Kiwamu Akagi

Three DNA Methylation Epigenotypes in Human Colorectal Cancer

Purpose: Whereas the CpG island methylator phenotype (CIMP) in colorectal cancer associates with microsatellite instability (MSI)-high and BRAF-mutation(+), the existence of an intermediate-methylation subgroup associated with KRAS-mutation(+) is controversial, and suitable markers for the subgroup have yet to be developed. Our aim is to clarify DNA methylation epigenotypes of colorectal cancer more comprehensively. Experimental Design: To select new methylation markers on a genome-wide scale, we did methylated DNA immunoprecipitation-on-chip analysis of colorectal cancer cell lines and re-expression array analysis by 5-aza-2′-deoxycytidine/Trichostatin A treatment. Methylation levels were analyzed quantitatively in 149 colorectal cancer samples using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Colorectal cancer was epigenotyped by unsupervised two-way hierarchical clustering method. Results: Among 1,311 candidate silencing genes, 44 new markers were selected and underwent quantitative methylation analysis in colorectal cancer samples together with 16 previously reported markers. Colorectal cancer was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two major groups: group 1 showing methylation in high-methylation epigenotype, and group 2 showing methylation in high- and intermediate-methylation epigenotypes. A two-step marker panel deciding epigenotypes was developed with 95% accuracy: the 1st panel consisting of three group-1 markers (CACNA1G, LOX, SLC30A10) to extract high-methylation epigenotype, and the 2nd panel consisting of four group-2 markers (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. The high-methylation epigenotype correlated significantly with MSI-high and BRAF-mutation(+) in concordance with reported CIMP. Intermediate-epigenotype significantly correlated with KRAS-mutation(+). KRAS-mutation(+) colorectal cancer with intermediate-methylation epigenotype showed significantly worse prognosis. Conclusions: Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis. Clin Cancer Res; 16(1); 21–33

Estrogen receptor β is expressed in human stomach adenocarcinoma

AbstractPurpose. In stomach adenocarcinoma, the role of the hormonal receptor, estrogen receptor (ER), has been controversial. Recently, a new estrogen receptor, called estrogen receptor β (ERβ), was found to be expressed in various tissues including normal gastrointestinal tract. In this paper, the expression of ERβ in stomach adenocarcinomas has been investigated for the first time, specifically in signet ring cell adenocarcinomas, together with surrounding non-cancerous tissues. Methods. By immunohistochemistry the expression of ERα and β was studied in 29 stomach adenocarcinomas, ten signet ring cell adenocarcinomas, and 19 other adenocarcinomas. Western blotting was performed to examine the immunohistochemical result. Statistical studies (Students t test and χ2-test) explored the relation between the immunohistochemical result and clinicopathological characteristics. Results. All 29 adenocarcinomas, including the signet ring cell ones, demonstrated clear ERβ nucleus staining. Lymphocytes, venous endothelial cells, smooth muscle, and non-cancerous stomach glands also showed strong ERβ staining, while no staining was observed in the immunohistochemistry of ERα. Western blotting showed equivalent ERβ protein levels in cancerous and non-cancerous tissues, which was consistent with the results of immunohistochemical staining. Among signet ring cell adenocarcinomas of the stomach, cytoplasm were stained in addition to nuclei, specifically in patients under the age of 40 years. Conclusions. Our results imply that the effects of estrogen in stomach cancer, as well as those in normal stomach, may be mediated by ERβ, and that the role of ERβ may differ by the subtype of stomach adenocarcinoma – specifically signet ring cell adenocarcinomas and other ones – although large scale samples are needed to confirm these findings.

