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Macrophages are a pivotal immune cell population in the tumor microenvironment of colorectal cancer (CRC). Differently-polarized macrophages could be exploited to yield naturally-tailored biomimetic nanoparticles for CRC targeting. Here, membrane proteins were isolated from the THP-1 cell line, and anti-tumor macrophages (M1) were obtained from differentiation of THP-1. Membrane proteins were isolated from THP-1 and M1 and used to produce lipid nanovesicles (LNVs; T-LNVs and M1-LNVs) by microfluidic process, which were loaded with doxorubicin (DOXO). The DOXO loaded T-LNVs and M1-LNVs showed similar size (120-145 nm), PDI (0.11-0.28), zeta potential (-15 to -30 mV) and drug loading efficiency (65-75 %). Mass-spectrometry confirmed the presence of the membrane proteins in the LNVs. The abundance of proteins related to stealth properties, cancer targeting, endothelial adhesion and immune-related markers was significantly different in T-LNVs and M1-LNVs. Cell culture studies showed that M1-LNVs possessed higher cancer cell targeting, uptake and cytotoxicity compared to T-LNVs. In vivo studies performed with zebrafish embryos showed that M1-LNVs yielded higher cancer cell targeting and cytotoxicity while systemic cytotoxicity was lower compared to free DOXO. These findings confirm the potentiality and versatility of M1-LNVs for cancer treatment, which could be exploited as new avenue of nanoparticles-based therapies for precision medicine., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Edoardo D’Angelo reports administrative support, article publishing charges, and equipment, drugs, or supplies were provided by University of Padua Department of Surgical Oncological and Gastroenterology Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper]., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer., Methods: Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry., Results: In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p = .008) and tumor-infiltrating lymphocyte infiltration was higher (p = .02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p = .001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p = .027 and p = .034, respectively)., Conclusions: These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC., (© 2024 American Cancer Society.)

Background & Aims: Multiple colorectal adenomas (MCRAs) can result from APC (AFAP) or biallelic MUTYH (MAP) mutations, but most patients are wild type and referred to as non-APC/MUTYH polyposis (NAMP). We aim to examine the risk of colorectal cancer (CRC) and the role of endoscopy in managing patients with MCRAs, with a specific focus on clinical features and genotype., Methods: Records of MRCAs between 2000 and 2022 were retrospectively analysed. Patients were divided according to the genotype (MAP vs. NAMP) and the number of categorised polyps' burden (group 1: 10-24, group 2: 25-49, and group 3: 50-99 adenomas). Predictors of outcome were CRC-free survival (CRC-FS) and Surgery free-survival (S-FS)., Results: 220 patients were enrolled (NAMP n = 178(80.0%)). CRC at diagnosis was more frequent in group 3 (p = 0.01), without significant differences between the genotypes (p = 0.20). At a follow-up of 83(41-164) months, 15(7%) patients developed CRC during surveillance. CRC-FS was not correlated to genotype (p = 0.07) or polyps' number (p = 0.33), while S-FS was similar in MAP and NAMP (p = 0.22) and lower in groups 2 and 3 (p = 0.0001)., Conclusions: MAP and NAMP have the same CRC risk and no difference in treatment. Endoscopic surveillance compared favorably with surgery in avoiding CRC risk, even in patients with more severe colorectal polyposis., Competing Interests: Conflict of interest Matteo Fassan received research funding from Astellas Pharma, Macrophage Pharma, and QED Therapeutics and had roles as consultant or advisor for Astellas Pharma, GSK-Tesaro, MSD, Roche, and Astra Zeneca. The other authors have no conflicts of interest to declare regarding the present manuscript., (Copyright © 2023 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

