Sub-classification of type VI pit patterns in colorectal tumors: relation to the depth of tumor invasion

We analyzed pit patterns of colorectal tumors with magnifying colonoscopy, focusing on the relationship between the VI pattern sub-classification and the depth of tumor invasion. The VI pattern was divided into the well-demarcated and poorly demarcated subtypes. The percentage of tumors with a depth of invasion of over 1,000 microm was in the order of non-V types (type I-IV) < type VI < type VN. Most well-demarcated VI lesions (80.8%) showed a depth of invasion of <1,000 microm compared to only 21.1% of poorly demarcated VI lesions. Both the frequency of degeneration or prolapse of the epithelial lining and the frequency of desmoplastic reaction occurred in the order of non-V types < type VI < type VN, while the degree of histological differentiation and the frequency of residual muscularis mucosae occurred in the order of non-V types > type VI > type VN. Furthermore, the poorly demarcated VI subtype showed a higher frequency of epithelial lining degeneration or prolapse and a lower frequency of residual muscularis mucosae than the well-demarcated VI subtype. Tumors with a depth of invasion of >1,000 microm showed a higher frequency of epithelial lining degeneration or prolapse, a lower degree of histological differentiation, a higher frequency of desmoplastic reaction, and a lower frequency of residual muscularis mucosae than did tumors with <1,000 microm of invasion. These results suggest that VI pit pattern sub-classification is a useful indicator of the depth of colorectal tumor invasion and treatment selection. A prospective study on this subject would be useful.     

Pks-positive Escherichia coli in tumor tissue and surrounding normal mucosal tissue of colorectal cancer patients

A significant association exists between the gut microbiome and colorectal carcinogenesis, as well as cancer progression. It has been reported that Escherichia coli (E. coli) containing polyketide synthetase (pks) island contribute to colorectal carcinogenesis by producing colibactin, a polyketide-peptide genotoxin. However, the functions of pks⁺ E. coli in initiation, proliferation, and metastasis of colorectal cancer (CRC) remain unclear. We investigated the clinical significance of pks⁺ E. coli to clarify its functions in CRC. This study included 413 patients with CRC. Pks⁺ E. coli of tumor tissue and normal mucosal tissue were quantified using droplet digital PCR. Pks⁺ E. coli was more abundant in Stages 0–I tumor tissue than in normal mucosal tissue or in Stages II–IV tumor tissue. High abundance of pks⁺ E. coli in tumor tissue was significantly associated with shallower tumor depth (hazard ratio [HR] = 5.0, 95% confidence interval [CI] = 2.3–11.3, p < 0.001) and absence of lymph node metastasis (HR = 3.0, 95% CI = 1.8–5.1, p < 0.001) in multivariable logistic analyses. Pks⁺ E. coli-low and -negative groups were significantly associated with shorter CRC-specific survival (HR = 6.4, 95% CI = 1.7–25.6, p = 0.005) and shorter relapse-free survival (HR = 3.1, 95% CI = 1.3–7.3, p = 0.01) compared to the pks⁺ E. coli-high group. Pks⁺ E. coli was abundant in Stages 0–I CRC and associated with CRC prognosis. These results suggest that pks⁺ E. coli might contribute to carcinogenesis of CRC but might not be associated with tumor progression.     

Genome-wide differences between microsatellite stable and unstable colorectal tumors

Genomic copy number changes are frequently found in cancers and they have been demonstrated to contribute to carcinogenesis; and it is widely accepted that tumors with microsatellite instability (MSI) are genetically stable and mostly diploid. In the present study we compared the copy number alterations and the gene-expression profiles of microsatellite stable (MSS) and MSI colorectal tumors. A total number of 31 fresh-frozen primary tumors (16 MSS and 15 MSI) were used. Twenty-eight samples (15 MSS and 13 MSI) were analyzed with metaphase comparative genomic hybridization (CGH), nine of which plus one additional sample (4 MSS and 6 MSI) were further analyzed by cDNA-based array-CGH. Gene expression analysis was performed with six samples [3 MSS and 3 MSI, four of these used in metaphase CGH (mCGH) analysis] to identify differentially expressed genes possibly located in the lost or amplified regions found by CGH, stressing the biological significance of copy number changes. Metaphase and array-CGH analysis of two colon cancer cell lines (HTC116 and SW480, reported as MSI and MSS archetypes) gave comparable results. Alterations found by mCGH in MSS tumors were +20, +8q, -8p and -18q. Interestingly, 1p22, 4q26 and 15q21 were found deleted preferentially in MSS tumors, while 22q13 was found gained in MSI tumors. The regions of alterations identified by array-CGH were gains at 8q24, 16q24.3 and 20q13, and the loss of 5q21, appearing in the both types of tumors. Gene expression analysis revealed genes with specific associations with the copy number changes of the corresponding genomic regions. As a conclusion, colorectal cancer is a heterogeneous disease, demonstrated by the genomic profiles of individual samples. However, our data shows that copy number changes do not occur exclusively in the MSS phenotypes.     