Intermediate Methylation Epigenotype and Its Correlation to KRAS Mutation in Conventional Colorectal Adenoma

A subset of colorectal cancer shows significant accumulation of aberrant promoter methylation. Previously, we developed two groups of methylation markers that classified colorectal cancer into three epigenotypes: i) high-, ii) intermediate-, and iii) low-methylation epigenotypes. High-methylation epigenotype, with methylation of both group 1 and group 2 markers, correlates to BRAF-mutation((+)). Intermediate-methylation epigenotype, with methylation of group 2 markers, but not group 1, correlates to KRAS-mutation((+)). To gain insight into epigenotype development in colorectal carcinogenesis, especially intermediate-methylation epigenotype and its correlation to KRAS-mutation((+)) in adenoma, we analyzed methylation levels of group 1 and group 2 markers quantitatively by matrix assisted laser desorption ionization-time of flight mass spectrometry, in 51 adenomas, 13 aberrant crypt foci, and 26 normal mucosa samples, and we compared them to 149 previously analyzed colorectal cancer samples. Three serrated adenomas were all BRAF-mutation((+)), showing great methylation of group 1 and group 2 markers, thus high-methylation epigenotype. Forty-eight conventional adenomas were not methylated in group 1 markers and were classified into two clusters with higher and lower methylation of group 2 markers, thus into intermediate- and low-methylation epigenotypes, respectively. Adenoma with intermediate-methylation epigenotype correlated to KRAS-mutation((+)). Methylation levels of group 2 markers in adenoma were higher than aberrant crypt foci and normal samples, but similar to cancer. These data suggested that epigenotype development occur at an earlier stage than carcinoma formation, and already be completed at the adenoma stage. Intermediate methylation epigenotype and its correlation to KRAS-mutation((+)) were developed in conventional adenoma.

Identification of chromosomal aberrations of metastatic potential in colorectal carcinoma.

In colorectal cancer (CRC) care, treatment decisions depend on the efforts to estimate the metastatic potential of tumors. The liver is one of the most common metastatic sites of CRC and the prognosis of CRC patients often reflects metastases to distant sites. To identify chromosomal aberrations associated with liver metastasis, we performed allelic copy number analysis for CRC with or without synchronous liver metastasis using genotyping arrays. By allelic copy number analysis of CRC samples, we observed common aberrations in 14 chromosomal arms in two groups, that is, gains on 7p22.3‐p11.2, 8q22.3‐q24.3, 13q12.12‐q34, and 20q11.22‐q13.33 and loss of heterozygosity (LOH) on 4q12‐q35.1, 5q11.2‐q35.3, 8p23.3‐p12, 15q11.2‐q26.3, 17p13.3‐p11.2, 17q11.2‐q25.1, 18p11.32‐p11.21, 18q11.2‐q23, 20p13‐p12.1, and 22q11.1‐q13.32. We found that gains on 20p13‐p12.1 and 20q11.21‐q13.33 and LOH on 6q14.1‐q25.1 were more frequent in CRC with liver metastasis. We also compared chromosomal aberrations in primary CRC lesions with those of the corresponding liver metastasis and found that the allelic genome imbalance status of a metastatic lesion is similar to that of the primary cancer, which suggests that chromosomal aberrations are largely maintained on hematogenous spread. Intriguingly, several chromosomal aberrations in CRC were found in the primary cancer but not in the corresponding liver metastasis, thus suggesting heterogeneity of cancer cells within solid tumors or the presence of events uniquely developed in primary tumors. Consequently, CRC with and without liver metastasis harbor similar chromosomal aberrations, and chromosomal aberration at 6q, 20p, and 20q may be involved in the process of liver metastasis of CRC.

Prognostic value of KRAS and BRAF mutations in curatively resected colorectal cancer.

