Showing papers by "Francesco Salvatore published in 2012"

Combined CD133/CD44 Expression as a Prognostic Indicator of Disease-Free Survival in Patients With Colorectal Cancer

Abstract: Hypothesis Because of some inconsistencies in the traditional model of human colorectal carcinogenesis, the cancer stem cell (CSC) model was recently proposed, in which tumor results from neoplastic transformation of stem cells, which become CSCs. Identification of CSCs by expression of surface antigens remains a critical issue because no biomarker has been shown to be completely reliable. CD133 and CD44 are commonly used as CSC markers, and correlation of their expression with colorectal cancer (CRC) clinicopathological features and outcomes may be useful. Design Pilot study. Setting University hospital. Patients Thirty-six consecutive patients with CRC.CD133 and CD44 expression (alone or combined) was determined in nontumor cells and in tumor cells by flow cytometry, which identified viable cells only. Main Outcome Measures Correlation of CD133 and CD44 expression with each other, with other prognostic indicators, and with disease-free survival. Results CD133 and CD44 expression was significantly higher in tumor cells than in nontumor cells, and expression of one did not necessarily correlate with expression of the other. CD133 or CD44 expression alone was variable, while combined CD133/CD44 expression identified a small subset of cells positive for CRC. CD133 or CD44 overexpression was not associated with CRC recurrence; only high frequencies of CD133 + /CD44 + cells were a strong indicator of worse disease-free survival and an independent risk factor for CRC recurrence. Conclusion Evaluation of combined CD133/CD44 expression could be useful to identify putative colorectal CSCs and tumors with a poor prognosis.

Protein cross-talk in CD133+ colon cancer cells indicates activation of the Wnt pathway and upregulation of SRp20 that is potentially involved in tumorigenicity

Abstract: The cancer stem cell (CSC) theory represents a breakthrough in cancer research. We characterized the protein pattern of CSCs to identify specific intracellular pathways in this subpopulation of tumor cells. We studied colon CSCs using two different colon cancer cell lines: CaCo-2 and HCT-116. Putative CSCs were separated from non-CSCs by flow cytometry using CD133 as stemness marker. Total protein extracts of CD133+ cells were then compared to protein extracts of CD133- cells by 2D DIGE. The protein spots differentially expressed in the two subpopulations of cells were analyzed by mass spectrometry. Bioinformatics analysis of the identified proteins indicated alteration of two main processes: energy metabolism and the Wnt pathway. Interestingly, we observed upregulation of the splicing factor SRp20, a newly identified target gene of the Wnt/β-catenin pathway, and we demonstrated a direct cause-effect relationship between Wnt pathway activation and the increased SRp20 expression. Our results also show that SRp20 influences cell proliferation, which suggests it plays a role in the tumorigenicity of CD133+ cells. In conclusion, activation of the Wnt pathway in CD133+ cells and upregulation of SRp20, which is implicated in tumorigenesis, raises the possibility of a sequential series of molecular events occurring in connection with this process.

Cytometric and biochemical characterization of human breast cancer cells reveals heterogeneous myoepithelial phenotypes.

Abstract: To determine whether cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes, we evaluated the expression of a cell surface marker panel in breast cancer cell cultures derived from various subtypes of human breast carcinoma. We used a four-color flow cytometry strategy to immunophenotype seven human breast cancer cell cultures and four reference breast cancer cell lines. We analyzed 28 surface markers selected based on their potential to distinguish epithelial or mesenchymal lineage, to identify stem cell populations, and to mediate cell adhesion and migration. We determined their ability to form mammospheres and analyzed luminal cytokeratins CK18, CK19, and myoepithelial/basal CK5, SMA (alpha-smooth muscle actin), and vimentin expression by western blot. All cell surface markers showed a unimodal profile. Ten/28 markers were homogenously expressed. Four (CD66b, CD66c, CD165, CD324) displayed negative/low expression. Six (CD29, CD55, CD59, CD81, CD151, CD166) displayed homogenous high expression. Eighteen (CD9, CD10, CD24, CD26, CD44, CD47, CD49b, CD49f, CD54, CD61, CD90, CD105, CD133, CD164, CD184, CD200, CD227, CD326) were heterogeneously expressed. Spearman's rank test demonstrated a significant correlation (p< 0.001) between mesenchymal phenotype and breast cancer cell cultures. Breast cancer cell cultures, all CD44+, displayed concomitant high expression of only three antigens (CD10, CD54, CD90), and low expression of CD326; cell cultures formed mammospheres and expressed CK5, SMA and vimentin, and were weakly CK19-positive. We demonstrate that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry. Therefore, cell cultures can be used for in vitro modeling of breast cancer subtypes; immunophenotyping may mirror breast cancer heterogeneity and reveal molecular characteristics of individual tumors useful for testing target therapy.

