Makoto Eizuka

Tumor budding at the invasive front of colorectal cancer may not be associated with the epithelial-mesenchymal transition

Tumor budding is thought to reflect the epithelial-mesenchymal transition (EMT). However, the molecular mechanism linking tumor buds and the EMT remains unclear. Here, we examined the induction of tumor budding and EMT and their association with EMT-related proteins (ZEB1, TWIST, SNAIL, and SLUG) in colorectal cancer (CRC). Immunohistochemical expression of pan-cytokeratin was examined for identification of tumor budding in 101 CRCs. Grading of tumor budding was classified into low- and high-grade groups. Tissue microarray was conducted to identify tumor budding sites. The expression of E-cadherin, ZEB1, TWIST, SNAIL, and SLUG was examined in areas of tumor budding and the surrounding tumor stroma using a double-immunostaining method. Specifically, pan-cytokeratin and EMT-related proteins were assessed by double immunostaining. Low or no expression of E-cadherin was found in areas of tumor budding. Moreover, ZEB1, TWIST, SNAIL, and SLUG were not expressed in regions of tumor budding. However, the expression level of ZEB1 in the stromal cells surrounding tumor budding was significantly more frequent than that of TWIST, SNAI, and SLUG. In addition, the expression of EMT-related proteins in surrounding stromal cells was significantly greater in areas of high-grade tumor budding than in low-grade areas. Our present results suggest that EMT-related proteins play a minor role in forming tumor buds. In addition, our findings suggest the existence of subtypes of stromal cells in CRC with phenotypical and functional heterogeneity.

Molecular differences in the microsatellite stable phenotype between left-sided and right-sided colorectal cancer

Differences in the pathogenesis of microsatellite stable (MSS) sporadic colorectal cancers (CRCs) between left‐sided CRC (LC) and right‐sided CRC (RC) have not been clarified. To identify pathogenesis‐related genomic differences between MSS CRCs within the two locations, we performed a comprehensive molecular analysis using crypt isolation with samples from 92 sporadic CRCs. Microsatellite instability (MSI; high and low/negative) and DNA methylation status (low methylation epigenome; intermediate methylation epigenome [IME] or high methylation epigenome [HME]) were determined using polymerase chain reaction (PCR) microsatellite analysis and PCR‐bisulfite pyrosequencing, respectively. Additionally, mutations in the TP53, KRAS, BRAF and PIK3CA genes were examined using PCR‐bisulfite pyrosequencing (for KRAS and BRAF mutations) or PCR‐single conformation polymorphism (for TP53 and PIK3CA mutations), followed by sequencing of aberrant bands. Finally, a genome‐wide study using a copy number alteration (CNA)‐targeted single nucleotide polymorphism array was performed. Ninety‐two CRCs were classified into 71 MSS and 21 MSI phenotypes. We examined 71 CRCs with the MSS phenotype (LC, 56; RC, 15). Mutations in KRAS were associated with RC with the MSS phenotype, whereas mutations in TP53 were more frequently found in LC with the MSS phenotype. There were significant differences in the frequencies of KRAS and TP53 mutations in the IME between LC and RC with the MSS phenotype. Although CNA gains were associated with LC with the MSS phenotype, CNA losses were not major alterations associated with the MSS phenotype. These findings suggested that the molecular pathogenesis of the MSS phenotype in LC was different from that in RC.

Clinicopathological and molecular alterations in early gastric cancers with the microsatellite instability‐high phenotype

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)‐high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal‐type adenocarcinoma). Tumors were classified as MSI‐high (45 cases), MSI‐low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)‐microsatellite assays and PCR‐pyrosequencing to detect chromosomal allelic imbalances in multiple cancer‐related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI‐high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features.