Different expression of calgizzarin (S100A11) in normal colonic epithelium, adenoma and colorectal carcinoma

The aim of the study was to detect proteomic markers usable to distinguish colorectal carcinoma from colon adenoma for a better understanding of the molecular mechanisms in the process of tumourigenesis. Therefore, we microdissected colon carcinoma tissue, epithelial colon adenoma tissue as well as normal adjacent colon epithelium and determined protein profiles by SELDI-TOF MS. A multitude of significantly different signals was detected. For their identification colon biopsis were lysed and subjected to a two-dimensional gel electrophoresis for separation. Subsequently, we identified nearly 100 proteins by tryptic digestion, peptide fingerprint mapping and database search. Calgizzarin (S100A11; S100C) identified by peptide fingerprint mapping correlated very well with a significantly differentially expressed signal found in prior protein profiling. Using an immunodepletion assay we confirmed the identity of this signal as calgizzarin. To localise calgizzarin in tissues we performed immunohistochemistry. For further confirmation of the identity of calgizzarin we re-analysed IHC-positive as well as IHC-negative tissue sections on ProteinChip arrays. This work demonstrates that biomarkers in colorectal cancer can be detected, identified and assessed by a proteomic approach comprising tissue-microdissection, protein profiling and immunological techniques.     

Correlation of Skp2 with carcinogenesis, invasion, metastasis, and prognosis in colorectal tumors

In colorectal tumors, S-phase kinase-associated protein 2 (Skp2) still has numerous important questions unanswered: its expression in adenomas, its correlation with key clinicopathological indices, its association with patient prognosis, its variation in lymph node metastases, and its association with many cell-cycle regulators. To answer these questions in colorectal tumors, Skp2, cyclin A, cyclin B1, cyclin E, CDK2, and Ki67 were immunohistochemically stained in 12 normal mucosa, 36 adenomas, 11 carcinomas in adenomas, 102 primary carcinomas, and 12 paired lymph node metastases; and Skp2 was examined by Western blot in 8 pairs of normal mucosa and carcinomas. Situated in nuclei, Skp2 expression significantly increased from normal mucosa through adenoma to primary carcinoma (p<0.0001), from mild through moderate to severe dysplasia in adenomas (p=0.038), from peripheral adenoma to paired central carcinoma (p=0.0033), and from primary carcinoma to lymph node metastasis (p=0.015), and these increases were confirmed by Western blot. Expression, however, relatively declined significantly in the primary carcinomas showing deep invasion (p=0.0113), lymph nodal metastases (p=0.0268), and poor prognosis for all (p=0.0104) or stage III patients (p=0.0119). High Skp2 was also significantly linked with elevated cyclin A, cyclin B1, cyclin E, CDK2 (in primary carcinomas only), and Ki67 in both adenomas and primary carcinomas. Thus, overexpression of Skp2 is associated with colorectal carcinogenesis and late metastasis to lymph nodes, whereas relative reduction of Skp2 is correlated with local invasion of primary carcinoma.     

Expression of p-glycoprotein in the normal colorectal epithelium and in colorectal-carcinoma

Expression of P-glycoprotein (pgp) was analyzed by immunohistochemical staining with monoclonal antibody JSB-1 in 145 frozen specimens (67 were samples of normal colorectal mucosa from sites adjacent to the tumor, 66 were colorectal carcinomas, 5 colorectal polyps, 5 metastatic lymph nodes, and 2 samples of metastatic liver tumors) of 67 patients with colorectal carcinoma and polyps. All 72 specimens of normal colorectal mucosa and adenomatous polyps expressed pgp to various degrees. By contrast, 18 of 39 (46.2%) samples from cases of well differentiated adenocarcinoma were positive for pgp but only 3 of 21 (14.3%) samples from cases of moderately differentiated adenocarcinoma and none of the 4 samples from cases of poorly differentiated adenocarcinoma were positive for pgp. There was no correlation between the clinicopathological stage of colorectal carcinoma and the expression of pgp. These findings indicate that the expression of P-glycoprotein is closely related to the differentiation of cells. In normal colorectal epithelium, pgp was expressed normally and in well differentiated adenocarcinomas, pgp was still expressed. However, expression of pgp was no longer detectable in carcinomas with moderate or poor differentiation.     

