Ex-vivo characterization of circulating colon cancer cells distinguished in stem and differentiated subset provides useful biomarker for personalized metastatic risk assessment.

BACKGROUND: Circulating tumor cells (CTCs) represent one of the most interesting target in improving diagnosis, prognosis and treatment. Herein we evaluate the possibility of using an emo-cytometric approach on the evaluation of the heterogeneous population of CTCs to improve personalized metastatic risk assessment. We benchmarked ex vivo behavior of distinct subsets of circulating colon tumor cells with correspondent clinical behavior of patients from which we isolated CTCs. METHODS: Isolation and CTC expansion were performed by a gradient protocol. In vitro characterization was determined by flow cytometry, immunofluorescence, western blotting and proteomic profiling. Cell sorter was performed with immunomagnetic beads. Confocal microscopy was used to evaluate tissue sections. Kaplan Mayer curves was cared for through Medcalc program. RESULTS: We collected heterogeneous CTCs, derived from the whole blood of seven patients affected by colon cancer, expressing CD133(pos)CD45(neg) (5 ± 1) and (2 ± 1) and CK20(pos)CD45(neg) of (29 ± 3) (11 ± 1) cells/ml in Dukes D and A stage respectively. Proliferation rate of 57 ± 16 %, expression for CXCR4(pos) of 18 ± 7 % and detectable levels of IL-6, IL-8 and SDF-1 cytokines in conditioned culture medium characterized short-time expanded-CTCs (eCTCs). ECTCs organized in tumor sphere were CD45(neg)CD133(pos) while in adhesion were CXCR4(pos)CK20(pos). These two subsets were separately injected in mice. The first group of xenografts developed superficial lesions within 2 weeks. In the second group, in absence of growing tumour, the survival of injected eCTCs was monitored through SDF-1 serum levels detection. The detection of human cancer cells expressing CK20, in mice tissues sections, suggested a different biological behaviour of injected eCTC-subsets: tumorigenic for the first and disseminating for the second. The benchmarking of the experimental data with the clinical course highlights that patients with prevalence of circulating cancer stem cells (CD45(neg)CD133(pos)) have a lower overall survival. Conversely, patients with prevalence of circulating differentiated cells (CXCR4(pos)CK20(pos)) have a low disease-free survival. CONCLUSION: On the basis of the heterogeneous composition and despite the low number of CTCs, it was possible to distinguish two subgroups of CTCs, suggesting a different clinical outcome. CTC-subsets detailing is useful to better define the metastatic-risk personalized score thus improving disease management and reducing patient care cost.

Nasopharyngeal carcinoma: review of the literature with a focus on therapeutical implications.

Nasopharyngeal carcinoma (NPC) is a rare neoplasm which is associated with Epstein-Barr virus (EBV). First of all, we reviewed the literature on NPC treatment. Radio/chemotherapy is currently the gold standard but unfortunately is affected by rates of failure ranging from 7% up to 58%. Because NPC development is promoted by the EBV latent life cycle, EBV-targeted treatments were investigated. Firstly, forcing cytolytic virus activation through administration of gemcitabine and/or valproic acid before administration of a nucleoside analogue showed anti-tumoral activity in vitro as well as in murine model and it was also well tolerated in humans. Secondly, the association of autologous EBV-specific cytotoxic T lymphocytes with chemotherapy correlated with an improved median survival and was safe but not effective versus metastatic lesions. Thirdly, suppression of late membrane protein-1 in the clinic proved controversial because it gave resistance to chemotherapy and, on the other hand, increased radiosensitivity. Finally, we suggest future perspectives for clinical research which should include both prospective and observational cohort studies to assess the role of different risk factors in the development of NPC and the effectiveness of new investigational treatments.

Folic acid functionalized surface highlights 5-methylcytosine-genomic content within circulating tumor cells.

Although the detection of methylated cell free DNA represents one of the most promising approaches for relapse risk assessment in cancer patients, the low concentration of cell-free circulating DNA constitutes the biggest obstacle in the development of DNA methylation-based biomarkers from blood. This paper describes a method for the measurement of genomic methylation content directly on circulating tumor cells (CTC), which could be used to deceive the aforementioned problem. Since CTC are disease related blood-based biomarkers, they result essential to monitor tumor's stadiation, therapy, and early relapsing lesions. Within surface's bio-functionalization and cell's isolation procedure standardization, the presented approach reveals a singular ability to detect high 5-methylcytosine CTC-subset content in the whole CTC compound, by choosing folic acid (FA) as transducer molecule. Sensitivity and specificity, calculated for FA functionalized surface (FA-surface), result respectively on about 83% and 60%. FA-surface, allowing the detection and characterization of early metastatic dissemination, provides a unique advance in the comprehension of tumors progression and dissemination confirming the presence of CTC and its association with high risk of relapse. This functionalized surface identifying and quantifying high 5-methylcytosine CTC-subset content into the patient's blood lead significant progress in cancer risk assessment, also providing a novel therapeutic strategy.

