Defective generation and maturation of dendritic cells from monocytes in colorectal cancer patients during the course of disease.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in Western countries. Today, the role of the host's immune system in controlling the progression and spread of solid tumors is broadly established. Tumor immunosurveillance escape mechanisms, such as those involving dendritic cells (DCs), the most important antigen-presenting cells, are likewise recognized processes involved in cancer. The present study evaluates the ability of CRC patients to generate DCs in vitro from circulating monocytes at both pre- and post-operative timepoints; the results are correlated with the stage of disease to shed light on the systemic immune statuses of CRC patients. Our data showed that patients' DCs had lower co-stimulatory molecule expression and were less able to present antigens to allogeneic T cells compared to healthy controls' (HC) DCs. Furthermore altered cytokine secretion, such as increased IL-10 and reduced IL-12 and TNF-α, was observed. At the post-operative timepoints we observed a recovery of the patients' ability to generate immature DCs, compared to HCs, but the maturational capacity remained affected. Our study conclusively highlights the persistently impaired in vitro generation of fully mature and functional DCs, which appears to be more altered during advanced stages. This work sheds light on a dendritic cell-based tumor immune escape mechanism that could be useful for the development of more effective immunotherapeutic strategies.

Efficacy and feasibility of the epithelial cell adhesion molecule (EpCAM) immunomagnetic cell sorter for studies of DNA methylation in colorectal cancer.

The aim of this work was to assess the impact on measurements of methylation of a panel of four cancer gene promoters of purifying tumor cells from colorectal tissue samples using the epithelial cell adhesion molecule (EpCAM)-immunomagnetic cell enrichment approach. We observed that, on average, methylation levels were higher in enriched cell fractions than in the whole tissue, but the difference was significant only for one out of four studied genes. In addition, there were strong correlations between methylation values for individual samples of whole tissue and the corresponding enriched cell fractions. Therefore, assays on whole tissue are likely to provide reliable estimates of tumor-specific methylation of cancer genes. However, tumor cell tissue separation using immunomagnetic beads could, in some cases, give a more accurate value of gene promoter methylation than the analysis of the whole cancer tissue, although relatively expensive and time-consuming. The efficacy and feasibility of the immunomagnetic cell sorting for methylation studies are discussed.

Genetics, cytogenetics, and epigenetics of colorectal cancer.

Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80-85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype.

The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches.

BACKGROUND: Because colorectal cancer is a significant cause of morbidity and mortality in the Western population, knowledge of the molecular and biological alterations associated with its development is important. Since primary human colon cancer cultures from fresh tumor tissue are technically difficult to obtain, experiments in most laboratories are performed on colon epithelial cell lines, but these represent just one stage of tumor progression. Only primary cultures of neoplastic colonocytes may reflect the actual responsiveness of tumors at certain developmental stages to antitumor agents. METHODS: This paper analyzes several critical points concerning primary cultures, ranging from cell isolation to culture conditions, and compares different methodological approaches to isolate and cultivate a pure fraction of viable tumor cells. Samples of resected colorectal cancers were collected from 20 patients (stage T3 or T4). We compared in vitro several approaches of tissue disaggregation including mechanical disaggregation and enzymatic dissociation with trypsin or collagenase. Isolated cells were maintained in a short-term serum-free culture system. Evaluation of the purity and tumoral nature of isolated cells was performed by immunochemistry. RESULTS: We established the antibiotic concentration necessary during transport and washing of the specimens to prevent microbial overgrowth. We demonstrated that the number of viable cells was dependent on the dissociation method used. Mechanical disaggregation is not a valid dissociation method because of the high mortality of cells and might be used only in samples for molecular analysis. Comparison of the enzymatic digestion procedures showed that digestion with trypsin allowed the highest recovery of viable cells. CONCLUSION: In this paper we analyzed several critical aspects of cell culture procedures and designed a methodological approach suitable for functional studies of colorectal cancer.

[Colorectal cancer: tissutal explantation and primary cell culture]. / Carcinoma colon-rettale: espianto tissutale e colture primarie.

BACKGROUND: Setting of cellular cultures extracted from colorectal cancer tissue represents a valid model for in vitro study of biological and molecular characteristics of each single tumor finalized to obtain a tailored chemiotherapy. The end point of this study is to create primary cellular cultures from "fresh" cancer tissue in different stages of evolution. METHODS: Cancer tissue samples are obtained by means of surgical excisional biopsy or by means of semi-automatic biopsy instrument (Sprig-Cut). After having compared different approaches, two experimental protocols have been selected to have the highest number or intact cells: enzimatic digestion with trypsin and explantation. RESULTS AND CONCLUSIONS: Primary cell culture free of microbic contamination, obtained mainly by means of Spring-Cut methods, underwent immunohistochemical analysis to evaluate what kind of cell have been grown in vitro by measuring the expression of CK20 and GFAP both resulted positive. The possibility of setting a primary cell culture which represents the cancer of each patient allows a pharmacologic and biomolecular study which can contribute to the development of a tailored adjuvant therapy with many advantages for the patient in terms of positive answer to the treatment and reduced toxicity.