Genome-wide analysis of DNA copy number alterations in early and advanced gastric cancers

To better understand progressive changes in gastric cancer (GC), early and advanced GCs (EGC and AGC, respectively) were examined for copy number alterations (CNAs). A crypt isolation method was used to isolate DNA from tumors and normal glands in 20 AGCs, and fresh tumor samples were obtained from 45 EGCs. We assessed CNAs for differentiated‐type GCs using an Infinium HumanCytoSNP‐12v2.1 BeadChip in EGCs and AGCs. The most frequent aberrations in EGC were gains at 8q23.3 (42.2%) and 8q23.2 (40%), and loss of heterozygosity (LOH) at 3p14.2 (24.2%), suggesting that these CNAs were involved in the development of EGC. On the other hand, the highest frequencies of gains in AGC were found at 8q24.21 (65%) and 8q24.3 (60%). The most frequent LOHs in AGC were at 11q24.3‐25, 11q23.2‐24.1, 11q14.1, and 12p11.21‐13.33, whereas that in EGC was at 3p14.2. In addition, regions of copy‐neutral LOHs in AGC were detected at 11q21, 11q13.3‐14.3, 11q11, 11p13‐15.3, 12q21.1, 12q12‐13.3 and 5q33.3‐35.1. Comparisons of gains in EGC and AGC showed significant differences at 12q22‐q23.2, 12q21.33, 11p12, 11p14.1, 12q21.31‐32.32, 3p12.3, 3p14.1, 10p15.1, 1q24.2 and 2q12.1. Copy neutral LOHs were significantly higher in AGC than in EGC at 14q32.11‐32.33, 14q21.3, 14q11.2, 5q11.2, 5q 13.3, 14q21.1‐23.2, 14q13.2‐13.3, 5q12.1‐12.3, 5q11.1, and 17p13.3. The total lengths of the CNAs were significantly greater in AGC than in EGC. We found that the pattern of CNAs in AGC was quite different from that in EGC. We suggest that increasing numbers of CNAs are associated with disease progression from EGC to AGC.

Vascular Invasion and Stromal S100A4 Expression at the Invasive Front of Colorectal Cancer are Novel Determinants and Tumor Prognostic Markers

Object: The aim of the present study was to investigate the clinicopathological characteristics and prognostic factors associated with sporadic colorectal cancer (CRC). We examined the clinicopathological findings and immunohistochemical expression of tumor prognostic markers at tumor budding sites to determine their predictive value for patient prognosis. Materials and Methods: Immunohistochemical examination was performed by tissue microarray (TMA) of specimens from 106 patients with CRC. On hematoxylin and eosin (H&E)-stained tumor tissue slides, a representative area of tumor budding at the invasive front was selected for the construction of a TMA. Immunostaining for matrix metalloproteinase-7 (MMP7), the laminin-5 (ln-5) γ2 chain and S100A4 was performed to determine the association between patient survival and these markers. Results: Clinicopathological variables were also assessed. Tumor location, histological type, degree of lymphatic invasion and vascular invasion, tumor stage, epithelial expression of S100A4, stromal cell expression of S100A4 and expression of the ln-5γ2 chain were associated with an increased risk of mortality. Five factors were retained in the multivariate logistic regression analysis. Specifically, the tumor location, degree of lymphatic invasion and vascular invasion, tumor stage and stromal cell expression of S100A4 remained significant predictors of patient survival after controlling for the other variables. Conclusion: Vascular invasion and stromal expression of S100A4 in the tumor budding areas correlated with patient survival. Stromal immunostaining of S100A4 may be useful for identifying high-risk patients with advanced CRC.

