutant melanoma, has only had a modest response rate in CRC [80]. A RNA-interference-based genetic screen was utilized in cancer cells treated with vemurafenib, to explore whether the knockdown of selective kinases would synergize with vemurafenib [81]. Interestingly, EGFR blockade and BRAF mutation inhibition had strong synergy. Despite inhibition of BRAF mutation there was continued proliferation due to the feedback activation of EGFR. A clinical trial testing the combination of BRAF inhibition and anti-EGFR therapy would be indicated in patients with EGFR expressing and BRAF mutant tumors. BRAF mutation is also indicative of MAPK pathway activation, and thus MEK inhibition is another targeted therapy that has been under investigation [71]. In BRAF/KRAS mutant CRC cell lines, the MEK inhibitor, CI-1040, has been shown to impair anchorage-independent growth [71]. There is an ongoing phase I/II trial of selumetinib, a MEK inhibitor, in combination with irinotecan in KRAS and BRAF mutant CRC. Also, there are trials with single-agent MEK162 or AZD6244, both MEK inhibitors in BRAF mutant cancers. Regorafenib is a multikinase inhibitor that was recently reported to have improved survival outcomes versus placebo in patients with metastatic CRC (although patients were not selected for BRAF mutation) [82].
5. Potential Interactions between MSI and the Presence of CIMP and BRAF Mutation
There is considerable overlap among the tumors characterized as MSI, CIMP, and BRAF mutant [53]. Tumors that harbor BRAF mutation, MSI, and CIMP are generally proximal and mucinous tumors [83]. In a population-based study of 1315 patients with colon cancer, cigarette smokers had a higher incidence of MSI, CIMP, and BRAF mutant tumors [58]. Cigarette smoking was associated with CIMP and BRAF mutant tumors, odds ratio (OR) 2.85 (95% CI 1.53–5.29), and MSI cancer OR 3.43 (95% CI 1.57–7.50). This suggests that genetic mutation and epigenetic alterations due to environmental insults could concomitantly contribute to carcinogenesis. When Weisenberger et al. first classified CRC tumors as CIMP positive and CIMP negative, they observed that all BRAF mutant tumors were CIMP positive and that sporadic MSI cancers had CIMP methylation of MLH1 [52]. The presence of all three features suggests that they contribute to colon carcinogenesis. Sessile serrated polyps, which account for 40% of colon cancers, are precursor lesions that lead to CIMP carcinoma and Lynch syndrome cancers [84]. BRAF mutation is likely the activating mutation in the serrated pathway, as it is commonly present in serrated adenomas and inhibits normal apoptosis of colon cells [71, 85, 86]. In turn, the serrated adenomas are prone to methylation, especially the MLH1 gene. In a study that assayed 79 sporadic polyps, it was demonstrated that as histological changes were more advanced, from polyp to carcinoma, there was a correlation with increased methylation levels [87]. Also, to support that BRAF mutation is the first initiating step in carcinogenesis, in vitro studies have demonstrated that that transfection of BRAF mutant plasmids into CaCO2 cells induced MSI, resulting in a lower DNA content, cell cycle distribution, and downregulation of DNA repair genes [88]. However, transfecting CIMP-negative, BRAF wild-type colorectal cell lines with BRAF mutant expressing vector was not sufficient to cause DNA hypermethylation [89].
Serrated pathway syndrome is a familial colorectal cancer that is predominantly BRAF mutant, in contrast to Lynch syndrome. In the initial report, 43 individuals from 11 families were studied and all patients met Bethesda guidelines [90]. There were BRAF mutations in 63% polyps and 70% cancers. Eighty percent of the patients had MINT31 hypermethylation and MSI status was variable, which suggested a differing carcinogenesis pathway than sporadic MSI tumors which are BRAF mutant and CIMP (with hypermethylation at MLH1). Their polyps were serrated in description and the cancers were early in onset. A separate study screened 194 colorectal tumors from patients with family history of colon cancer [91]. BRAF mutation was identified in 100% () MSI tumors, and BRAF mutation correlated with extracolonic tumors among the families. BRAF mutation may be the initiating step in carcinogenesis for MSI tumors that are not due to HNPCC, in both sporadic and familial CRC, and may warrant specific targeting in this patient population in future trials.
BRAF mutation occurs at a higher frequency in CIMP tumors than non-CIMP tumors and can be used as a surrogate marker for CIMP in many instances. One study examining CIMP and BRAF-mutation in 460 tumors showed that CIMP occurred at a higher frequency in BRAF mutated tumors (68%) than BRAF wild-type tumors (5.2–12%) [92]. BRAF mutations are also very common in MSI tumors (50%) but rarely occur in HNPCC tumors [66, 93]. The evaluation of MSI and BRAF mutation together is a useful tool for screening HNPCC in the colon cancer population. For example, all patients who have colon cancer are screened for HNPCC in our institution with the following algorithm: samples are tested for the presence of MMR proteins by immunohistochemistry. Recognizing that the absence of MMR proteins could be due to either epigenetic methylation or germ line mutation, we then proceed with the detection of BRAF mutations as a surrogate for CIMP or hMLH1 methylation. If the tumor is BRAF wild type, we then perform gene mutation analysis to separate the tumors with germ line mutation from tumors that have MSI phenotype due to hMLH1 methylation and are BRAF wild type.
Although CIMP, MSI, and BRAF mutation may be present in the same tumor, there are also clearly distinct clinical outcomes observed across the 3 categories (Table 2). MSI is associated with a better prognosis for patients with stage II and III CRC and in contrast BRAF mutation is associated with worse prognosis. It is unclear what that may imply on tumors that are MSI and BRAF mutant. Likewise, it is uncertain what level of interaction exists between CIMP and BRAF mutation when they tend to be present together in a number of tumors. In a study of 649 colon cancers, patients with MSI tumors had superior cancer-specific mortality when compared to MSS tumors in univariate analysis, HR 0.38 (95% CI 0.22–0.66). However, in the multivariate analysis accounting for MSI, CIMP, and BRAF mutation, this advantage was attenuated [35]. BRAF mutation was a marker for poorer prognosis, and no protective effect was seen with MSI versus MSS in BRAF mutant tumors. Patients with CIMP high tumors fared better than CIMP low tumors regardless of MSI status or BRAF mutation status. However, the majority of studies have identified CIMP as a marker for worse outcome (Table 2).