Lessons to be learned from the clinical management of a MEN 2A patient bearing a novel 634/640/700 mutation of the RET proto‐oncogene

Abstract: A cluster of germline gain-of-function mutations of the RET proto-oncogene are responsible for Multiple Endocrine Neoplasia type 2A (MEN 2A), an autosomal dominant, inherited disorder characterized by medullary thyroid carcinoma (MTC), phaeochromocytoma (Phaeo) and hyperparathyroidism. Genetic screening of MEN 2A patients has been available for the past decade and useful genotype–phenotype correlations have been established: specific RET mutations are associated with age at first diagnosis and tumour aggressiveness. Accordingly, MEN 2A patients can be stratified into three risk groups depending on the RET mutation. Management uncertainties remain regarding patients bearing uncommon RET mutations or genetic variations for which mutation-specific risk profiles and treatment recommendations are unavailable. Here we report the thirteen-year clinical and surgical follow-up of a patient with MEN 2A bearing three de novo RET mutations at codons 634, 640 and 700 (p. C634R, p.A640G and p.M700L) in exon 11: a combination of mutations which has not previously been described. In May 1998, a 26-year-old female patient was admitted to our unit because of recurrent episodes of hypertension, tachycardia and headache. She had recently been diagnosed with a MTC which had been removed surgically, with associated cervical central lymphectomy. Elevated urinary catecholamine levels and imaging examinations showing a left adrenal mass were suggestive of a Phaeo. Hyperplasia of the contralateral gland was also detected. The patient underwent left “open” adrenalectomy 3 months later; post-operative catecholamine levels decreased to normal values, while the serum calcitonin remained elevated (Fig. 1). The diagnosis of MEN 2A was confirmed by testing the patient’s DNA for RET mutations. Two heterozygous germline mutations were identified: a transition at position c.1900 replaced a T with a C and a transversion at position c.1919 replaced a C with a G, resulting in the substitution of a cysteine with an arginine and an alanine with a glycine at positions p.634 and p.640, respectively. The patient presented with MTC and Phaeo without parathyroid gland involvement, so we speculated that this clinical picture could be correlated with the two RET mutations identified and the unusual feature of calcitonin production by the adrenal tissue. No clinical symptoms suggestive of MEN 2A and no RET mutations were found in either parent or any of the available relatives tested (three sisters and one brother). In 2003, at

Reversal of metabolic and neurological symptoms of phenylketonuric mice treated with a PAH containing helper-dependent adenoviral vector.

Abstract: Phenylketonuria (PKU) is one of the most common inborn errors of metabolism and is due to a deficit of phenylalanine hydroxylase, the enzyme that converts phenylalanine (Phe) into tyrosine (Tyr). The resultant hyperphenylalaninemia (HPA) leads to severe neurological impairment, whose pathogenesis has not been entirely elucidated. Treatment of PKU consists essentially in lifelong protein restriction and, in mild cases, in tetrahydrobiopterin supplementation. However, compliance to both strategies, particularly to the long-term diet, is low and therefore other therapies are desirable. We explored a gene therapy approach aimed at long-term correction of the pathologic phenotype of BTBR-PahEnu2 mice, a mouse model of PKU. To this aim, we developed a helper-dependent adenoviral (HD-Ad) vector expressing phenylalanine hydroxylase and administered it to 3-week-old PKU mice. This resulted in complete normalization of Phe and Tyr levels and reversal of coat hypopigmentation that lasted throughout the observation period of six months. The spatial learning deficits observed in PKU mice were also reversed and hippocampus levels of the N-methyl-D-Aspartate and 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl) propanoic acid receptor subunits returned to normal. Long-term potentiation, which is impaired in PKU mice, was also restored by treatment. Therefore, HD-Ad vector-mediated gene therapy is a promising approach to PKU treatment.