[Show abstract][Hide abstract] ABSTRACT: Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated by OFFGEL electrophoresis, and analyzed by LC-coupled tandem MS. Non-redundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples, and cancer cell lines from HCEC cells. Two hundred twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change vs. normal mucosa +/-1.4), which correlated (r=0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (~25%) proteins displayed directionally similar expression changes in colorectal cancer cells (vs. HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein upregulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase (SORD), a key enzyme in the polyol pathway. Validation studies revealed dramatically increased SORD concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism may represent a novel facet of the metabolome remodeling associated with tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: Biological processes are controlled by transcription networks. Expression changes of transcription factor (TF) genes in precancerous lesions are therefore crucial events in tumorigenesis. Our aim was to obtain a comprehensive picture of these changes in colorectal adenomas.
Using a 3-pronged selection procedure, we analyzed transcriptomic data on 34 human tissue samples (17 adenomas and paired samples of normal mucosa, all collected with ethics committee approval and written, informed patient consent) to identify TFs with highly significant tumor-associated gene expression changes whose potential roles in colorectal tumorigenesis have been under-researched. Microarray data were subjected to stringent statistical analysis of TF expression in tumor vs. normal tissues, MetaCore-mediated identification of TF networks displaying enrichment for genes that were differentially expressed in tumors, and a novel quantitative analysis of the publications examining the TF genes' roles in colorectal tumorigenesis.
The 261 TF genes identified with this procedure included DACH1, which plays essential roles in the proper proliferation and differentiation of retinal and leg precursor cell populations in Drosophila melanogaster. Its possible roles in colorectal tumorigenesis are completely unknown, but it was found to be markedly overexpressed (mRNA and protein) in all colorectal adenomas and in most colorectal carcinomas. However, DACH1 expression was absent in some carcinomas, most of which were DNA mismatch-repair deficient. When networks were built using the set of TF genes identified by all three selection procedures, as well as the entire set of transcriptomic changes in adenomas, five hub genes (TGFB1, BIRC5, MYB, NR3C1, and TERT) where identified as putatively crucial components of the adenomatous transformation process.
The transcription-regulating network of colorectal adenomas (compared with that of normal colorectal mucosa) is characterized by significantly altered expression of over 250 TF genes, many of which have never been investigated in relation to colorectal tumorigenesis.
BMC Cancer 01/2014; 14(1):46. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Since there are few prospective studies on colorectal endoscopic resection to date, we aimed to prospectively assess safety and efficacy of endoscopic resection in a cohort of Italian patients.
Prospective multicentre assessment of resection of sessile polyps or non-polypoid lesions ≥ 10 mm in size or smaller (if depressed). Outcome measures included complete excision, morbidity, mortality, and residual/recurrence at 12 months.
Overall, 1012 resections in 928 patients were analysed (62.4% sessile polyps, 28.8% laterally spreading tumours, 8.7% depressed non-polypoid lesions). Lesions were prevalent in the proximal colon. Enbloc resection was possible in 715/1012 cases (70.7%), whereas piecemeal resection was required in 297 (29.3%). Endoscopically complete excision was achieved in 866 cases (85.6%). Adverse events occurred in 83 (8.2%), and no deaths occurred. Independent predictors of 12-month residual/recurrence were the location of the lesion in the proximal colon (OR 2.22 [95% CI 1.16–4.26]; p = 0.015) and piecemeal endoscopic resection (OR 2.76 [95% CI 1.56–4.87]; p = 0.0005). Limitations of the study were: potential expertise bias, no data on eligible and potentially resectable excluded lesions, high percentage of lesions < 20 mm, follow-up limited to 1 year.
In this registry study the endoscopic resection of colorectal lesions was safe and achieved high rates of long-term endoscopic clearance.