Investigation of GHSR and GHRL methylation in colorectal cancer.

AIM: To investigate GHSR and GHRL methylation in 73 pairs of colorectal cancer (CRC) tissues and healthy adjacent mucosa. METHODS: Methylation was assessed with methylation-sensitive high-resolution melting. RESULTS: GHSR was significantly hypermethylated in CRC tissues than in healthy mucosa (p < 1 × 10-5), but no significant changes of GHRL methylation were observed. GHSR hypermethylation was already detectable at the adenoma stage and maintained in later stages independently of age, gender, anatomical location, histological grading, MLH1 deficiency, as well as of major polymorphisms in folate-pathway genes, yielding an area under the curve of 0.824 for discriminating cancers from respective non-neoplastic mucosa specimens. CONCLUSION: GHSR hypermethylation occurs early in CRC, but is not paralleled by significant changes of GHRL methylation.

Duodenal Crohn's disease.

The Authors report on an uncommon case of duodenal Crohn's disease in an adult man. The patient was admitted for a history of epigastric pain, recurrent vomiting, weight loss and low grade fever. He was evaluated with esophagogastroduodenoscopy and with radiological double-contrast technique. Then, due to these untreatable clinical manifestations, he underwents a surgical treatment.

Use of virtual reality simulator in the training of postgraduated surgical residents.

AIM OF THE STUDY: The purpose of this research project, granted by the Italian University and Scientific Research Ministry (MIUR) and carried on among four Surgical Departments in Padua, Verona, Pisa and Rome-Tor Vergata Universities, is to study the effectiveness of a virtual reality simulator as a tool for surgical residents training and as a method for measuring the surgical skills. MATERIALS AND METHODS: The residents performances on the computer were compared with those obtained by other training groups: medical students with no surgical background, senior surgeons with experience in the laparoscopy field and non medical students with referred ability in videogames. The residents were also sent to a well certified live animal laboratory where they could perform a cholecystectomy in a pig. Their operation was assessed by two independent observers using a new scoring methods for assessing the operative errors. RESULTS: Statistically significant differences among the groups could be seen only after several tasks sessions and in the more surgical specific tasks. The first data analysis shows a fair correlation between the residents rank positions after the training tasks on the simulator and those obtained in the surgical operation. CONCLUSION: Even if the data are not conclusive and the surgical simulators must develop greater levels of fidelity and operational diversity, the potential value of simulation in the educational surgical field appears to be impressive.

Application of artificial neural networks to link genetic and environmental factors to DNA methylation in colorectal cancer.

AIMS: We applied artificial neural networks (ANNs) to understand the connections among polymorphisms of genes involved in folate metabolism, clinico-pathological features and promoter methylation levels of MLH1, APC, CDKN2A(INK4A), MGMT and RASSF1A in 83 sporadic colorectal cancer (CRC) tissues, and to link dietary and lifestyle factors with gene promoter methylation. MATERIALS & METHODS: Promoter methylation was assessed by means of methylation-sensitive high-resolution melting and genotyping by PCR-RFLP technique. Data were analyzed with the Auto Contractive Map, a special kind of ANN able to define the strength of the association of each variable with all the others and to visually show the map of the main connections. RESULTS: We observed a strong connection between the low methylation levels of the five CRC genes and the MTR 2756AA genotype. Several other connections were revealed, including those between dietary and lifestyle factors and the methylation levels of CRC genes. CONCLUSION: ANNs revealed the complexity of the interconnections among factors linked to DNA methylation in CRC.

Sorafenib in the treatment of thyroid cancer.

Sorafenib has been evaluated in several Phase II and III studies in patients with locally advanced/metastatic radioactive iodine-refractory differentiated thyroid carcinomas (DTCs), reporting partial responses, stabilization of the disease and improvement of progression-free survival. Best responses were observed in lung metastases and minimal responses in bone lesions. On the basis of these studies, sorafenib was approved for the treatment of metastatic DTC in November 2013. Few studies suggested that reduction of thyroglobulin levels, or of average standardized uptake value at the fluorodeoxyglucose-PET, could be helpful for the identification of responding patients; but further studies are needed to confirm these results. Tumor genetic marker levels did not have any prognostic or predictive role in DTC patients.The most common adverse events observed included skin toxicity and gastrointestinal and constitutional symptoms. Encouraging results have also been observed in patients with medullary thyroid cancer. Many studies are ongoing to evaluate the long-term efficacy and tolerability of sorafenib in DTC patients.