Endoscopic and molecular characterization of colorectal sessile serrated adenoma/polyps with cytologic dysplasia

BACKGROUND AND AIMS Sessile serrated adenoma/polyps (SSA/Ps), which are precursor lesions of colorectal cancer (CRC) with BRAF mutation and the CpG island methylator phenotype (CIMP), develop cytologic dysplasia (CD) during the progression of colorectal tumorigenesis. In the present study we aimed to clarify the endoscopic and molecular signatures of SSA/Ps, with and without CD. METHODS A series of 208 serrated lesions, including 41 hyperplastic polyps, 90 SSA/Ps, 33 SSA/Ps with CD, and 44 traditional serrated adenomas, were observed and resected using magnifying endoscopy. BRAF and KRAS mutations and methylation of CIMP markers (MINT1, MINT2, MINT12, MINT31, and p16) were analyzed through pyrosequencing. Molecular alterations were then compared with endoscopic and pathologic characteristics. RESULTS Among SSA/Ps without CD, the Type II-Open pit pattern (Type II-O), BRAF mutation, and CIMP were tightly associated with a proximal colon location. SSA/Ps in the distal colon infrequently exhibited Type II-O and CIMP. By contrast, most SSA/Ps with CD showed Type II-O plus adenomatous pit patterns (Type III or IV), BRAF mutation, and CIMP, irrespective of their locations. CONCLUSIONS Our results suggest that the Type II-O plus III/IV pit pattern is a common feature of SSA/Ps with CD in both the proximal and distal colon and that this pit pattern is a hallmark of serrated lesions at high risk of developing into CRCs.

Genetic differences stratified by PCR-based microsatellite analysis in gastric intramucosal neoplasia.

BackgroundAlthough genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood.MethodsWe evaluated genetic differences in 158 IMNs, including 51 low-grade dysplasias, 58 high-grade dysplasias (HGDs), 30 intramucosal cancers (IMCs), and 19 mixed tumors (composed of IMC and HGD within the same tumor), using PCR-based microsatellite analysis [allelic imbalance (AI) and microsatellite instability (MSI)]. We classified the DNA methylation status as a hypermethylated epigenome, a moderately methylated epigenome, or a hypomethylated epigenome. In addition, p53 overexpression, β-catenin nuclear localization, and mucin expression were also examined.ResultsFrom cluster analysis, the IMNs examined were categorized into four subgroups as follows. Tumors in subgroup 1 were characterized by MSI-high status, a hypermethylated epigenome, and loss or reduction of expression of MLH-1. Tumors in subgroup 2 showed a mixed pattern consisting of AI and MSI. In contrast, tumors in subgroup 3, which showed accumulation of multiple AIs, were closely associated with HGD, IMC, or mixed tumor and exhibited nuclear expression of β-catenin. Tumors in subgroup 4, which were generally low-grade dysplasias, exhibited a low frequency of AIs and no MSI. Although the mucin phenotype was not correlated with any subgroup, expression of mucin was associated with some subgroups. Overexpression of p53 was common in all subgroups.ConclusionThe approach described herein was useful for studying genetic differences in IMNs. In addition, we suggest that stratification of genetic differences may help to identify genetic molecular profiles in IMNs.

Analysis of the DNA methylation level of cancer-related genes in colorectal cancer and the surrounding normal mucosa

BackgroundTwo molecular pathways promote the development of colorectal cancer (CRC). One is termed “microsatellite stable” (MSS) whereas the other is characterized by “microsatellite instability” (MSI or MIN). In addition, the CpG island methylation phenotype is known to be an important alteration as a third molecular type. Thus, DNA methylation is thought to provide potential biomarkers for assessment of cancer risk in normal mucosa. In addition, it is also known that colonic location is an important parameter in the development of CRC.MethodsWe examined the surrounding normal mucosa in three parts of the colon. Next, we quantified DNA methylation levels of SFRP1, SFRP2, SFRP5, DKK2, DKK3, mir34b/c, RASSF1A, IGFBP7, CDKN2A, and MLH1 in isolated cancerous glands and crypts of normal colorectal mucosa adjacent to CRCs using a pyrosequencer.ResultsDNA methylation levels of SFRP1, SFRP2, DKK2, and mir34b/c were significantly higher in CRCs with an MSS phenotype than in those with an MSI phenotype. The average level of methylation in normal crypts did not decrease with the distance from the tumor, irrespective of microsatellite status or the tumor location. DNA methylation levels in SFRP1 and SFRP2 genes in normal crypts were significantly higher in left-side than right-side CRC with an MSS phenotype. Finally, the genes were classified into three types based on the methylation frequencies in normal crypts, including type I (SFRP1 and SFRP2I), type II (DKK2 and mir34b/c), and type III (others).ConclusionsOur results showed that DNA methylation of SFRP1 and SFRP2 might be useful to predict cancer risk of surrounding normal mucosa. In addition, a field effect may be present in CRC, affecting both adjacent and non-adjacent normal mucosa.