6. Conclusion and Future Directions
Studies examining interactions between MSI and the presence of CIMP and BRAF mutation and their clinical outcomes are summarized in Table 2. However, due to the retrospective nature of the majority of the studies, dependence on tissue availability, and low incidence of BRAF mutations and MSI tumors, the disease stages are not matched with treatment and clinical outcome. Nonetheless, a few broad conclusions can be made. MSI tumors consistently predict for better clinical outcome than MSS tumors. BRAF mutation is a poor prognostic marker; however, its interaction with MSI has to be further clarified. Patients with MSS tumors that harbored BRAF mutation or CIMP fared worse. It is interesting that one study reported CIMP to be associated with better patient outcomes, which is contrary to the other reported studies [35]. This could be due to different panel markers that they used to define CIMP or due to the different patient population in their study. Whether CIMP tumors respond or are resistant to 5FU-based chemotherapy is confounding as well and likely points to the heterogeneity of the tumors studied, due to CIMP criteria and differing disease stages. Clearly, there is a great paucity of information in the analysis of the tumor-subtype interactions.
Further information needs to be gleaned from these distinct but occasionally overlapping tumor subtypes, with regards to clinical outcomes in the different disease stages and sensitivity to various therapies. In stage II disease, oncologists already utilize MSI status to help guide their decision in adjuvant 5FU therapy. Other prognostic but necessarily predictive tools that are being developed for stage II cancer include Oncotype DX, a 12-gene assay using real-time reverse-transcriptase PCR, which examines 7 cancer genes and 5 reference genes to determine a recurrence score. This technology has been validated with the QUASAR trial, a phase III trial of stage II colon cancer patients who were randomized to adjuvant 5FU or observation alone [94]. Characterizing tumors further by BRAF mutation and CIMP status could potentially dissect out the differences the tumors may have and be used as prognostic and predictive biomarkers. We have already begun utilizing MSI and BRAF mutational status in a systematic way to screen for HNPCC in patients with colon cancer. However, investigating the different behavior of MSI tumors that are the result of germ line mutation versus hypermethylation has not been established, largely due to small numbers of HNPCC patients. There are likely significant differences that could alter the way we treat our patients. Additionally, it is essential to have a consensus on a standardized panel of loci to define CIMP similar to the standardized panel utilized to identify MSI.
In conclusion, personalized medicine has become a significant part of the modern management of colon cancer. One example is the selection of patients who are more likely to respond to anti-EGFR therapy by excluding KRAS mutant tumors. Based on the current data available, our recommendations are for all patients with CRC to be tested for MSI status. If the tumor is dMMR, then follow-up tests including BRAF mutation and genetic testing will identify patients with HNPCC. Also, for stage II colon cancer patients, the presence of dMMR along with favorable histopathological features would justify no adjuvant chemotherapy. Patients with mCRC should have their tumor checked for BRAF mutation if they do not have KRAS mutation, to see if they are eligible for clinical trials for BRAF mutation inhibitors or drugs targeting the MAPK pathway. Routine CIMP testing is not recommended. Further unraveling of the CIMP, BRAF, and MSI findings will enable us to target selected patients, identify their recurrence risk, and make decisions about their treatment options across all stages of colon cancer.
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An Exclusive Sneak Peek at Lauren Conrad's New Book, Infamous: A Fame Game Novel
The newest addition to Lauren Conrad's best-selling series The Fame Game is quite the page-turner. While delving into the drama between fictional TV stars Madison Parker, Kate Hayes, and Carmen Curtis, Infamous explores the grittier side of celebrity life and the high price of fame. We have an exclusive excerpt from the book that will keep you hanging off of every page. Be sure to pick up Infamous on June 11. Click through Lauren's glamorous July cover shoot with Marie Claire!
When Madison had offered to give her some reality TV pointers, Kate had eagerly agreed. She was tired of being called "boring" and "vanilla" and "ho-hum." But so far she was having a hard time understanding why Madison was apparently so great at TV while she herself was apparently not. Besides the whole camera-hog thing, obviously; she knew Madison was better at that.
Madison explained, "You make vague but important-sounding pronouncements. 'I feel like things are going to change for her.' Or 'What could possibly go wrong?' Or 'Everything is going to be different this time around.' Those are good because Trevor can use them as teasers in the commercials they play all week long, and as a cliff-hanger at the end of an episode. Which means that your one sentence will get played about a hundred times."
"But it's sort of an unspecific and boring sentence?" Kate said hesitatingly.
"Unspecific, yes. But boring, no. Having your sentence be the tease puts you at the center of the drama. The one who's either right—or spectacularly wrong. Which it is doesn't matter." Madison fast-forwarded as she talked. "As you can see, as far as the Gaby prediction goes, I was correct. Things changed for her all right. She spent a month in lockdown."
Kate reached down and picked up Lucinda as she waited for Madison to find what she was looking for. She'd promised herself she'd practice six hours a day until the showcase, but she'd only strummed two chords before Madison whirled around.
"Ahem. What are you doing?" Madison demanded.
"Um, playing a guitar?"
"Would you play a guitar in calculus class?"
"What? No."
"Well, then put that thing away, because this is a lot more important than calculus. Honestly, you're lucky I'm even taking the time to help you with this."

