Comprehensive Analyses of Somatic Copy Number Alterations and Mutations Based on the Adenoma-Carcinoma Sequence.

AIMS: Identifying molecular alterations in the adenoma and carcinoma components within the same tumor would greatly contribute to understanding the neoplastic progression of early colorectal cancer. METHODS AND RESULTS: We examined somatic copy number alterations (SCNAs) and mutations involved in the adenoma and carcinoma components obtained from the same tumor in 46 cases of microsatellite-stable carcinoma in adenoma, using a genome-wide SNP array and gene mutation panel. In addition, we also performed hierarchical clustering to determine the SCNA frequencies in the tumors, resulting in stratification of the samples into two subgroups according to SCNA frequency. Subgroup 1 was characterized by multiple SCNAs and carcinoma components exclusively, while Subgroup 2 was characterized by a low frequency of SCNAs and both the adenoma and carcinoma components. The numbers of total genes and genes with gains were higher in the carcinoma than adenoma components. The three most frequent gains in both components were located at 1p36.33-1q44, 2p25.3-2q37.3, and 3p26.3-3q29. However, no candidate genes mapped to these regions. APC and KRAS mutations were common in both components, whereas the frequency of TP53 mutations was statistically higher in the carcinoma than adenoma component. However, TP53 mutations were not correlated with SCNA frequency. CONCLUSIONS: We suggest that considerable SCNAs and TP53 mutations are required for progression from adenoma to carcinoma within the same intramucosal neoplastic lesion.

The Regulation Pathway of VEGF Gene Expression Is Different between 2D Cells and 3D Spheroids in Human Lung Cancer Cells.

Angiogenesis is involved in the malignant transformation of cancers. Vascular endothelial growth factor (VEGF) is important in inducing angiogenesis. Cultured cells play an important role in analyzing the regulation of VEGF expression, and it is revealed that VEGF expression is induced under hypoxia. However, it has been shown that there are differences in the pathway for gene expression between two-dimensional (2D) cells and in vivo cells. Three-dimensional (3D) spheroids constructed in 3D culture with a gene expression pattern more similar to that of in vivo cells than 2D cells have been used to solve this problem. This study analyzed the VEGF gene expression pathway in 3D spheroids of human lung cancer cells, A549 and H1703. Hypoxia-inducible factor-1α (HIF-1α) and aryl hydrocarbon receptor nuclear translocator (ARNT) regulated VEGF gene expression in 3D spheroids. However, VEGF gene expression was not regulated by HIF-1α in 2D cells. To conclude, we found that the regulatory pathway of VEGF gene expression is different between 2D cells and 3D spheroids in human lung cancer cells. These results suggest the possibility of a new VEGF gene expression regulation pathway in vivo. In addition, they show useful knowledge for the analysis of angiogenesis induction mechanisms and also demonstrate the usefulness of 3D spheroids.

Gastroblastoma mimics the embryonic mesenchyme of the foregut: a case report.

BACKGROUND: Gastroblastoma is a rare gastric tumor composed of epithelial and spindle cell components. The characteristic MALAT-GLI1 fusion gene has only been identified in 5 reported cases. We report the morphological characterization of gastroblastoma with the MALAT1-GLI1 fusion gene in a young Japanese woman. CASE PRESENTATION: A 29-year-old Japanese woman visited Iwate Medical University Hospital with upper abdominal pain. Computed tomography revealed a tumor in expansive lesions involving the gastric antrum. Histologically, we observed a biphasic morphology composed of epithelial and spindle cell components. The epithelial components appeared as slit-like glandular structures with tubular or rosette-like differentiation. The spindle cell components consisted of short spindle-shaped oval cells. Immunohistochemical (IHC) analysis revealed that the spindle cell component was positive for vimentin, CD10, CD56, GLI1, and HDAC2, and focally positive for PD-L1. The epithelial component was positive for CK AE1/AE3, CAM5.2, and CK7, and negative for CK20 and EMA. Both components were negative for KIT, CD34, DOG1, SMA, desmin, S100 protein, chromogranin A, synaptophysin, CDX2, and SS18-SSX. The MALAT-GLI1 fusion gene was detected molecularly. CONCLUSIONS: We report the following new findings with this case: (i) gastric tumors mimic the gastrointestinal mesenchyme in the embryonic period; (ii) nuclear expression of PD-L1 and HDAC2 were observed in the spindle cell component of a gastroblastoma. We speculate that histone deacetylase (HDAC) inhibitors may offer a promising treatment option for gastroblastoma.