Role of systemic chemotherapy in the management of resected or resectable colorectal liver metastases: a systematic review and meta-analysis of randomized controlled trials.

Liver metastases are a common event in patients with colorectal cancer. Surgical resection, if feasible, produces a survival benefit. We performed a systematic review of randomized clinical trials (RCT) and meta-analysis to address the question if current available studies support the use of systemic chemotherapy as an adjunct to surgery in resected/resectable patients. The search was based on major databases (Pubmed, CancerLit, Embase, Medscape and Cochrane) of published literature and selecting abstracts from major cancer meetings. We performed a literature for the January 1982-May 2010 time frame. The hazard ratios (HRs), with confidence intervals, as presented in retrieved studies, referred to the disease- and/or progression-free (DFS and/or PFS) and overall survival (OS) were extracted. The meta-analysis was carried out by the fixed-effect and the random-effects model. Three studies randomizing combined treatment vs. surgery alone for a total of 666 patients (642 evaluable for survival analysis) were selected and included in the final analysis. Evidence for chemotherapy-induced benefit in terms of both DFS (pooled HR, 0.71; CI, 0.582-0.878; p=0.001) and PFS (pooled HR, 0.75; CI, 0.620-0.910; p=0.003) was demonstrated. However, our meta-analysis failed to demonstrate a significant advantage of combined treatment in terms of OS (pooled HR, 0.743; CI, 0.527-1.045; p=0.088). Chemotherapy combined with surgical resection of colorectal liver metastases improves DFS and PFS whereas the benefit in OS is not demonstrated on the basis of the available results of RCTs. New prospective trials in the era of targeted therapy are eagerly awaited on this specific topic.

Mass spectrometry-based identification of the tumor antigen UN1 as the transmembrane CD43 sialoglycoprotein.

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100-120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation. In this study, we have identified the UN1 antigen as CD43, a transmembrane sialoglycoprotein involved in cell adhesion, differentiation, and apoptosis. Indeed, mass spectrometry detected two tryptic peptides of the membrane-purified UN1 antigen that matched the amino acidic sequence of the CD43 intracellular domain. Immunological cross-reactivity, migration pattern in mono- and bi-dimensional electrophoresis, and CD43 gene-dependent expression proved the CD43 identity of the UN1 antigen. Moreover, the monosaccharide GalNAc-O-linked to the CD43 peptide core was identified as an essential component of the UN1 epitope by glycosidase digestion of specific glycan branches. UN1-type CD43 glycoforms were detected in colon, sigmoid colon, and breast carcinomas, whereas undetected in normal tissues from the same patients, confirming the cancer-association of the UN1 epitope. Our results highlight UN1 monoclonal antibody as a suitable tool for cancer immunophenotyping and analysis of CD43 glycosylation in tumorigenesis.

Efficacy of boron neutron capture therapy on liver metastases of colon adenocarcinoma: optical and ultrastructural study in the rat.

The effect of neutron boron capture therapy (BNCT) was studied in rat tumor liver cells after induction of the liver metastases by splenic inoculation of cells from DHA/K12/TRb line. Ten days following the treatment, the BPA was injected into rats and therefore the animals were sacrificed, the liver was exposed to neutron irradiation and processed. In some experiments the liver was reimplanted (after irradiation) in syngenic animals and studied 3 days later, following sacrifice. Samples of tissue obtained from metastasised and non-metastasised areas of the liver parenchyma, before and after the neutron irradiation, were examined in light microscopy and electron microscopy. The analysis pointed out damages induced by the neutron treatment in single tumor cells mostly localised in the synusoidal blood stream. Debris and apoptotic cells were sometimes observed in the neoplastic nodules before treatment, while the tumor cell death (apoptosis) increased in the tumor cells following BNCT treatment. An intense scavenger activity of Kupffer cells after irradiation was accompanied by a strong acid phosphatase reaction detectable in wide cytoplasmic areas. In the liver parenchyma of reimplanted animals, the presence of large collagen bundles spread among the hepatocytes was observed at electron microscopy.