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.

Circulating tumor cells in metastatic colorectal cancer: do we need an alternative cutoff?

PURPOSE: To assess the prognostic and predictive value of circulating tumor cells (CTCs) in metastatic colorectal cancer (mCRC) irrespective of detection level. MATERIALS AND METHODS: We evaluated the prognostic and predictive significance of CTC count at baseline and under treatment in 119 mCRC subjects and compared the standard cutoff (≥3 CTCs/7.5 mL to ≥1 CTCs/7.5 mL). RESULTS: An overall comparison was made between patients with 0, 1-2 and ≥3 CTC (median PFS 8, 4 and 5 months, respectively). Two poor prognostic groups were found, including patients with ≥1 CTCs before and during treatment and patients with 0 CTC at baseline who converted to ≥1 CTCs (p = 0.014). CONCLUSIONS: The presence of at least 1 CTC at baseline count is predictive for poor prognosis in mCRC patients. Patients with 1-2 CTC should be switched from the favorable prognostic group--conventionally defined by the presence of <3 CTC--to the unfavorable, deserving a more careful monitoring.

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.

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.

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.

Circulating tumor cells in cancer therapy: are we off target?

What clinical oncologists learned about metastatic process, is that it is the main cause of cancer-related deaths. What scientists learned about metastatic disease, is that it is due to a highly selective process, which involves a minority of tumor cells that are able to survive within the bloodstream, and to initiate a new growth in distant sites. These cells "in transit" are known as circulating tumor cells (CTCs). Although their nature is not fully understood, what is widely accepted, is that they are drug resistant, and that their presence may represent the main reason for treatment failure. Despite this body of evidence, the pharmacological approach against cancer, with both chemotherapic and biological drugs, is still targeted on the primary tumor, raising the question as to whether we are missing the target. Targeting circulating tumor cells, may represent a new promising approach to indivisualize anticancer therapy.

Cancer stem cells and epithelial-mesenchymal transition: revisiting minimal residual disease.

The cancer stem cell (CSC) hypothesis provides an attractive model of tumour development and progression, holding that solid tumours are hierarchically organized and sustained by a minority of the tumour cell population with stem cell properties, such as self-renewal, tumorigenicity and multilineage differentiation capacity. Therapeutic resistance, underlying tumour recurrence and the lack of curative treatments in metastatic disease, raise the question if conventional anticancer therapies target the right cells. Indeed, these treatments might miss CSCs, which represent a more chemoresistant and radioresistant subpopulation within cancer. Recently, a direct link between the epithelial-mesenchymal transition process and the gain of stem cell competence were demonstrated in cultured breast cells. In particular, it was shown that the induction of EMT program not only allow cancer cells to disseminate from the primary tumor, but also promotes their self-renewal capability. Furthermore, the expression of stemness and EMT markers in CTCs were associated with resistance to conventional anti-cancer therapies and treatment failure, highlighting the urgency of improving tools for detecting and eliminating minimal residual disease.

Celecoxib upregulates multidrug resistance proteins in colon cancer: lack of synergy with standard chemotherapy.

Recent phase II randomised trials in colorectal cancer failed to demonstrate any advantage of celecoxib combined with standard chemotherapy; some authors even reported that the addition of celecoxib to irinotecan and oxaliplatin in colon cancer results in an inferior response rate. This observation leads to the hypothesis that there are pharmacokinetic interactions between celecoxib and chemotherapeutic drugs. The aim of the study was to investigate the induction by celecoxib of some multidrug resistance proteins, MRP1, MRP2, MRP4 and MRP5, involved in the transport of irinotecan and 5-FU. WiDr and COLO-205 cells were treated with celecoxib at a clinically relevant concentration. A viability assay was performed by treating cells with chemotherapy alone and chemotherapy plus celecoxib. The expression of MRP1, MRP2, MRP4 and MRP5 was analysed by RT-PCR and Western blot analysis. The sub cellular localization of MRP4 and MRP5 was investigated by cryoimmunoelectron microscopy. In both cell lines celecoxib induced MRP4 and MRP5 over-expression at RNA and protein levels. No induction of MRP1 and MRP2 was observed in treated cells compared to controls. Cryoimmunoelectron microscopy showed increased MRP4 and MRP5 immunolabeling in celecoxib treated cells both at cytoplasmic level and along the plasma membrane. Our findings suggest that the low response rate observed in clinical trials using celecoxib added to 5-fluorouracil and irinotecan may reflect celecoxib-mediated extrusion of chemotherapeutic drugs from cancer cells through the up regulation of ATP-binding cassette proteins. Our findings, together with the results of clinical trials, may suggest that the combined use of celecoxib and drugs that are substrate for MRP4/MRP5 should be avoided.