subset: commoncrawl


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Mophie Juice Pack Air cutouts - Mophie Juice Pack Air
The Mophie Juice Pack Air has cut-outs that allow you to access your iPhone controls.
|‹Mophie Juice Pack Air back
|Mophie Juice Pack Air apart›
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Home >> Saudi Arabia >> The Diplomat
About The Diplomat Magazine
The Diplomat is a bimonthly magazine published by the Institute of Diplomatic Studies in Saudi Arabia. It was founded in 2007 and covers international relations, the politics of the Middle East and the Persian Gulf, the arts, global finance and economics.
The editor in chief is Dr. Turki Alawwad, under the supervision of Prof. Abdullkarim Aldekhayel. Recent contributors include Saudi Arabia's Foreign Minister Saud bin Faisal bin Abdul Aziz, Professor Paul Krugman, Robin Wells Krugman and Fawaz Gerges.
The Diplomat Magazine Details
The Diplomat Publisher : Institute of Diplomatic Studies
The Diplomat Language : English
The Diplomat Magazine URL : http://the-diplomat.com/
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WASHINGTON -- The violent crimes rate declined in 2003 despite a third consecutive yearly increase in the number of homicides, according to preliminary FBI statistics released yesterday.
The rate for violent crimes -- rape, robbery, aggravated assault, and homicides, including murder and manslaughter -- dropped 3.2 percent compared with 2002, fueled mostly by sharp declines in rape and assault.
Homicide was the only category to increase, rising nationwide last year by about 178 cases, or 1.1 percent. In the previous two years, murder and manslaughter edged up 1 percent in 2002 and 2 percent in 2001.
''I think it's very impressive," said Jack Levin, a criminologist who is director of the Brudnick Center on Violence and Conflict at Northeastern University. ''We've gone through a recession, we've gone through a period where the stock market plummeted, and we came out in pretty good shape."
Specialists said that the decrease in violent crime should be interpreted carefully, because police have wider discretion in reporting rape and assault, the categories showing the biggest declines last year.
Even so, the rise in homicide is relatively small, compared to the double-digit increases of the early 1990s, sparked by crack cocaine, and in the tumultuous 1960s, a period marked by discord over the Vietnam War and civil rights.
Meanwhile, the increase in homicides in recent years might be attributed to increased drug activity and more guns on the streets, specialists said.
''We've had plenty of disagreements, but nothing like the huge social movements in the past," said Gary LaFree, a criminal justice professor at the University of Maryland in College Park. ''And the fact that robbery is going down suggests there may be a partial law enforcement effect."
LaFree said the overall drop in violent crime is a bit surprising, given cutbacks in police forces in recent years. He attributed it in part to smarter policing.
Rates for violent crimes have been dropping steadily over the past five years, particularly in the nation's largest cities. The report yesterday found a 6.5 percent decrease from 2002 to 2003 in those crimes in cities with more than 1 million inhabitants. Decreases were reported in all regions.


