Growth rate and myofibroblast differentiation of desmoid fibroblast-like cells are modulated by TGF-ß signaling.

Desmoid-like fibromatosis (DF) is a rare myofibroblastic benign tumor, often associated with local and repeated injuries, spontaneous regression and stabilization of disease progression suggesting the involvement of altered Wnt/ß-catenin signaling activation and/or aberrant response of the DF cells to external environmental stimuli. The aim of this study was to investigate the response of DF cells to microenvironmental factors such as inflammatory and growth factors or hormones. We observed that the inflammatory cytokine, transforming growth factor-ß (TGF-ß1) stimulated cell growth and myofibroblast differentiation of DF cells regardless of the presence of a ß-catenin mutation. The role of TGF-ß1 in cell growth and myogenic differentiation of in vitro cultures of primary DF cells and normal fibroblasts was investigated by gene and protein expression analyses. We demonstrated that TGF-ß1 exerted its role via the canonical Smad pathway with the phosphorylation of Smad3 being crucial for TGF-ß1 dependent DF cell growth and myofibroblastic differentiation. Furthermore we demonstrated that cell confluence is a critical determinant of TGF-ß1 inducing the DF myofibroblast differentiation, implying that the intercellular communications have an important role on the DF myofibroblast behavior. We observed the formation of an increased stress-fiber pattern in DF cells with increased projected cell area and stronger cell-cell contacts in presence of TGF-ß1. These results demonstrated that TGF-ß1 plays a crucial role in the DF cells growth and, together with cell-cell interactions, in DF myofibroblast conversion; we also highlighted that the cellular sensitivity to this cytokine was an intrinsic feature of the DF cells.

Diagnostic and prognostic role of cell-free DNA testing for colorectal cancer patients.

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.

13 novel putative mutations in ATP7A found in a cohort of 25 Italian families.

ATP7A is a copper-transporting P-type adenosine triphosphatase whose loss of function leads to the Menkes disease, an X-linked copper metabolism multi-organ disorder (1 in 100.000 births). Here we document our experience with the ATP7A linked diseases in Italy. We analyzed the exonic structure of the ATP7A gene in 25 unrelated Italian families and studied the variants of unknown significance. We identified 22 different DNA alterations, 13 of which first reported in this study. The classical Menkes phenotype was present in 21 of the 25 families and was linked with highly damaging mutations (7 nonsense; 4 frame-shift; 2 small in-frame deletions, 2 splice site alterations, 2 gross deletions, and 1 gross duplication). Of the 4 cases with milder variants of the Menkes disease two had a missense mutation, one a leaky splice site alteration and one a nonsense mutation in exon 22. We determined in silico that all the mutations leading to the classical Menkes disease leave no residual activity of ATP7A including the apparently less severe in-frame deletions. Whereas milder forms of the disease are characterized by mutations that allow a limited residual activity of ATP7A, including the nonsense mutation observed.

Clinical predictive circulating peptides in rectal cancer patients treated with neoadjuvant chemoradiotherapy.

Preoperative chemoradiotherapy is worldwide accepted as a standard treatment for locally advanced rectal cancer. Current standard of treatment includes administration of ionizing radiation for 45-50.4 Gy in 25-28 fractions associated with 5-fluorouracil administration during radiation therapy. Unfortunately, 40% of patients have a poor or absent response and novel predictive biomarkers are demanding. For the first time, we apply a novel peptidomic methodology and analysis in rectal cancer patients treated with preoperative chemoradiotherapy. Circulating peptides (Molecular Weight <3 kDa) have been harvested from patients' plasma (n = 33) using nanoporous silica chip and analyzed by Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometer. Peptides fingerprint has been compared between responders and non-responders. Random Forest classification selected three peptides at m/z 1082.552, 1098.537, and 1104.538 that were able to correctly discriminate between responders (n = 16) and non-responders (n = 17) before therapy (T0) providing an overall accuracy of 86% and an area under the receiver operating characteristic (ROC) curve of 0.92. In conclusion, the nanoporous silica chip coupled to mass spectrometry method was found to be a realistic method for plasma-based peptide analysis and we provide the first list of predictive circulating biomarker peptides in rectal cancer patients underwent preoperative chemoradiotherapy.

Circulating cell-free DNA: a promising marker of pathologic tumor response in rectal cancer patients receiving preoperative chemoradiotherapy.

PURPOSE: The circulating cell-free DNA (cfDNA) in plasma has been reported to be a marker of cancer detection. The aim of this study was to investigate whether the cfDNA has a role as response biomarker in patients receiving preoperative chemoradiotherapy (CRT) for rectal cancer. METHODS: Sixty-seven patients (median age 61 years; male/female 42/25) who underwent CRT for rectal cancer were evaluated. After tumor regression grade (TRG) classification was made, the patients were classified as having disease that responded (TRG 1-2) and that did not respond (TRG 3-5) to therapy. Plasma samples were obtained from patients before and after CRT. The cfDNA levels were analyzed by quantitative real-time polymerase chain reaction of ß-globin. On the basis of the Alu repeats, the cfDNA was considered as either total (fragments of 115 bp, Alu 115) or tumoral (fragments of 247 bp, Alu 247). The association between the pre- or post-CRT levels and between variations during CRT of the Alu 247, Alu 115 repeat, and Alu 247/115 ratio (cfDNA integrity index) and the pathologic tumor response was analyzed. RESULTS: The baseline levels of cfDNA were not associated with tumor response. The post-CRT levels of the cfDNA integrity index were significantly lower in responsive compared to nonresponsive disease (P = 0.0009). Both the median value of the Alu 247 repeat and the cfDNA integrity index decreased after CRT in disease that responded to therapy (P < 0.005 and P < 0.005, respectively) compared to disease that did not respond to therapy (P = 0.83 and P = 0.726, respectively). The results of the multivariable logistic regression analysis showed that only the cfDNA integrity index was significantly and independently associated with tumor response to treatment. CONCLUSIONS: The plasma levels of the longer fragments (Alu 247) of cfDNA and the cfDNA integrity index are promising markers to predict tumor response after preoperative CRT for rectal cancer.

[Compound heterozygosis with novel AQP2 gene mutation in sisters affected by autosomal congenital nephrogenic diabetes insipidus].

Congenital nephrogenic diabetes insipidus (CNDI) is a rare inherited disorder, mostly caused by antidiuretic hormone receptor type 2 (ADHR2) gene mutations, which are inherited as X-linked traits. Less than 10% of cases are due to mutations in the aquaporin-2 (AQP2) gene, inherited in autosomal recessive or dominant manner. We report the case of two adult sisters, of 30 and 27 years of age, diagnosed in early infancy with X-linked CNDI. The patients' sex and family history did not fit in well with this diagnosis, so we sequenced the coding regions of the ADHR2 and AQP2 genes. As expected, no mutations were found in the ADHR2 gene, while we found a compound heterozygosis for two different mutations in the AQP2 gene. A missense mutation (c. 439G>A, p.Ala147Thr), an already known cause of CNDI, and a novel missense putative mutation of an adenine to cytosine at position 551 (c.551A>C), resulting in the substitution of asparagine with threonine at amino acid position 184 (p.Asn184Thr). This second mutation changes a fundamental extracellular Asn-Pro-Ala motif (NPA) of the AQP2 protein, inhibiting its function. Its pathogenicity has been confirmed by in silico predictions and is in line with comparable alterations to the intracellular NPA motif of the AQP2 protein.

A novel ABCC6 variant causative of pseudoxanthoma elasticum.

Pseudoxanthoma elasticum is an autosomal recessive heritable disorder caused by mutations in ABCC6. We describe two siblings showing typical skin lesions and a clinical diagnosis of pseudoxanthoma elasticum. Genetic analysis of ABCC6 revealed a novel homozygous c.4041G > A variant located in the last position of exon 28 that compromises the splicing donor site, resulting in a shorter messenger RNA. The deletion impairs the nucleotide-binding fold region, which is crucial for ABCC6 function.

The Wnt/ß-catenin pathway in human fibrotic-like diseases and its eligibility as a therapeutic target.

The canonical Wnt signaling pathway is involved in a variety of biological processes like cell proliferation, cell polarity, and cell fate determination. This pathway has been extensively investigated as its deregulation is linked to different diseases, including various types of cancer, skeletal defects, birth defect disorders (including neural tube defects), metabolic diseases, neurodegenerative disorders and several fibrotic diseases like desmoid tumors. In the "on state", beta-catenin, the key effector of Wnt signaling, enters the nucleus where it binds to the members of the TCF-LEF family of transcription factors and exerts its effect on gene transcription. Disease development can be caused by direct or indirect alterations of the Wnt/ß-catenin signaling. In the first case germline or somatic mutations of the Wnt components are associated to several diseases such as the familial adenomatous polyposis (FAP) - caused by germline mutations of the tumor suppressor adenomatous polyposis coli gene (APC) - and the desmoid-like fibromatosis, a sporadic tumor associated with somatic mutations of the ß-catenin gene (CTNNB1). In the second case, epigenetic modifications and microenvironmental factors have been demonstrated to play a key role in Wnt pathway activation. The natural autocrine Wnt signaling acts through agonists and antagonists competing for the Wnt receptors. Anomalies in this regulation, whichever is their etiology, are an important part in the pathogenesis of Wnt pathway linked diseases. An example is promoter hypermethylation of Wnt antagonists, such as SFRPs, that causes gene silencing preventing their function and consequently leading to the activation of the Wnt pathway. Microenvironmental factors, such as the extracellular matrix, growth factors and inflammatory mediators, represent another type of indirect mechanism that influence Wnt pathway activation. A favorable microenvironment can lead to aberrant fibroblasts activation and accumulation of ECM proteins with subsequent tissue fibrosis that can evolve in fibrotic disease or tumor. Since the development and progression of several diseases is the outcome of the Wnt pathway cross-talk with other signaling pathways and inflammatory factors, it is important to consider not only direct inhibitors of the Wnt signaling pathway but also inhibitors of microenvironmental factors as promising therapeutic approaches for several tumors of fibrotic origin.

Circulating cell-free DNA, SLC5A8 and SLC26A4 hypermethylation, BRAF(V600E): A non-invasive tool panel for early detection of thyroid cancer.

PURPOSE: In the latest years, high levels of circulating cell-free DNA (cf-DNA) have been found to be associated with cancer diagnosis and progression, and cf-DNA has become a potential candidate as biomarker for tumor detection. cf-DNA has been investigated in plasma or serum of many tumor patients affected by different malignancies, but not yet in thyroid cancer (TC). Furthermore, in TC cells the capability to metabolize iodine is frequently lost. SLC5A8 and SLC26A4 genes are both involved in the iodine metabolism, and SLC5A8 hypermethylation status is associated with the BRAF(V600E) mutation, which is the most frequent genetic event underlying the development of papillary TC. The aim of our study is the development of a new non-invasive tool for the diagnosis and prognosis of TC based on cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis. METHODS: cf-DNA was measured by quantitative real-time PCR in nine cases of anaplastic thyroid cancer (ATC), 58 medullary thyroid cancers (MTC), five of synchronous medullary and follicular thyroid cancers (SMFC), 23 follicular adenomas (FA), 86 papillary thyroid cancers (PTC). A control group of 19 healthy subjects was taken. Moreover, in the PTC group we analyze the state of hypermethylation of SLC5A8 and SLC26A4, BRAF(V600E) mutation, and their involvement in the loss of function of the thyroid. RESULTS: cf-DNA showed a high ability to discriminate healthy individuals from cancer patients. cf-DNAALU83 and cf-DNAALU244 values were significantly correlated with the histological type of TC (P-value < 0.0001). A significant increase in the amount of cf-DNAALU83 and cf-DNAALU244 when methylation occurs was observed (P-value = 0.02). A correlation between BRAF(V600E) and cf-DNAALU244/ALU83 was also found (P-value = 0.02). CONCLUSIONS: According to our experimental results, the panel including cf-DNA, SLC5A8 and SLC26A4 hypermethylation, and BRAF(V600E) analysis appears easy, reproducible, and non-invasive for the diagnosis on TC. Its possible implication in clinical setting remains to be elucidated.

Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions.

The therapeutic efficacy of systemic drug-delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. White blood cells of the immune system--known as leukocytes--possess all of these properties and exert their targeting ability through cellular membrane interactions. Here, we show that nanoporous silicon particles can successfully perform all these actions when they are coated with cellular membranes purified from leukocytes. These hybrid particles, called leukolike vectors, can avoid being cleared by the immune system. Furthermore, they can communicate with endothelial cells through receptor-ligand interactions, and transport and release a payload across an inflamed reconstructed endothelium. Moreover, leukolike vectors retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in a tumour.

A ten markers panel provides a more accurate and complete microsatellite instability analysis in mismatch repair-deficient colorectal tumors.

UNLABELLED: Tumour microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC) with defective DNA mismatch repair (MMR) genes. A reference Bethesda panel has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumours with MMR deficiencies. We developed a multiplex PCR assay with additional four mononucleotide markers and one dinucleotide marker (NR-21, NR-24, BAT-40, TGF-BetaR and D18S58) for a rapid and proper classification of MSI-H, MSI-L and MSS colorectal cancers. Two tetranucleotide markers were added to identify sample mix-ups and/or contamination. RESULTS: all the 44 cases test cases were in agreement with previous classification except for three cases: one case MSI-H-Bethesda unstable only for dinucleotides markers shifted to MSI-L category and two cases MSI-L-Bethesda unstable for mononucleotide markers shifted to MSI-H category. Immunohistochemistry analysis revealed that these two MSI-H cases did not expressed hMLH1 and they were found to be methylated at the MLH1 promoter, while the first one that shifted to MSI-L showed MMR protein expression. CONCLUSION: a complete panel of ten markers including four dinucleotide and six mononucleotide microsatellites allows accurate evaluation of tumor MSI status.

The role of MYH gene in genetic predisposition to colorectal cancer: another piece of the puzzle.

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.