Digestive and Liver Disease 01/2014; 46(2):146–151. · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epigenetic silencing of protein-encoding genes is common in early-stage colorectal tumorigenesis. Less is known about the methylation-mediated silencing of genes encoding microRNAs (miRNAs), which are also important epigenetic modulators of gene expression. Using quantitative PCR, we identified 56 miRNAs that were expressed in normal colorectal mucosa and in HT29 colorectal cancer cells treated with demethylating agents but not in untreated HT29 cells, suggesting that they probably undergo methylation-induced silencing during colorectal tumorigenesis. One of these, miR-195, had recently been reported to be underexpressed in colorectal cancers and to exert tumor-suppressor effects in colorectal cancer cells. We identified the transcription start site (TSS) for primary miRNA (pri-miR)-497/195, the primary precursor that yields miR-195 and another candidate on our list, miR-497, and a single CpG island upstream to the TSS, which controls expression of both miRNAs. Combined bisulfite restriction analysis and bisulfite genomic sequencing studies revealed monoallelic methylation of this island in normal colorectal mucosa (50/50 samples) and full methylation in most colorectal adenomas (38/50; 76%). The hypermethylated precancerous lesions displayed significantly downregulated expression of both miRNAs. Similar methylation patterns were observed at two known imprinted genes, MEG3 and GNAS-AS1, which encode several of the 56 miRNAs on our list. Imprinting at these loci was lost in over half the adenomas (62% at MEG3 and 52% at GNAS-AS1). Copy-number alterations at MEG3, GNAS-AS1 and pri-miR-497/195, which are frequent in colorectal cancers, were less common in adenomas and confined to tumors displaying differential methylation at the involved locus. Our data show that somatically acquired, epigenetic changes at monoallelically methylated regions encoding miRNAs are relatively frequent in sporadic colorectal adenomas and might contribute to the onset and progression of these tumors.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The malignant transformation of precancerous colorectal lesions involves progressive alterations at both the molecular and morphologic levels, the latter consisting of increases in size and in the degree of cellular atypia. Analyzing preinvasive tumors of different sizes can therefore shed light on the sequence of these alterations. METHODS: We used a molecular pathway-based approach to analyze transcriptomic profiles of 59 colorectal tumors representing early and late preinvasive stages and the invasive stage of tumorigenesis. Random set analysis was used to identify biological pathways enriched for genes differentially regulated in tumors (compared with 59 samples of normal mucosa). RESULTS: Of the 880 canonical pathways we investigated, 112 displayed significant tumor-related upregulation or downregulation at one or more stages of tumorigenesis. This allowed us to distinguish between pathways whose dysregulation is probably necessary throughout tumorigenesis and those whose involvement specifically drives progression from one stage to the next. We were also able to pinpoint specific changes within each gene set that seem to play key roles at each transition. The early preinvasive stage was characterized by cell-cycle checkpoint activation triggered by DNA replication stress and dramatic downregulation of basic transmembrane signaling processes that maintain epithelial/stromal homeostasis in the normal mucosa. In late preinvasive lesions, there was also downregulation of signal transduction pathways (e.g., those mediated by G proteins and nuclear hormone receptors) involved in cell differentiation and upregulation of pathways governing nuclear envelope dynamics and the G2>M transition in the cell cycle. The main features of the invasive stage were activation of the G1>S transition in the cell cycle, upregulated expression of tumor-promoting microenvironmental factors, and profound dysregulation of metabolic pathways (e.g., increased aerobic glycolysis, downregulation of pathways that metabolize drugs and xenobiotics). CONCLUSIONS: Our analysis revealed specific pathways whose dysregulation might play a role in each transition of the transformation process. This is the first study in which such an approach has been used to gain further insights into colorectal tumorigenesis. Therefore, these data provide a launchpad for further exploration of the molecular characterization of colorectal tumorigenesis using systems biology approaches.
BMC Cancer 12/2012; 12(1):608. · 3.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND AIMS: Advances in colonoscopy, such as the Pentax i-Scan electronic technique, have the potential to improve the early detection of colorectal cancer. The aim of this multicentre study was to assess the interobserver agreement in the visualization of the surface and margins of colorectal polyps and in distinguishing neoplastic from non-neoplastic polyps. PATIENTS AND METHODS: Eight expert endoscopists examined 400 mixed previously recorded images of polyps taken with different Pentax i-Scan settings in order to give an evaluation of the surface of the polyp and regular colonic mucosa, the pit-pattern and the nature of the lesion. RESULTS: A total of 400 mixed images of polyps with a diameter >5mm and <10mm were stored for analysis. Overall, there was a K(f) agreement of 0.370 (p<0.001) and 0.306 (p<0.001) regarding pit-pattern and margins, respectively. The K(f) agreement for the difference between neoplastic and non-neoplastic lesions was of 0.446 (p<0.001). CONCLUSIONS: We observed good interobserver agreement in the evaluation of neoplastic and non-neoplastic lesions and poor agreement in the evaluation of pit-pattern and margins. Adequate training is required in order to interpret images acquired with the i-Scan technique.
Digestive and Liver Disease 10/2012; · 3.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Laterally spreading tumors (LSTs) are increasingly recognized as important precursors of colorectal carcinoma. The clinical behavior of these large nonpolypoid lesions is still uncertain. The aim of the present study was to assess prevalence and clinico-pathological features of LSTs in a large Italian cohort of patients.
The study was a subgroup analysis of a large database of patients undergoing total colonoscopy. The database originated from a multicenter cross-sectional observational study involving 80 centers throughout Italy.
Data from 27,400 total colonoscopies were analyzed. Precancerous lesions were detected in 5609 patients. Of these, LSTs were identified in 254 patients (4.5%; 95% confidence interval [CI] 3.5-6.2). Granular-type LSTs (G-LSTs) accounted for 83% of the cases (211/254). LSTs were predominant in the proximal colon (154, 60.6%). A total 231 lesions were endoscopically removed, with histology being available for 242. Neoplasia was confirmed in 225 lesions (93.4%) (143 low grade adenoma, 76 high grade adenoma, and six submucosal cancer). The six cases of submucosally invasive carcinoma were diagnosed in five G-LST and one nongranular LST (NG-LST). The risk of containing advanced histology was not increased in G-LST compared with NG-LST (odds ratio [OR] 1.55, 95%CI 0.73-3.27); it was significantly higher in lesions with large nodules (OR 3.09, 95%CI 1.05-9.04; P = 0.041) or depressed surface (OR 4.27, 95%CI 1.24-14.61; P = 0.021).
