Takeshi Nagasaka

Publications of Takeshi Nagasaka

• Characteristic pattern of DNA methylation alterations predict emergence of human hepatocellular carcinoma.

  Authors: Naoshi Nishida, Masatoshi Kudo, Takeshi Nagasaka, Iwao Ikai, Ajay Goel

  Hepatology (Baltimore, Md.). 03/2012;

  We aimed to identify the specific subset of tumor suppressor genes (TSGs) that are methylation-silenced during the earliest steps of hepatocarcinogenesis, and to further evaluate whether these genesWe aimed to identify the specific subset of tumor suppressor genes (TSGs) that are methylation-silenced during the earliest steps of hepatocarcinogenesis, and to further evaluate whether these genes can serve as predictive biomarkers of HCC emergence. A total of 482 liver tissues including 177 pairs of HCCs and matched non-tumor livers and 128 liver biopsies from chronic hepatitis C (CHC) patients were analyzed for quantitative methylation analysis in 24 tumor suppressor gene (TSG) promoters and 3 MINT loci. The tumors were classified as early, less-progressed, and highly-progressed HCCs using histology and radiological approaches. A subset of TSGs, which harbored distinctly high levels of methylation in early HCCs were selected. Based upon the methylation profiles of these genes, Kaplan-Meier analyses were performed to determine time-to-HCC occurrence in CHC patients. Subsequently, multivariate analysis was performed using age, gender, fibrosis stage and number of methylated TSGs as covariates. Among TSGs analyzed, a subset of 8 TSGs (HIC1, GSTP1, SOCS1, RASSF1, CDKN2A, APC, RUNX3 and PRDM2) demonstrated a distinct cluster by hierarchical clustering and receiver operating characteristic analyses. This subset of TSGs showed significantly higher methylation levels in the early HCCs (p < 0.0001). In the CHC patients, methylation frequencies in these TSGs were associated with shorter time-to-HCC occurrence (p < 0.0001), and number of methylated genes was an independent risk factor for HCC (hazard ratio = 5.21, 95% CI = 2.25 - 11.76, p = 0.0002). CONCLUSION: Epigenetic inactivation of a subset of TSGs plays a critical role in the earliest steps of hepatocarcinogenesis. Furthermore, epigenetic inactivation of these genes in CHC provides a prognostic value for determining the risk for developing HCC later in life. (HEPATOLOGY 2012.).

• Branched-chain amino acid-enriched nutrients improve nutritional and metabolic abnormalities in the early post-transplant period after living donor liver transplantation.

  Authors: Ryuichi Yoshida, Takahito Yagi, Hiroshi Sadamori, Hiroaki Matsuda, Susumu Shinoura, Yuzo Umeda, Daisuke Sato, Masashi Utsumi, Takeshi Nagasaka, Nami Okazaki, Ai Date, Ayako Noguchi, Akemi Tanaka, Yuko Hasegawa, Yachiyo Sakamoto, Toshiyoshi Fujiwara

  Journal of hepato-biliary-pancreatic sciences. 09/2011;

  BACKGROUND/PURPOSE: Malnutrition and metabolic disorder of patients undergoing living donor liver transplantation (LDLT) can affect post-transplant prognosis. The aim of this study was to establishBACKGROUND/PURPOSE: Malnutrition and metabolic disorder of patients undergoing living donor liver transplantation (LDLT) can affect post-transplant prognosis. The aim of this study was to establish whether perioperative usage of branched-chain amino-acid (BCAA)-enriched nutrients improve metabolic abnormalities of patients undergoing LDLT. METHODS: We designed a randomized pilot study (UMIN registration number; 000004323). Twenty-five consecutive adult elective LDLT recipients were enroled and divided into two groups: the BCAA group (BCAA-enriched nutrients, n = 12) and the control group (standard diet, n = 13). Metabolic and nutritional parameters, including BCAA-to-tyrosine ratio (BTR), retinol binding protein (RBP), and prealbumin were regularly measured from 1 week before to 4 weeks after LDLT. Non-protein respiratory quotient (npRQ) was measured before and 4 weeks after LDLT. RESULTS: BTR and RBP improved considerably in the BCAA group compared with the controls. npRQ significantly increased from 1 week before LDLT to 4 weeks after LDLT in the BCAA group (0.77 ± 0.05 to 0.84 ± 0.06, P = 0.002), but not in the control group (0.78 ± 0.04 to 0.81 ± 0.05). CONCLUSIONS: Supplementation with BCAA-enriched nutrients might improve persistent nutritional and metabolic disorders associated with end-stage liver disease in the early post-transplant period, and consequently shorten the post-transplant catabolic phase after LDLT. A larger multicenter trial is needed to confirm these findings.