The targeted analysis of free fatty acids (FFAs) is attracting interest since several years with a plenty of studies. However, most of them are devoted to the solely determination of the short-chain fatty acids (SCFAs) arising from the symbiotic gut microbiota metabolism. Recently, the FFAs analysis highlighted changes in the plasma levels of octanoic and decanoic acids (medium-chain fatty acids or MCFAs) may be associated to gastrointestinal diseases, including colorectal cancer (CRC). Then, the simultaneous quantification of both SCFAs and MCFAs could be useful to put in evidence the interconnection between microbiota and metabolic alterations during hosts' disease. To this aim, it was developed an isotopic dilution gas-chromatography coupled mass spectrometry (ID/GC-MS) method for the targeted analysis of both linear and branched FFAs (SCFAs, MCFAs, and LCFAs) in human plasma samples as specific markers for both microbiota and host metabolic alterations. In order to minimize sample manipulation procedures, an efficient, sensible and low time-consuming procedure is presented, which relies in a simple liquid-liquid extraction before the determination of underivatized free acids (FFAs) by Single Ion Monitoring (SIM) acquisition. The reached detection limits (LODs) were less than 100 μg L -1 for most of analytes, except for acetic, hexadecanoic and octadecanoic acids that showed a LOD > 1 mg L -1 . Methods accuracy and precision, obtained by the analysis of the FFAs mixtures showed accuracy values between 84% and 100% and precision (RSD %) between 0.1% and 12.4% at the concentration levels tested. The proposed ID/GC-MS method was applied in a case study to evaluate the FFAs as specific markers for both microbiota and host alterations in CRC patients. Obtained results highlight the advantage of present method for its rapidity, simplicity, and robustness., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: An increased risk of metachronous colorectal cancer is usually associated with microsatellite instability occurring in Lynch syndrome. However, not all patients with metachronous colorectal cancer have microsatellite instability. The density of tumor-infiltrating lymphocytes is an independent predictor of outcome in patients with colorectal cancer, and a fascinating hypothesis is that they can be involved in the onset of metachronous colorectal cancer. The aim of this study was to analyze the tumor microenvironment and tumor mutation frequency in sporadic and metachronous colorectal cancer., Methods: The clinical and pathological records of a series of consecutive colorectal cancer patients who were operated on from 2015 to 2019 were retrieved for this retrospective study. We defined metachronous colorectal cancer as a second colorectal cancer that appeared at least 1 year after the primary one, and sporadic colorectal cancer as those that did not have a metachronous colorectal cancer. Histology for the infiltration of intratumoral lymphomononuclear cells, immunohistochemistry for MLH1, PMS2, MSH2, and MSH6, and mutational analysis of BRAF, KRAS, and NRAS were all performed. Sporadic colorectal cancer and metachronous colorectal cancer were compared. Nonparametric tests were used for small sample size comparison., Results: In the study, 238 patients were operated on for colorectal cancer at the General Surgery Unit of the Azienda Ospedaliera di Padova from 2015 to 2019. We identified 26 patients with metachronous colorectal cancer, and only 3 of them had had adjuvant therapy after the primary colorectal cancer. No difference was observed in terms of cancer stage between metachronous and sporadic colorectal cancer. Mismatch repair gene deficiencies and microsatellite instability frequency was similar in metachronous colorectal cancer and in sporadic colorectal cancer (P = .77). Likewise, the mutation frequency of BRAF and KRAs was similar in the 2 groups (P = .75 and P = .21, respectively). To the contrary, the absence of infiltration of lymphomononuclear cells within the tumor (P = .004) in patients with metachronous colorectal cancer was more frequent and they tended to have a higher frequency of NRAS mutation (P = .06)., Conclusion: Our study showed that, rather unexpectedly, microsatellite instability frequency was similar in metachronous and sporadic colorectal cancer. Moreover, our data suggest that an altered immune microenvironment may be a crucial factor, permitting the occurrence of metachronous colorectal cancer. In fact, the absence of lymphomononuclear cells can be the substrate for a weak immune response to cancer neoantigens, opening the way to a second primary colorectal cancer., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance., Methods: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines., Results: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines., Conclusions: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell lines, animal models and clinical research. Here, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D-bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural and secreted proteins. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy tissue, caused by direct effect of DEFA3. We demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings., (© 2017 Wiley Periodicals, Inc.)