Selective use of vandetanib in the treatment of thyroid cancer.

Vandetanib is a once-daily orally available tyrosine kinase inhibitor that works by blocking RET (REarranged during Transfection), vascular endothelial growth factor receptor (VEGFR-2, VEGFR-3), and epidermal growth factor receptor and to a lesser extent VEGFR-1, which are important targets in thyroid cancer (TC). It is emerging as a potentially effective option in the treatment of advanced medullary thyroid cancer (MTC) and in dedifferentiated papillary thyroid cancer not responsive to radioiodine. The most important effect of vandetanib in aggressive MTC is a prolongation of progression-free survival and a stabilization of the disease. Significant side effects have been observed with the vandetanib therapy (as fatigue, hypertension, QTc prolongation, cutaneous rash, hand-and-foot syndrome, diarrhea, etc), and severe side effects can require the suspension of the drug. Several studies are currently under way to evaluate the long-term efficacy and tolerability of vandetanib in MTC and in dedifferentiated papillary TC. The efficacy of vandetanib in patients with MTC in long-term treatments could be overcome by the resistance to the drug. However, the effectiveness of the treatment could be ameliorated by the molecular characterization of the tumor and by the possibility to test the sensitivity of primary TC cells from each subject to different tyrosine kinase inhibitor. Association studies are evaluating the effect of the association of vandetanib with other antineoplastic agents (such as irinotecan, bortezomib, etc). Further research is needed to determine the ideal therapy to obtain the best response in terms of survival and quality of life.

Genetic and epigenetic biomarkers for diagnosis, prognosis and treatment of colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer worldwide and results from the accumulation of mutations and epimutations in colonic mucosa cells ultimately leading to cell proliferation and metastasis. Unfortunately, CRC prognosis is still poor and the search of novel diagnostic and prognostic biomarkers is highly desired to prevent CRC-related deaths. The present article aims to summarize the most recent findings concerning the use of either genetic or epigenetic (mainly related to DNA methylation) biomarkers for CRC diagnosis, prognosis, and response to treatment. Recent large-scale DNA methylation studies suggest that CRC can be divided into several subtypes according to the frequency of DNA methylation and those of mutations in key CRC genes, and that this is reflected by different prognostic outcomes. Increasing evidence suggests that the analysis of DNA methylation in blood or fecal specimens could represent a valuable non-invasive diagnostic tool for CRC. Moreover, a broad spectrum of studies indicates that the inter-individual response to chemotherapeutic treatments depends on both epigenetic modifications and genetic mutations occurring in colorectal cancer cells, thereby opening the way for a personalized medicine. Overall, combining genetic and epigenetic data might represent the most promising tool for a proper diagnostic, prognostic and therapeutic approach.

Dendritic cell defects in the colorectal cancer.

Colorectal cancer (CRC) results from the accumulation of both genetic and epigenetic alterations of the genome. However, also the formation of an inflammatory milieu plays a pivotal role in tumor development and progression. Dendritic cells (DCs) play a relevant role in tumor by exerting differential pro-tumorigenic and anti-tumorigenic functions, depending on the local milieu. Quantitative and functional impairments of DCs have been widely observed in several types of cancer, including CRC, representing a tumor-escape mechanism employed by cancer cells to elude host immunosurveillance. Understanding the interactions between DCs and tumors is important for comprehending the mechanisms of tumor immune surveillance and escape, and provides novel approaches to therapy of cancer. This review summarizes updated information on the role of the DCs in colon cancer development and/or progression.

Quantification of blood dendritic cells in colorectal cancer patients during the course of disease.

Colorectal cancer is a malignancy with poor prognosis that might be associated with defective immune function. The aim of the present study was to investigate circulating dendritic cells in colorectal cancer patients, in order to contribute to elucidate tumor-escape mechanisms and to point out a possible correlation with the clinical condition of the disease. Therefore, we enumerated ex vivo myeloid and plasmacytoid dendritic cells, through multicolor flow cytometry, in 26 colorectal patients and 33 healthy controls. Furthermore we performed several analyses at determined time points in order to define the immunological trend of cancer patients after surgery and other conventional treatments. At the pre-operative time point the absolute number of plasmacytoid dendritic cells in cancer patients was significantly reduced in comparison to controls, this result being mainly referred to stage III-IV patients. The number of myeloid dendritic cells did not show any significant difference compared to healthy controls; interestingly the expression of the tolerogenic antigen CD85k was significantly higher on cancer patients' myeloid dendritic cells than controls'. At the following samplings, circulating dendritic cell absolute number did not show any difference compared to controls. Conclusively the impairment of the number of circulating dendritic cells may represent one of the tumor escape mechanisms occurring in colorectal cancer. These alterations seem to be correlated to cancer progression. Our work sheds light on one of dendritic cell-based tumor immune escape mechanisms. This knowledge may be useful to the development of more effective immunotherapeutic strategies.