• Expansion of CpG methylation in the SFRP2 promoter region during colorectal tumorigenesis.

  Authors: Masanori Takeda, Takeshi Nagasaka, Sun Dong-Sheng, Hiroyuki Nishie, Tetsuhiro Oka, Eiji Yamada, Yoshiko Mori, Kunitoshi Shigeyasu, Tatsuya Morikawa, Satoshi Mizobuchi, Toshiyoshi Fujiwara

  Acta medica Okayama. 06/2011; 65(3):169-77.

  Secreted frizzled-related protein 2, (SFRP2) is a Wnt inhibitor whose promoter CpGs were recently found to be methylated at high frequency in colorectal cancers (CRCs). We hypothesized that theSecreted frizzled-related protein 2, (SFRP2) is a Wnt inhibitor whose promoter CpGs were recently found to be methylated at high frequency in colorectal cancers (CRCs). We hypothesized that the pattern of SFRP2 methylation may differ throughout the promoter during progressive tumorigenesis. Using combined bisulfite restriction analysis (COBRA), two methylation-sensitive regions (Regions A and B) of the SFRP2 promoter were investigated in 569 specimens of colorectal tissue:222 CRCs, 103 adenomatous polyps (APs), 208 normal colonic mucosa from CRC patients (N-Cs), and 36 normal colonic mucosa from subjects with no evidence of colorectal neoplasia at colonoscopy (N-Ns). Extensive (including both Regions A and B) and partial (either Region A or B) SFRP2 methylation levels were found in 61.7% and 24.8% of CRCs, 8.7% and 37.9% of APs, 3.9% and 39.9% of N-Cs, and 0% and 30.6% of N-Ns, respectively. Extensive methylation of the SFRP2 promoter was present primarily in CRCs, while partial methylation was common in APs. Whereas APs with the KRAS mutant showed no correlation to any pattern of SFRP2 methylation, extensive methylation of the SFRP2 promoter was significantly associated with KRAS mutant CRCs (p＜.0001), suggesting that genetic alteration in the RAS-RAF pathway might precede the spread of CpG methylation through the SFRP2 promoter, which is observed in over 60% of advanced colorectal tumors.

• De novo constitutional MLH1 epimutations confer early-onset colorectal cancer in two new sporadic Lynch syndrome cases, with derivation of the epimutation on the paternal allele in one.

  Authors: Ajay Goel, Thuy-Phuong Nguyen, Hon-Chiu E Leung, Takeshi Nagasaka, Jennifer Rhees, Erin Hotchkiss, Mildred Arnold, Pia Banerji, Minoru Koi, Chau-To Kwok, Deborah Packham, Lara Lipton, C Richard Boland, Robyn L Ward, Megan P Hitchins

  International journal of cancer. Journal international du cancer. 02/2011; 128(4):869-78.

  Lynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations ofLynch syndrome is an autosomal dominant cancer predisposition syndrome classically caused by germline mutations of the mismatch repair genes, MLH1, MSH2, MSH6 and PMS2. Constitutional epimutations of the MLH1 gene, characterized by soma-wide methylation of a single allele of the promoter and allelic transcriptional silencing, have been identified in a subset of Lynch syndrome cases lacking a sequence mutation in MLH1. We report two individuals with no family history of colorectal cancer who developed that disease at age 18 and 20 years. In both cases, cancer had arisen because of the de novo occurrence of a constitutional MLH1 epimutation and somatic loss-of-heterozygosity of the functional allele in the tumors. We show for the first time that the epimutation in one case arose on the paternally inherited allele. Analysis of 13 tumors from seven individuals with constitutional MLH1 epimutations showed eight tumors had lost the second MLH1 allele, two tumors had a novel pathogenic missense mutation and three had retained heterozygosity. Only 1 of 12 tumors demonstrated the BRAF V600E mutation and 3 of 11 tumors harbored a mutation in KRAS. The finding that epimutations can originate on the paternal allele provides important new insights into the mechanism of origin of epimutations. It is clear that the second hit in MLH1 epimutation-associated tumors typically has a genetic not epigenetic basis. Individuals with mismatch repair-deficient cancers without the BRAF V600E mutation are candidates for germline screening for sequence or methylation changes in MLH1.

• Low frequency of Lynch syndrome among young patients with non-familial colorectal cancer.

  Authors: Ajay Goel, Takeshi Nagasaka, Jennifer Spiegel, Richard Meyer, Warren E Lichliter, C Richard Boland

  Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association. 11/2010; 8(11):966-71.