Colorectal cancer (CRC), the third most common cancer diagnosed in both men and women in the United States, shows a highly ineffective therapeutic management. In these years neither substantial improvements nor new therapeutic approaches have been provided to patients. Performing the early lead discovery phases of new cancer drugs in cellular models, resembling as far as possible the real in vivo tumor environment, may be more effective in predicting their future success in the later clinical phases. In this review, we critically describe the most representative bioengineered models for anticancer drug screening in CRC from the conventional two-dimensional models to the new-generation three-dimensional scaffold-based ones. The scaffold aims to replace the extracellular matrix, thus influencing the biomechanical, biochemical, and biological properties of cells and tissues. In this scenario, we believe that reconstitution of tumor condition is mandatory for an alternative in vitro methods to study cancer development and therapeutic strategies., (© 2018 Wiley Periodicals, Inc.)

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C>T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C>T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Colorectal cancer (CRC) whit more than a million of new cases per year is one of the most common registered cancers worldwide with few treatment options especially for advanced and metastatic patients.The tumor microenvironment is composed by extracellular matrix (ECM), cells, and interstitial fluids. Among all these constituents, in the last years an increased interest around the ECM and its potential role in cancer tumorigenesis is arisen. During cancer progression the ECM structure and composition became disorganized, allowing cellular transformation and metastasis. Up to now, the focus has mainly been on the characterization of CRC microenvironment analyzing separately structural ECM components or cell secretome modifications. A more extensive view that interconnects these aspects should be addressed. In this review, biochemical (secretome) and biomechanical (structure and architecture) changes of tumor microenvironment will be discussed, giving suggestions on how these changes can affect cancer cell behavior. J. Cell. Physiol. 232: 967-975, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Circulating cell-free DNA (cfDNA) was found in increased amounts in cancer patients and tumor-associated molecular alteration can be detected in cancer patient's samples. For this reason, the cfDNA analysis is actually considered as a new concept of liquid biopsy. We evaluated the presence and integrity of plasma cfDNA by ALU-based qPCR and the methylation profile of OSMR and SFRP1 genes promoter in a large cohort of colorectal cancer (CRC) patients (n = 114) in comparison to healthy subjects (n = 56) and patients with adenomatous lesions (n = 22). Moreover, we studied the prognosis value focusing on histopathological staging and survival. The cfDNA concentration and the integrity index were increased in CRC patients. The ALU83 and ALU244 fragment dosage showed a moderate discriminant capacity between CRC patients and controls and CRC and adenoma patients. Especially, cfDNA was significantly higher in CRC patients at advanced histopathological stage. In addition, the increased cfDNA level was associated with poor prognosis. A comparison of methylation profile in matched tissue and plasma on 25 CRC patients was performed and only three mismatched cases were observed. A lower methylation quantification was observed in cfDNA than tissue DNA. The cfDNA methylation frequency was statistically different in controls, adenoma and CRC patients and this frequency increased with the histopathological stage of tumor. The adenoma and CRC patients methylated cfDNA showed a higher quantity of ALU83 and ALU244. An integrated approach, combining the detection of ALU fragments and cancer type-specific epigenetic alteration, can improve diagnostic efficiency and better define the prognostic value for CRC disease., (© 2016 UICC.)