MUC1 and nuclear beta-catenin are coexpressed at the invasion front of colorectal carcinomas and are both correlated with tumor prognosis.

PURPOSE: Overexpression of MUC1 and cytosolic interaction of the mucin with beta-catenin are claimed to be involved in colorectal carcinogenesis. In vitro data published recently suggest that MUC1 overexpression results in an increase of steady state levels of nuclear beta-catenin. We tried to elucidate the coexpression of both molecules in colorectal cancer to demonstrate possible correlations with clinical, pathological, and prognostic data. EXPERIMENTAL DESIGN: An immunohistochemical double staining study was performed to characterize the expression and subcellular distribution of MUC1 and beta-catenin in a series of 205 patients with colorectal carcinoma. The results were correlated with clinicopathological variables as well as overall survival. RESULTS: MUC1 was strongly expressed in the tumor center and at the invasion front in approximately 50% of the cases. Similar results were obtained with regard to nuclear accumulation of beta-catenin at the invasive tumor parts. MUC1 protein expression in the tumor center correlated significantly with a low grade of differentiation, and nuclear beta-catenin in the tumor periphery was more frequent in carcinomas of the left colon and rectum. Overexpression of MUC1 and beta-catenin, as well as their nuclear coexpression at the invasion front correlated with a worse overall survival in an univariate analysis. However, only pathological tumor-node-metastasis staging and MUC1 at the invasion front revealed as independent prognostic factors. CONCLUSIONS: These results suggest that MUC1 and beta-catenin are coexpressed at the invasion front of colorectal carcinomas and that this feature is associated with an accelerated course of disease and worse prognosis.     

Genome-wide expression patterns associated with oncogenesis and sarcomatous transdifferentation of cholangiocarcinoma

Background  

The molecular mechanisms of CC (cholangiocarcinoma) oncogenesis and progression are poorly understood. This study aimed to determine the genome-wide expression of genes related to CC oncogenesis and sarcomatous transdifferentiation.     

Distribution of cells expressing myc proteins in human colorectal epithelium, polyps, and malignant tumors.

The myc gene family encodes nuclear phosphoproteins that are thought to play a role in the control of cellular proliferation and differentiation. We have undertaken an immunohistochemical study assessing the expression of myc gene family proteins in individual cells of normal colonic mucosa, colorectal polyps, and colorectal adenocarcinomas. We screened a panel of mouse monoclonal antibodies that we raised against recombinant human c-myc and N-myc proteins for recognition of myc proteins in paraffin tissue sections. Two of these antibodies, H120C69 and H8C150, were selected for indirect immunoperoxidase staining of tissue sections from 16 normal mucosas, 24 polyps, and 30 adenocarcinomas. In normal colon, about 25% of the cells in the lower one-third of the crypts of Lieberkühn stain for myc-related protein. This distribution resembles that of proliferating cells in the crypt. Benign hyperplastic polyps resemble normal mucosa in their myc staining pattern, with about 25% of the cells positive. In adenomatous polyps, the putative precursors of adenocarcinomas, from 50 to 100% of the cells stain positively for myc protein. In these cases, stained cells extend to the luminal surface, consistent with the previously reported expansion of the proliferation zone in these lesions. All adenocarcinomas examined had increased levels of myc protein relative to normal mucosa. The tumor cells exhibited markedly heterogeneous myc staining patterns, both among different tumors and, in some cases, within a single tumor. Comparison with Ki-67 monoclonal antibody staining indicates that myc protein expression in many tumors is uncoupled from cellular proliferation. Surprisingly, we observed increased numbers of myc-expressing cells and increased levels of myc protein in histologically normal colon directly adjacent to tumor, suggesting that many colorectal carcinomas secrete growth factors that activate gene expression in neighboring normal mucosa.     