Aryl hydrocarbon receptor as a DNA methylation reader in the stress response pathway.

The aryl hydrocarbon receptor (AhR) mediates various cellular responses upon exposure to exogenous and endogenous stress factors. In these responses, AhR plays a dual role as a stress sensor for detecting various AhR ligands and as a transcription factor that upregulates the expression of downstream effector genes, such as those encoding drug-metabolizing enzymes. As a transcription factor, it selectively binds to the unmethylated form of a specific sequence called the xenobiotic responsive element (XRE). We suggest that AhR is a novel DNA methylation reader, unlike classical methylation readers, such as methyl-CpG-binding protein 2, which binds to methylated sequences. Under physiological conditions of continuous exposure to endogenous AhR ligands, such as kynurenine, methylation states of the individual target XREs must be strictly regulated to select and coordinate the expression of downstream genes responsible for maintaining homeostasis in the body. In contrast, long-term exposure to AhR ligands frequently leads to changes in the methylation patterns around the XRE sequence. These data indicate that AhR may contribute to the adaptive cellular response to various stresses by modulating DNA methylation. Thus, the DNA methylation profile of AhR target genes should be dynamically controlled through a balance between robustness and flexibility under both physiological and stress conditions. AhR is a pivotal player in the regulation of stress response as it shows versatility by functioning as a stress sensor, methylation reader, and putative methylation modulator.

ß-naphthoflavone-induced upregulation of CYP1B1 expression is mediated by the preferential binding of aryl hydrocarbon receptor to unmethylated xenobiotic responsive elements.

Human cytochrome P450 1 (CYP1) enzymes are transcriptionally induced by specific xenobiotics through a mechanism that involves the binding of aryl hydrocarbon receptors (AhR) to target xenobiotic responsive element (XRE) sequences. To examine the effect of DNA methylation on the AhR-mediated pathway, reverse transcription-quantitative PCR analysis was performed. ß-naphthoflavone (ßNF)-induced CYP1B1 expression was found to be potentiated by pre-treatment of human HepG2 liver cancer cells with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, but not HuH7 cells. It was hypothesized that this increase is mediated by the demethylation of CpG sites within XRE2/XRE3 sequences, suggesting that methylation of these sequences inhibits gene expression by interfering with the binding of AhR to the target sequences. To test this hypothesis, a novel method combining the modified chromatin immunoprecipitation of AhR-XRE complexes with subsequent DNA methylation analysis of the XRE regions targeted by activated AhR was applied to both liver cancer cell lines treated with ßNF. XRE2/XRE3 methylation was found to be exclusively observed in the input DNA from HepG2 cells but not in the precipitated AhR-bound DNA. Furthermore, sub-cloning and sequencing analysis revealed that the two XRE sites were unmethylated in the samples from the AhR-bound DNA even though the neighboring CpG sites were frequently methylated. To the best of our knowledge, the present study provides the first direct evidence that ligand-activated AhR preferentially binds to unmethylated XRE sequences in the context of natural chromatin. In addition, this approach can also be applied to assess the effects of DNA methylation on target sequence binding by transcription factors other than AhR.

A genome-wide analysis of the molecular alterations occurring in the adenomatous and carcinomatous components of the same tumor based on the adenoma-carcinoma sequence.

Identification of molecular alterations occurring in the adenomatous and carcinomatous components within the same tumor would greatly enhance understanding of the neoplastic progression of colorectal cancer. We examined somatic copy number alterations (SCNAs) and mRNA expression at the corresponding loci involved in the adenoma-carcinoma sequence in the isolated adenomatous and cancer glands of the same tumor in 15 cases of microsatellite-stable "carcinoma in adenoma," using genome-wide SNP and global gene expression arrays. Multiple copy-neutral loss of heterozygosity events were detected at 4q13.2, 15q15.1, and 14q24.3 in the adenomatous component and at 4q13.2, 15q15.1, and 14q24.3 in the carcinomatous component. There were significant differences in the copy number (CN) gain frequencies at 20q11.21-q13.33, 8q13.3, 8p23.1, and 8q21.2-q22.2 between the adenomatous and carcinomatous components. Finally, we found a high frequency of five genotypes involving CN gain with upregulated expression of the corresponding gene (RPS21, MIR3654, RSP20, SNORD54, or ASPH) in the carcinomatous component, whereas none of these genotypes were detected in the adenomatous component. This finding is interesting in that CN gain with upregulated gene expression may enhance gene function and play a crucial role in the progression of an adenoma into a carcinomatous lesion.

