_:b6672448 "Interestingly, in the proposed serrated pathway to CIMP+ tumors, both BRAF mutations and CIMP+ have been observed in early precursor lesions, whereas MSI+ has not [6], [>>10<<], [41]. Thus, inactivation of MLH1 might occur at a later stage of tumor development. Minoo and colleagues observed the DNA hypermethylation of CDKN2A and 15 other CIMP-associated markers (IGFBP7 was not examined) in parent NCM460 cells," . _:b421483696 . _:b421483729 "2"^^ . _:b6672427 . _:b421483782 . _:b421483796 . _:b6672367 _:b6672384 . . _:b421483699 . _:b6672367 _:b6672385 . _:b6672436 . _:b6672375 . _:b421483728 "2"^^ . _:b6672367 _:b6672386 . _:b6672367 _:b6672387 . _:b6672367 _:b6672388 . _:b6672427 . _:b421483698 . _:b6672367 _:b6672389 . _:b6672437 . _:b421483731 "2"^^ . _:b6672367 _:b6672390 . . _:b421483739 . _:b6672367 _:b6672391 . _:b421483709 . . _:b421483709 . _:b421483730 "2"^^ . _:b6672438 . _:b421483784 . _:b421483785 . _:b421483786 . _:b421483787 . _:b6672436 "The distinct features found in CIMP are important clues in understanding this phenotype [>>3<<], [5], [7], [8]." . _:b421483708 . _:b421483741 "2"^^ . _:b6672439 . _:b421483805 . . _:b421483788 . _:b421483789 . _:b421483790 . _:b6672394 . _:b6672394 "Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [15]\u2013[17], [22], [>>23<<], making it a reliable source for DNA methylation measurements across 1,505 loci." . _:b421483765 . _:b6672395 "materials and methods" . _:b421483791 . _:b421483711 . _:b421483740 "2"^^ . _:b6672432 . _:b421483776 . _:b421483777 . _:b421483778 . _:b421483779 . _:b421483710 . . _:b421483743 "2"^^ . _:b6672403 . _:b421483780 . . _:b6672433 . _:b421483781 . _:b421483782 . _:b421483783 . _:b421483705 . _:b421483742 "2"^^ . _:b6672434 . . _:b421483800 . _:b421483801 . _:b421483802 . _:b6672455 . _:b421483803 . _:b421483704 . _:b421483737 "2"^^ . _:b6672453 . _:b6672435 . _:b421483804 . _:b421483805 . _:b6672422 . _:b421483806 . _:b421483807 . _:b6672387 "However, recent studies have indicated that mutations of these genes might play distinct roles in tumor initiation and/or maintenance [>>10<<], [14]." . _:b421483707 . _:b421483736 "2"^^ . _:b6672444 . _:b421483792 . _:b421483793 . _:b421483794 . . _:b421483706 . _:b421483795 . _:b421483739 "2"^^ . _:b6672445 . _:b6672426 "BRAFV600E has been shown to induce cellular senescence [>>30<<]\u2013[32]. Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [33]." . _:b421483796 . _:b421483797 . _:b421483798 . _:b421483799 . _:b421483809 . _:b421483717 . . _:b421483738 "2"^^ . _:b6672446 . _:b421483752 . _:b421483753 . . . _:b6672390 "The GoldenGate DNA methylation assay has been widely used in various studies and is now a standard method for DNA methylation analysis [15]\u2013[>>24<<]. Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [15]\u2013[17], [22], [23], making it a reliable source for DNA methylation" . _:b421483754 . _:b421483755 . _:b421483758 . _:b421483716 . _:b421483756 . _:b6672447 . _:b6672378 . _:b421483749 "2"^^ . _:b421483757 . _:b6672418 . . _:b421483758 . _:b421483759 . _:b421483719 . _:b421483744 . _:b6672440 . _:b421483748 "2"^^ . _:b421483745 . _:b421483746 . . . _:b6672422 "The functional consequence of DNA hypermethylation of such tumor suppressor genes linked with CIMP+ and BRAFV600E remains speculative [>>25<<]\u2013[28]." . _:b421483718 . _:b421483747 . _:b421483748 . _:b6672441 . _:b421483751 "2"^^ . _:b421483749 . _:b421483750 . _:b421483751 . _:b421483713 . _:b421483768 . _:b6672442 . _:b421483750 "2"^^ . _:b421483769 . _:b421483770 . _:b421483771 . _:b421483712 . _:b421483772 . _:b6672443 . _:b421483745 "2"^^ . _:b421483773 . _:b421483774 . . _:b421483775 . . _:b421483715 . _:b421483760 . _:b6672452 . _:b421483744 "2"^^ . _:b421483761 . _:b421483762 . _:b421483763 . _:b421483714 . _:b421483764 . _:b6672459 . _:b6672453 . _:b6672374 "CIMP in colorectal cancer may arise through a distinct pathway originating in certain subtypes of serrated polyps [6] and is observed in approximately 15% of all colorectal cancer cases [>>5<<], [7]. Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [3], [5], [7], [8]." . _:b421483747 "2"^^ . _:b421483765 . . _:b421483766 . _:b421483767 . _:b6672413 "We used MethyLight to assess the DNA methylation status of five CIMP-defining markers previously identified in our laboratory [>>5<<]. Using a PMR (percent of methylated reference) of \u226510 as a threshold for positive methylation, we identified six cell lines that lacked DNA methylation for all five CIMP-specific markers (Figure 1). To test our hypothesis, we initially" . _:b421483725 . _:b421483720 . _:b6672454 . _:b421483746 "2"^^ . _:b421483721 . _:b421483722 . _:b421483723 . _:b421483724 . _:b421483724 . _:b6672455 . _:b421483757 "2"^^ . . _:b421483725 . _:b421483726 . _:b421483727 . _:b6672390 . _:b421483727 . _:b421483712 . _:b6672448 . _:b421483756 "2"^^ . _:b421483752 . _:b421483713 . _:b421483753 . . _:b6672382 . _:b421483714 . _:b421483715 . _:b6672428 . . _:b421483726 . _:b421483716 . _:b6672449 . _:b421483759 "2"^^ . _:b421483717 . _:b421483718 . . _:b421483719 . _:b421483721 . _:b421483736 . _:b6672450 . _:b421483763 . _:b421483758 "2"^^ . . _:b421483737 . _:b421483738 . _:b421483739 . _:b421483720 . _:b421483740 . _:b6672451 . _:b421483753 "2"^^ . _:b421483741 . _:b6672424 . _:b421483742 . _:b421483664 . _:b6672438 . _:b421483723 . _:b421483743 . _:b421483728 . _:b6672460 . _:b6672437 "The distinct features found in CIMP are important clues in understanding