Kiyoko Takane

Genetic and epigenetic aberrations occurring in colorectal tumors associated with serrated pathway

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high‐methylation CRC. The methylation levels of six Group‐1 and 14 Group‐2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group‐1 and Group‐2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high‐methylation CRC. But TSA showed low‐methylation levels of Group‐1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high‐methylation CRC. High‐methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1‐methylated SSA/P showed lower methylation level of MLH1 compared with high‐methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1‐methylated and MLH1‐unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF‐β and BMP signaling (but not in TP53 signaling) were significantly involved in high‐methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.

Aberrant promoter methylation of PPP1R3C and EFHD1 in plasma of colorectal cancer patients

Aberrant DNA methylation is a common epigenetic alteration involved in colorectal cancer (CRC). In our previous study, we performed methylated DNA immunoprecipitation‐on‐chip analysis combined with gene re‐expression analysis by 5‐aza‐2′‐deoxycytidine treatment, to identify methylation genes in CRC genome widely. Among these genes, 12 genes showed aberrant hypermethylation frequently in >75% of 149 CRC samples but did not in normal samples. In this study, we aim to find out any of these methylation genes to be utilized for CRC detection using plasma DNA samples. Primers for methylation‐specific PCR and pyrosequencing were designed for seven of the 12 genes. Among them, PPP1R3C and EFHD1 were rarely hypermethylated in peripheral blood cells, but frequently hypermethylated in 24 CRC tissue samples and their corresponding plasma samples. In plasma samples, PPP1R3C was methylated in 81% (97/120) of CRC patients, but only in 19% (18/96) of noncancer patients (P = 6 × 10−20, Fishers exact test). In combined analysis with EFHD1, both genes were methylated in 53% (64/120) of CRC patients, but only in 4% (4/96) of noncancer patients (P = 2 × 10−16), giving high specificity of 96%. At least one of the two genes was methylated in 90% (108/120) of CRC patients, and 36% (35/96) of control patients, giving high sensitivity of 90%. Compared with low sensitivity of carcinoembryonic antigen (17% at stage I, 40% at stage II) and CA19‐9 (0% at stage I, 13% at stage II) for early‐stage CRCs, sensitivity of aberrant methylation was significantly higher: PPP1R3C methylation at 92% (11/12) for stage I and 77% (23/30) for stage II, and methylation of at least one gene at 100% (12/12) for stage I and 87% (26/30) for stage II. PPP1R3C methylation or its combined use of EFHD1 methylation was highly positive in CRC plasma samples, and they might be useful in detection of CRC, especially for early‐stage CRCs.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

TP53 mutation at early stage of colorectal cancer progression from two types of laterally spreading tumors

Although most sporadic colorectal cancers (CRC) are thought to develop from protruded adenomas through the adenoma–carcinoma sequence, some CRC develop through flat lesions, so‐called laterally spreading tumors (LST). We previously analyzed epigenetic aberrations in LST and found that LST are clearly classified into two molecular subtypes: intermediate‐methylation with KRAS mutation and low‐methylation with absence of oncogene mutation. Intermediate‐methylation LST were mostly granular type LST (LST‐G) and low‐methylation LST were mostly non‐granular LST (LST‐NG). In the present study, we conducted a targeted exon sequencing study including 38 candidate CRC driver genes to gain insight into how these genes modulate the development of LST. We identified a mean of 11.5 suspected nonpolymorphic variants per sample, including indels and non‐synonymous mutations, although there was no significant difference in the frequency of total mutations between LST‐G and LST‐NG. Genes associated with RTK/RAS signaling pathway were mutated more frequently in LST‐G than LST‐NG (P = 0.004), especially KRAS mutation occurring at 70% (30/43) of LST‐G but 26% (13/50) of LST‐NG (P < 0.0001). Both LST showed high frequency of APC mutation, even at adenoma stage, suggesting its involvement in the initiation stage of LST, as it is involved at early stage of colorectal carcinogenesis via adenoma‐carcinoma sequence. TP53 mutation was never observed in adenomas, but was specifically detected in cancer samples. TP53 mutation occurred during development of intramucosal cancer in LST‐NG, but during development of cancer with submucosal invasion in LST‐G. It is suggested that TP53 mutation occurs in the early stages of cancer development from adenoma in both LST‐G and LST‐NG, but is involved at an earlier stage in LST‐NG.