Cellular irinotecan resistance in colorectal cancer and overcoming irinotecan refractoriness through various combination trials including DNA methyltransferase inhibitors: a review.

Treatment with pharmacological drugs for colorectal cancer (CRC) remains unsatisfactory. A major cause of failure in pharmacotherapy is the resistance of colon cancer cells to the drugs, creating an urgent issue. In this review, we summarize previous studies on the resistance of CRC cells to irinotecan and discuss possible reasons for refractoriness. Our review presents the following five major causes of irinotecan resistance in human CRC: (1) cellular irinotecan resistance is induced mainly through the increased expression of the drug efflux transporter, ABCG2; (2) cellular irinotecan resistance is also induced in association with a nuclear receptor, pregnane/steroid X receptor (PXR/SXR), which is enriched in the CYP3A4 gene enhancer region in CRC cells by exposing the cells to SN-38; (3) irinotecan-resistant cells possess either reduced DNA topoisomerase I (Top1) expression at both the mRNA and protein levels or Top1 missense mutations; (4) alterations in the tumor microenvironment lead to drug resistance through intercellular vesicle-mediated transmission of miRNAs; and (5) CRC stem cells are the most difficult targets to successfully treat CRC. In the clinical setting, CRC gradually develops resistance to initially effective irinotecan-based therapy. To solve this problem, several clinical trials, such as irinotecan plus cetuximab vs. cetuximab monotherapy, have been conducted. Another clinical trial on irinotecan plus guadecitabine, a DNA-methyltransferase inhibitor, has also been conducted.

Recent Progress in Prediction Systems for Drug-induced Liver Injury Using In vitro Cell Culture.

BACKGROUND: In order to avoid drug-induced liver injury (DILI), in vitro assays, which enable the assessment of both metabolic activation and immune reaction processes that ultimately result in DILI, are needed. OBJECTIVE: In this study, recent progress in the application of in vitro assays using cell culture systems is reviewed for potential DILI-causing drugs/xenobiotics and a mechanistic study on DILI, as well as on the limitations of in vitro cell culture systems for DILI research, was carried out. METHODS: Information related to DILI was collected through a literature search of the PubMed database. RESULTS: The initial biological event for the onset of DILI is the formation of cellular protein adducts after drugs have been metabolically activated by drug metabolizing enzymes. The damaged peptides derived from protein adducts lead to the activation of CD4+ helper T lymphocytes and recognition by CD8+ cytotoxic T lymphocytes, which destroy hepatocytes through immunological reactions. Because DILI is a major cause of drug attrition and drug withdrawal, numerous in vitro systems consisting of hepatocytes and immune/inflammatory cells or spheroids of human primary hepatocytes containing non-parenchymal cells have been developed. These cellular-based systems have identified DILI-inducing drugs, with approximately 50% sensitivity and 90% specificity. CONCLUSION: Different co-culture systems consisting of human hepatocyte-derived cells and other immune/inflammatory cells have enabled the identification of DILI-causing drugs and of the actual mechanisms of action.

Molecular alterations in gastric cancer and the surrounding intestinal metaplastic mucosa: an analysis of isolated glands.

BACKGROUND: Intestinal metaplasias (IMs) are generally regarded as pre-neoplastic gastric lesions. However, molecular alterations including genetic and epigenetic changes occurring in individual IM glands are not well defined. AIMS: We sought to identify DNA methylation status, microsatellite instability (MSI) and allelic imbalance (AI) occurring in individual IM glands and non-IM glands within the same mucosa. METHODS: We divided examined isolated gland obtained from GC into 4 components: isolated cancer, antral isolated intestinal metaplastic tissue, antral isolated non-metaplastic gland and isolated non-metaplastic gland derived from the greater curvature of the most distant gastric body without mucosal atrophy. We examined AI and microsatellite instability statuses using PCR-based microsatellite analysis. Next, the DNA methylation status (high methylation epigenome [HME], intermediate methylation epigenome [IME], and low methylation epigenome [LME]) was investigated. DNA methylation analysis of CDKN2A, mir34-b/c and MLHI genes was also performed. RESULTS: Although antral isolated IM glands were characterized by IME, isolated non-IM glands showed LME. In isolated cancer glands, HME was frequently found, compared with isolated non-IM glands. DNA methylation of mir34-b/c was common in isolated cancer and IM glands, whereas DNA methylation of CDKN2A was a rare event in isolated samples. The MLH1 gene was not methylated in isolated non-IM glands. Although multiple AIs were frequently found in isolated cancer glands, a few AIs were detected in isolated IM glands. CONCLUSIONS: We suggest that the DNA methylation status and the status of the mir34-b/c gene among isolated samples of IMs and isolated non-IM glands have an impact on IM development.