  Colorectal cancer (CRC) is uncommon in individuals <50 years old. Lynch syndrome is caused by germline mutations in DNA mismatch repair (MMR) genes and associated with early-onset CRC, but little isColorectal cancer (CRC) is uncommon in individuals <50 years old. Lynch syndrome is caused by germline mutations in DNA mismatch repair (MMR) genes and associated with early-onset CRC, but little is known about the proportion of young patients with apparently sporadic CRC who actually have Lynch syndrome. We examined patterns of microsatellite instability (MSI) and MMR genes among patients <50 years old with non-familial CRC (patients with not more than 1 family member with CRC). Tissue specimens were collected from 75 CRC patients <50 years old (mean age, 34.5 years) and analyzed using immunohistochemical analyses of MLH1, MSH2, MSH6, and PMS2. MSI and mutations in BRAF and KRAS were also analyzed. Most cancers (72%) arose in the distal colon. MSI was detected in 21% of the samples, and loss of 1 or more MMR proteins was observed in 21%. Interestingly, only 38% of the MMR-deficient CRCs lost either MLH1 or MSH2, whereas 63% of the MMR-deficient CRC samples lost either PMS2 or MSH6. All 11 CRC samples that had lost MSH2, MLH1, or PMS2 had MSI, but only 2 of the 5 tumors that lost only MSH6 had MSI. There were no BRAF mutations in any tumor. In young patients with apparently sporadic CRC, most tumors arise in the distal colon; only 21% have features of Lynch syndrome. Loss of MSH6 or PMS2 occurred in 13.3% of these tumors. Most tumors that lose MSH6 will not be detected in screens for MSI; CRC screening might be modified to identify more patients with Lynch syndrome.

• Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis.

  Authors: Francesc Balaguer, Alexander Link, Juan Jose Lozano, Miriam Cuatrecasas, Takeshi Nagasaka, C Richard Boland, Ajay Goel

  Cancer research. 08/2010; 70(16):6609-18.

  Global downregulation of microRNAs (miRNA) is a common feature in colorectal cancer (CRC). Whereas CpG island hypermethylation constitutes a mechanism for miRNA silencing, this field largely remainsGlobal downregulation of microRNAs (miRNA) is a common feature in colorectal cancer (CRC). Whereas CpG island hypermethylation constitutes a mechanism for miRNA silencing, this field largely remains unexplored. Herein, we describe the epigenetic regulation of miR-137 and its contribution to colorectal carcinogenesis. We determined the methylation status of miR-137 CpG island in a panel of six CRC cell lines and 409 colorectal tissues [21 normal colonic mucosa from healthy individuals (N-N), 160 primary CRC tissues and their corresponding normal mucosa (N-C), and 68 adenomas]. TaqMan reverse transcription-PCR and in situ hybridization were used to analyze miR-137 expression. In vitro functional analysis of miR-137 was performed. Gene targets of miR-137 were identified using a combination of bioinformatic and transcriptomic approaches. We experimentally validated the miRNA:mRNA interactions. Methylation of the miR-137 CpG island was a cancer-specific event and was frequently observed in CRC cell lines (100%), adenomas (82.3%), and CRC (81.4%), but not in N-C (14.4%; P < 0.0001 for CRC) and N-N (4.7%; P < 0.0001 for CRC). Expression of miR-137 was restricted to the colonocytes in normal mucosa and inversely correlated with the level of methylation. Transfection of miR-137 precursor in CRC cells significantly inhibited cell proliferation. Gene expression profiling after miR-137 transfection discovered novel potential mRNA targets. We validated the interaction between miR-137 and LSD-1. Our data indicate that miR-137 acts as a tumor suppressor in the colon and is frequently silenced by promoter hypermethylation. Methylation silencing of miR-137 in colorectal adenomas suggests it to be an early event, which has prognostic and therapeutic implications.

• Fecal MicroRNAs as novel biomarkers for colon cancer screening.

  Authors: Alexander Link, Francesc Balaguer, Yan Shen, Takeshi Nagasaka, Juan José Lozano, C Richard Boland, Ajay Goel

  Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 07/2010; 19(7):1766-74.