Colorectal cancer is characterized by aberrant Cyclooxigenase-2 (COX-2) and β-Catenin pathways. Recently, the protease inhibitor SerpinB3 has been described overexpressed in more advanced stages of this tumor. Aim of the study was to explore the possible relationship between these molecules in this setting. We evaluated colorectal cancer specimens from 105 patients and a positive correlation between SerpinB3, COX-2 and β-Catenin expression was observed, with higher levels in tumor than in adjacent tissue. The highest levels were associated with pathologic parameters of poor prognosis, including vascular invasion, lymph node metastasis and perineural invasion. The molecular and protein profiles of COX-2 and β-Catenin were analyzed in cell lines with different expression of SerpinB3. In those with high expression of SerpinB3, COX-2 and β-Catenin were higher than in controls. Cells with high levels of SerpinB3 showed higher proliferation and invasion compared to controls. In conclusion, in colorectal cancer SerpinB3, COX-2 and β-Catenin are positively correlated and associated with more advanced tumor stage. The in vitro experimental results support a driving role of SerpinB3 in the upregulation of COX-2/ β-Catenin positive loop, associated with a more aggressive cellular phenotype.

Colorectal cancer (CRC) is one of the most common tumors in developed countries. The five-year survival rate decreases depending on how advanced the CRC is when first diagnosed. Screening has been proven to greatly reduce mortality from colorectal cancer, but an ideal screening tool is far from being established. Here, we aimed to discover and validate early CRC biomarkers by means of an untargeted/targeted metabolomic approach. A preliminary untargeted analysis of plasma lipids performed on a small patient cohort (30 plasma samples) revealed some alterations that occurred in the presence of this tumor. In particular, medium-chain fatty acids with between six and twelve carbon atoms (C6-C12) were found to be the lipid class that showed the most marked changes upon the development of CRC. In order to evaluate the utility of this lipid class as diagnostic CRC biomarkers, a further study based on a wider cohort of patients (117 plasma samples) was performed. Using a targeted approach, these fatty acids were quantified in plasma samples by means of fast gas chromatography coupled to a time-of-flight analyzer. Plasma samples from patients with CRCs at different tumor stages were analyzed and compared to those from healthy subjects, ulcerative colitis patients, high-grade dysplasia adenoma patients, and breast cancer patients in order to test the specificity and sensitivity of these possible biomarkers. Results revealed significant differences among the considered groups in terms of their C6, C8, C10, and C12 fatty acid plasma concentrations. In particular, receiver operating characteristic (ROC) curves obtained for the C10 fatty acid gave an area under the curve of 0.8195 along with a sensitivity of 87.8 % and a specificity of 80 %, strongly suggesting that it could be a valuable early diagnostic biomarker of CRC.

Irinotecan is a widely used antineoplastic drug, mostly employed for the treatment of colorectal cancer. This drug is a feasible candidate for therapeutic drug monitoring due to the presence of a wide inter-individual variability in the pharmacokinetic and pharmacodynamic parameters. In order to determine the drug concentration during the administration protocol, we developed a quantitative MALDI-MS method using CHCA as MALDI matrix. Here, we demonstrate that MALDI-TOF can be applied in a routine setting for therapeutic drug monitoring in humans offering quick and accurate results. To reach this aim, we cross validated, according to FDA and EMA guidelines, the MALDI-TOF method in comparison with a standard LC-MS/MS method, applying it for the quantification of 108 patients' plasma samples from a clinical trial. Standard curves for irinotecan were linear (R (2) ≥ 0.9842) over the concentration ranges between 300 and 10,000 ng/mL and showed good back-calculated accuracy and precision. Intra- and inter-day precision and accuracy, determined on three quality control levels were always <12.8 % and between 90.1 and 106.9 %, respectively. The cross-validation procedure showed a good reproducibility between the two methods, the percentage differences within 20 % in more than 70 % of the total amount of clinical samples analysed.