The clinicopathological and molecular features of sporadic gastric foveolar type neoplasia.

Gastric intraepithelial foveolar type neoplasia (IEFN) is not well defined. In addition, atrophic mucosa (AM) is an important issue to consider when evaluating gastric tumorigenesis. Here, we assessed the clinicopathological characteristics and molecular alterations contributing to the development of IEFN compared with intestinal type neoplasia. We examined the clinicopathological and molecular features of 42 cases of IEFN with low-grade dysplasia (LGD) and those of 77 cases of intraepithelial intestinal type neoplasia (IEIN) with LGD. The clinicopathological and molecular features examined included the AM status, mucin phenotype expression, CDX2 expression, p53 overexpression, ß-catenin intranuclear accumulation, microsatellite instability (MSI), DNA methylation status (low methylation epigenotype [LME], intermediate ME, or high ME), allelic imbalances (AIs), and APC promoter 1B mutations. There were no differences in the frequencies of AM and rates of CDX2 expression between IEFN and IEIN cases. Although no differences in the frequencies of p53 overexpression and MSI were observed between the two histological types, intranuclear expression of ß-catenin was significantly higher in IEIN than in IEFN. In addition, although the rate of LME was significantly higher in IEFN cases than in IEIN cases, IEFN was characterized by AIs at multiple foci. Finally, mutation of the APC promoter 1B, which is a characteristic of gastric adenocarcinoma and proximal polyposis of the stomach (potentially resembling IEFN), was detected in only one IEFN case. These findings suggested that IEFN may be an independent entity in terms of molecular alterations including the presence of multiple AIs and LME.

Aryl Hydrocarbon Receptor Mediates Cell Proliferation Enhanced by Benzo[a]pyrene in Human Lung Cancer 3D Spheroids.

The aryl hydrocarbon receptor (AhR) is activated by the ligand, benzo[a]pyrene (B[a]P), a component of smoke that is implicated in lung carcinogenesis in humans. However, the role of B[a]P and AhR in lung cancer malignancy is not well known. In this study, we analyzed the effects of B[a]P and AhR in the 3 D spheroids of human lung cancer cells in vitro. In these spheroids, B[a]P and AhR enhanced cancer cell proliferation. These results suggest that the AhR-dependent effects of B[a]P on cell proliferation may contribute to the adverse effects of continuous smoking with respect to lung cancer malignancy.

The regulation mechanism of AhR activated by benzo[a]pyrene for CYP expression are different between 2D and 3D culture of human lung cancer cells.

Most of cytochrome P450 (CYP) expressions are regulated by nuclear receptors. The regulation pathways of transcription are activated by binding of the ligand to the receptor. Many combination of CYPs and nuclear receptors in transcriptional regulation have been reported. However, we have reported that the combination changes depending on culture condition on the same type of cells. The regulation pathway of CYP1A expression is different between 2D monolayer cultured cells and 3D spheroids of human liver cancer cells. Aryl hydrocarbon receptor (AhR) is one of the transcription factors for CYP1A and CYP1B1 expression, and this pathway is important for inducing human lung cancer. CYP1B1 expression in human lung cancer cells are regulated by AhR in 2D and 3D cells. But CYP1A expression are not induced by AhR in 3D cells. As with liver cancer cells, the function of AhR in lung cancer cells is different between 2D cells and 3D spheroids. These results important for understanding relationship between AhR and CYP expression before and after cell neoplastic formation in human lung.

Comprehensive molecular analysis based on somatic copy number alterations in intramucosal colorectal neoplasias and early invasive colorectal cancers.

It is unclear whether somatic copy number alterations (SCNAs) contribute to the development of colorectal cancer (CRC). Here, we aimed to identify the molecular profiles of early colorectal carcinogenesis based on SCNAs and determine the associations of other molecular abnormalities for the detection of neoplasia in both intramucosal neoplasia (IMN) and invasive CRC with invasion into the muscular layer without metastasis (early invasive CRC). A single nucleotide polymorphism array was used to examine 100 colorectal IMNs (low-grade adenoma [LGA], 40; high-grade adenoma [HGA], 25; intramucosal adenocarcinoma [IMA], 35) and early invasive CRC (20 tumors). In addition, genetic mutations (KRAS, BRAF), TP53 overexpression, microsatellite instability (MSI), and DNA methylation (low, intermediate, high) were examined. Hierarchical clustering analysis based on the SCNA pattern was carried out to identify molecular profiles in IMNs and early invasive CRC. Colorectal tumors were classified into three subgroups based on SCNA patterns. Subgroup 1 was characterized by multiple SCNAs, subgroup 3 was closely associated with infrequent SCNAs, and subgroup 2 was an intermediate subgroup in SCNA pattern between subgroups 1 and 3. Although mutations in KRAS were commonly found in all three subgroups, overexpression of TP53 was observed primarily in subgroup 1 and 2. DNA methylation showed a low/intermediate type. Finally, no MSI was detected. Each subgroup was correlated with histology (subgroup 1, early invasive CRC; subgroup 2, LGA; subgroups 2 and 3, HGA and IMA). Considerable SCNAs may be required for acquisition of invasive ability in CRC. Our results provide novel insights into early CRC.

Differential sensitization of two human colon cancer cell lines to the antitumor effects of irinotecan combined with 5-aza-2'-deoxycytidine.

Irinotecan (CPT-11) is a key therapeutic drug used in the treatment of colorectal cancer, although acquired or constitutive resistance to CPT-11 (and its activated metabolite SN-38) can lead to tumor progression. Since the acquisition of drug resistance can result from DNA hypermethylation, the antitumor activity of CPT-11 and SN-38 was assessed in combination with a known DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, also known as decitabine (DAC). DAC potentiated the antitumor activity of CPT-11 additively, and that of SN-38 synergistically, as measured by colony formation in the human colorectal cancer HCT116 cell line. No DAC potentiation of these antitumor effects was observed with another human colorectal cancer HT29 cell line. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein expression was reduced to 50-67% of the control following a single treatment with CPT-11, SN-38, or DAC, and was markedly reduced to 7-8% following the combination of CPT-11/SN-38 with DAC. By contrast, Bcl-2 protein expression was barely detected in HT29. Wilms' tumor protein (WT1), which has been shown to be a positive regulator of Bcl-2 in HCT116 cells through WT1-kncokdown experiments, was downregulated in HCT116 and HT29 cells when treated with CPT-11/SN-38 combined with DAC, with decreases greater than any single administration of CPT-11, SN-38, or DAC. The extent of CPT-11/SN-38 potentiation by DAC may depend on Bcl-2 expression levels in human colorectal cancer cells.

Molecular profiling and comprehensive genome-wide analysis of somatic copy number alterations in gastric intramucosal neoplasias based on microsatellite status.

BACKGROUND: We attempted to identify the molecular profiles of gastric intramucosal neoplasia (IMN; low-grade dysplasia, LGD; high-grade dysplasia, HGD; intramucosal cancer, IMC) by assessing somatic copy number alterations (SCNAs) stratified by microsatellite status (microsatellite stable, MSS; microsatellite instable, MSI). Thus, microsatellite status was determined in 84 tumors with MSS status and 16 tumors with MSI status. METHODS: One hundred differentiated type IMNs were examined using SCNAs. In addition, genetic mutations (KRAS, BRAF, PIK3CA, and TP53) and DNA methylation status (low, intermediate and high) were also analyzed. Finally, we attempted to identify molecular profiles using a hierarchical clustering analysis. RESULTS: Three patterns could be categorized according to SCNAs in IMNs with the MSS phenotype: subgroups 1 and 2 showing a high frequency of SCNAs, and subgroup 3 displaying a low frequency of SCNAs (subgroup 1 > 2 > 3 for SCNA). Subgroup 1 could be distinguished from subgroup 2 by the numbers of total SCNAs (gains and losses) and SCN gains (subgroup 1 > 2). The SCNA pattern of LGD was different from that of HGD and IMC. Moreover, IMNs with the MSI phenotype could be categorized into two subtypes: high frequency of SCNAs and low frequency of SCNAs. Genetic mutations and DNA methylation status did not differ among subgroups in IMNs. CONCLUSION: Molecular profiles stratified by SCNAs based on microsatellite status may be useful for elucidation of the mechanisms of early gastric carcinogenesis.