AIM To investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population. METHODS We assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stage I-III CRC and examined associations of these mutations with disease-free survival (DFS) and overall survival (OS) using uni- and multivariate Cox proportional hazards models. RESULTS KRAS and BRAF mutations were detected in 312 (38%) of 812 and 40 (5%) of 811 tumors, respectively. KRAS mutations occurred more frequently in females than in males (P=0.02), while the presence of BRAF mutations was significantly associated with the female gender (P=0.006), proximal tumor location (P<0.001), mucinous or poorly differentiated histology (P<0.001), and MSI-high tumors (P<0.001). After adjusting for relevant variables, including MSI status, KRAS mutations were associated with poorer DFS (HR=1.35; 95%CI: 1.03-1.75) and OS (HR=1.46; 95%CI: 1.09-1.97). BRAF mutations were poor prognostic factors for DFS (HR=2.20; 95%CI: 1.19-4.06) and OS (HR=2.30; 95%CI: 1.15-4.71). Neither the BRAF by MSI interaction test nor the KRAS by MSI interaction test yielded statistically significant results for DFS and OS. CONCLUSION KRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

MYND-less splice variants of AML1–MTG8 (RUNX1–CBFA2T1) are expressed in leukemia with t(8;21)

The AML1–MTG8 fusion gene is generated by chromosome translocation t(8;21), which is frequently observed in acute myeloid leukemia. The fusion gene produces a chimeric transcription factor that suppresses the expression of AML1‐target genes via the MTG8 part of the chimeric protein, which is thought to be the primary cause of leukemia. The C‐terminal region of MTG8 contains the MYND domain, represented by highly conserved zinc‐finger‐like protein motifs, and is known to interact with corepressor proteins. We found that, instead of the MYND domain, an alternative last exon of MTG8 encoding 27 amino acids in‐frame is expressed naturally in human adult testis and in several leukemia cell lines. This type of alternative splicing also occurred in the AML1–MTG8 fusion gene at high levels in leukemia cell lines with t(8;21), as well as in blast cells of leukemia patients with t(8;21). The variant proteins of both MTG8 and AML1–MTG8 reduced transcriptional repressor activity in a mammalian two‐hybrid assay. However, mixed expression of these variants with wild‐type MTG8 recovered their repressor activity, suggesting that these variants also act as repressors in vivo where wild‐type MTG8 and other family members exist in abundance. On the other hand, the MYND‐less variants acquired a higher affinity for binding to MTG8 and formed a multimer, whereas the wild‐type protein forms a dimer. Thus, expression of the MYND‐less variants by the dysregulation of splicing machinery, which stimulates the oligomerization of fusion proteins in leukemia cells, may enhance malignant conversion of hematopoietic cells.

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.

12-Gene Recurrence Score Assay Stratifies the Recurrence Risk in Stage II/III Colon Cancer With Surgery Alone: The SUNRISE Study

PURPOSE The 12-gene Recurrence Score assay has been validated in resected stage II colon cancer treated with or without chemotherapy and resected stage III disease treated with chemotherapy. This study evaluated the 12-gene Recurrence Score assay for stage II and III colon cancer without chemotherapy to reveal the natural course of recurrence risk in stage III disease. METHODS A cohort-sampling design was used. From 1,487 consecutive patients with stage II to III disease who had surgery alone, 630 patients were sampled for inclusion with a 1:2 ratio of recurrence and nonrecurrence. Sampling was stratified by stage (II v III). The assay was performed on formalin-fixed, paraffin-embedded primary cancer tissue. Association of the Recurrence Score result with recurrence-free interval (RFI) was assessed by using weighted Cox proportional hazards regression. RESULTS Overall, 597 of 630 patients were analyzable-247 patients had stage II, and 350 had stage III colon cancer. The continuous Recurrence Score was significantly associated with RFI after adjustment for disease stage (hazard ratio for a 25-unit increase in Recurrence Score, 2.05; 95% CI, 1.47 to 2.86; P < .001). With respect to prespecified subgroups, as defined by low (< 30), intermediate (30 to 40), and high (≥ 41) Recurrence Score risk groups, patients with stage II disease in the high-risk group had a 5-year risk of recurrence similar to patients with stage IIIA to IIIB disease in the low-risk group (19% v 20%), whereas patients with stage IIIA to IIIB disease in the high-risk group had a recurrence risk similar to that of patients with stage IIIC disease in the low-risk group (approximately 38%). CONCLUSION To our knowledge, this study provides the first validation of the 12-gene Recurrence Score assay in stage III colon cancer without chemotherapy and showed the heterogeneity of recurrence risks in stage III as well as in stage II colon cancer.