DNA methylation epigenotype and clinical features of NRAS-mutation(+) colorectal cancer

Sporadic colorectal cancer (CRC) is classified into several molecular subtypes. We previously established two groups of DNA methylation markers through genome‐wide DNA methylation analysis to classify CRC into distinct subgroups: high‐, intermediate‐, and low‐methylation epigenotypes (HME, IME, and LME, respectively). HME CRC, also called CpG island methylator phenotype (CIMP)‐high CRC, shows methylation of both Group 1 markers (CIMP markers) and Group 2 markers, while IME/CIMP‐low CRC shows methylation of Group 2, but not of Group 1 markers, and LME CRC shows no methylation of either Group 1 or Group 2 markers. While BRAF‐ and KRAS‐mutation(+) CRC strongly correlated with HME and IME, respectively, clinicopathological features of NRAS‐mutation(+) CRC, including association with DNA methylation, remain unclear. To characterize NRAS‐mutation(+) CRC, the methylation levels of 19 methylation marker genes (6 Group 1 and 13 Group 2) were analyzed in 61 NRAS‐mutation(+) and 144 NRAS‐mutation(−) CRC cases by pyrosequencing, and their correlation with clinicopathological features was investigated. Different from KRAS‐mutation(+) CRC, NRAS‐mutation(+) CRC significantly correlated with LME. NRAS‐mutation(+) CRC showed significantly better prognosis than KRAS‐mutation(+) CRC (P = 3 × 10−4). NRAS‐mutation(+) CRC preferentially occurred in elder patients (P = 0.02) and at the distal colon (P = 0.006), showed significantly less lymph vessel invasion (P = 0.002), and correlated with LME (P = 8 × 10−5). DNA methylation significantly accumulated at the proximal colon. NRAS‐mutation(+) CRC may constitute a different subgroup from KRAS‐mutation(+) CRC, showing significant correlation with LME, older age, distal colon, and relatively better prognosis.

Gastrointestinal stromal tumor with nephrotic syndrome as a paraneoplastic syndrome: a case report

IntroductionParaneoplastic syndromes are disorders associated with clinical signs and symptoms caused by substances produced by malignant disease and are not directly related to the physical effects of a primary or metastatic tumor. We describe a patient with gastrointestinal stromal tumor of the stomach accompanied by nephrotic syndrome as paraneoplastic syndrome in whom symptomatic treatment was ineffective. Nephrotic syndrome caused by gastrointestinal stromal tumors is quite rare, and to the best of our knowledge this is the first time that such a case has been documented.Case presentationWe describe a 69-year-old Asian woman with a gastrointestinal stromal tumor of the stomach accompanied by paraneoplastic syndrome. The patient had severe hypoalbuminemia and proteinuria, which were apparently attributed to a gastrointestinal stromal tumor. After preoperative treatment for hypoalbuminemia, the tumor was resected and nephrotic syndrome improved. Two years after her operation, she is still alive with neither tumor recurrence nor nephrotic syndrome.ConclusionPatients with refractory nephrotic syndrome caused by a malignant tumor should be treated aggressively, even if they are in poor general condition. Otherwise, the opportunity for potentially curative surgery may be missed.

Inhibition of DNA Methylation at the MLH1 Promoter Region Using Pyrrole–Imidazole Polyamide

Aberrant DNA methylation causes major epigenetic changes and has been implicated in cancer following the inactivation of tumor suppressor genes by hypermethylation of promoter CpG islands. Although methylated DNA regions can be randomly demethylated by 5-azacytidine and 5-aza-2′-deoxycytidine, site-specific inhibition of DNA methylation, for example, in the promoter region of a specific gene, has yet to be technically achieved. Hairpin pyrrole (Py)–imidazole (Im) polyamides are small molecules that can be designed to recognize and bind to particular DNA sequences. In this study, we synthesized the hairpin polyamide MLH1_–16 (Py-Im-β-Im-Im-Py-γ-Im-Py-β-Im-Py-Py) to target a CpG site 16 bp upstream of the transcription start site of the human MLH1 gene. MLH1 is known to be frequently silenced by promoter hypermethylation, causing microsatellite instability and a hypermutation phenotype in cancer. We show that MLH1_–16 binds to the target site and that CpG methylation around the binding site is selectively inhibited in vitro. MLH1_non, which does not have a recognition site in the MLH1 promoter, neither binds to the sequence nor inhibits DNA methylation in the region. When MLH1_–16 was used to treat RKO human colorectal cancer cells in a remethylating system involving the MLH1 promoter under hypoxic conditions (1% O2), methylation of the MLH1 promoter was inhibited in the region surrounding the compound binding site. Silencing of the MLH1 expression was also inhibited. Promoter methylation and silencing of MLH1 were not inhibited when MLH1_non was added. These results indicate that Py–Im polyamides can act as sequence-specific antagonists of CpG methylation in living cells.

The frequency of promoter DNA hypermethylation is decreased in colorectal neoplasms of familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an inherited disorder characterized by numerous colorectal adenomatous polyps with predisposition to the development of colorectal cancer (CRC). Here, we conducted genome-wide DNA methylation analysis of FAP neoplasms, including seven cancer samples and 16 adenoma samples, using an Infinium 450K BeadArray. As controls for sporadic colorectal neoplasms and mucosae, we used Infinium 450k data from 297 CRC samples, 45 colorectal adenoma samples, and 37 normal mucosa samples with reference to The Cancer Genome Atlas and other databases. Unsupervised two-way hierarchical clustering analysis of FAP and sporadic CRC/adenoma revealed that CRC was classified into four DNA methylation epigenotypes (MEs): high-ME (HME), intermediate-ME (IME), low-ME (LME), and normal-like ME (NME). Five FAP neoplasms (two cancer and three adenoma) were clustered with IME, whereas 18 FAP neoplasms (five cancer and 13 adenoma) were clustered into NME. IME FAP neoplasms significantly correlated with KRAS mutations, similar to sporadic CRC. Within IME cases, however, aberrant DNA methylation was significantly less frequent in FAP neoplasms than sporadic neoplasms, and these unmethylated genes included WNT family genes and several types of oncogenes. In summary, FAP neoplasms were classified into at least two molecular subtypes, i.e., NME in the majority of cases showing mostly no aberrant methylation and IME in some cases accompanied by KRAS mutations but less frequent aberrant DNA methylation than sporadic neoplasms, suggesting that FAP may follow a tumorigenesis pathway different from that of sporadic CRC.

Two subtypes of colorectal tumor with distinct molecular features in familial adenomatous polyposis

While sporadic colorectal cancer (CRC) is classified into several molecular subtypes, stratification of familial colorectal tumors is yet to be well investigated. We previously established two groups of methylation markers through genome-wide DNA methylation analysis, which classified sporadic CRC and adenoma into three distinct subgroups: high-, intermediate-, and low-methylation epigenotypes. Here, we investigated familial adenomatous polyposis (FAP), through quantitative methylation analysis of 127 samples (16 cancers, 96 adenomas, and 15 benign mucosa from 14 patients with FAP) using six Group-1 and 14 Group-2 methylation markers, APC, BRAF, and KRAS mutation analysis, and CTNNB1 and TP53 immunohistochemical analysis. All the 14 patients presented with APC germline mutation. Three were from the same family and presented the same APC mutation. FAP tumors lacked BRAF-mutation(+) high-methylation epigenotype and were classified into two methylation epigenotypes. While 24 of 112 tumor samples showed intermediate-methylation epigenotype significantly correlating with KRAS-mutation(+) (P=3×10-4), 88 tumor samples showed low-methylation epigenotype correlating with the absence of KRAS- and BRAF-mutations. Similar to sporadic CRC, CTNNB1 was frequently activated at the adenoma stage, and TP53 mutation occurred during cancer development from adenoma. Whereas some patients showed a single epigenotype in all tumors throughout the colon, tumors with two distinct epigenotypes developed within a family with the same APC mutation or even within one patient. Methylation accumulation significantly correlated with proximal location and older age. These results indicate that there are at least two distinct molecular subtypes of FAP tumors, resembling sporadic CRC and independent from the APC germline mutation status.

Abstract 3000: New epigenetic biomarker for colorectal cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA is released and circulate in the blood of cancer patients. Epigenetic changes in the levels of circulating DNA has been associated with tumor burden and malignant progression. Public physical examination recommends occult fecal blood test for colorectal cancer (CRC) screening but its false positive rate is too high. As for serum marker for CRC including CEA and CA19-9, their sensitivity are nor satisfactory especially in early stage CRC patients and therefore, the potential use of circulating DNA for CRC screening has emerged. The aim of this study is the clinical utility of cell-free DNA as blood biomarkers. Based on our previous report about the new methylation markers for CRC on a genome-wide scale, we selected 4 methylation markers positive for CRC and established CRC screening panel. First, we collected plasma DNA from blood samples of 100 CRC patients preoperatively and 50 age-matched healthy volunteers. Then, we performed pyrosequence about plasma DNA after bisulfite treatment. Using this cancer screening panel, 82 CRC patients were positive for at least 1 marker. On the other hand, no control samples were positive for any markers and therefore, sensitivity and specificity using this cancer screening panel for CRC were 82.0% and 100%, respectively. According to TNM classification, positive rate for earlier stage including 1 and 2 patients were lower than advanced CRC patients. Similarly, the patients with abnormal serum CEA level harbored higher positive rate for the cancer panel, but serum CA19-9 level which is another CRC marker was not associated with positive rate. Two markers of the panel were relatively high in old age although they were negative for control samples. Considering that no hypermethylation was detected in control samples, this cancer panel is superior to preexisting blood tumor markers for CRC and available for cancer screening. Citation Format: Kiyoko Takane, Yutaka Midorikawa, Koichi Yagi, Ayako Sakai, Hiroyuki Aburatani, Tadatoshi Takayama, Atsuhi Kaneda. New epigenetic biomarker for colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2013-3000