Early detection of colorectal cancer (CRC) remains a challenge. It has been highlighted that the pathological alterations within an organ and tissues might be reflected in serum or plasma proteomic/peptidic patterns. The aim of the study was to follow the changes in the plasma peptides associated to colorectal cancer progression by mass spectrometry. This study included 27 adenoma, 67 CRC (n = 33 I-II stage and n = 34 III-IV stage), 23 liver metastasis from CRC patients and 34 subjects disease-free as controls. For plasma peptides analysis, samples purification was performed on the Nanoporous Silica Chips technology followed by matrix-assisted laser desorption/ionisation-time of flight analysis. Since the high complexity of the obtained dataset, multivariate statistical analysis, and discriminant pattern recognition were performed for study groups classification. Forty-four of 88 ionic species were successfully identified as fragments of peptides and proteins physiologically circulating in the blood and belonging to immune and coagulation systems and inflammatory mediators. Many peptides clustered into sets of overlapping sequences with ladder-like truncation clearly associated to proteolytic processes of both endo- and exoproteases activity. Comparing to controls, a different median ion intensity of the group-type fragments distribution was observed. Moreover, the degradation pattern obtained by proteolytic cleavage was different into study groups. This pattern was specific and characteristic of each group: controls, colon tumour disease (including adenoma and CRC), and liver metastasis, revealing a role as biomarker in early diagnosis and prognosis. Our findings highlighted peculiar changes in protease activity characteristic of CRC progression from pre-cancer lesion to metastatic disease. J. Cell. Physiol. 231: 915-925, 2016. © 2015 Wiley Periodicals, Inc., (© 2015 Wiley Periodicals, Inc.)

MiR-182 expression was evaluated by qRT-PCR and in situ hybridization in 20 tubular adenomas, 50 colorectal carcinoma (CRC), and 40 CRC liver metastases. Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor., (©2014 AACR.)

Background: Colorectal cancer (CRC) is an important cause of cancer-related death. Prediction of recurrence is an important issue in the treatment of disease, particularly for stage II patients. The level of telomere-specific reverse transcriptase (hTERT), the catalytic component of the telomerase complex, increases along with CRC progression, but its prognostic value is still unclear., Methods: One hundred and thirty-seven CRC patients were studied for hTERT expression in tumour cells by real-time PCR. hTERT level was evaluated as a prognostic factor of overall survival (OS) in all patients and of disease recurrence in a subgroup of 50 stage II patients., Results: The median hTERT level was 93.8 copies (interquartile range 48-254). Patients with high hTERT levels (above the median) showed a significantly worse survival than those with low hTERT levels (below the median; log-rank test P<0.0001; hazard ratio (HR)=3.30 (95% confidence interval (CI) 1.98-5.52); P<0.0001). The negative prognostic value of high hTERT level is independent of the pathological stage and microsatellite instability (HR=2.09 (95% CI 1.20-3.64), P=0.009). Moreover, in stage II CRC, high hTERT levels identified patients with a higher risk of disease recurrence (HR=3.06 (95% CI 1.03-9.04), P=0.043) and death (HR=3.24 (95% CI 1.37-7.71), P=0.008)., Conclusion: hTERT level is an independent prognostic marker of OS in CRC patients. In addition, assessment of hTERT level could improve stratification of stage II CRC patients for the risk of disease recurrence.

Purpose: Germline nonsense and frameshift mutations in the adenomatous polyposis coli (APC) gene are found in approximately 90% of individuals affected by familial adenomatous polyposis (FAP) and a genotype-phenotype relationship has been observed. Missense mutations have also been found in a few cases, even if their role in FAP is still unknown. An association between a missense mutation, APC I1307K, and the risk of sporadic colorectal cancer (CRC) has been reported. In order to improve the knowledge about the genetic effect of APC I1307K on the phenotype, we tried a new approach using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS)., Experimental Design: An APC mutation (I1307K) was found in an index case of a non-Jewish woman and her son with attenuated familial adenomatous polyposis (A-FAP) and no family history of cancer. In order to evaluate whether the presence and abundance of the ionic species are related to the presence of cancer or the presence of mutation, comparative analyses of 11 healthy clean-colon subjects, 59 patients with CRC (stage II n=19, stage III n=23, stage IV n=17) without polyps, and 9 FAP patients, carriers of a nonsense mutation in the APC gene, were evaluated., Results: Comparative analysis of serum protein profiles of the index patient and her healthy son, FAP and sporadic CRC patients, and subjects with preneoplastic lesions showed a characteristic abundance of ionic species at m/z 905, which was not present in healthy controls. Two peptides were identified from MALDI/MS/MS spectra of m/z 905 belonging to the kininogen-1 precursor and the human forkhead box protein 01A (FOXO1A). FOXO1A was present in only two subjects carrying I1307K, but not in other patients., Conclusions: Our findings seem to suggest a relationship between m/z 905, FOXO1A and the development and growth of colorectal cancer. FOXO1A fragment determination in serum with MALDI/MS might be a promising approach for early detection of colon carcinoma or for the development of targeted therapies.