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A few days ago, I contacted an acquaintance high up in the Fatah Party hierarchy and asked him how he thought the present crisis in Gaza could be resolved.
"The Hamas people," he said, "have climbed up a very tall tree and don't know how to get down."
Then, he added, "Next to that tree is another tall tree, and on top of it I can see [Israeli Prime Minister Ehud] Olmert and [Defense Minister Amir] Peretz, and they also don't know how to get down."
That is a good, if simplistic, description of our predicament.
Both we and the Palestinians have, to a large extent, become prisoners of our own public opinion.
The Palestinian leaders may wish that Cpl. Gilad Shalit had never been kidnapped, but now that he's in their hands, they fear that Palestinian public opinion won't allow them to return him without the release of some of the prisoners in Israeli hands.
Olmert also knows that public opinion will never forgive him if he's not seen as doing everything possible to obtain Shalit's release.
"Everything possible," however, does not include giving in to the demands of his captors. Surrender to their ultimatum would have meant "open season" for a spate of additional kidnappings, and would have had disastrous consequences. Public opinion demanded action that would make the lives of the inhabitants of Gaza so unbearable that they would pressure the captors into giving up the young soldier.
The trouble is that we have seen time and time again that this formula doesn't work. We destroy their power station? The Gazans grit their teeth, say terrible things about Israel, and hunker down. We bomb the prime minister's office? Ismail Haniyeh simply gains in popularity among his people.
Past experience has shown that the more we pressure the civilian population, the greater the hatred against us and the determination to withstand any pressure that we bring to bear. We've been through that scenario so many times that we must know by now that we have very little to gain from "taking out" power stations and bridges, or by firing salvos against questionable targets.
Yet the public demands action, and I don't envy the prime minister. Olmert knows that the only hope to end the standoff is via the efforts of the negotiators — Egyptians, Turks, Saudis, Europeans and Americans. He knows that eventually a deal will be made — with an attached price tag, though that price need not be paid immediately upon the release of our kidnapped corporal.
The prime minister had, after all, been planning to release prisoners as a gesture of good will toward Palestinian Authority President Mahmoud Abbas. Yet in his present weak position, he has no choice but to play to the public.
The continued launching of Kassam rockets against Israeli towns and kibbutzim has compounded the problem, especially now that two of them have landed in the heart of Ashkelon.
Kassams landed in our midst when our troops were stationed in the Gaza Strip, and when Gush Katif had not yet been evacuated. The truth is that in this sort of warfare, the underdog has advantages, illustrated by the continued terror attacks in Iraq, under the noses of the American army.
An incursion into Gaza will not necessarily bring an end to the Kassams, although it will quench the thirst of the public for action, and will probably provide a partial and temporary respite.
The worst possible scenario for Israel is complete and absolute chaos in the Palestinian territories — a situation where we have no one to talk to, and where every armed gang is a law unto itself. Unfortunately, we seem to be well on the way to reaching such a state of affairs, at least in the Gaza Strip.
The alternative can only come through negotiations with Abbas. That, however, can only take place after the return of Shalit.
Let us hope, for all of us — Israelis and Palestinians alike — that this particular episode of our difficult history will end speedily, and successfully.
David Kimche is a former director general of Israel's Foreign Ministry.


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Watch The New U.C.S Music Video, Because You're In It!
First I gotta say "What a hell of a show"! I can't tell ya how proud we are of local boys, Universal Choke Sign! They have posted another epic video from their first performance at the Wilma. If you attended the show (with Drowning Pool, ANew Revolution, and Static Cycle) than more than likely you are in it!
The boys put on a hell of a performance, any you were (as always) the best effing crowd in the world! Thanks to everyone for getting out and supporting your local Metal Music Scene!


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"Thank You So Much! Our wedding day was made so special because of your love & thoughtful gift." A flexible design, this lovely teal wedding thank you card showcases 1-3 photos surrounded by custom teal & ivory colors. We can recolor the border, text background, text, and ribbon graphic in different colors). We can leave any of your photos in original color or tint them in black & white (shown here below) or sepia for you, when you fill out the form at: Lovely Photograph Wedding Thank You Card.
For more design ideas, please see our Modern Elegance Wedding Thank You Card.


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FP3: Alonso sets the pace in Bahrain
Ferrari's Fernando Alonso beat Red Bull Racing's Sebastian Vettel to the top of the timesheet in the final free practice session ahead of qualifying for the Bahrain Grand Prix.
Most of the hour-long session was spent on the hard tyre compound, with Vettel bossing proceedings with a lap of 1:33.348 set after half an hour. And when the field began to move on to the medium tyre, the defending champion's P1 time remained the benchmark. Mercedes' Lewis Hamilton went closest to the Red Bull Racing man's time with a lap of 1:33.455 on the quicker tyre.
It was only when Alonso bolted on a set of medium tyres in the last 10 minutes of the session that Vettel's time was eclipsed. The Ferrari driver immediately logged a best lap of 1:33.247 to bump Vettel to P2, with Red Bull Racing team-mate Mark Webber third, 0.133 adrift of top spot with his best lap on medium tyres.
Vettel, though, had emerged from pit lane on medium tyres and set about chasing down an improvement. The lap didn't come together, however, as traffic in the final sector forced him to back off, with the result that Alonso's lap stood.
It was a similar story across the track in the closing moments. With most of the field circling as the clock ticked down, few of the front runners were able to get the best out of their medium tyre runs, with the result that the order remained largely static by the time the chequered flag was displayed.
In the end, Alonso held top spot, ahead of the Red Bull Racing pair. Kimi Raikkonen was third for Lotus ahead of Hamilton, Romain Grosjean and Adrian Sutil, who took P7 for Force India. The top ten order was completed by Paul Di Resta, Nico Rosberg and Nico Hulkenberg.
The major drama of the session came right at the end of the hour when Lewis Hamilton got on the radio and informed his Mercedes crew that he had a problem with the rear left of the car. The Briton was forced to limp back to the pits with what looked like a broken suspension.
There was trouble too for Max Chilton. The Marussia driver was forced to sit out the second half of the session as mechanics worked to repair a water pump on his car's KERS unit.
2013 Bahrain Grand Prix – Free Practice 3 Times
1 Fernando Alonso Ferrari 1:33.247
2 Sebastian Vettel Red Bull Racing 1:33.348
3 Mark Webber Red Bull Racing-Renault 1:33.380
4 Kimi Räikkönen Lotus-Renault 1:33.446
5 Lewis Hamilton Mercedes 1:33.455
6 Romain Grosjean Lotus-Renault 1:33.464
7 Adrian Sutil Force India-Mercedes 1:33.596
8 Paul di Resta Force India-Mercedes 1:33.700
9 Nico Rosberg Mercedes 1:33.764
10 Nico Hulkenberg Sauber 1:33.922
11 Felipe Massa Ferrari 1:33.949
12 Jenson Button McLaren 1:34.117
13 Sergio Perez McLaren 1:34.282
14 Daniel Ricciardo Toro Rosso 1:34.577
15 Valtteri Bottas Williams 1:34.611
16 Jean-Eric Vergne Toro Rosso 1:34.678
17 Pastor Maldonado Williams 1:34.833
18 Charles Pic Caterham 1:35.816
19 Jules Bianchi Marussia 1:36.731
20 Giedo van der Garde Caterham 1:36.939
21 Max Chilton Marussia 1:37.630
22 Esteban Gutierrez Sauber 1:39.592