Association of germline or somatic TP53 missense mutation with oncogene amplification in tumors developed in patients with Li-Fraumeni or Li-Fraumeni-like syndrome.

Germline TP53 mutations are found in Li‐Fraumeni syndrome (LFS) patients, predisposed to soft tissue sarcoma and other malignancies. The mutations and succeeding genetic events are thought to cause LFS‐associated cancer, whose genetic alterations have rarely been investigated. Here, we study two LFS or Li‐Fraumeni‐like syndrome (LFLS) patients whose cancers showed aggressive phenotypes. Patient 1 with LFS and TP53R273H developed a rhabdomyosarcoma twice at the ages of 18 months and 21 years. A single‐nucleotide polymorphism array‐based analysis revealed two amplicons in the second tumor; one at 5q11.2 containing MAP3K1 and the other at 11q22.2 containing BIRC2/3 and YAP1. Increase of kinase signaling of MAP3K1 along with anti‐apoptosis function of BIRC2/3 may have facilitated progression of this tumor. Patient 2 with LFLS and wild‐typeTP53 suffered from acute myeloid leukemia. The leukemic cells had TP53I195T and two amplicons; one at 8q24.1 containing DEPDC6 and the other at 8q24.2 containing TRIB1, MYC, and PVT1. Quantitative PCR confirmed amplification of the genes and FISH revealed co‐amplification of DEPDC6 and PVT1 in the same double minutes. Quantitative RT‐PCR revealed increased expression levels of TRIB1, but no or little expression of DEPDC6, MYC, and PVT1. The results indicate that TRIB1 may be the target gene in the amplicon in the leukemia cells. Mutant TP53 can be engaged in pathways triggering gene amplification through impairment of DNA double‐stranded break repair. The amplified candidate oncogenes identified in this study may have played a part in cancer development and lead to the poor outcome of LFS or LFLS‐associated tumors.

Prevalence of Lynch syndrome and Lynch-like syndrome among patients with colorectal cancer in a Japanese hospital-based population

Objective: We investigated the prevalence of Lynch syndrome and Lynch‐like syndrome among Japanese colorectal cancer patients, as there have been no credible data from Japan. Methods: Immunohistochemical analyses for mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) were carried out in surgically resected, formalin‐fixed paraffin‐embedded specimens obtained from 1,234 newly diagnosed colorectal cancer patients between March 2005 and April 2014. The presence/absence of the BRAF V600E mutation and hypermethylation of the MLH1 promoter was analyzed where necessary. Genetic testing was finally undertaken in patients suspected as having Lynch syndrome. Results: By the universal screening approach with immunohistochemical analysis for mismatch repair proteins followed by analyses for the BRAF V600E mutation and MLH1 promoter methylation status, 11 (0.9%) of the 1,234 patients were identified as candidates for genetic testing. Out of the 11 patients, 9 (0.7%) were finally diagnosed as having Lynch syndrome; the responsible genes included MLH1 (n = 1), MSH2 (n = 4), EPCAM (n = 1) and MSH6 (n = 3). The remaining two patients (0.2%) were regarded as having Lynch‐like syndrome, since biallelic somatic deletion of the relevant mismatch repair genes was detected in the absence of germline mismatch repair alterations. None of the cases was identified as having germline MLH1 epimutation. Conclusions: The prevalence of Lynch syndrome among all newly diagnosed cases of colorectal cancer in Japan is in the same range as that recently reported by studies in Western population. The prevalence of Lynch‐like syndrome seems to be extremely low.