  Colorectal cancer (CRC) is the second leading cause of cancer-related deaths, but currently available noninvasive screening programs have achieved only a modest decrease in mortality. MicroRNAsColorectal cancer (CRC) is the second leading cause of cancer-related deaths, but currently available noninvasive screening programs have achieved only a modest decrease in mortality. MicroRNAs (miRNA) play an important role in a wide array of biological processes and are commonly dysregulated in neoplasia. We aimed to evaluate the feasibility of fecal miRNAs as biomarkers for colorectal neoplasia screening. Total RNA was extracted from freshly collected stool samples from 8 healthy volunteers and 29 samples collected via fecal occult blood testing from subjects with normal colonoscopies, colon adenomas, and CRCs. miRNA expression analyses were done with TaqMan quantitative reverse transcription-PCR for a subset of miRNAs. Illumina miRNA microarray profiling was done to evaluate the differences in expression patterns between normal colonic mucosa tissues and stool samples from healthy subjects. We efficiently extracted miRNAs from stool specimens using our developed protocol. Data from independent experiments showed high reproducibility for miRNA extraction and expression. miRNA expression patterns were similar in stool specimens among healthy volunteers, and reproducible in stool samples that were collected serially in time from the same individuals. miRNA expression profiles from 29 patients showed higher expression of miR-21 and miR-106a in patients with adenomas and CRCs compared with individuals free of colorectal neoplasia. Our data indicate that miRNAs could be extracted from stool easily and reproducibly. The stools of patients with colorectal neoplasms have unique and identifiable patterns of miRNA expression. Fecal miRNAs may be an excellent candidate for the development of a noninvasive screening test for colorectal neoplasms.

• Somatic hypermethylation of MSH2 is a frequent event in Lynch Syndrome colorectal cancers.

  Authors: Takeshi Nagasaka, Jennifer Rhees, Matthias Kloor, Johannes Gebert, Yoshio Naomoto, C Richard Boland, Ajay Goel

  Cancer research. 04/2010; 70(8):3098-108.

  Heritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. It is not known whether somatic MSH2 methylation occurs inHeritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. It is not known whether somatic MSH2 methylation occurs in MSH2 mutation-positive Lynch syndrome subjects or sporadic colorectal cancers (CRC). Therefore, we determined the methylation status of the MSH2 gene in 268 CRC tissues, including 222 sporadic CRCs and 46 Lynch syndrome tumors that did not express MSH2. We also looked for microsatellite instability (MSI), germline mutations in the MSH2 and EpCAM genes, somatic mutations in BRAF and KRAS, and the CpG island methylator phenotype (CIMP). We observed that somatic MSH2 hypermethylation was present in 24% (11 of 46) of MSH2-deficient (presumed Lynch syndrome) tumors, whereas no evidence for MSH2 methylation existed in sporadic CRCs (MSI and microsatellite stable) or normal colonic tissues. Seven of 11 (63%) patients with MSH2 methylation harbored simultaneous pathogenic germline mutations in the MSH2 gene. Germline EpCAM deletions were present in three of four patients with MSH2 methylation but without pathogenic MSH2 germline mutations. The mean methylation scores at CIMP-related markers were significantly higher in Lynch syndrome tumors with MSH2 methylation than MSH2-unmethylated CRCs. In conclusion, our data provide evidence for frequent MSH2 hypermethylation in Lynch syndrome tumors with MSH2 deficiency. MSH2 methylation in this subset of individuals is somatic and may serve as the "second hit" at the wild-type allele. High levels of aberrant methylation at CIMP-related markers in MSH2-methylated tumors raise the possibility that MSH2 is a target susceptible to aberrant methylation in Lynch syndrome.

• The establishment of a new mouse model with orthotopic esophageal cancer showing the esophageal stricture.

  Authors: Toshiaki Ohara, Munenori Takaoka, Kazufumi Sakurama, Kaori Nagaishi, Haruo Takeda, Yasuhiro Shirakawa, Tomoki Yamatsuji, Takeshi Nagasaka, Junji Matsuoka, Noriaki Tanaka, Yoshio Naomoto

  Cancer letters. 02/2010; 293(2):207-12.

  We established a promising new experimental animal model with an orthotopic xenograft of esophageal cancer that successfully represents poor oral intake, a major clinical feature of esophagealWe established a promising new experimental animal model with an orthotopic xenograft of esophageal cancer that successfully represents poor oral intake, a major clinical feature of esophageal cancer. The advantage of this model is that no surgical technique is required, only the injection of a cell suspension by a needle and syringe via the esophageal lumen from the mouth, which provides a high reproducibility of tumor implantation and a rapid progress of outcome. We propose that this model is useful to study cancer-related outcomes and for developing new therapies for esophageal cancer, and we expect it to make a contribution to clinical practice.

• Correction: an optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers.

  Authors: Ajay Goel, Takeshi Nagasaka, Richard Hamelin, C Richard Boland

  PloS one. 01/2010; 5(3).

  [This corrects the article on p. e9393 in vol. 5.].

• An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers.

  Authors: Ajay Goel, Takeshi Nagasaka, Richard Hamelin, C Richard Boland

  PloS one. 01/2010; 5(2):e9393.

  Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNAMicrosatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI. We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA. Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using > or = 2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1-98.1%) and a positive predictive value of 100% (95% CI = 96.6%-100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI. An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.

• Analysis of Fecal DNA Methylation to Detect Gastrointestinal Neoplasia.

  Authors: Takeshi Nagasaka, Noriaki Tanaka, Harry M Cullings, Dong-Sheng Sun, Hiromi Sasamoto, Takuyuki Uchida, Minoru Koi, Naoshi Nishida, Yoshio Naomoto, C Richard Boland, Nagahide Matsubara, Ajay Goel

  Journal of the National Cancer Institute. 08/2009;

  Background The development of noninvasive screening tests is important to reduce mortality from gastrointestinal neoplasia. We sought to develop such a test by analysis of DNA methylation fromBackground The development of noninvasive screening tests is important to reduce mortality from gastrointestinal neoplasia. We sought to develop such a test by analysis of DNA methylation from exfoliated cancer cells in feces. Methods We first analyzed methylation of the RASSF2 and SFRP2 gene promoters from 788 primary gastric and colorectal tissue specimens to determine whether methylation patterns could act as stage-dependent biomarkers of gastrointestinal tumorigenesis. Next, we developed a novel strategy that uses single-step modification of DNA with sodium bisulfite and fluorescence polymerase chain reaction methodology to measure aberrant methylation in fecal DNA. Methylation of the RASSF2 and SFRP2 promoters was analyzed in 296 fecal samples obtained from a variety of patients, including 21 with gastric tumors, 152 with colorectal tumors, and 10 with non-neoplastic or inflammatory lesions in the gastrointestinal lumen. Results Analysis of DNA from tissues showed presence of extensive methylation in both gene promoters exclusively in advanced gastric and colorectal tumors. The assay successfully identified one or more methylated markers in fecal DNA from 57.1% of patients with gastric cancer, 75.0% of patients with colorectal cancer, and 44.4% of patients with advanced colorectal adenomas, but only 10.6% of subjects without neoplastic or active diseases (difference, gastric cancer vs undiseased = 46.5%, 95% confidence interval (CI) = 24.6% to 68.4%, P < .001; difference, colorectal cancer vs undiseased = 64.4%, 95% CI = 53.5% to 75.2%, P < .001; difference, colorectal adenoma vs undiseased = 33.8%, 95% CI = 14.2% to 53.4%, P < .001). Conclusions Methylation of the RASSF2 and SFRP2 promoters in fecal DNA is associated with the presence of gastrointestinal tumors relative to non-neoplastic conditions. Our novel fecal DNA methylation assay provides a possible means to noninvasively screen not only for colorectal tumors but also for gastric tumors.

• Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

  Authors: Hitoshi Nakagawa, Takeshi Nagasaka, Harry M Cullings, Kenji Notohara, Naoko Hoshijima, Joanne Young, Henry T Lynch, Noriaki Tanaka, Nagahide Matsubara

  Oncology reports. 07/2009; 21(6):1577-83.

  It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes.It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

• JC Virus Mediates Invasion and Migration in Colorectal Metastasis.

  Authors: Alexander Link, Sung Kwan Shin, Takeshi Nagasaka, Francesc Balaguer, Minoru Koi, Barbara Jung, C Richard Boland, Ajay Goel

  PloS one. 01/2009; 4(12):e8146.

  INTRODUCTION: JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functionalINTRODUCTION: JC Virus (JCV), a human polyomavirus, is frequently present in colorectal cancers (CRCs). JCV large T-Ag (T-Ag) expressed in approximately half of all CRC's, however, its functional role in CRC is poorly understood. We hypothesized that JCV T-Ag may mediate metastasis in CRC cells through increased migration and invasion. MATERIAL AND METHODS: CRC cell lines (HCT116 and SW837) were stably transfected with JCV early transcript sequences cloned into pCR3 or empty vectors. Migration and invasion assays were performed using Boyden chambers. Global gene expression analysis was performed to identify genetic targets and pathways altered by T-Ag expression. Microarray results were validated by qRT-PCR, protein expression analyses and immunohistochemistry. Matching primary CRCs and liver metastases from 33 patients were analyzed for T-Ag expression by immunohistochemistry. RESULTS: T-Ag expressing cell lines showed 2 to 3-fold increase in migration and invasion compared to controls. JCV T-Ag expression resulted in differential expression of several genetic targets, including genes that mediate cell migration and invasion. Pathway analysis suggested a significant involvement of these genes with AKT and MAPK signaling. Treatment with selective PI3K/AKT and MAPK pathway inhibitors resulted in reduced migration and invasion. In support of our in-vitro results, immunohistochemical staining of the advanced stage tumors revealed frequent JCV T-Ag expression in metastatic primary tumors (92%) as well as in their matching liver metastasis (73%). CONCLUSION: These data suggest that JCV T-Ag expression in CRC associates with a metastatic phenotype, which may partly be mediated through the AKT/MAPK signaling pathway. Frequent expression of JCV T-Ag in CRC liver metastasis provides further clues supporting a mechanistic role for JCV as a possible mediator of cellular motility and invasion in CRC.