this phenotype [3], [>>5<<], [7], [8]." . _:b421483752 "2"^^ . . _:b421483729 . _:b6672449 "Interestingly, in the proposed serrated pathway to CIMP+ tumors, both BRAF mutations and CIMP+ have been observed in early precursor lesions, whereas MSI+ has not [6], [10], [>>41<<]. Thus, inactivation of MLH1 might occur at a later stage of tumor development. Minoo and colleagues observed the DNA hypermethylation of CDKN2A and 15 other CIMP-associated markers (IGFBP7 was not examined) in parent NCM460 cells, which" . _:b6672425 . _:b6672405 "A 58 sample set included five CIMP+ tumors, five CIMP\u2013 tumors and 48 randomly selected tumors, as indicated previously [>>5<<]. A 235 sample set included the same 48 randomly selected samples described above along with an additional 187 randomly collected tumors described previously [5]. CIMP status and mutation status of BRAF and KRAS for each tumor sample was" . _:b421483730 . _:b421483731 . _:b421483812 . _:b421483722 . _:b421483732 . _:b6672461 . _:b421483755 "2"^^ . _:b421483733 . _:b421483734 . _:b421483735 . _:b421483735 . _:b421483733 . _:b421483688 . _:b6672462 . _:b421483754 "2"^^ . _:b421483689 . _:b6672442 . _:b421483690 . _:b421483691 . _:b6672368 . _:b421483732 . _:b421483692 . _:b6672463 . _:b421483765 "2"^^ . _:b421483693 . _:b421483694 . . _:b421483695 . _:b421483735 . _:b421483680 . _:b6672456 . _:b421483764 "2"^^ . _:b421483681 . _:b421483682 . _:b421483683 . _:b421483734 . _:b421483684 . _:b6672457 . _:b421483767 "2"^^ . _:b421483685 . _:b421483686 . _:b421483687 . _:b421483729 . _:b421483728 . _:b421483704 . _:b6672458 . _:b421483766 "2"^^ . _:b421483705 . _:b421483706 . _:b421483707 . _:b421483808 . _:b421483728 . _:b421483708 . _:b6672459 . _:b421483761 "2"^^ . _:b421483709 . _:b421483710 . _:b421483711 . _:b6672432 "These tumors have been described as CIMP-low or CIMP2 [35], [>>36<<]. We did not find an association between IGFBP7 DNA hypermethylation and KRAS mutations when we excluded tumors with mutant BRAF (P value \u200A=\u200A0.85)." . _:b421483731 . _:b421483696 . _:b6672468 . _:b421483788 . _:b421483760 "2"^^ . _:b421483697 . _:b421483690 . _:b6672458 "In order for BRAFV600E to promote its oncogenic effects, additional cooperative events are required to bypass senescence [>>33<<]. Recently, the molecular basis of BRAFV600E-induced senescence and apoptosis has been studied in detail. Wajapeyee et al. identified IGFBP7 as a mediator of BRAFV600E-induced senescence in human primary fibroblasts using a genome-wide" . _:b421483698 . _:b421483699 . _:b421483730 . _:b421483700 . _:b421483763 "2"^^ . _:b6672469 . _:b421483701 . _:b421483702 . _:b421483703 . _:b421483741 . . _:b6672470 . _:b421483762 "2"^^ . _:b421483713 . _:b421483740 . . _:b421483773 "2"^^ . _:b421483663 . _:b421483743 . _:b421483772 "2"^^ . _:b6672464 . . _:b421483740 . _:b421483801 . _:b421483742 . _:b421483775 "2"^^ . _:b6672465 . _:b421483737 . _:b421483672 . _:b6672466 . _:b421483774 "2"^^ . _:b421483673 . _:b421483674 . _:b421483675 . _:b421483736 . _:b421483676 . _:b6672467 . _:b421483769 "2"^^ . _:b421483677 . _:b421483678 . _:b421483679 . _:b421483772 . _:b421483739 . _:b421483664 . _:b421483768 "2"^^ . _:b421483665 . _:b6672444 . _:b421483666 . _:b6672433 "discussion" . _:b421483738 . _:b421483667 . . _:b421483771 "2"^^ . _:b421483668 . _:b421483669 . _:b421483670 . . _:b421483749 . _:b421483671 . _:b421483770 "2"^^ . _:b421483748 . _:b421483781 "2"^^ . . _:b421483751 . _:b421483780 "2"^^ . _:b6672443 . _:b421483750 . _:b421483783 "2"^^ . . _:b421483745 . _:b421483782 "2"^^ . _:b421483710 . . _:b421483744 . . _:b6672450 . _:b421483777 "2"^^ . _:b421483747 . _:b6672457 . _:b421483776 "2"^^ . _:b421483746 . . _:b421483779 "2"^^ . . _:b421483697 . _:b421483757 . _:b421483778 "2"^^ . . _:b6672458 . _:b421483756 . _:b421483789 "2"^^ . _:b421483759 . _:b421483742 . _:b421483788 "2"^^ . _:b421483671 . _:b421483758 . _:b421483769 . _:b421483791 "2"^^ . _:b6672433 . . _:b421483753 . _:b6672411 "We selected 100 CpG sites with P<0.001 after a correction for multiple-comparison [45] and mean |\u0394\u03B2|>0.17, the estimated error in \u03B2 [>>16<<]." . _:b421483790 "2"^^ . _:b421483751 . . _:b421483752 . _:b421483692 . _:b421483785 "2"^^ . _:b421483792 . . _:b421483755 . _:b421483667 . _:b6672392 . _:b421483784 "2"^^ . _:b421483798 . _:b421483754 . _:b6672369 "Distinct subgroups of several types of human cancers have been proposed to have a CpG island methylator phenotype (CIMP) in which an exceptionally high frequency of cancer-specific DNA hypermethylation is found [>>2<<], [3]. Although this concept has been controversial [4], we have confirmed the existence of CIMP in colorectal cancer in a large-scale comprehensive study [5]." . _:b421483787 "2"^^ . . _:b421483765 . _:b421483786 "2"^^ . _:b421483764 . _:b421483797 "2"^^ . _:b421483767 . _:b421483796 "2"^^ . _:b6672412 . _:b421483766 . _:b421483799 "2"^^ . _:b421483761 . _:b421483798 "2"^^ . _:b6672419 "We confirmed the recently observed associations between DNA hypermethylation of BMP3 and MCC with CIMP+ and BRAFV600E in colorectal cancer [25], [>>26<<]. We also found CIMP-specific DNA hypermethylation of BMP6." . _:b6672374 . . _:b421483760 . _:b421483793 "2"^^ . _:b421483763 . _:b6672395 . _:b421483792 "2"^^ . _:b6672372 . _:b421483762 . _:b6672447 "Interestingly, in the proposed serrated pathway to CIMP+ tumors, both BRAF mutations and CIMP+ have been observed in early precursor lesions, whereas MSI+ has not [>>6<<], [10], [41]." . _:b421483795 "2"^^ . _:b421483770 . _:b421483773 . _:b421483787 . . _:b421483794 "2"^^ . _:b421483711 . _:b421483772 . _:b421483694 . _:b6672443 "One caveat of our system is that BRAFV600E could play a role in inducing DNA methylation only early in colorectal tumorigenesis, as BRAF mutations have been described at the earliest stage of tumor development [10], [>>38<<], [39]. It is possible that a unique set of genetic and/or epigenetic changes that occurred in COLO 320DM cells might have created an unfavorable environment for BRAFV600E to induce DNA hypermethylation. Experiments similar to those" . _:b421483805 "2"^^ . _:b6672377 . _:b6672469 "supporting information" . _:b421483775 . _:b421483804 "2"^^ . . _:b421483774 . _:b421483807 "2"^^ . _:b421483769 . _:b421483806 "2"^^ . _:b421483768 . _:b421483801 "2"^^ . _:b6672381 . _:b421483771 . _:b421483800 "2"^^ . _:b6672380 . _:b6672452 "Our average intra-clonal (within clones at different passages) R2 correlation is 0.97\u00B10.01 and the R2 correlation between technical replicates in Illumina GoldenGate DNA methylation analysis is 0.98\u00B10.02 [>>16<<]. Consequently, we found some large differences in DNA methylation at several loci even among control clones (Figures 2B and 3). This emphasizes the importance of using multiple clones for this type of studies." . _:b421483770 . _:b421483803 "2"^^ . _:b421483802 "2"^^ . _:b421483679 . _:b421483668 . _:b421483812 "2"^^ . . _:b6672430 "to be epigenetically silenced by CpG island promoter hypermethylation specifically in primary melanoma samples carrying BRAFV600E, indicating that loss of IGFBP7 expression is critical in the development of BRAFV600E-positive melanoma [>>34<<]." . _:b421483799 . _:b421483675 . . _:b6672469 . _:b6672385 . _:b421483809 "2"^^ . . _:b6672457 "Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [>>33<<]. In order for BRAFV600E to promote its oncogenic effects, additional cooperative events are required to bypass senescence [33]. Recently, the molecular basis of BRAFV600E-induced senescence and apoptosis has been studied in detail." . _:b421483808 "2"^^ . . _:b6672462 "However, an increasing number of genes have recently been reported to be involved in OIS, and cooperation of multiple different signals appears to be critical for OIS [>>44<<]. It is therefore possible that CIMP-associated DNA hypermethylation events may impair OIS by affecting other components of the OIS signaling pathway in colorectal cancer." . _:b421483811 "2"^^ . _:b421483810 "2"^^ . _:b421483794 . _:b6672407 "CIMP status and mutation status of BRAF and KRAS for each tumor sample was previously determined [>>5<<]. BRAF mutations and KRAS mutations were found in 15% (33/235) and 33% (74/221) of the colorectal tumor samples, respectively. The KRAS mutation status of 14 tumor samples was not available. BRAF mutations and KRAS mutations were mutually" . _:b6672387 . . . _:b6672375 "CIMP in colorectal cancer may arise through a distinct pathway originating in certain subtypes of serrated polyps [6] and is observed in approximately 15% of all colorectal cancer cases [5], [>>7<<]. Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [3], [5], [7], [8]." . _:b6672393 . _:b421483716 . . _:b421483705 . _:b421483737 . _:b421483669 . _:b6672433 _:b6672434 . _:b6672433 _:b6672435 . _:b6672433 _:b6672436 . _:b6672433 _:b6672437 . _:b6672419 . _:b6672433 _:b6672438 . . _:b6672433 _:b6672439 . _:b6672433 _:b6672440 . _:b421483780 . _:b6672433 _:b6672441 . _:b6672433 _:b6672442 . _:b6672446 . _:b6672433 _:b6672443 . _:b6672433 _:b6672444 . _:b6672433 _:b6672445 . _:b421483789 . _:b6672433 _:b6672446 . _:b6672433 _:b6672447 . _:b6672453 "Intriguingly, we observed CIMP-dependent DNA hypermethylation and transcriptional inactivation of IGFBP7, which has been shown to mediate BRAFV600E-induced cellular senescence and apoptosis [>>34<<]. BRAFV600E has been shown to induce cellular senescence in cultured and primary human cells [30], [31], as well as mouse model [32]. Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [33]. In" . _:b421483755 . . . _:b421483804 . . _:b6672470 . . _:b6672449 . _:b6672438 "The distinct features found in CIMP are important clues in understanding this phenotype [3], [5], [>>7<<], [8]. Particularly striking is the extremely tight association between CIMP and BRAFV600E [5]. Mechanisms linking epigenetic (CIMP) and genetic (BRAF mutation) events and the temporal sequence in which these two events take place have" . _:b421483683 . . _:b6672370 "Distinct subgroups of several types of human cancers have been proposed to have a CpG island methylator phenotype (CIMP) in which an exceptionally high frequency of cancer-specific DNA hypermethylation is found [2], [>>3<<]. Although this concept has been controversial [4], we have confirmed the existence of CIMP in colorectal cancer in a large-scale comprehensive study [5]." . "PMC0" . . _:b6672440 "Particularly striking is the extremely tight association between CIMP and BRAFV600E [>>5<<]. Mechanisms linking epigenetic (CIMP) and genetic (BRAF mutation) events and the temporal sequence in which these two events take place have attracted interest [37]." . . _:b6672442 "One caveat of our system is that BRAFV600E could play a role in inducing DNA methylation only early in colorectal tumorigenesis, as BRAF mutations have been described at the earliest stage of tumor development [>>10<<], [38], [39]." . . . _:b421483722 . _:b421483712 . _:b421483762 . _:b6672426 . _:b6672459 "Moreover, they observed loss of IGFBP7 in primary BRAFV600E-positive melanoma samples and concluded that silencing of IGFBP7 expression is a critical step in the development of a melanoma harboring BRAFV600E [>>34<<]." . _:b6672433 _:b6672448 . _:b6672433 _:b6672449 . . _:b421483723 . . _:b6672433 _:b6672450 . _:b6672433 _:b6672451 . _:b6672433 _:b6672452 . _:b6672433 _:b6672453 . _:b6672372 "Although this concept has been controversial [4], we have confirmed the existence of CIMP in colorectal cancer in a large-scale comprehensive study [>>5<<]." . _:b6672433 _:b6672454 . _:b6672433 _:b6672455 . _:b421483681 . _:b6672433 _:b6672456 . . _:b6672367 . _:b6672433 _:b6672457 . _:b6672433 _:b6672458 . _:b6672433 _:b6672459 . _:b6672433 _:b6672460 . _:b6672433 _:b6672461 . _:b6672433 _:b6672462 . . _:b6672433 _:b6672463 . _:b6672433 _:b6672464 . _:b421483745 . . _:b6672433 _:b6672465 . _:b421483768 . _:b421483744 . . _:b6672433 _:b6672466 . _:b6672433 _:b6672467 . _:b6672433 _:b6672468 . . _:b6672372 . _:b6672435 "CIMP in colorectal cancer provides a unique opportunity to study molecular mechanisms that lead to epigenetic changes in cancer and the contributions of these changes in the development of the disease [3], [>>37<<]. The distinct features found in CIMP are important clues in understanding this phenotype [3], [5], [7], [8]." . . _:b6672434 "CIMP in colorectal cancer provides a unique opportunity to study molecular mechanisms that lead to epigenetic changes in cancer and the contributions of these changes in the development of the disease [>>3<<], [37]. The distinct features found in CIMP are important clues in understanding this phenotype [3], [5], [7], [8]." . _:b6672373 . _:b421483734 . _:b421483743 . _:b6672399 . . _:b6672374 . _:b421483673 . . _:b6672375 . _:b6672388 "However, recent studies have indicated that mutations of these genes might play distinct roles in tumor initiation and/or maintenance [10], [>>14<<]." . . _:b6672412 _:b6672413 . _:b6672399 "Genomic DNA was treated with sodium bisulfite and subsequently analyzed by MethyLight as previously described [5], [>>52<<]. The primer and probe sequences for the MethyLight reactions were described previously [5]." . _:b6672412 _:b6672414 . _:b6672412 _:b6672415 . _:b6672439 "The distinct features found in CIMP are important clues in understanding this phenotype [3], [5], [7], [>>8<<]. Particularly striking is the extremely tight association between CIMP and BRAFV600E [5]. Mechanisms linking epigenetic (CIMP) and genetic (BRAF mutation) events and the temporal sequence in which these two events take place have" . _:b6672368 . _:b6672417 "We previously identified the CIMP status and BRAF mutation status of 235 primary colorectal tumor samples [>>5<<]. We found BRAFV600E in 33 tumors (14.0%); 31 of these were classified as CIMP+ and only 2 as CIMP\u2212. We performed the Illumina GoldenGate DNA methylation assay on these samples, and identified 60 genes, represented by 89 CpG sites, that" . . _:b6672369 . _:b421483701 . _:b6672421 "Moreover, we found an association of SLC5A8 and TIMP3 DNA methylation with BRAFV600E in our colorectal tumor samples, as had been previously reported in papillary thyroid carcinomas [>>28<<]. The functional consequence of DNA hypermethylation of such tumor suppressor genes linked with CIMP+ and BRAFV600E remains speculative [25]\u2013[28]." . _:b6672451 "It is interesting to note that a selection drug in cultured cells has been described to result in changes in global chromatin structure [>>42<<], and a similar process may be associated with our observations here." . . _:b6672370 . . _:b6672371 . . . _:b6672380 . . _:b421483741 . _:b6672381 . . . _:b6672382 . . _:b6672412 _:b6672432 . . _:b6672383 . . _:b6672376 . _:b6672377 . _:b6672412 _:b6672420 . _:b6672429 "The underlying molecular mechanism of BRAFV600E-induced senescence and apoptosis has been elucidated in a recent study [>>34<<]. It has been demonstrated that expression of IGFBP7 is both necessary and sufficient to induce senescence and apoptosis mediated by BRAFV600E. Intriguingly, IGFBP7 was shown to be epigenetically silenced by CpG island promoter" . _:b6672412 _:b6672421 . _:b6672412 _:b6672422 . _:b6672412 _:b6672423 . _:b6672378 . _:b6672412 _:b6672416 . _:b6672412 _:b6672417 . _:b6672412 _:b6672418 . _:b6672412 _:b6672419 . _:b6672379 . _:b6672412 _:b6672428 . _:b6672468 "Notably, it appeared that the expression of SMO was silenced in colorectal cancer cell lines harboring BRAFV600E [>>29<<]. Our data in primary colorectal tumors indicate that the CIMP-specific promoter DNA hypermethylation may result in a different effect of the Hedgehog (Hh) signaling pathway on colorectal tumorigenesis (Table S3)." . _:b6672412 _:b6672429 . _:b6672412 _:b6672430 . _:b421483778 . _:b6672412 _:b6672431 . _:b6672412 _:b6672424 . _:b6672412 _:b6672425 . _:b6672418 "We confirmed the recently observed associations between DNA hypermethylation of BMP3 and MCC with CIMP+ and BRAFV600E in colorectal cancer [>>25<<], [26]. We also found CIMP-specific DNA hypermethylation of BMP6." . _:b6672412 _:b6672426 . _:b6672412 _:b6672427 . _:b421483663 . . _:b6672395 _:b6672396 . _:b6672395 _:b6672397 . _:b6672395 _:b6672398 . _:b6672395 _:b6672399 . . _:b6672389 "The GoldenGate DNA methylation assay has been widely used in various studies and is now a standard method for DNA methylation analysis [>>15<<]\u2013[24]. Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [15]\u2013[17], [22], [23], making it a reliable source for DNA methylation" . . "10.1371%2Fjournal.pone.0008357" . _:b6672395 _:b6672408 . _:b6672395 _:b6672409 . . _:b6672395 _:b6672410 . . _:b6672395 _:b6672411 . . . . _:b6672460 "DNA methylation inhibitor 5-aza-2\u2032-deoxycytidine has been shown to restore IGFBP7 expression in colorectal cancer cell lines, indicating that the DNA hypermethylation plays a major role in silencing of this gene in colorectal cancer [>>43<<]. However, its association with BRAF mutation and CIMP+ status in human colorectal cancers has not been explored." . _:b6672395 _:b6672400 . _:b6672395 _:b6672401 . . _:b421483727 . _:b6672395 _:b6672402 . _:b6672395 _:b6672403 . _:b421483670 . _:b6672395 _:b6672404 . _:b6672395 _:b6672405 . _:b6672395 _:b6672406 . _:b6672395 _:b6672407 . _:b6672386 "Mutations in KRAS and BRAF are generally mutually exclusive, implying equivalent downstream effects in tumorigenesis [>>13<<]. However, recent studies have indicated that mutations of these genes might play distinct roles in tumor initiation and/or maintenance [10], [14]." . _:b6672432 . . . _:b6672434 . . . . _:b6672382 "Both CIMP and BRAF mutations have been reported in the earliest stages of colorectal neoplasia: CIMP in apparently normal mucosa of patients predisposed to multiple serrated polyps [>>9<<] and BRAF mutations in aberrant crypt foci [10]." . . _:b6672448 . _:b421483771 . . . _:b6672373 "CIMP in colorectal cancer may arise through a distinct pathway originating in certain subtypes of serrated polyps [>>6<<] and is observed in approximately 15% of all colorectal cancer cases [5], [7]." . _:b6672402 . _:b6672436 . _:b6672393 "Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [15]\u2013[17], [>>22<<], [23], making it a reliable source for DNA methylation measurements across 1,505 loci." . . . _:b6672439 . _:b421483781 . _:b6672398 . _:b421483780 . _:b421483666 . _:b421483807 . . _:b6672446 "Minoo et al. reported MLH1 DNA methylation upon stable transfection of BRAFV600E into the NCM460 cell line [>>40<<]. In our system, we did not detect such an increase in MLH1 DNA methylation (data not shown). Moreover, of the 33 BRAFV600E primary tumors we examined only 42% (14/33) showed MLH1 DNA hypermethylation. Therefore, BRAFV600E may affect DNA" . _:b6672381 "Furthermore, we found a strong association of CIMP with the presence of an activated mutant form of BRAF (BRAFV600E) [>>5<<]. Both CIMP and BRAF mutations have been reported in the earliest stages of colorectal neoplasia: CIMP in apparently normal mucosa of patients predisposed to multiple serrated polyps [9] and BRAF mutations in aberrant crypt foci [10]." . _:b421483783 . _:b6672396 . . _:b421483782 . _:b421483767 . _:b421483777 . _:b421483721 . _:b6672464 . _:b421483776 . _:b6672445 "Previously, we described CIMP-associated methylation of MLH1 as the underlying basis for mismatch repair deficiency (MSI+) in sporadic colorectal cancer [>>5<<]. Minoo et al. reported MLH1 DNA methylation upon stable transfection of BRAFV600E into the NCM460 cell line [40]." . . _:b6672401 . _:b421483779 . _:b421483761 . _:b6672400 . _:b421483736 . _:b421483802 . _:b421483778 . . _:b421483775 . _:b6672407 . _:b421483789 . _:b6672406 . _:b6672455 "BRAFV600E has been shown to induce cellular senescence in cultured and primary human cells [30], [>>31<<], as well as mouse model [32]." . _:b421483788 . _:b421483793 . _:b6672383 . _:b6672405 . _:b421483791 . _:b421483677 . _:b421483749 . _:b6672404 . _:b421483790 . _:b6672386 . . _:b421483785 . _:b421483777 . _:b6672391 "Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [>>15<<]\u2013[17], [22], [23], making it a reliable source for DNA methylation measurements across 1,505 loci." . _:b421483691 . _:b421483790 . _:b421483784 . _:b6672428 "Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [>>33<<]. The underlying molecular mechanism of BRAFV600E-induced senescence and apoptosis has been elucidated in a recent study [34]. It has been demonstrated that expression of IGFBP7 is both necessary and sufficient to induce senescence and" . . _:b6672466 "Somatic mutations or overexpression of these genes has been implicated in colorectal tumorigenesis, which may involve the activation of the RAS-RAF-MEK-ERK signaling [45]\u2013[>>50<<]. The potential inactivation of these genes in CIMP may lead to the development of tumors dependent on oncogenic BRAF-driven hyperactivation of the RAS-RAF-MEK-ERK signaling pathway. Furthermore, we also found that DNA methylation of SMO" . _:b6672456 "BRAFV600E has been shown to induce cellular senescence in cultured and primary human cells [30], [31], as well as mouse model [>>32<<]. Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [33]. In order for BRAFV600E to promote its oncogenic effects, additional cooperative events are required to bypass senescence [33]." . _:b421483685 . _:b421483787 . _:b6672408 . . _:b421483783 . _:b421483786 . _:b421483764 . _:b6672415 . _:b421483730 . _:b421483756 . _:b421483797 . . _:b421483796 . . . _:b6672413 . _:b421483799 . _:b6672400 "The primer and probe sequences for the MethyLight reactions were described previously [>>5<<]. The results of MethyLight analyses were scored as PMR (Percent of Methylated Reference) values as previously defined [5]." . . _:b6672379 "Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [3], [5], [7], [>>8<<]. Our study using a newly developed CIMP marker panel in colorectal cancers demonstrated that sporadic microsatellite instability (MSI+) occurs as a consequence of CIMP-associated MLH1 DNA hypermethylation [5]. Furthermore, we found a" . _:b421483800 . _:b421483773 . _:b6672450 "the DNA hypermethylation of CDKN2A and 15 other CIMP-associated markers (IGFBP7 was not examined) in parent NCM460 cells, which limited their ability to study further the role of BRAFV600E inducing CIMP in their experimental system [>>40<<]." . _:b421483798 . _:b6672379 . _:b421483793 . . . _:b421483792 . . . _:b6672417 . _:b421483795 . _:b421483699 . _:b6672416 . _:b421483794 . _:b421483805 . _:b6672466 . . . _:b421483804 . . . _:b6672420 "The simultaneous epigenetic inactivation of BMP3 and BMP6 was shown to be associated with the activation of the RAS-RAF-MEK-ERK signaling pathway in non-small-cell lung cancer [>>27<<]. Moreover, we found an association of SLC5A8 and TIMP3 DNA methylation with BRAFV600E in our colorectal tumor samples, as had been previously reported in papillary thyroid carcinomas [28]. The functional consequence of DNA" . _:b421483807 . . _:b421483732 . _:b6672427 "BRAFV600E has been shown to induce cellular senescence [30]\u2013[>>32<<]. Oncogene-induced senescence (OIS) has been recognized as an important tumor suppressor mechanism [33]." . _:b421483806 . . _:b6672410 . . _:b421483719 . _:b421483801 . . _:b6672367 _:b6672376 . _:b421483750 . _:b421483689 . _:b6672367 _:b6672377 . _:b6672367 _:b6672378 . _:b421483800 . _:b421483733 . _:b6672367 _:b6672379 . _:b6672367 _:b6672380 . _:b6672367 _:b6672381 . _:b6672367 _:b6672382 . _:b421483803 . . _:b421483672 . _:b6672367 _:b6672383 . . _:b6672367 _:b6672368 . _:b6672367 _:b6672369 . . _:b421483802 . _:b6672367 _:b6672370 . _:b6672367 _:b6672371 . _:b6672367 _:b6672372 . _:b6672367 _:b6672373 . _:b6672367 _:b6672374 . _:b6672367 _:b6672375 . _:b6672462 . . _:b421483812 . . _:b6672425 "However, Arimura et al. also showed that colorectal cancer cell lines harboring BRAFV600E, including COLO 205, HT-29 and RKO, did not appear to show expression of SMO [>>29<<]. Our data indicates that CIMP-specific promoter DNA hypermethylation might be involved in the repression of SMO in colorectal tumors carrying BRAFV600E (Table S3)." . _:b6672467 "It has been demonstrated that elevated expression of SMO might contribute to colorectal tumorigenesis through activation of the Wnt signaling pathway in a mouse model and colorectal cancer cell lines [>>29<<]. Notably, it appeared that the expression of SMO was silenced in colorectal cancer cell lines harboring BRAFV600E [29]. Our data in primary colorectal tumors indicate that the CIMP-specific promoter DNA hypermethylation may result in a" . . _:b421483809 . _:b421483808 . _:b421483720 . _:b421483811 . _:b6672444 "One caveat of our system is that BRAFV600E could play a role in inducing DNA methylation only early in colorectal tumorigenesis, as BRAF mutations have been described at the earliest stage of tumor development [10], [38], [>>39<<]. It is possible that a unique set of genetic and/or epigenetic changes that occurred in COLO 320DM cells might have created an unfavorable environment for BRAFV600E to induce DNA hypermethylation. Experiments similar to those described" . _:b421483686 . _:b421483810 . . . _:b421483680 . _:b421483779 . _:b421483725 . _:b6672437 . _:b421483674 . . _:b6672435 . _:b6672403 "The MSI status of each cell line was based on the Sanger Institute Cancer Genome Project (http://www.sanger.ac.uk/) and based on a previous study [>>53<<]." . . . _:b6672385 "KRAS mutations occur most frequently in 30\u201340% of all colorectal cancers [11] and BRAF mutations are present at a frequency of 5\u201322%, in which the constitutively activated BRAFV600E variant accounts for \u223C90% of all the BRAF mutations [>>12<<]. Mutations in KRAS and BRAF are generally mutually exclusive, implying equivalent downstream effects in tumorigenesis [13]." . _:b6672440 . _:b6672456 . _:b6672463 . _:b6672454 . _:b6672441 . _:b6672416 "for this discrepancy could be that these reactions are designed around the transcription start site of RUNX3 isoform 1, whereas our CIMP-specific RUNX3 MethyLight reaction is designed at the promoter CpG island of the RUNX3 isoform 2 [>>5<<]. We saw no apparent difference in DNA methylation between BRAFV600E transfected clones and EVCs at these CIMP-associated CpG sites (Figure 3)." . _:b421483718 . . _:b6672445 . _:b421483774 . _:b421483715 . . . . . _:b6672380 "Our study using a newly developed CIMP marker panel in colorectal cancers demonstrated that sporadic microsatellite instability (MSI+) occurs as a consequence of CIMP-associated MLH1 DNA hypermethylation [>>5<<]. Furthermore, we found a strong association of CIMP with the presence of an activated mutant form of BRAF (BRAFV600E) [5]. Both CIMP and BRAF mutations have been reported in the earliest stages of colorectal neoplasia: CIMP in apparently" . _:b6672401 "The results of MethyLight analyses were scored as PMR (Percent of Methylated Reference) values as previously defined [>>5<<]." . _:b6672469 _:b6672470 . _:b421483702 . . _:b421483810 . _:b421483665 . . . _:b421483791 . . . . . _:b421483757 . . _:b421483760 . . . . _:b6672408 "The KRAS mutation status of 14 tumor samples was not available. BRAF mutations and KRAS mutations were mutually exclusive [>>5<<]." . _:b421483706 . . . _:b6672421 . _:b421483695 . _:b421483704 . _:b6672423 . . _:b421483663 "13"^^ . _:b6672410 "We selected 100 CpG sites with P<0.001 after a correction for multiple-comparison [>>45<<] and