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Davide Prina wrote:
In Sid stat http://ddtp.debian.net/stats/stats-sid.htmlI see German team has translated the initramfs-tools, but how has it done that?
Well, now I have to share how to cheat ;) - go to the DDTP package page (1) - from there go to the latest version (2) - download your version (3) - sed -i 's/Description-it:/Description-it.UTF-8:/' - translate the headline - send a mailBut before starting - think whether you really want to make this effort to gain
leadership in a worthless statistic. To me it seems more sensible to dosomething else: Translate squeeze descriptions and hope for the repair of the system. The system will propagate the headlines of your work to newer descriptions
with unchanged headlines. MAX_VALID_INDEX=91153 for (i=MAX_VALID_INDEX, b=0; b < LIMIT; i--, b++) { examine http://ddtp.debian.net/ddt.cgi?desc_id=$i if [no-translation for $i]; then pull $i into DDTSS (using curl) translate as usual fi } Cheers, Martin 1: http://ddtp.debian.net/ddt.cgi?package=initramfs-tools 2: http://ddtp.debian.net/ddt.cgi?desc_id=125363 3: http://ddtp.debian.net/ddt.cgi?desc_id=125363&getuntrans=it


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*Rates shown are per day, based upon a 7-day car rental. Taxes and additional surcharges may apply.
Besides the obvious draw of nearby metropolitan London, there are many reasons to tour England with a rental car in Bracknell. Adjacent to the ruins of the ancient Roman town Cavella Atrebatum, complete with intact town wall and amphitheater, the Beale Wildlife Park in nearby Lower Basildon, and the Mapledurham House and Watermill, Bracknell is well suited to exploration by car. Whether you desire the vibrant city energy of London, or the quaint village feel of the English countryside, Bracknell offers itself up as the perfect jumping-off point for your vacation in England. Have questions about driving in Bracknell? Our rental car driving information page should provide answers.
For over 60 years Auto Europe has thrilled customers with unbeatable rates and top-notch service on rental cars in Bracknell and around the world. We leverage our experience and our global network of 10,000 car rental locations to guarantee the lowest rates in the industry and we are positive that once you book with Auto Europe you'll be a customer for life. Reserve your car online in minutes using our secure online booking engine or, for more information, review our car rental FAQ or call Auto Europe toll-free, 24/7 at 1-888-223-5555 to reserve the perfect car.


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Senator Dilan Announces New Leadership Role Within Senate Minority Conference
State Senator Martin Malavé Dilan (D-Brooklyn) today announced his new appointment as Chair of the SenateMinority Conference. In this new capacity Senator Dilan will be the third most powerful Democrat in the Senate.
"I am honored to have been chosen by Senator Malcolm Smith, the newly elected Minority Leader, and my colleagues to serve as Chair of theMinority Conference. I look forward to working with each of my fellow Senators to advance a forward thinking and effective agenda," Senator Dilan said.
Senator Dilan has been a member of the Senate since 2002 and it is very rare for someone to be elevated to this position after only four years. It is testament to his hard work and the high level of respect he has been able to earn from his colleagues in Albany.
As the Conference Chair, Senator Dilan will have a significant role as a member of the SenateMinority Leadership team. This will allow Senator Dilan to be an integral part of key decisions that will shape the Senate for years to come.
"As we move toward taking over as the Majority party in the Senate, I am excited to have this opportunity to work closely with our new Governor and with our new leader. We are in a position to institute some real positive changes within our state government, and as Chair of theMinority Conference, I believe I can help to instill some useful and effective policies," said Senator Dilan.


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Fort Myers, Fla. — Chico's FAS Inc. is reportedly considering taking itself private. According to Financial Times, the women's apparel retailer has been holding talks with several private equity firms in recent weeks.
While sources indicated Chico's has been having conversations about going private, they have not clarified whether the retailer would go into partnership with a private equity investor or simply sell the business to a private interest. Chico's current market value is $2.36 billion, and experts believe the company could get about 30% of that figure, or roughly $700 million, if it went private.


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FMC Agricultural Products Group (APG) is introducing Marvel herbicide for soybean growers. This new herbicide provides growers with a "more aggressive tank-mix partner for maximum postemergence control of glyphosate-resistant weeds," the company claims.
"Glyphosate resistance continues to make weed control more difficult," said Brent Neuberger, FMC Agricultural Products North America technical service advisor for Marvel herbicide. "Marvel herbicide was designed as a premix to offer a broader spectrum postemergence herbicide that provides growers with an excellent product to fit into their weed management programs."
With speed and use flexibility, Marvel herbicide controls resistant broadleaves such as waterhemp, palmer pigweed, lambsquarters, morningglory and velvetleaf. Visual control symptoms can be seen in as quickly as 24 hours. This "ideal tank-mix partner" can be used from preplant to full flowering of soybeans.
More information on FMC crop protection products can be found at www.FMCcrop.com.