Purpose: The risk of cancer or severe polyposis of the rectal stump after total colectomy for MutYH-associated polyposis is scarcely defined. To evaluate this risk, we describe the findings of endoscopic surveillance of the rectal stump in a series of patients with biallelic MutYH mutations and polyposis., Methods: This is a retrospective, observational, multicenter case series derived from 2 familial cancer registries. Biallelic, germ-line MutYH mutations were found in 14 patients with no adenomatous polyposis coli gene mutations. Eleven of them underwent total colectomy with ileorectal anastomosis and yearly proctoscopic surveillance thereafter. Phenotype and histology of rectal polyps were recorded at diagnosis and during follow-up. Development of adenomas and carcinomas during endoscopic surveillance of the rectal stump was observed., Results: At diagnosis, 6 patients had attenuated polyposis (10-100 adenomas), 5 patients had classical polyposis, 8 patients had colon carcinoma, and no patient had rectal carcinoma. The mean number of rectal polyps at diagnosis was 2.64 ± 2.11 (range, 0-6). No patients had rectal cancer. The most frequent MutYH mutations were Y165C/Y165C and G382D/G382D in 6 and 2 patients, respectively. During surveillance of the rectal stump after surgery (median duration, 5 y; range, 2-23 y), no patient developed rectal cancer. The mean number of adenomas per proctoscopy was 1.23 ± 2.19 (range, 0-10 adenomas per proctoscopy). This study was limited by the small size and retrospective nature of the case series., Conclusion: Total colectomy with ileorectal anastomosis may be appropriate for patients with MutYH-associated polyposis, provided that they have no rectal cancer or severe rectal polyposis at presentation and that they undergo yearly endoscopic surveillance thereafter.

Purpose: Colorectal cancer (CRC) is one of the most common cancers in western countries. Identification of circulating markers for CRC would optimize early stage diagnosis and the monitoring for disease recurrence. Expression of telomerase reverse transcriptase (hTERT) is essential to the oncogenic process and might be used as a molecular marker of neoplastic disease., Experimental Design: Eighty-five CRC samples (25 stage I, 15 stage II, 15 stage III, and 30 stage IV), the available corresponding noncancerous mucosa (n = 42), and plasma collected at the time of surgery (n = 49) were analyzed. Control plasma samples were obtained from 43 age-matched healthy subjects. All hTERT transcripts (hTERT-AT) and transcripts encoding the functional protein (hTERT-FL) were quantified by real-time PCR., Results: hTERT-AT was found to correlate with hTERT-FL (r = 0.849; P < 0.0001) mRNA levels in tumors. Both hTERT mRNAs were significantly higher in tumors than in adjacent noncancerous mucosa and both significantly increased with tumor progression (P < 0.0001). In contrast to controls, all but two plasma samples from CRC patients were positive for hTERT mRNAs. Using the cutoff value of 180 copies hTERT-AT/mL, the sensitivity and specificity of the assay for CRC detection were 92% and 100%, respectively. Furthermore, hTERT-AT mRNA levels in plasma significantly correlated with hTERT-AT mRNA levels in tumors (r = 0.702, P < 0.0001)., Conclusions: These findings indicate that quantification of hTERT mRNA in plasma may be used as a marker for detection and monitoring of neoplastic colorectal disease.