Cathepsin D protein levels in colorectal tumors: divergent expression patterns suggest complex regulation and function

Cathepsin D protein patterns were analyzed in 59 colorectal tumors by Western blotting, glycosylation and immunohistochemical assays. Measurement of protein content by laser densitometry of tumor/normal pairs on Western blots revealed loss of cathepsin D protein in more than 50% of colorectal tumors. Independent loading controls and statistical estimates of reproducibility on duplicate assays confirmed frequent decreases in cathepsin D. For cases having a tumor/normal ratio (T/N) <1, the average T/N was 0.50+/-0.19, equivalent to the loss of one cathepsin D allele. However, 2-fold increases in cathepsin D protein levels were also observed in approximately 1/3 of tumors, supporting the concept that colorectal cancers develop via divergent molecular pathways and that cathepsin D may function differently in different cancers. Although normal cathepsin D expression was detected in some earlier stage tumors, protein levels became increasingly bimodal with progression such that cathepsin D levels were increased in 1/3 but decreased in 2/3 of stage III and IV cancers. Other laboratories have reported both significant loss and gain of chromosome 11 (site of the cathepsin D gene) in different colorectal tumors, providing a possible mechanism for our observations on cathepsin D. However, differential regulation of cathepsin D expression by mutant versus wild-type p53 may also contribute to variable cathepsin D levels in colorectal cancers. Immunohistochemical studies demonstrated a shift from a predominantly punctate distribution of cathepsin D protein in normal mucosa to a more diffuse cytoplasmic distribution in tumor tissues. Mutant forms of cathepsin D were not detected in tumors either as changes in electrophoretic mobility or altered glycosylation but minor changes in protein sequence could not be ruled out. Loss of cathepsin D protein may provide an advantage to colorectal tumors related to a loss of cathepsin D function in proapototic or antiangiogenic pathways while increased cathepsin D may promote cancer cell proliferation or invasion.     

粘液性大肠癌组织蛋白酶B、D表达与肿瘤浸润

目的组织蛋白酶B(Cathepsin B,CB)在粘液性大肠癌的表达尚未明了.组织蛋白酶D(Cathepsin D,CD)在肿瘤浸润和转移中的作用仍存在争议.本实验目的系探讨粘液性大肠癌CB和CD表达及其与肿瘤浸润转移的关系.方法采用免疫组化方法对48例人不同类型大肠癌CB及CD表达进行半定量检测.结果伴淋巴结转移大肠癌CB和CD表达率高于不伴淋巴结转移大肠癌,差异具显著性(P＜0.05;P＜0.005).粘液性大肠癌CB表达高于非粘液性者,差别具显著性(P＜0.05),但CD表达差异却无显著性意义(P＞0.05).结论提示大肠癌CB和CD表达与肿瘤浸润转移有关,CB在粘液性大肠癌浸润转移中作用可能更为重要.     

CD44s expression in resectable colorectal carcinomas and surrounding mucosa

Background: CD44s (standard isoform) is a cell adhesion molecule belonging to the family of the hyaluronan-binding proteins. The CD44 family has been found to be overexpressed in epithelial tumors, where they are generally in relationship with tumor growth and metastasic properties. The aim of this work was to evaluate the membranous CD44s content in colorectal cancer and in healthy surrounding mucosa, its possible relationship with clinicopathological parameters, and its potential prognostic significance. Materials and Methods: Membranous CD44s levels were measured by an immunoenzymatic assay in tumors and surrounding mucosa samples from 72 patients with resectable colorectal carcinomas. The patients were followed for a mean time period of 30 months. Results: There was a wide variability of CD44s levels in tumor-surrounding mucosal samples (26.6-727 ng/mg protein) as well as in tumors (28.5-381 ng/mg protein). Tumor samples showed significantly higher CD44s levels (median 115.3 ng/mg protein) than surrounding mucosal samples (81 ng/mg protein) (p = 0.03). In the same way, CD44s levels in tumors as well as in surrounding mucosal samples were significantly higher in high S-phase tumors than in low S-phase tumors (p = 0.001 for both). There was no significant relationship between tumor CD44s levels and patient's outcome. However, high levels of the glycoprotein in nonneoplastic surrounding mucosa were significantly (p = 0.018) associated with a poor overall patient survival. Conclusion: CD44s may play a role in the tumorogenesis of colorectal carcinomas. In addition, CD44s levels in tumor-surrounding mucosa may provide, in concert with some clinicopathological parameters, important information about prognostic evaluation of patients with resectable colorectal carcinomas.     

MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells

Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.     

结直肠肿瘤微血管计数及血管内皮细胞生长因子表达

目的 探讨血管生成与结直肠肿瘤的发生,发展关系,评估微血管计数（ＭＶＤ值）及血管内皮细胞生长因子（ＶＥＧＦ）表达与结直肠肿瘤预后的相关性。方法 应用免疫组化法回顾性地研究了３２例结直肠肿瘤石蜡包埋的病理组织。结果 正常粘膜,腺瘤,癌组织的ＭＶＤ值递增。不同病理状态下的结直肠癌ＭＶＤ值有差异,ＶＥＧＦ阴性组ＭＶＤ值低于ＶＥＧＦ阳性组,低ＭＶＤ值主ＶＥＧＦ阴性组生存率高于高ＭＶＤ值组及ＶＥＧＦ阳性组。     

Sucrase-isomaltase expression and enterocytic ultrastructure of human colorectal tumors

We report the relative frequency of sucrase-isomaltase (SI) antigen expression in human colonic adenocarcinoma (22/57), in peritumoral mucosa taken next to the tumor (31/41) or distant from it (29/42) as well as in 21/23 polyps. Our results are based on indirect immunofluorescence with a monoclonal antibody (MAb) specific for human intestinal SI. A regular and intense expression of SI occurred only in 6 tumor specimens. In the remaining 16 SI-positive tumor samples, labelling was heterogeneous, i.e., scattered over more or less extensive areas. A similar irregular staining pattern was also found in polyps and in peritumoral mucosa, irrespective of its distance from the tumor. Electron microscopic examination of 19 carcinomas mostly revealed altered brush-border membrane features, irrespective of histological SI staining pattern. Brush-border enzyme activities of sucrase, alkaline phosphatase and maltase showed no difference between tumor specimens and peritumoral mucosa, but aminopeptidase was depressed in the former. Sucrase activity was extremely low (mean values 1.1 to 1.8 mU/mg protein) and rose only exceptionally to 17.5 mU/mg prot.     

表皮-钙黏附素和Snail蛋白的表达与大肠癌侵袭转移及预后的关系

目的 研究表皮-钙黏附素(E-CD)和Snail蛋白在大肠癌组织中的表达及其与大肠癌侵袭、转移和预后之间的关系.方法 采用免疫组化EnVision法,检测E-CD和Snail蛋白在30例正常大肠黏膜、30例大肠腺瘤及142例大肠癌组织中的表达.结果 E-CD蛋白在正常大肠黏膜组织中的强阳性表达率为90.0%,明显高于其在大肠腺瘤和大肠癌组织中的强阳性表达率(63.3%和41.5%,P<0.05).Snail蛋白在大肠癌组织中的阳性表达率为52.1%,明显高于其在正常大肠黏膜和大肠腺瘤组织中的阳性表达率(6.7%和26.7%,P<0.05).E-CD蛋白的表达与大肠癌的分化程度、浸润深度、静脉侵犯、淋巴管侵犯、淋巴结转移以及Duke's分期有关(均P<0.05),而与患者的年龄、性别、肿瘤大小以及病理组织学类型无关(均P0.05).Snail蛋白的表达与大肠癌的分化程度、浸润深度、静脉侵犯、淋巴管侵犯、淋巴结转移、病理组织学类型以及Duke's分期有关(均P<0.05),而与患者的年龄、性别以及肿瘤大小无关(均P>0.05).E-CD蛋白与Snail蛋自在大肠癌组织中的表达呈负相关(r=-0.508).E-CD蛋白阳性表达者的术后1、3和5年生存率明显高于阴性表达者(P<0.05),Snail蛋白阴性表达者的术后1、3和5年生存率明显高于阳性表达者(P<0.05).淋巴结转移、Duke's分期、E-CD和Snail蛋白的表达可作为大肠癌独立的预后指标(均P<0.05).结论 E-CD和Snail蛋白的表达与大肠癌的侵袭、转移和预后明显相关,E-CD蛋白表达降低和Snail蛋白表达增强的大肠癌患者病期晚、预后差.