Molecular profiling and genome-wide analysis based on somatic copy number alterations in advanced colorectal cancers.

To characterize somatic alterations in colorectal cancer (CRC), we conducted a genome-scale analysis of 106 CRC specimens. We assessed comprehensive somatic copy number alterations (SCNAs) in these CRC specimens. In addition, we examined microsatellite instability (MSI; low and high), genetic mutations (KRAS, BRAF, TP53, and PIK3CA), and DNA methylation status (classified into low, intermediate, and high type). We stratified molecular alterations in the CRCs using a hierarchical cluster analysis. The examined CRCs could be categorized into three subgroups using hierarchical cluster analysis. Tumors in subgroup 1 were characterized by a low frequency of SCNAs and a high frequency of MSI-high status, whereas tumors in subgroups 2 and 3 were closely associated with a high frequency of SCNAs. Tumors in subgroup 1 were preferentially present in the right-sided colon and showed frequent MSI-high status. Subgroup 3 was distinguished by specific alterations, including gains at 1q23-44, 1p11-36, 10q11-26, 10p11-13, 12q24-24, and 13q33-33. In contrast, tumors in subgroup 2 were characterized by copy-neutral LOH at 12p12-13, 1q24-25, and 10q22. In addition, KRAS mutations were more frequently found in subgroup 3 than in subgroup 1. TP53 mutations and intermediate levels of DNA methylation were common alterations in the three subgroups. SCNAs contributed to sporadic CRC, and there were three subgroups based on SCNAs that played a different role in driving the development of this disease.

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

BACKGROUND: Two 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. METHODS: We 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. RESULTS: DNA 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). CONCLUSIONS: Our 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.

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

BACKGROUND: We examined colorectal adenomas and intramucosal adenocarcinomas (IMAs) to develop a genome-wide overview of copy number alterations (CNAs) during colorectal tumorigenesis. METHODS: We 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. RESULTS: Although 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. CONCLUSIONS: Genomic alterations play an essential role in early colorectal carcinogenesis. CNAs in colorectal tumours provide new insights for evaluation of colorectal tumorigenesis.

Molecular subtypes of colorectal cancers determined by PCR-based analysis.

Tumor tissue consists of a heterogeneous cell population. The allelic imbalance (AI) ratio, determined in isolated tumor glands, is a good index of tumor heterogeneity. However, associations of the patterns of AI and microsatellite instability (MSI) development, observed in most cases of colorectal cancer (CRC), with tumor progression have not been reported previously. In this study, we examined whether CRC genetic profiles stratified by a combination of the AI ratio and MSI facilitate categorization of CRC, and whether these genetic profiles are associated with specific molecular alterations in CRC. A crypt isolation method was used to isolate DNA from tumors and normal glands obtained from 147 sporadic CRCs. AI and MSI statuses were determined using PCR-based microsatellite analysis and stratified based on AI ratio and MSI status. DNA methylation status (high methylation, intermediate methylation and low methylation status and mutations in KRAS, BRAF, and TP53 were examined. In addition, mucin markers were immunostained. Based on this analysis, four subgroups were categorized. Subgroup 1 was characterized by a high MSI status and BRAF mutation; subgroup 2 was closely associated with a high AI ratio, which accumulated during the early phases of colorectal carcinogenesis, and TP53 mutation; subgroup 3 was associated with a low AI ratio, seen during the later phases of colorectal carcinogenesis, and KRAS mutation; and subgroup 4 was defined as a minor subgroup. These results confirmed that classification of distinct molecular profiles provides important insights into colorectal carcinogenesis.

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

BACKGROUND: Although genetic alterations in patients with advanced gastric cancer have been extensively studied, those in patients with intramucosal neoplasia (IMN) are still poorly understood. METHODS: We 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, ß-catenin nuclear localization, and mucin expression were also examined. RESULTS: From 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 ß-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. CONCLUSION: The 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.