Biallelic germline mutations in the MYH gene cause MYH-Associated Polyposis but patients with a single mutation possibly have an increased colorectal cancer (CRC) risk. Using DNA from consecutive CRC patients we carried out a case-control study, with the aim to contribute data on the Italian population. Genotyping of four MYH mutations found two biallelic and two monoallelic carriers among 439 cases, and only one heterozygous individual among 247 age-matched controls. The frequencies of the mutant alleles were 0.68% (6/878) and 0.20% (1/494), respectively. These differences were not statistically significant. Results on the monoallelic carriers were combined with those from 11 studies on other populations, and the risk of developing a CRC was estimated with an OR=1.11 (95% CI=0.90; 1.36), yet not reaching a significant evidence of increased CRC risk.

In an investigation devoted to the search for plasma markers for colorectal cancer (CRC), carried out by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a series of overexpressed peptides were identified in the plasma of patients. Among them the peptide with molecular weight 903 Da was the most abundant one, with a mean +/- (SD) relative abundance of 37 +/- 17% and a frequency over 60%. Interestingly, also in plasma samples of ten subjects affected by familial adenomatous polyposis (FAP), the peptide with molecular weight 903 was overexpressed. In this investigation, MALDI/MS/MS experiments were carried out on the ion at m/z 904 detected in the MALDI mass spectra of CRC and FAP patients. The data analysis by SwissProt.2007.01.09 indicates that this peptide is due to the sequence RPPGFSPF, found in the kininogen-1 precursor, which is an alpha-2-thiol proteinase inhibitor. In the case of subjects affected by a particular FAP syndrome, the MALDI/MS/MS spectra were quite different from those obtained from CRC and FAP patients. In fact, two sequences have been evidenced: RPPGFSPF belonging to kininogen-1 precursor, and PRKSSSSR belonging to Forkhead box protein 01A.

The aim of the present study was to identify the pattern of plasma protein species of interest as markers of colorectal cancer (CRC). Using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), the plasma protein profile was determined in nine stage IV CRC patients (study group) and nine clean-colon healthy subjects (control group). Multivariate analysis methods were employed to identify distinctive disease patterns at protein spectrum. In the study and control groups, cluster analysis (CA) on the complete MALDI-MS spectra plasma protein profile showed a distinction between CRC patients and healthy subjects, thus allowing the identification of the most discriminating ionic species. Principal component analysis (PCA) and linear discriminant analysis (LDA) yielded similar grouping results. LDA with leave-one-out cross validation achieved a correct classification rate of 89% in both the patients and the healthy subjects.

Purpose: To analyze the genetic variability in a variable number of tandem repeats (VNTR) in the thymidylate synthase (TS) enhancer promoter region and assess the influence of functional alterations in mismatch repair genes by analyzing constitutional and tumoral DNA from patients with colorectal adenocarcinoma with a high microsatellite instability (MSI-H) or microsatellite stability (MSS) status., Patients and Methods: Patients who underwent surgery for colorectal adenocarcinoma were selected from the colorectal database of our institute and, on the basis of MSI status, assigned to a study group and a control group: group A, MSI-H; group B, MSS. Microsatellite status was investigated using the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, D17S250). In MSI-H patients an additional analysis was made of the microsatellite loci D18S61 and D18S58, both mapping in the region containing the TS gene (18p11.2-11.32). Based on the number of altered microsatellites (> or = 2, 1, or 0), tumors were considered as having high (MSI-H) or low (MSI-L) instability or microsatellite stability (MSS), respectively. Genotyping for thymidylate synthase promoter polymorphism was carried out on constitutional and tumor DNA of each patient by PCR amplification of the polymorphic region., Results: MSI-H was found in 55 patients (group A) and MSS in 50 patients (group B). In none of the MSI-H patients was microsatellite instability found in the additional D18S61 and D18S58 loci. In five group A and ten group B cases the analysis was not performed because constitutional DNA and/or tumoral DNA were not amplifiable. Homozygotes for the triple repeat variant (3R/3R) displayed only the large PCR product, homozygotes for the double repeat variant (2R/2R) displayed only the smaller PCR product, while heterozygotes (2R/3R) displayed both the larger and smaller PCR products. In 3/50 (6%) group A patients and 5/40 (12%) group B patients repeat variations were found in tumoral DNA., Conclusion: Our findings demonstrate that there is genetic homogeneity between constitutional and tumoral DNA but do not support the hypothesis that mismatch repair genes are involved in VNTR recombinant events in TS gene variability.