• Microsatellite instability, MLH1 promoter methylation, and BRAF mutation analysis in sporadic colorectal cancers of different ethnic groups in Israel.

  Authors: Alex Vilkin, Yaron Niv, Takeshi Nagasaka, Sarah Morgenstern, Zohar Levi, Zvi Fireman, Florentine Fuerst, Ajay Goel, C Richard Boland

  Cancer. 01/2009;

  BACKGROUND:: The molecular mechanisms that underlie colorectal cancer (CRC) include microsatellite instability (MSI), chromosomal instability, and the CpG island methylator phenotype. There isBACKGROUND:: The molecular mechanisms that underlie colorectal cancer (CRC) include microsatellite instability (MSI), chromosomal instability, and the CpG island methylator phenotype. There is evidence to suggest that CRC incidence varies among different ethnic populations worldwide. The authors of this report hypothesized that environmental factors and lifestyle differences among various ethnic groups may differentially influence the epigenetic regulation of tumor suppressor genes in CRC. METHODS:: In the current study, microdissection and DNA extraction were performed on 128 samples of CRC from Israeli patients (85 Jews and 43 Arabs). MSI analysis, mutL homolog 1 (MLH1) and mutS homolog 2 (MSH2) protein expression levels, and MLH1 promoter methylation were investigated by combined bisulfite restriction analysis. The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) valine-to-glutamic acid mutation at residue 600 was investigated by direct DNA sequencing. RESULTS:: High MSI (MSI-H), MLH1 methylation, and BRAF mutations were observed in 11.6%, 9.4%, and 23.5% of Jews, respectively, and in 16.2%, 17.6%, and 20.9% of Arabs, respectively (P value nonsignificant). MLH1 promoter methylation was observed in 22.6% of microsatellite-stable (MSS) tumors and in 53.8% of MSI-H tumors (P < .015). Extensive methylation (covering both 5' and 3' promoter regions) was present in all MSI-H tumors with loss of MLH1 expression. BRAF mutation was observed in 15.6% and 46.1% of MSS tumors and MSI-H tumors, respectively (P < .007). BRAF mutation was observed in 66%, 22.2%, and 14.7% of patients who had tumors with extensive MLH1 promoter methylation, methylation of the 5' region alone, or without methylation, respectively (P < .006). CONCLUSIONS:: There was no difference in molecular signatures examined between Jewish and Arab patients with CRC in Israel. Extensive promoter methylation was associated with MLH1 inactivation, MSI, and BRAF mutation. Cancer 2009. (c) 2009 American Cancer Society.

• Genetic instability caused by loss of MutS homologue 3 in human colorectal cancer.

  Authors: Astrid C Haugen, Ajay Goel, Kanae Yamada, Giancarlo Marra, Thuy-Phuong Nguyen, Takeshi Nagasaka, Shinsaku Kanazawa, Junichi Koike, Yoshinori Kikuchi, Xiaoling Zhong, Michitsune Arita, Kazutoshi Shibuya, Mitsuo Oshimura, Hiromichi Hemmi, C Richard Boland, Minoru Koi

  Cancer research. 11/2008; 68(20):8465-72.

  Microsatellite instability (MSI) is a hallmark of mismatch repair (MMR) deficiency. High levels of MSI at mononucleotide and dinucleotide repeats in colorectal cancer (CRC) are attributed toMicrosatellite instability (MSI) is a hallmark of mismatch repair (MMR) deficiency. High levels of MSI at mononucleotide and dinucleotide repeats in colorectal cancer (CRC) are attributed to inactivation of the MMR genes, hMLH1 and hMSH2. CRC with low levels of MSI (MSI-L) exists; however, its molecular basis is unclear. There is another type of MSI--elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)--where loci containing [AAAG](n) or [ATAG](n) repeats are unstable. EMAST is frequent in non-CRCs; however, the incidence of EMAST and its cause in CRC is not known. Here, we report that MutS homologue 3 (MSH3) knockdown or MSH3-deficient cells exhibit the EMAST phenotype and low levels of mutations at dinucleotide repeats. About 60% of 117 sporadic CRC cases exhibit EMAST. All of the cases defined as MSI-H (16 cases) exhibited high levels of EMAST. Among 101 non-MSI-H cases, all 19 cases of MSI-L and 35 of 82 cases of MSS exhibited EMAST. Although non-MSI-H CRC tissues contained MSH3-negative tumor cells ranging from 2% to 50% of the total tumor cell population, the tissues exhibiting EMAST contained more MSH3-negative cells (average, 31.5%) than did the tissues not exhibiting EMAST (8.4%). Taken together, our results support the concept that MSH3 deficiency causes EMAST or EMAST with low levels of MSI at loci with dinucleotide repeats in CRC.