mean |\u0394\u03B2|>0.17, the estimated error in \u03B2 [16]." . . _:b6672451 . _:b421483797 . . _:b6672392 "Findings obtained from the commercially available \u201CGoldenGate Methylation Cancer Panel I\u201D, in particular, have been validated using various other techniques [15]\u2013[>>17<<], [22], [23], making it a reliable source for DNA methylation measurements across 1,505 loci." . _:b421483746 . _:b421483748 . _:b421483729 . _:b6672465 "Somatic mutations or overexpression of these genes has been implicated in colorectal tumorigenesis, which may involve the activation of the RAS-RAF-MEK-ERK signaling [>>45<<]\u2013[50]. The potential inactivation of these genes in CIMP may lead to the development of tumors dependent on oncogenic BRAF-driven hyperactivation of the RAS-RAF-MEK-ERK signaling pathway. Furthermore, we also found that DNA methylation of" . _:b6672414 . . . _:b6672398 "Genomic DNA was treated with sodium bisulfite and subsequently analyzed by MethyLight as previously described [>>5<<], [52]. The primer and probe sequences for the MethyLight reactions were described previously [5]." . . . _:b421483669 "5"^^ . _:b421483668 "5"^^ . _:b6672373 . _:b421483682 . _:b6672412 "results" . _:b421483671 "4"^^ . _:b6672470 "We performed the Illumina GoldenGate DNA methylation assay on 235 primary colorectal tumor samples, whose CIMP status and BRAF mutation status have been determined previously [>>5<<]. We dichotomized the DNA methylation \u03B2-value (methylated or unmethylated) for each locus. The dichotomization threshold was chosen for each locus using the mean \u03B2-value + 3SD (standard deviations) from ten normal mucosal samples. The" . . _:b6672411 . _:b421483670 "4"^^ . _:b421483707 . _:b6672397 "Stably expressing clones were maintained in 500 \u00B5g/ml of G418. Genomic DNA from each clone was isolated as previously described [>>51<<]." . . _:b6672409 . _:b6672376 "Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [>>3<<], [5], [7], [8]." . _:b421483667 "5"^^ . _:b6672431 . _:b421483676 . _:b421483664 "10"^^ . _:b421483687 . _:b421483665 "7"^^ . _:b421483666 "5"^^ . _:b421483677 "4"^^ . . _:b421483724 . _:b421483676 "4"^^ . . _:b421483698 . _:b421483679 "4"^^ . _:b6672397 . _:b6672389 . . _:b421483678 "4"^^ . _:b421483785 . _:b6672370 . . . _:b421483688 . _:b421483673 "4"^^ . _:b421483806 . _:b6672391 . _:b421483672 "4"^^ . _:b421483754 . _:b421483675 "4"^^ . _:b6672420 . _:b421483784 . . _:b421483674 "4"^^ . _:b421483781 . _:b6672468 . _:b421483685 "4"^^ . _:b421483703 . . _:b421483684 "4"^^ . _:b6672396 "All patients provided written informed consent for the collection of samples and subsequent analysis. DNA from these patients was also analyzed in a previous publication [>>5<<]." . _:b421483687 "4"^^ . _:b6672414 "The DNA methylation data were scored as \u03B2-values as previously defined [>>16<<]. Each row corresponds to an individual CpG locus and the data were sorted by average \u03B2-value across all samples." . _:b6672467 . _:b421483686 "4"^^ . . _:b421483681 "4"^^ . . _:b6672409 "Illumina GoldenGate DNA methylation analyses were performed as previously described [>>16<<] at the USC Epigenome Center Core Facility." . _:b6672388 . _:b6672367 . _:b421483680 "4"^^ . _:b6672376 . _:b6672389 . _:b421483683 "4"^^ . _:b6672454 "BRAFV600E has been shown to induce cellular senescence in cultured and primary human cells [>>30<<], [31], as well as mouse model [32]." . . _:b6672390 . _:b421483682 "4"^^ . _:b6672391 . _:b421483693 "3"^^ . _:b421483663 . _:b6672384 . _:b421483692 "4"^^ . _:b6672441 "Mechanisms linking epigenetic (CIMP) and genetic (BRAF mutation) events and the temporal sequence in which these two events take place have attracted interest [>>37<<]." . _:b6672415 "The mutation status of BRAF and KRAS in these samples had been determined previously [>>5<<] (Table S1)." . _:b6672385 . _:b421483695 "3"^^ . _:b6672461 . _:b6672465 . _:b6672386 . _:b421483694 "3"^^ . _:b6672460 . . _:b6672387 . _:b6672377 "Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [3], [>>5<<], [7], [8]." . _:b421483689 "4"^^ . . . _:b6672396 . _:b6672378 "Features associated with CIMP in colorectal cancer include gender (female), proximal location, and poorly differentiated or mucinous histology [3], [5], [>>7<<], [8]. Our study using a newly developed CIMP marker panel in colorectal cancers demonstrated that sporadic microsatellite instability (MSI+) occurs as a consequence of CIMP-associated MLH1 DNA hypermethylation [5]. Furthermore, we found a" . . _:b421483731 . _:b421483688 "4"^^ . _:b6672423 "The functional consequence of DNA hypermethylation of such tumor suppressor genes linked with CIMP+ and BRAFV600E remains speculative [25]\u2013[>>28<<]." . _:b6672397 . _:b421483691 "4"^^ . _:b6672431 "These tumors have been described as CIMP-low or CIMP2 [>>35<<], [36]. We did not find an association between IGFBP7 DNA hypermethylation and KRAS mutations when we excluded tumors with mutant BRAF (P value \u200A=\u200A0.85)." . _:b421483669 . _:b6672463 "Disruption of the BMP signaling pathway has been proposed to play a role in colorectal tumorigenesis [>>25<<]. Concurrent epigenetic inactivation of BMP3 and BMP6 was shown to be associated with the hyperactivation of the RAS-RAF-MEK-ERK signaling pathway in non-small-cell lung cancer [27]. Furthermore, receptor tyrosine kinases (RTKs) such as" . _:b6672398 . _:b421483690 "4"^^ . _:b421483668 . _:b6672399 . _:b421483701 "3"^^ . _:b6672406 "A 235 sample set included the same 48 randomly selected samples described above along with an additional 187 randomly collected tumors described previously [>>5<<]. CIMP status and mutation status of BRAF and KRAS for each tumor sample was previously determined [5]. BRAF mutations and KRAS mutations were found in 15% (33/235) and 33% (74/221) of the colorectal tumor samples, respectively. The KRAS" . _:b421483717 . _:b421483671 . _:b6672392 . _:b421483700 "3"^^ . _:b421483678 . _:b421483726 . _:b421483670 . _:b6672393 . _:b421483703 "3"^^ . _:b6672367 "introduction" . _:b421483665 . . _:b6672394 . . _:b421483702 "3"^^ . . _:b421483664 . _:b6672395 . _:b421483811 . _:b421483697 "3"^^ . _:b421483667 . _:b421483738 . _:b6672404 . _:b421483696 "3"^^ . _:b6672388 . _:b421483666 . _:b421483776 . . . _:b6672464 "Concurrent epigenetic inactivation of BMP3 and BMP6 was shown to be associated with the hyperactivation of the RAS-RAF-MEK-ERK signaling pathway in non-small-cell lung cancer [>>27<<]. Furthermore, receptor tyrosine kinases (RTKs) such as EPHA3, KIT, and FLT1 also showed CIMP-associated DNA hypermethylation (Table S3). Somatic mutations or overexpression of these genes has been implicated in colorectal tumorigenesis," . _:b6672405 . _:b421483699 "3"^^ . _:b421483677 . _:b421483803 . . _:b6672406 . _:b421483698 "3"^^ . _:b421483676 . _:b421483684 . _:b6672407 . _:b421483709 "3"^^ . _:b6672452 . _:b421483679 . _:b6672400 . _:b421483708 "3"^^ . _:b421483678 . _:b6672401 . _:b421483711 "3"^^ . _:b421483673 . _:b6672402 . _:b421483710 "3"^^ . _:b421483714 . _:b421483696 . _:b421483672 . _:b6672403 . _:b421483705 "3"^^ . _:b421483675 . _:b6672371 . _:b6672424 "Intriguingly, it has been recently reported that increased expression of SMO contributes to colorectal tumorigenesis [>>29<<]. However, Arimura et al. also showed that colorectal cancer cell lines harboring BRAFV600E, including COLO 205, HT-29 and RKO, did not appear to show expression of SMO [29]. Our data indicates that CIMP-specific promoter DNA" . _:b6672412 . _:b6672371 "Although this concept has been controversial [>>4<<], we have confirmed the existence of CIMP in colorectal cancer in a large-scale comprehensive study [5]." . _:b421483704 "3"^^ . _:b421483674 . . _:b6672413 . _:b421483707 "3"^^ . . _:b6672383 "Both CIMP and BRAF mutations have been reported in the earliest stages of colorectal neoplasia: CIMP in apparently normal mucosa of patients predisposed to multiple serrated polyps [9] and BRAF mutations in aberrant crypt foci [>>10<<]." . _:b421483685 . _:b6672414 . _:b421483706 "3"^^ . _:b421483684 . _:b6672415 . _:b421483717 "3"^^ . _:b421483747 . . _:b6672384 . _:b421483687 . _:b421483708 . _:b6672430 . _:b6672408 . _:b421483716 "3"^^ . . _:b421483686 . . _:b6672409 . _:b421483719 "3"^^ . _:b421483681 . _:b6672410 . _:b421483718 "3"^^ . _:b421483700 . . . _:b421483680 . _:b6672411 . _:b6672429 . _:b421483713 "3"^^ . _:b421483683 . . _:b6672420 . _:b421483712 "3"^^ . _:b6672368 "Promoter CpG island hypermethylation associated with inactivation of selected tumor suppressor genes appears to be critical in tumors from inception through to maintenance of the tumor phenotype [>>1<<]. Distinct subgroups of several types of human cancers have been proposed to have a CpG island methylator phenotype (CIMP) in which an exceptionally high frequency of cancer-specific DNA hypermethylation is found [2], [3]. Although this" . _:b421483682 . _:b6672421 . _:b421483715 "3"^^ . . . _:b421483693 . _:b6672422 . _:b421483714 "3"^^ . _:b6672404 "Primary colorectal tissue samples were collected and DNA was extracted as previously described [>>5<<]. A 58 sample set included five CIMP+ tumors, five CIMP\u2013 tumors and 48 randomly selected tumors, as indicated previously [5]." . . _:b6672384 "KRAS mutations occur most frequently in 30\u201340% of all colorectal cancers [>>11<<] and BRAF mutations are present at a frequency of 5\u201322%, in which the constitutively activated BRAFV600E variant accounts for \u223C90% of all the BRAF mutations [12]." . _:b421483692 . _:b421483725 "2"^^ . _:b6672423 . _:b421483695 . _:b421483724 "2"^^ . _:b6672416 . _:b421483795 . _:b421483694 . . _:b6672447 . _:b6672417 . _:b421483727 "2"^^ . _:b6672369 . _:b421483689 . _:b421483726 "2"^^ . _:b6672418 . . . _:b421483688 . _:b6672419 . _:b421483721 "3"^^ . _:b421483759 . . _:b421483786 . _:b421483691 . _:b6672428 . _:b421483720 "3"^^ . _:b421483690 . _:b6672461 "They did not find mutations associated with inactivation of IGFBP7 in their cell lines [>>43<<]. However, an increasing number of genes have recently been reported to be involved in OIS, and cooperation of multiple different signals appears to be critical for OIS [44]. It is therefore possible that CIMP-associated DNA" . _:b421483723 "2"^^ . _:b6672429 . _:b421483693 . _:b421483701 . _:b421483722 "2"^^ . _:b6672430 . . _:b6672402 "Primer sequences and PCR conditions for direct sequencing of BRAF at codon 600 in exon 15 and at codons 12 and 13 of KRAS in exon 2 were reported previously [>>13<<]. The MSI status of each cell line was based on the Sanger Institute Cancer Genome Project (http:" . _:b421483700 . _:b421483733 "2"^^ . _:b6672431 . . _:b421483766 . _:b421483703 . _:b421483732 "2"^^ . _:b6672424 . _:b421483808 . _:b421483809 . _:b421483810 . _:b421483811 . _:b421483702 . . . _:b421483735 "2"^^ . _:b6672425 . _:b421483812 . _:b6672367 _:b6672392 . _:b421483697 . _:b6672367 _:b6672393 . _:b6672426 . _:b421483734 "2"^^ . _:b6672367 _:b6672394 . .