Purpose: Early-age-at-onset colorectal cancer and microsatellite instability are characteristic features of hereditary nonpolyposis colorectal cancer. Our aim was therefore to investigate whether these features might be useful markers in screening for hereditary nonpolyposis colorectal cancer and mismatch repair gene mutations., Methods: From 1,132 consecutive patients who underwent surgery for colorectal cancer at our department between 1980 and 1999, we selected all patients 40 years of age or younger (study group, n = 59) and a subset of patients 40 years of age or older (control group, n = 60) who were matched for gender and pathologic TNM stage. Patients for whom a complete family cancer history or microsatellite status was unavailable were excluded from the study. Family cancer histories, retrieved from archival charts, were reassessed. Microsatellite status was investigated with the five microsatellites from the Bethesda recommended panel (BAT-26, BAT-25, D2S123, D5S346, and D17S250). On the basis of the number of altered microsatellites (> or = 2, 1, or 0), tumors were considered as having high or low instability or microsatellite stability, respectively. Mutation analysis for MLH1 and MSH2 genes was performed only in cases of high instability. DNA was investigated for mutations by single-strand conformational polymorphism and sequencing analysis., Results: Data from 95 patients (study group: n = 37, 18 males, mean age 35 years; control group: n = 58, 29 males, mean age 62 years) were available for analysis. Four patients (study group, n = 3; control group, n = 1) fulfilled the Amsterdam II criteria for hereditary nonpolyposis colorectal cancer. Of the 37 study group tumors, 12 (32.4 percent) showed high-frequency microsatellite instability, and 25 had microsatellite stability, whereas among the 58 control group tumors, 4 (7 percent) showed high-frequency microsatellite instability, and 54 had microsatellite stability (P < 0.002). Mismatch repair gene mutation analysis was performed in 12 cases (study group, n = 7; control group, n = 5). We found four mutations (MSH2 119delG, MLH1 ex9 684insT, MSH2 Gln239Stop, and MLH1 del0.8 Kb) in the study group patients and none in the control group. Of four hereditary nonpolyposis colorectal cancer patients who underwent mismatch repair gene mutation analysis, one had a mutation., Conclusions: Early-age-at-onset colorectal cancer is significantly correlated with high-frequency microsatellite instability tumor status and is a useful criterion to identify hereditary nonpolyposis colorectal cancer patients. Moreover, when used in association with high-frequency microsatellite instability status, it is effective in selecting patients for mismatch repair gene mutation analysis.

Thirteen patients with a diagnosis of primary colorectal tumour and 68 patients previously operated for colon cancer underwent an immunoscintigraphy carried out with monoclonal antibodies anti-CEA F(ab')2, labelled with 131I or 111In. These studies led to the detection of all primary tumours and of most of their associated lesions (7/8). In this group 15 neoplastic deposits previously undetected were demonstrated, allowing an improvement in the patient staging before the operation. In the group of the 68 operated patients, immunoscintigraphy succeeded in imaging 96 out of 121 known lesions: the best outcomes were obtained for abdominal and pelvic recurrences as well as for lymph nodes lesions. The lowest levels of sensitivity were observed for liver metastases. An enhancement of the immunoscintigraphy sensitivity was obtained when the radiopharmaceutical was injected intraperitoneally. In 50 patients, this alternative administration route was successful in detecting 91/107 cancer deposits and, in particular, liver metastases were found out in 36/42 cases.