LSTs represent approximately 5% of all precancerous colorectal lesions in the Italian population and are prevalent in the proximal colon. These lesions are no more likely to harbor advanced histology than similar-sized polypoid lesions. Large nodularity or depressed surface are risk factors for advanced histology.
[Show abstract][Hide abstract] ABSTRACT: Improved colonoscopy is revealing precancerous lesions that were frequently missed in the past, and ∼30% of those detected today have nonpolypoid morphologies ranging from slightly raised to depressed. To characterize these lesions molecularly, we assessed transcription of 23,768 genes in 42 precancerous lesions (25 slightly elevated nonpolypoid and 17 pedunculated polypoid), each with corresponding samples of normal mucosa. Nonpolypoid versus polypoid morphology explained most gene expression variance among samples; histology, size, and degree of dysplasia were also linked to specific patterns. Expression changes in polypoid lesions frequently affected cell-cycling pathways, whereas cell-survival dysregulation predominated in nonpolypoid lesions. The latter also displayed fewer and less dramatic expression changes than polypoid lesions. Paradigmatic of this trend was progressive loss through the normal > nonpolypoid > polypoid > cancer sequence of TMIGD1 mRNA and protein. This finding, along with TMIGD1 protein expression patterns in tissues and cell lines, suggests that TMIGD1 might be associated with intestinal-cell differentiation. We conclude that molecular dysregulation in slightly elevated, nonpolypoid, precancerous colorectal lesions may be somewhat less severe than that observed in classic adenomatous polyps.
EMBO Molecular Medicine 06/2011; 3(6):334-47. · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Colorectal tumorigenesis is one of the best known processes of cellular transformation in humans. Its characterization has
moved ahead by leaps and bounds during the last three decades thanks to major advances in the fields of endoscopy, histology
and molecular pathology. And as often happens when a human disease is subjected to in-depth investigation, what originally
appeared to be a single entity turns out to include several distinct clinical, histologic, and molecular phenotypes. Among
other things, tumor phenotypes can tell us a great deal about the route taken by the tumor cells on their journey toward malignancy.
Not surprisingly, some tumors develop along pathways that are “heavily trafficked” (and for this reason, relatively well
known); others follow the “roads less traveled.” But if obstacles arise along the way, tumor cells are adept at exploiting
alternative routes that permit them to continue their journey toward cancer, and these deviations can give rise to mixed phenotypes.
These phenotypes are nonetheless consistent with the concept of carcinogenesis as a nonrandom – and therefore, predictable
– process. Each pathway, each crossroads is the result of a specific set of genetic or epigenetic alterations. Many are already
well defined, others are only partially characterized, and some are still in the realm of hypothesis. Thus far, we have fairly
reliable maps of at least two of the major pathways to colorectal cancer, but with increasingly sophisticated molecular analysis
of preinvasive lesions, there is little doubt that we will eventually identify variants of these pathways and uncover others
whose existence was not even suspected.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to assess the prevalence of nonpolypoid lesions (NPLs) in Italy and their risk of containing neoplasia or advanced histology.
This was a multicenter cross-sectional observational study on consecutive patients undergoing total colonoscopy over a 3-month period in 80 Italian centers.
In all, 27,400 total colonoscopies were analyzed. Cancer was diagnosed in 801 patients (2.9 %). A total of 6553 precancerous lesions were detected in 5609 patients. Of these, 4154 patients (74.1 %) had polypoid lesions and 1455 patients (25.9 %) had NPLs. Therefore, the prevalence of NPLs was 5.3 % (95 %CI 5.0 - 5.6). NPLs larger than 10 mm were detected in 254 patients (17.5 %). NPLs were more predominant in the proximal colon (OR 2.92, 95 %CI 2.56 - 3.43; P < 0.0001 vs. polypoid lesions). Neoplastic tissue was diagnosed in 79.0 % and advanced histology (high-grade intraepithelial neoplasia or more) in 20.9 % of resected lesions. The risk of advanced histology was similar for polypoid and nonpolypoid lesions when adjusted for size. Depressed lesions had the highest risk of advanced histology (OR 10.56, 95 %CI 6.02 - 18.55; P < 0.0000 vs. flat-elevated). Age was an independent predictor of both neoplasia and advanced histology ( P = 0.0001).
NPLs are relatively common in the Italian population, with a prevalence similar to that in other Western series. NPLs are not more aggressive than polypoid lesions, except for those with depressed morphology.