• Molecular characteristics and predictors of survival in patients with malignant neuroendocrine tumors.

  Authors: Christian N Arnold, Takeshi Nagasaka, Ajay Goel, Iris Scharf, Patricia Grabowski, Andrea Sosnowski, Annette Schmitt-Gräff, C Richard Boland, Rudolf Arnold, Hubert E Blum

  International journal of cancer. Journal international du cancer. 10/2008; 123(7):1556-64.

  To better understand the molecular pathogenesis of neuroendocrine tumors (NET), we investigated the molecular and clinical characteristics of malignant poorly differentiated colorectal NET andTo better understand the molecular pathogenesis of neuroendocrine tumors (NET), we investigated the molecular and clinical characteristics of malignant poorly differentiated colorectal NET and compared these findings with sporadic CRC and well-differentiated benign and malignant fore-/midgut NET. Tumors were analyzed and correlated for microsatellite instability (MSI) and the CpG island methylator phenotype (CIMP). NET were scored for proliferation using Ki-67. A total of 34 malignant poorly differentiated colorectal NET, 38 well-differentiated benign and malignant fore-/midgut-NET and 150 sporadic colorectal cancers (CRC) with known MSI status were investigated. Among the sporadic CRC, CIMP was significantly correlated with MSI-high (MSI-H) (p < 0.001). Of the 34 colorectal NET, 0/1 of the MSI-H, 3/5 (60%) of the MSI-L and 13/19 (68%) of the MSS tumors were CIMP+ (p = 0.17). Of the fore-/midgut-NET, none was MSI-H. 20/34 (59%) colorectal NET vs. 11/38 (29%) fore-/midgut-NET were CIMP+ (p = 0.01). The Ki-67 index was significantly higher in poorly differentiated colorectal NET compared to the less malignant fore-/midgut-NET (p < 0.0001). Besides the location in the colon, Ki-67 predicted poor outcome in NET (p < 0.0001). CIMP status did not affect survival. In NET, p16 methylation predicted a poor outcome (p = 0.0004). We conclude that molecular pathogenesis in sporadic CRC and poorly differentiated colorectal NET is different despite some similarities. Main differences between malignant well-differentiated and poorly differentiated NET are the Ki-67 proliferation rate and differential methylation in tumor-associated genes. Predictors of a poor outcome in patients with NET are poor differentiation, a high Ki-67 index and p16 methylation.

• Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis.

  Authors: Takeshi Nagasaka, Ajay Goel, Kenji Notohara, Takaomi Takahata, Hiromi Sasamoto, Takuyuki Uchida, Naoshi Nishida, Noriaki Tanaka, Clement Richard Boland, Nagahide Matsubara

  International journal of cancer. Journal international du cancer. 07/2008; 122(11):2429-36.

  O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpGO(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair gene which is frequently methylated in colorectal cancer (CRC). However, it remains controversial whether methylation of specific CpG sequences within MGMT promoter leads to loss of its protein expression, and if MGMT methylation correlates with G to A transition mutations in KRAS. Two methylation sensitive regions (Mp and Eh region) of MGMT promoter were investigated in 593 specimens of colorectal tissue: 233 CRCs, 104 adenomatous polyps (AP), 220 normal colonic mucosa from CRC patients (N-C) and 36 normal colonic mucosa specimens obtained from subjects without colorectal neoplasia (N-N) by combined bisulfite restriction analysis (COBRA). The region-specific methylation data were compared to the MGMT protein expression, spectrum of KRAS mutations and other clinical features. Extensive (including both Mp and Eh) and partial (either Mp or Eh) MGMT methylation were found in 24.5% and 11.6% of CRCs, 3.8% and 27.9% of APs, 0.5% and 7.7% of C-Ns and 2.8% and 2.8% of N-Ns, respectively. Extensive methylation of MGMT promoter was primarily present in CRCs while partial methylation was common in APs. Extensive methylation of MGMT promoter was associated with loss/reduced protein expression (p < 0.0001), as well as with G to A mutations in KRAS (p = 0.0017). We herein provide first evidence that extensive methylation of MGMT promoter region is essential for methylation-induced silencing of this gene. Our data suggest that MGMT methylation may evolve and spread throughout the promoter in a stepwise manner as the colonic epithelial cells progress through the classical-adenoma-cancer multistep cascade.

• Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer.

  Authors: Takeshi Nagasaka, Minoru Koi, Matthias Kloor, Johannes Gebert, Alex Vilkin, Naoshi Nishida, Sung Kwan Shin, Hiromi Sasamoto, Noriaki Tanaka, Nagahide Matsubara, C Richard Boland, Ajay Goel

  Gastroenterology. 06/2008; 134(7):1950-60, 1960.e1.

  BACKGROUND & AIMS: Colorectal cancers (CRCs) with the CpG island methylator phenotype (CIMP) often associate with epigenetic silencing of hMLH1 and an activating mutation in the BRAF gene. However,BACKGROUND & AIMS: Colorectal cancers (CRCs) with the CpG island methylator phenotype (CIMP) often associate with epigenetic silencing of hMLH1 and an activating mutation in the BRAF gene. However, the current CIMP criteria are ambiguous and often result in an underestimation of CIMP frequencies in CRCs. Because BRAF and KRAS belong to same signaling pathway, we hypothesized that not only mutations in BRAF but mutant KRAS may also associate with CIMP in CRC. METHODS: We determined the methylation status in a panel of 14 markers (7 canonical CIMP-related loci and 7 new loci), microsatellite instability status, and BRAF/KRAS mutations in a collection of 487 colorectal tissues that included both sporadic and Lynch syndrome patients. RESULTS: Methylation analysis of 7 CIMP-related markers revealed that the mean number of methylated loci was highest in BRAF-mutated CRCs (3.6) vs KRAS-mutated (1.2, P < .0001) or BRAF/KRAS wild-type tumors (0.7, P < .0001). However, analyses with 7 additional markers showed that the mean number of methylated loci in BRAF mutant tumors (4.4) was the same as in KRAS mutant CRCs (4.3, P = .8610). Although sporadic microsatellite instability high tumors had the highest average number of methylated markers (8.4), surprisingly, Lynch syndrome CRCs also demonstrated frequent methylation (5.1). CONCLUSIONS: CIMP in CRC may result from activating mutations in either BRAF or KRAS, and the inclusion of additional methylation markers that correlate with mutant KRAS may help clarify CIMP in future studies. Additionally, aberrant DNA methylation is a common event not only in sporadic CRC but also in Lynch syndrome CRCs.

• Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma.

  Authors: Naoshi Nishida, Takeshi Nagasaka, Takafumi Nishimura, Iwao Ikai, C Richard Boland, Ajay Goel

  Hepatology (Baltimore, Md.). 03/2008; 47(3):908-18.

  Aberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution ofAberrant DNA methylation is an important epigenetic alteration in hepatocellular carcinoma (HCC). However, the molecular processes underlying the methylator phenotype and the contribution of hepatitis viruses are poorly understood. The current study is a comprehensive methylation analysis of human liver tissue specimens. A total of 176 liver tissues, including 77 pairs of HCCs and matching noncancerous liver and 22 normal livers, were analyzed for methylation. Methylation of 19 epigenetic markers was quantified, and the results were correlated with different disease states and the presence or absence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. Based on methylation profiles, the 19 loci were categorized into 3 groups. Normal liver tissues showed methylation primarily in group 1 loci (HIC-1, CASP8, GSTP1, SOCS1, RASSF1A, p16, APC), which was significantly higher than group 2 (CDH1, RUNX3, RIZ1, SFRP2, MINT31) and group 3 markers (COX2, MINT1, CACNA1G, RASSF2, MINT2, Reprimo, DCC) (P < 0.0001). Noncancerous livers demonstrated increased methylation in both group 1 and group 2 loci. Methylation was significantly more abundant in HCV-positive livers compared with normal liver tissues. Conversely, HCC showed frequent methylation at each locus investigated in all 3 groups. However, the group 3 loci showed more dense and frequent methylation in HCV-positive cancers compared with both HBV-positive cancers and virus-negative cancers (P < 0.0001). CONCLUSION: Methylation in HCC is frequent but occurs in a gene-specific and disease-specific manner. Methylation profiling allowed us to determine that aberrant methylation is commonly present in normal aging livers, and sequentially progresses with advancing stages of chronic viral infection. Finally, our data provide evidence that HCV infection may accelerate the methylation process and suggests a continuum of increasing methylation with persistent viral infection and carcinogenesis in the liver.