Significance of a white opaque substance under magnifying narrow-band imaging colonoscopy for the diagnosis of colorectal epithelial neoplasms.

BACKGROUND AND AIMS The aim of this study was to examine the significance of a white opaque substance (WOS) found on magnifying narrow-band imaging (M-NBI) for the diagnosis of colorectal neoplastic lesions. METHODS We retrospectively reviewed colonoscopy records from 2006 to 2012 at our institution and identified cases of endoscopically or surgically resected colorectal epithelial neoplasms observed by M-NBI colonoscopy. The colonoscopic and histologic characteristics of the lesions were compared between WOS-positive and WOS-negative lesions. We further classified the WOS as regular or irregular and compared the histologic characteristics between the two types of lesions. RESULTS There were 105 WOS-positive lesions and 451 WOS-negative lesions. The former were subdivided into lesions with regular and irregular WOS. The incidence of high-grade dysplasia or carcinoma was significantly higher in WOS-positive lesions (61.9%) than in WOS-negative lesions (28.6%) (P < .05). Among the WOS-positive lesions, massive submucosal invasion was more frequent in lesions with irregular WOS (82.4%) than in those with regular WOS (1.4%) (P < .05). Among cancers with massive submucosal invasion, lymph node metastasis was more frequent in cancers with irregular WOS (17.4%) than in those with regular WOS or without the WOS (0%) (P < .05). CONCLUSIONS A WOS in colorectal neoplasms may be an optical marker for high-grade dysplasia and cancer. An irregular WOS may be indicative of massive submucosal invasion and lymph node metastasis.

Molecular alterations in colorectal adenomas and intramucosal adenocarcinomas defined by high-density single-nucleotide polymorphism arrays

BackgroundWe examined colorectal adenomas and intramucosal adenocarcinomas (IMAs) to develop a genome-wide overview of copy number alterations (CNAs) during colorectal tumorigenesis.MethodsWe analysed CNAs using a high-resolution SNP array of isolated tumour glands obtained from 55 colorectal adenomas (35 low-grade adenomas and 20 high-grade adenomas) and 30 IMAs. Next, we examined whether frequent CNAs differed between low-grade and high-grade adenomas or high-grade adenomas and IMAs. Finally, we investigated the total lengths of the CNAs in low-grade adenomas, high-grade adenomas, and IMAs.ResultsAlthough no frequent CNAs were found in low-grade adenomas, the most frequent alterations of high-grade adenomas were gains of 7q11, 7q21 and 9p13 and loss of 5q14.3-35. High levels of gains were detected at 13q, 7q, 8p, 20q, 7p, 18p and 17p in IMAs. Although no frequent alteration differed between low-grade and high-grade adenomas, significant differences of gains at 13q, 17p and 18p were found between high-grade adenoma and IMAs. Although the total lengths of all CNAs (gains and losses), copy number gains, and losses of heterozygosity were significantly greater in high-grade adenomas than in low-grade adenomas, no significant differences in the lengths of CNAs were found between high-grade adenomas and IMAs.ConclusionsGenomic alterations play an essential role in early colorectal carcinogenesis. CNAs in colorectal tumours provide new insights for evaluation of colorectal tumorigenesis.