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The Benicia High boys basketball team defeated Vanden 48-46 on Thursday to pull into first place in the Solano County Athletic Conference.
The Panthers (13-7, 3-0) were led by Derek Johnstone, who had 19 points, eight rebounds, two assists and two blocks. Koree Cox had 13 points, eight rebounds and five assists while Jamal Easterling had 10 points and 15 rebounds.
Benicia travels to Bethel on Tuesday night.


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Amazing time lapse video of Everest
01. Jul, 2013 0 CommentsWritten by Manny
From the video description: Via [Bad Astronomy].
Enjoy a good week-start with this awesome time-lapse video created by Elia Saikaly. Have fun watching it and be ready for a new day!
This time lapse video is comprised of thousands of photographs, processed and assembled on Mt. Everest.
Shot on a Canon 5D Mark II
-Canon 2.8 16-35mm
-Canon 2.8 24-70mm
-Canon 2.8 70-200mm (which was way to heavy to carry beyond 6400M)
-TL Remote was purchased off eBay
Edited in Final Cut Pro
Processed in Adobe LightRoom
Movies compiled in Quicktime
From the video description:
Via [Bad Astronomy].


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The need to speak, even if one has nothing to say, becomes more pressing when one has nothing to say, just as the will to live becomes more urgent when life has lost its meaning.Jean Baudrillard
On this Veterans Day, let us remember the service of our veterans, and let us renew our national promise to fulfill our sacred obligations to our veterans and their families who have sacrificed so much so that we can live free.Dan Lipinski
You will never be happy if you continue to search for what happiness consists of. You will never live if you are looking for the meaning of life.Albert Camus
Now that the wars are coming to an end, I wish you to prosper in peace. May all mortals from now on live like one people in concord and for mutual advancement. Consider the world as your country, with laws common to all and where the best will govern irrespective of tribe. I do not distinguish among men, as the narrow-minded do, both among Greeks and Barbarians. I am not interested in the descendance of the citizens or their racial origins. I classify them using one criterion: their virtue. For me every virtuous foreigner is a Greek and every evil Greek worse than a Barbarian. If differences ever develop between you never have recourse to arms, but solve them peacefully. If necessary, I should be your arbitrator. You must not consider God like an autocratic despot, but as a common Father of all; so your behavior may resemble the life siblings have in a family. On my part I should consider all equals, white or blacks, and wish you all to be not only subjects of the Commonwealth, but participants and partners. As much as this depends on me, I should try to bring about what I promised. The oath we made over tonights libations hold onto as a Contract of Love.Alexander III
But after observation and analysis, when you find that anything agrees with reason and is conducive to the good and benefit of one and all, then accept it and live up to it.Buddha
The world is a dangerous place to live, not because of the people who are evil, but because of the people who don't do anything about it.Albert Einstein
To know the Truth, to love the Truth, and to live the Truth is the whole duty of man.Benjamin Fish Austin
Ofttimes the test of courage becomes rather to live than to die.Mohamed Abdel Moneim Fayed
Live amongst people in such a manner that if you die they weep over you and if you are alive they crave for your company.Ali bin Abu-Talib
Spiritual superiority only sees the individual. But alas, ordinarily we human beings are sensual and, therefore, as soon as it is a gathering, the impression changes -- we see something abstract, the crowd, and we become different. But in the eyes of God, the infinite spirit, all the millions that have lived and now live do not make a crowd, He only sees each individual.Soren Kierkegaard
Though the lion and the antelope happen to live in the same forest, the antelope still has time to grow up. - Cape Coast, Ghana.African Proverb
People who will not sustain trees will soon live in a world that will not sustain people.Bryce Nelson
The difference between a Republican and a Democrat is the Democrat is a cannibal -- they have to live off each other--while the Republicans, why, they live off the Democrats.Will Rogers
To live without an aim is to wander in a wildernessShri Radhe Maa
Live every day as if it is a festival. Turn your life into a celebrationShri Radhe Maa


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RIO DE JANEIRO – Japan coach Alberto Zaccheroni insists his Asian champions will have no easy ride after being drawn in a tricky Group C with North Korea, Uzbekistan and Syria for the third qualifying round of the 2014 World Cup on Saturday.
"This is the third round of qualifying so there are no easy groups and no easy opponents," the Italian coach said Sunday.
"We are the Asian champions, so our opponents will be doing their homework and come at us firing on all cylinders, but we will just focus on each and every match. This is significant as it will be my first experience of World Cup qualifying. I am always eager for new challenges and am looking forward to this first experience."
The third round of Asian qualifiers begins Sept. 2. The top two from each of the five groups advance to the yearlong final qualifying campaign starting in June 2012.
Japan opens its campaign against North Korea in Saitama on Sept. 2 and then faces Uzbekistan away on Sept. 6. Zaccheroni's men face Syria in Osaka on Oct. 11 followed by away games against Syria and North Korea on Nov. 11 and 15, respectively. They wrap up the third round at home to the Uzbeks at Toyota Stadium on Feb. 29 next year.
The 10 teams in the final round will be split into two groups, with the top two from each group automatically qualifying for Brazil. The third-place sides face off for a spot in the intercontinental playoff against a team from South America for a potential fifth place from Asia.
Japan was one of five Asian seeds for Saturday's preliminary draw, but landed in a very competitive group — at the hands of former coach Zico, who assisted in the event.
While Japan — which has yet to lose in the 10 games Zaccheroni has been in charge — is easily the highest ranked team of the four at 16th, its three opponents are anything but pushovers.
North Korea (115) raised a few eyebrows at the 2010 World Cup, while Uzbekistan (83) reached the semifinals of this year's Asian Cup in Qatar where Syria (104) tested the Japanese despite a 2-1 defeat in the group phase.
Japan and Uzbekistan were also grouped together during the final qualifiers for South Africa.
"There are no easy groups. Whoever you play in Asia it is always a tough battle," Japan's Belgian-based goalkeeper Eiji Kawashima said. "The most important thing is that we all prepare well."
Added Borussia Dortmund midfielder Shinji Kagawa, "Whoever you play it is difficult and we cannot afford to be complacent against any of our opponents."
"Our first two games will be important," said Japan Football Association technical director Hiromi Hara. "This is World Cup qualifying so our opponents will be really up for it. We just have to try and pick up points from each of our matches."
North Korea striker Chong Tese, who used to play in Japan and is now at German second-division club Bochum, said on his blog, "We will definitely qualify with Japan. It's the group of death and Japan and Uzbekistan will both be feeling the heat."
Japan was drawn together with North Korea in the final qualifying round for the 2006 finals and won 2-0 behind closed doors in Bangkok to book its ticket to Germany.
That match had been scheduled to be played in Pyongyang but soccer's world governing body FIFA ordered North Korea to play the match at a neutral venue as punishment for crowd violence that marred its 2-0 qualifying defeat to Iran in the reclusive state's capital.
The draw in Rio determined the path of 166 teams in their bid to qualify for the tournament in three years' time. It was the first major World Cup event in Brazil since the South American nation was awarded the competition in 2007. As host, Brazil is the only nation that doesn't have to qualify.
"We are happy to be back in Brazil," FIFA president Sepp Blatter said.
Fifty-three European nations were divided into nine groups, with the winners automatically qualifying and the eight best group runnersup advancing to a playoff. The European qualifying matches will begin Sept. 7 and end Oct. 15, 2013.
Spain is in a difficult Group I, pitted against 1998 winner France, Georgia, Belarus and Finland.
The draw placed England with 2012 European Championship co-hosts Ukraine and Poland in Group H. Montenegro, Moldova and San Marino also are in the group.
"It will be interesting, there won't be easy matches," England coach Fabio Capello said. "England will have to be very careful, especially against Montenegro, Poland and Ukraine."
Italy will meet Denmark and the Czech Republic in Group B, while Germany faces Sweden, Austria and Ireland in Group C.


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Thanks to the terrific petition posted yesterday by Melissa Silverstein, we now have an answer to the question I asked earlier today:
And the answer: Surprise! Surprise!! "Cannes" is disproportionately male!!!
Not only that, but the men on the jury are primarily filmmakers (a more powerful role), whereas the women who are on the jury are primarily actresses (a less powerful role).
Here is the main thing I have learned these past ten years from Martha Lauzen: Don't look for justifications & don't question motives. Just keep track of the [dismal] numbers & force those who create them to live with the results.
These numbers really do speak, & sooner or later everyone will finally hear what they say!
Meanwhile over 200 people have already signed Melissa's online petition, so add your name to this important cause ASAP:
Let's go viral & hold their feet to the fire!
"Cannes 2012" chart copyright Jan Lisa Huttner (5/16/12). And click HERE for my most recent evidence that, yes, "the gender gap" matters!


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Get an overview of this hotelWaterfront hotel with an outdoor pool
Popular property highlights
Nestled on the beach, this Parrita hotel is in a regional park and within 20 mi (32 km) of Rainmaker, Marina Pez Vela, and Mid World Tour Office. La Playita and Manuel Antonio Beach are also within 25 mi (40 km).
Hotel Features
At Clandestino Beach Resort, relax by an outdoor pool or hit the beach where you can enjoy surfing/boogie boarding.
Room Amenities
All 12 soundproofed rooms feature free WiFi and minibars. For entertainment, LCD TVs come with cable channels, and guests can also appreciate comforts like premium bedding and down comforters.
Guest Highlights
- Spotless guestrooms
- Comfortable rooms
- Beach
- Breakfast
- Pool
- WiFi
- Helpful staff
Clandestino Beach Resort
Hotel Amenities
Hotel Amenities
Clandestino Beach Resort features an outdoor pool and a children's pool. The hotel offers a restaurant. Guests can unwind with a drink at one of the hotel's bars, which include a poolside bar and a bar/lounge. A complimentary breakfast is offered. Public areas are equipped with complimentary wireless Internet access.
This beach hotel also offers a rooftop terrace, a library, and tour/ticket assistance. For a surcharge, a roundtrip airport shuttle (available 24 hours) is offered to guests. Complimentary self parking is available on site.
Clandestino Beach Resort has designated areas for smoking.
- Beach towels
- Front desk (limited hours)
- Airport transportation (surcharge)
- Total number of rooms - 12
- Number of floors - 1
- Bar/lounge
- Free breakfast
- Express check-in
- Gift shops or newsstand
- Billiards or pool table
- Tours/ticket assistance
- Free WiFi
- Designated smoking areas
- Pool umbrellas
- Library
- Multilingual staff
- Garden
- Rooftop terrace
- Poolside bar
- Porter/bellhop
- Luggage storage
- Restaurant
- Year Built 2009
- Free self parking
- Number of buildings/towers - 4
- Children's pool
- Outdoor pool
- Television in common areas
- Wedding services
Family Friendly Amenities
- Family Hotel
- Outdoor pool
- Children's pool
- Free Wi-Fi
- Soundproofed rooms
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free self parking
Room Amenities
- Cribs/infant beds (surcharge)
- Connecting/adjoining rooms available
- Premium bedding
- Furnished patio
- Soundproofed rooms
- Air conditioning
- Ceiling fan
- Minibar
- Daily housekeeping
- Turndown service
- Private bathroom
- Free toiletries
- Hair dryer
- In-room massage available
- Shower only
- Rainfall showerhead
- Desk
- In-room safe
- Cable TV service
- Free WiFi
- Down comforter
- Rollaway/extra beds (free)
Where to Eat
A complimentary breakfast is offered. An additional continental breakfast is served daily (subject to charges).
Clandestino - This beachfront brasserie serves breakfast, brunch, lunch, and dinner. Guests can order drinks at the bar and enjoy alfresco dining (weather permitting).
Nearby Things to Do
An outdoor pool and a children's pool are on site.
Children under 10 years old are not allowed in the swimming pool or spa tub without adult supervision.
The recreational activities listed below are available either on site or nearby; fees may apply.
- Personal motorized watercraft on site
- Fishing on site
- Boat tours nearby
- Golfing nearby
- Ecotours nearby
- Golf lessons available nearby
- Surfing/boogie boarding on site
- Yoga classes/instruction on site
If you have requests for specific accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- In-room accessibility
Hotel Policies
Check-in time ends at 5 AM
Check-in time starts at 1 PM
Special check-in instructions:
Check-out time is 11:30 AM
Payment types
Children and extra beds
- Children are welcome.
- Free rollaway/extra beds!
- Cribs (infant beds) are available for CRC 7500 per night.
- Pets not allowed (service animals welcome)
You need to know
Extra-person charges may apply and vary depending on property policy.
Optional extras
- Airport shuttle fee: CRC 90500 per vehicle (one way)
- Crib (infant bed) fee: CRC 7500 per night
Hotel Name
- Clandestino Beach
- Clandestino Beach Parrita
- Clandestino Beach Resort
- Clandestino Beach Resort Parrita
- Clandestino Resort
- Clandestino Beach Resort Costa Rica/Parrita
We should mention
This property offers transfers from the airport (surcharges may apply). Guests must contact the property with arrival details before travel, using the contact information on the booking confirmation. The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation.


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Google, Inc. (GOOG) Technical Play Ahead of Earnings
Google.com, Inc. Credit Spread (Nasdaq: GOOG)
TheOptionPlayer.com sets up a Google.com (GOOG) short-term (4-day) option strategy. Investors could simultaneously:
Sell the January week-four expiration GOOG $1175 call for $2.30 (Friday's closing bid/ask mean)
Buy the $1180 call at $1.68 (Friday's closing bid/ask mean)
The difference between funds received and paid out is a $.62 per share credit which we keep if Google.com stock closes below $1175 on Friday January 24th, but immediately exit the position if it appears the price will end up higher. Another suggestion is if the price gaps up open the trade using higher strike prices. See Guidelines page at www.theoptionplayer.com/ for explanation on how trade is set up.
Why we recommend it:
We are basically playing Google.com (NasdaqGS: GOOG) stock to continue trading below its current resistance level until earnings announcement at the end of the month. As noted in the Google.com 2-hour chart below, after breaking through the previous short term resistance at the end of last week, the price has been contained between current resistance and support (defined as previous resistance). Looking at the daily chart you can easily observe that even though Google traded at a 52-week high last week, the price is stuck in a trading range with momentum indicators flashing neutral signs. Unless there is some unexpected news it is reasonable to expect the stock price to remain subdued until Google.com reports earnings at the end of the month. At the very least there is a high probability that shares will continue to trade below the $1175 target for a few more days.
52-Week High: $1160.63
52-Week Low: $695.52
Average Volume (3 month): 1,866,500
The following article is from one of our external contributors. It does not represent the opinion of Benzinga and has not been edited.
Posted-In: Technicals Tech Trading Ideas


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arah Jane and Peter end up in a white room, a void where Peter tells her that he nearly died but the Trickster saved him and struck up a deal if he married Sarah Jane. He had no idea that any harm would be done to her or her friends. The Trickster explains his plan - to give Sarah Jane a new life so that she would forget her old one as a guardian of the Earth and make it vulnerable to the likes of him. The Doctor, K9, Rani and the boys have been trapped in a similar void but eventually make contact with Sarah Jane. Sadly, the only way for the Trickster to be defeated is for Peter to end the deal and to sacrifice himself. He was only living a half-life but marriage to Sarah Jane would have made it whole. Strengthened by his love for Sarah Jane,he agrees to his death in the knowledge that it will make the world more secure. Sarah Jane is understandably heart-broken but Doctor Who consoles her, telling her that she still has a wonderful life ahead of her and asking her never to forget him.Resumen IMDb.com
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|Nombre del documento
|The Sarah Jane Adventures S03E06 The Wedding of Sarah Jane Smith Part 2-spa.srt