Sentinel node radioguided biopsy in surgical management of the medullary thyroid carcinoma A case report.

INTRODUCTION: Medullary thyroid cancer is a rare carcinoma. Surgery is the only curative treatment and since cervical lymphnodes metastases are frequent and can occur at an early stage, a standardized central lymphnode dissection is associated to total thyroidectomy. However, the extent of lymphadenectomy to the lateral neck lynphnodes remains debated. To reduce the extent of lymphnode excision, the sentinel node biopsy has been used as an accurate technique to assess the status of the lymphnodes in the regional drainage basin in solid tumors, and more recently, in thyroid carcinoma. In this case report, we show the utility of the radioguided biopsy of the sentinel lymphnode in the surgical management of the medullary thyrod cancer. CASE REPORT: We present the case of a 24-year-old Caucasian, Italian woman with a sporadic medullary thyroid microcarcinoma occasionally detected by neck ultrasound and diagnosed by high serum calcitonin level and fine needle aspiration cytology. There was no ultrasound evidence of lymphnode involvement both in central and lateral compartment of the neck. We performed a preoperative mapping of the the sentinel lymphnodes by the injection of technetium-99m radiolabelled albumin nanocolloids in the thyroid nodule. Then our patient underwent total thyroidectomy combined with radioguided biopsy of the sentinel lymphnodes. Histology confirmed the presence of the medullary thyroid cancer and revealed micrometastases only in two sentinel lymphnodes detected in right lateral compartment of the neck so an ipsilateral lateral neck dissection besides the central neck dissection was performed at the end of operation. Basal and pentagastrin-stimulated serum calcitonin level was undetectable during the follow-up investigations. CONCLUSION: This is the first reported case that shows the utility of the radioguided SLN biopsy for the accurate staging of the cervical lymphnode involvement in patient with sporadic medullary thyroid microcarcinoma. Total thyrodectomy and central neck dissection is recommended for all patients with medullary thyroid carcinoma, but the indication for the lateral neck dissection is still controversial. The radioguided SLN biopsy technique could be a useful tool to perform the dissection only in those patients with proven lateral neck lymphnode involvement and reduce the extention of the lateral lymphnode excision and the incidence of related complications.

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: correlation with physiological and pathological characteristics, and with biomarkers of one-carbon metabolism.

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.

Radioguided localization of occult colonic lesions during open or laparoscopic surgery: an update.

The applications of radioguided surgery, an approach to oncologic surgery involving a multidisciplinary team, are expanding at a rapid pace. The technique of radioguided occult lesion localization (ROLL) was originally introduced in the mid- 90s for applications in breast surgery, and later adapted also to other tumor lesions such as solitary pulmonary nodules (during either open or laparoscopic surgery) and colonic lesions. Concerning the latter, in particular, the technique called radioguided occult colonic lesion identification (ROCLI) consists of identifying, with the aid of intraoperative gammaprobe counts, small lesions that may escape colic intraoperative palpation, after prior tagging of the lesions performed endoscopically through peri- or intra-lesional injection of Technetium-99m-labeled human albumin macroaggregates (99mTc- MAA), a particulate radiopharmaceutical (25-100 µm) that does not migrate from the site of interstitial administration. Since September 2001, ROCLI has been employed in 12 patients, using a collimated gamma- probe measuring 11 mm in external diameter (Scintiprobe MR100 Pol.Hi.Tech.). All patients underwent preoperative colonoscopy in order to inject 0.2 mL of a 99mTc-MAA suspension (10-20 MBq) into the submucosa or intra-perilesionally; such tagging required only a few minutes. Eight of the 12 patients were then submitted to open laparotomy, while laparoscopic access was utilized in the remaining 4 patients. In all 12 patients, localization of the lesion with the ROCLI technique was technically feasible, safe, efficient and highly accurate, enabling quick detection of the lesion during surgery, with a 100% success rate. No complications occurred, and there was no risk of contamination by ionizing radiation.

Colorectal carcinoma and folate.

More than a million people a year worldwide develops colorectal cancer (CRC), with a mortality rate close to 33%. Most of the CRC cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CRC arises from an accumulation of genetic and epigenetic alterations such as DNA methylation, which is able to modulate gene expression. Several studies in the literature show a possible correlation between an altered methylation in the promoter of tumor suppressor genes, proto-oncogenes, genes involved in DNA repair and the CRC risk; it has also been observed a global DNA hypomethylation, especially in the presence of a low folate uptake. Epigenetic changes are reversible, then could be interesting to evaluate on their relationship with dietary factors (as well as folates) and the genetic background of the individuals, for the development of novel strategies for cancer prevention.

Comparison study of MS-HRM and pyrosequencing techniques for quantification of APC and CDKN2A gene methylation.

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing.