Prognostic value of circulating tumor cells in nonmuscle invasive bladder cancer: a CellSearch analysis.

BACKGROUND: Circulating tumor cells (CTCs) provide prognostic information in patients with metastatic tumors. Recent studies have shown that CTCs are released in circulation in an early phase of cancer disease so that their presence is under investigation in the adjuvant setting. Few studies investigated the prognostic significance of CTCs enumeration in patients with metastatic and advanced bladder cancer. The current study has analyzed the presence of CTC in patients with nonmuscle-invasive bladder cancer (NMIBC). PATIENTS AND METHODS: Forty-four NMIBC patients were enrolled and included in a 24-month follow-up program. Blood drawings were carried out in all patients at the first diagnosis. CellSearch system (Veridex; LLC, Raritan, NJ) was used for CTCs enumeration. RESULTS: CTC were detectable in 8/44 patients (18%). Presence of CTC was found significantly associated to shorter time to first recurrence (6.5 versus 21.7 months, P < 0.001). Median time to progression was not reached, due to the short follow-up period. CTC presence was found associated to concomitant carcinoma in situ and higher T category. CONCLUSION: The detection of CTC in this setting of disease may allow to distinguish patients with high risk of recurrence from those with high risk of progression, as well as to early identify patients candidate for adjuvant treatment.

Prognostic significance of tyrosinase expression in sentinel lymph node biopsy for ultra-thin, thin, and thick melanomas.

BACKGROUND: Investigate if the tyrosinase mRNA expression may be predictive of the outcome on ultra-thin, thin, and thick melanoma patients. AIM: In our study, we sought to correlate tyrosinase mRNA expression to the outcome in a group of 71 patients with thick, thin and ultra-thin melanomas. MATERIALS AND METHODS: 71 patients with melanomas underwent a SLNB (sentinel lymph node biopsy) at the "Sapienza" University of Rome. Among these, 38 patients had thin melanomas, while the other 33 patients had thick melanomas. In every patient's sample histology, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) was completed. We then correlated tyrosinase mRNA expression to the statistical analysis of the outcome of patients. RESULTS: Positivity of histology was found in one patient (1.4%), immunohistochemistry in five patients (7%), and tyrosinase in 52/71 (73.2%). Thickness and tyrosinase positivity were predictive for disease progression (p < 0.05). The median follow-up was 58.24 months. There were recurrences and/or deaths in both groups of patients. CONCLUSIONS: Nodal metastasis in melanoma is uncommon, especially in patients with thin melanomas. In this study, histology and immunohistochemistry were found to be non predictive for the risk of nodal metastases, while instead, tyrosinase m-RNA expression appeared to play a role in highlighting those patients with a risk of disease progression. Moreover, no differences among the thin melanoma groups of patients (0.30-0.75 mm and 0.76-1.00 mm) were observed.

Circulating tumor cells (CTCs) in metastatic breast cancer (MBC): prognosis, drug resistance and phenotypic characterization.

BACKGROUND: the expression of ATP-binding cassette transporters on circulating tumor cells (CTCs) is predictive of response to chemotherapy in cancer patients. We tested the hypothesis that drug-resistant CTCs might have predictive value in metastatic breast cancer (MBC) and possibly retain stem-like properties. PATIENTS AND METHODS: CTCs obtained from 42 MBC patients were evaluated for multidrug-resistance-related proteins (MRPs), aldehyde dehydrogenase 1 (ALDH1), estrogen receptor α (ERα) and human epidermal growth factor receptor 2 (HER2/neu). Primary objective was to evaluate the prognostic and predictive value of CTCs profile. Secondary end points were the level of concordance in ERα and HER2/neu status between primary tumors and CTCs and the correlation in CTCs between ALDH1, drug resistance profile and number of MRPs. RESULTS: A difference in progression-free survival (PFS) was found between CTCs-positive and CTCs-negative patients. PFS was shorter in patients with a 'drug resistance' CTCs profile and in patients whose CTCs expressed two or more MRPs. No correlation was found between tumor characteristics and ALDH1. ALDH1 correlated to negative ERα and positive HER2/neu status in CTCs. The correlation between the number of MRPs expressed in CTCs and ALDH1 was statistically significant. CONCLUSION: in MBC, the presence of CTCs expressing MRPs and ALDH1 is predictive of response to chemotherapy.

CD69 is expressed on platelets and mediates platelet activation and aggregation.

CD69, a surface dimer so far considered an early activation antigen restricted to lymphocytes, was found constitutively expressed on human platelets. Biochemical analysis revealed that platelet CD69 appears on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a broad 55-65-kD band, in which three 55-, 60-, and 65-kD components were detectable when nonreduced, and as two 28- and 32-kD bands when reduced, corresponding to the two disulfide-linked chains of the dimer. It therefore closely resembles lymphoid CD69, although the resolution of the three bands under nonreducing conditions is not usually seen in lymphoid cells. Moreover, as CD69 expressed on activated lymphocytes and CD3bright thymocytes, both chains are constitutively phosphorylated. CD69 stimulation by anti-Leu-23 monoclonal antibodies induced platelet aggregation in a dose-dependent fashion. This effect was associated with Ca2+ influx and platelet degranulation, as revealed by adenosine triphosphate release. In addition, CD69 stimulation in platelets induced production of thromboxane B2 and PGE2, suggesting activation of arachidonic acid metabolism by cycloxygenase. As observed for CD69-mediated T cell activation, platelet activation through CD69 requires molecular crosslinking. These results suggest that CD69 may function as an activating molecule on platelets, as on lymphocytes, and point toward a more general role of this surface dimer in signal transduction.

Contrast echocardiography for pulmonary arteriovenous malformations screening: does any bubble matter?

AIMS: To evaluate diagnostic accuracy of contrast echocardiography (CE) as compared with CT, for the screening of pulmonary arteriovenous malformations (PAVMs) in hereditary haemorrhagic telangiectasia (HHT); to evaluate the clinical significance of semi-quantitative analysis of a shunt on CE. METHODS AND RESULTS: A blinded prospective study was conducted in 190 consecutive subjects at risk of HHT who underwent screening for PAVMs, including clinical evaluation, pulse oximetry, standard and CE, and chest multirow CT without contrast medium. A semi-quantitative analysis of the shunt size was performed according to the contrast echo opacification of the left-sided chambers: Grade 0, no bubbles; 1, occasional filling with <20 bubbles; 2, moderate filling; 3, complete opacification. The first 100 patients were compared with 100 controls. A total of 119 (63%) patients had positive CE (32.2% Grade 1, 13.1% Grade 2, 11% Grade 3, 6.3% with patent foramen ovale). The overall diagnostic performance of CE was sensitivity 1.00, specificity 0.49, positive predictive value (PPV) 0.32, negative predictive value (NPV) 1.00. The PPV for the different grades was 0.00 for Grade 1, 0.56 for Grade 2, 1.00 for Grade 3; the NPV of Grade 0 was 1.00. A significant correlation was found between the CE grading and the number of PAVM, and complications (P < 0.0001). CONCLUSION: CE is an extremely sensitive procedure for the detection of PAVMs with substantial clinical impact.

EPAC-lung: pooled analysis of circulating tumour cells in advanced non-small cell lung cancer.

INTRODUCTION: We assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a pooled analysis of individual patient data. METHODS: Nine European NSCLC CTC centres were asked to provide reported/unreported pseudo-anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 to March 2017. We used Cox regression models, stratified by centres, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinicopathological models using likelihood ratio (LR) statistics and c-indices. RESULTS: Seven out of nine eligible centres provided data for 550 patients with prognostic information for overall survival. CTC counts of ≥2 and ≥ 5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: hazard ratio [HR] = 1.72, p < 0·001; ≥5 CTCs: HR = 2.21, p < 0·001) and overall survival (≥2 CTCs: HR = 2·18, p < 0·001; ≥5 CTCs: HR = 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinicopathological models (log-transformed CTCs p < 0·001; ≥2 CTCs p < 0·001; ≥5 CTCs p ≤ 0·001 for both survival end-points), whereas moderate improvements were observed with the use of c-index models. There was some evidence of between-centre heterogeneity, especially when examining continuous counts of CTCs. CONCLUSIONS: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC and also reveal some evidence of between-centre heterogeneity. CTC count improves prognostication when added to full clinicopathological predictive models.

Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer.

The metastatic lymph node 64 (MLN64), which is localized in the human chromosome 17, encodes a protein with strong homology with steroidogenic acute regulatory protein. Its overexpression in human breast carcinomas and MLNs led to the hypothesis that this protein could be involved in intraneoplastic steroidogenesis. In the present study, we investigated the expression of MLN64 in prostate cancer, another hormone-dependent tumor, and compared its expression with that of CYP17, the gene encoding for the key enzyme of androgen synthesis. We investigated by RT-PCR the expression of MLN64 and CYP17 in 60 prostatic tumors and compared their expression with the stage of disease and the appearance of relapses in a follow-up of 24 months. We found MLN64 and CYP17 expressed in all samples examined, with significantly higher expression in neoplastic tissues with respect to normal tissues (NTs). Moreover, only in neoplastic but not in NTs, a positive linear correlation was found between MLN64 and CYP17 gene expression. MLN64 and CYP17 expression seems to correlate with high stage, high Gleason score and short relapse-free time. These data, for the first time, demonstrate the presence of MLN64 and CYP17 expression in both normal and neoplastic prostatic tissues. The biological role of MLN64 in human prostate and, particularly, in neoplastic tissue is still unclear. Our findings concerning MLN64 and CYP17 gene expression and their significant positive correlation in human prostate cancer may suggest their possible role in intraneoplastic autonomous steroidogenesis.

Evaluation of the potential role of class II histocompatibility antigen HLA-DR in platelet/tumor cell interaction.

It has been reported that HLA-DR is a potent inducer of thrombin generation. Human colorectal cells (GEO, WiDr, DLD-1, and MIP) that lack the constitutive expression of HLA-DR cause platelet aggregation through a thrombin-dependent mechanism. Treatment with recombinant human gamma-interferon induced the expression of HLA-DR in the GEO, WiDr, and DLD-1 cells, whereas the MIP cell line remained HLA-DR negative. The concurrent analysis of tumor cell/platelet interaction after gamma-interferon treatment showed a decrease in platelet proaggregating activity of either the responsive GEO (highly expressing HLA-DR) or the unresponsive MIP (HLA-DR negative) cells. Furthermore, the DLD-1 (moderately expressing HLA-DR) cells showed an increase of proaggregating activity after gamma-interferon treatment, whereas WiDr (highly expressing HLA-DR) cells did not modify their activity. These results suggest a lack of a role of HLA-DR in the in vitro platelet proaggregating activity of human colorectal tumor cells.

Involvement of GD3 in platelet activation. A novel association with Fcgamma receptor.

Gangliosides are sialic acid-containing glycosphingolipids present in the outer leaflet of the plasma membrane of many cell types where they modulate adhesive processes. The main population of glycolipids in resting platelets is represented by ganglioside M3 (GM3). It has been demonstrated that following platelet activation ganglioside D3 (GD3) is rapidly formed from the GM3 pool. The present study was designed to evaluate the link between platelet activation and GD3 expression and to verify whether this ganglioside might play a role in modulating signal transduction events. Our results suggest that following platelet activation, GD3 is rapidly expressed on the platelet surface and internalised to the cytoskeleton where it transiently associates first with the Src family tyrosine kinase Lyn then with the Fc receptor gamma chain. This sequence of events ultimately leads to an enhanced CD32 (the Fc receptor isoform present in platelets) expression on the platelet membrane. These data drive us to speculate that GD3 might act as second messenger in the activatory cascade, which leads to CD32 expression and triggers platelet adhesion and spreading to the subendothelial matrix.

Detection of epidermal growth factor receptor mRNA in peripheral blood: a new marker of circulating neoplastic cells in bladder cancer patients.

Despite the large number of studies performed in solid tumors, few attempts at molecular detection of urothelial cells in blood have been made. Specifically, only uroplakin II (UP-II) and cytokeratin 20 (CK-20) have been suggested as tumor markers in the blood of bladder cancer patients. Epidermal growth factor receptor (EGFR) mRNA expression was found in the blood of patients with some types of carcinoma; nevertheless, its expression has been never investigated in the blood of patients with urothelial tumors. We used a EGFR-based reverse transcription-PCR assay for the detection of tumoral cells in the blood of 27 patients with bladder cancer, in 30 healthy donors, and in 9 patients with cystitis. EGFR expression was compared with that of known markers of circulating epithelial cells, CK-19 and CK-20, and to a urothelial-specific marker, UP-II. Analysis by reverse transcription-PCR and Southern blot hybridization showed no evidence of EGFR and UP-II mRNA expression in any of the samples used as controls. Analysis of healthy donors showed mRNA expression for CK-19 and CK-20 in 6 of 30 and in 4 of 30 samples, respectively. All patients with cystitis resulted negative for EGFR expression, whereas 3 of 9, 2 of 9, and 3 of 9 were found expressing CK-19, CK-20, and UP-II, respectively. Among blood samples from tumoral patients, 74% had EGFR mRNA and 41% had positive signals for CK-19, whereas positivity for CK-20 and UP-II was found in 15% and 37% of patients, respectively. These results seem to indicate that EGFR mRNA in the blood may be a useful tumor marker in bladder cancer patients, as well as in other patients with epithelial tumors.

Protein kinase C activation is not a key step in ADP-mediated exposure of fibrinogen receptors on human platelets.

UNLABELLED: A selective inhibitor of protein kinase C (PKC), Ro 31-8220, blocks pleckstrin (P47) phosphorylation in platelets activated with either ADP, ADP plus synthetic thromboxane agonist U46619 and ADP plus U46619 plus epinephrine, while inducing a weak inhibition of platelet aggregation, and no significant effect on the fibrinogen binding. In platelets activated by U46619 alone, P47 phosphorylation, platelet aggregation, fibrinogen binding and serotonin release are all inhibited by Ro 31-8220. In the presence of an ADP scavenger system, U46619 induces pleckstrin phosphorylation, serotonin release and calcium mobilization but not platelet aggregation and fibrinogen binding, unless epinephrine is added. IN CONCLUSION: (1) PKC activation is required for ADP secretion; (2) ADP or epinephrine are essential for fibrinogen receptor exposure induced by U46619; (3) fibrinogen receptor exposure induced by ADP is independent of activation of PKC.

Concomitant activation of Gi protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation.

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G(i) and G(q)) is essential for platelet aggregation: in fact, the thromboxane A2 synthetic agonist, U46619, which causes the selective activation of Gq, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct Gq activation is not required for platelet aggregation and that the activation of an enzyme downstream of Gq, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCgamma2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a Gi protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a Gi protein-coupled receptor is activated.

Concomitant activation of Gi and Gq protein-coupled receptors does not require an increase in cytosolic calcium for platelet aggregation.

U46619 is a potent platelet agonist, its binding to the thromboxane A2 receptor resulting in Gq-binding protein-mediated responses; nevertheless, it is unable to cause platelet aggregation, unless released ADP is present. In this study we demonstrate that Gi activation is the step U46619 lacks to cause platelet aggregation; in fact, when platelets were treated with an ADP scavenger system, the response to U46619 was restored by the addition of epinephrine, which activates platelets via a Gi protein. The concomitant activation of Gi and Gq proteins does not require increased cytosolic calcium to cause aggregation, as assessed by the fact that platelets treated with the intracellular calcium chelator BAPTA were able to respond to U46619 provided ADP or epinephrine was present. Moreover, as the calcium ionophore ionomycin, at low concentrations, potentiated the response to U46619 but not to epinephrine, we may conclude that calcium influx preferentially activates a Gi downstream signalling pathway.

The flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular production of hydrogen peroxide.

BACKGROUND: Epidemiologic studies have shown an inverse relation between moderate consumption of red wine and cardiovascular disease. Studies have shown that red wine and its component flavonoids inhibit in vivo platelet activation, but the underlying mechanism has not yet been identified. OBJECTIVE: Because we showed previously that collagen-induced platelet aggregation is associated with a burst of hydrogen peroxide, which in turn contributes to stimulating the phospholipase C pathway, the aim of this study was to investigate whether flavonoids synergize in inhibiting platelet function and interfere with platelet function by virtue of their antioxidant effect. DESIGN: We tested the effect of 2 flavonoids, quercetin and catechin, on collagen-induced platelet aggregation and hydrogen peroxide and on platelet adhesion to collagen. RESULTS: Catechin (50-100 micromol/L) and quercetin (10-20 micromol/L) inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. The combination of 25 micromol catechin/L and 5 micromol quercetin/L, neither of which had any effect on platelet function when used alone, significantly inhibited collagen-induced platelet aggregation and platelet adhesion to collagen. Such a combination strongly inhibited collagen-induced hydrogen peroxide production, calcium mobilization, and 1,3,4-inositol triphosphate formation. CONCLUSIONS: These data indicate that flavonoids inhibit platelet function by blunting hydrogen peroxide production and, in turn, phospholipase C activation and suggest that the synergism among flavonoids could contribute to an understanding of the relation between the moderate consumption of red wine and the decreased risk of cardiovascular disease.

Platelet proaggregating activity of human colorectal tumour cell lines does not correlate with the degree of differentiation.

Six human colorectal tumour cell lines with various degrees of differentiation were studied. Each of the cell lines studied showed an in vitro platelet proaggregating activity, represented by the induction of typical aggregation waves. This activity was dose-dependent and probably related to a thrombin-dependent mechanism. However, the degree of cell differentiation did not correlate with the proaggregating activity. In fact, significant differences were observed between the two well differentiated cell lines, while a comparison between well and poorly differentiated cell lines did not reveal any difference. These results were confirmed by the ultrastructural observations, indicating that similar platelet-tumour cell interaction may be found in all the cell lines studied. The present results suggest that platelet proaggregating activity of the human colorectal tumour cell lines under investigation is unrelated to their degree of differentiation.

Possible involvement of tumour cell membrane gangliosides in platelet-tumour cell interactions.

The possible correlation(s) between platelet proaggregating activity, and sialic acid content and ganglioside expression of six human colorectal tumour cell lines (CBS, GEO, HT-29, WiDr, MIP and DLD-1) was evaluated. The three cell lines (HT-29, WiDr and DLD-1) capable of inducing remarkable in vitro platelet aggregation, had significantly higher amounts of lipid-bound sialic acid than those cell lines characterised by a lower platelet proaggregating activity (GEO, CBS and MIP). High performance thin-layer chromatography demonstrated the presence of one band comigrating with GM3 in all cell lines, while GD1a and GT1b comigrating gangliosides were present only in HT-29, WiDr and DLD-1 cells. Finally, an increased platelet pro-aggregating activity of GEO and CBS cell lines was observed after the incorporation of exogenous gangliosides. The present data support the hypothesis that lipid-bound sialic acid may be involved in platelet-tumour cell interactions.

High levels of transforming growth factor-alpha (TGF-alpha) mRNA may predict local relapses in early stage urinary bladder cancer.

Elevated expression of transforming growth factor-alpha (TGF-alpha) gene has been previously reported in some types of human neoplasms, but its role in the pathogenesis of bladder cancer has still not been investigated. In the present study, we analysed 28 samples of early stage bladder tumours for the presence of TGF-alpha mRNA using reverse transcription-polymerase chain reaction (RT-PCR). We detected TGF-alpha mRNA in 71% (20/28) of these samples. When we related the expression levels of TGF-alpha with local relapses of patients during a follow-up of 2 years, we found that a high TGF-alpha expression level in bladder cancer was significantly associated with local relapses in patients with early stage tumours. The appearance of early relapses in tumours with high TGF-alpha expression levels may suggest the existence of an additional marker in the prediction of local relapses in patients with superficial disease.

Lipoprotein(a) serum levels in patients affected by chronic obstructive pulmonary disease.

A recent study has suggested that symptoms of chronic bronchitis predict the risk of coronary disease independently of the known major cardiovascular risk factors. High serum levels of lipoprotein(a) (Lp(a)) have also been considered as an independent risk factor for coronary heart disease. Therefore, the aim of the present study was to investigate the behaviour of Lp(a) in patients affected by chronic obstructive pulmonary disease (COPD). Serum levels of total-cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglycerides, apolipoprotein (Apo) B-100, and Lp(a) were measured in 90 COPD patients and in 90 normal subjects matched for age, sex and smoking habit. COPD patients showed lower serum levels of Apo B-100 (P<0.0001) and Lp(a) (P<0.003) compared to controls. Conversely, TC, HDL-C, LDL-C and triglycerides were similar between patients and controls. No significant differences were found in Apo B-100 and Lp(a) levels of patients either undergoing different therapeutic regimens, or with different smoking habits. A significant correlation between Apo B-100 and Lp(a) (rho=0.433, P<0. 0001) was also observed. In conclusion, COPD patients do not show an atherogenetic lipid pattern and their increased risk of coronary disease could be attributable to different factors, such as the ongoing hypercoagulability state often associated with COPD.

Evidence for platelet alphaIIb beta3 activation despite elevated cytosolic cAMP.

Cyclic nucleotides, such as cAMP, are known to inhibit the multistep cascade that results in platelet aggregation. In the present study we provide evidence that it is possible to bypass cAMP inhibitory effect on fibrinogen binding site exposure induced by the thromboxane A2 synthetic analogue U46619, the snake venom toxin convulxin, or by the direct PKC activator OAG, by concomitantly activating a G1-coupled receptor by means of epinephrine or by inducing cytosolic calcium influx by means of ionomycin. In fact, in our study we demonstrate that, in iloprost-treated platelets, the inhibition of both platelet aggregation and fibrinogen binding was overcome by adding epinephrine or ionomycin. To further confirm this, we used the cAMP analogue dibutyryl cAMP and we obtained platelet aggregation in response to U46619, convulxin or OAG plus epinephrine. Moreover, a complete inhibition of platelet aggregation in the presence of high concentrations of cAMP was observed only in the case of U46619, while a small percentage of aggregation persisted when convulxin or OAG were used, due to the small amount of ADP that both convulxin and OAG are able to release. Since PKC inhibition didn't allow platelet aggregation to occur in response to the concomitant activation of U46619 or convulxin, plus epinephrine or ionomycin, we can conclude that cAMP-induced inhibition of aggregation can be counteracted by the simultaneous activation of PKC in the presence of an activated G1-coupled receptor or of an induced calcium influx.