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.

Detection of melanoma cells in sentinel lymph nodes by reverse transcriptase-polymerase chain reaction: prognostic significance.

BACKGROUND: Recently reverse transcriptase-polymerase chain reaction (RT-PCR) has been proposed as a new sensitive method for the detection of submicroscopic melanoma nodal metastases. Sentinel lymph node (SLN) status is considered the most important prognostic factor for melanoma patients. Thus, in recent years, melanoma research has been focused on identifying new molecular markers of micrometastases. METHODS: In this study, 129 SLNs were collected and analyzed by RT-PCR for tyrosinase and melanoma inhibitory activity (MIA) messenger RNA (mRNA) expression. RESULTS from PCR analysis were then compared with those obtained by hematoxylin and eosin and immunohistochemistry and related to progression of disease. RESULTS: MIA gene expression was positive by RT-PCR in 27% of the tyrosinase-positive SLNs. When the correlation between tyrosinase and/or MIA mRNA expression and disease-free survival was evaluated by the Kaplan-Meier exact test, there was a statistically significant correlation between simultaneous tyrosinase and MIA gene expression in SLNs and progression of disease. CONCLUSIONS: RT-PCR analysis for both MIA and tyrosinase mRNA may identify a subset of melanoma patients with a worse prognosis whom the routine methods, such as histology and immunohistochemistry, fail to identify because of the poor sensitivity of these methods.

Expression and prognostic significance of LIVIN, SURVIVIN and other apoptosis-related genes in the progression of superficial bladder cancer.

BACKGROUND: It has been suggested that progression of superficial bladder cancer may be regulated at the molecular level by a typical pattern of expression of genes involved in apoptosis. Recently LIVIN, belonging to the inhibitors of apoptosis (IAP) family, has been found to be expressed in most solid tumors, where its expression is suggested to have prognostic significance. No data are available concerning the significance of LIVIN in the progression of bladder tumors. PATIENTS AND METHODS: In the present paper we used RT-PCR to investigate the expression of LIVIN isoforms alpha and beta, SURVIVIN, BCL-X and BCL-2/BAX expression ratio both in normal and tumoral bladder tissues, and correlated their expression with the emergence of early relapses in a follow-up of 4 years. This study shows that only the alpha isoform of LIVIN, which is not expressed in normal bladder tissue, is expressed in a proportion of tumors with a high risk of relapse. RESULTS: LIVIN was found in 7/30 patients (23%), SURVIVIN in 9/30 (30%), BCL-2/BAX ratio >1 in 16/30 (53%), BCL-2/BAX expression ratio <1 in 14/30 (46.6%) and BCL-X, only in isoform BCL-X(L), in 11/30 (36.6%). When we evaluated the dependence between each gene expression and relapse free time of patients, we found that LIVIN, high BCL-2/BAX ratio and BCL-X(L), but not SURVIVIN, reached statistical significance in order to predict relapses. CONCLUSIONS: Our findings suggest that LIVIN may be involved in the progression of superficial bladder cancer and used as a marker of early recurrence; while the expression of SURVIVIN cannot be used to identify patients with high risk of relapse.

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.

Effects of isolated limb perfusion with tumor necrosis factor-alpha on circulating levels of proinflammatory cytokines.

Hyperthermic isolated limb perfusion (ILP) with tumor necrosis factor-a (TNFalpha) and cytotoxic drugs is currently used for treatment of melanoma and sarcoma of the limbs. Tumor necrosis factor-alpha is involved in the systemic inflammatory response syndrome as a result of activation of inflammatory cells and production of bioactive substances. The goal of this study was to determine the circulating levels of proinflammatory cytokines and soluble adhesion molecules in 19 patients with limb melanoma or sarcoma undergoing ILP with (n = 9) or without TNFalpha (n = 10). The results obtained demonstrated that ILP with TNFalpha was responsible for a leakage of TNFalpha in the systemic circulation, followed by a rise in interleukin (IL)-6 and IL-8 levels within I h. Elevated soluble (s)P-selectin levels were found 1-3 h after ILP. Plasma sE-selectin peaked 6-9 h after ILP, and soluble vascular cell adhesion molecule (sVCAM) levels reached a maximum after 24 h. Significant correlations were observed among these variables, confirming the interdependence of all changes observed. On the other hand, ILP with cytotoxic drugs alone induced only a modest release of TNFalpha, which was not followed by an immediate rise in IL-6 and IL-8. Four of the 9 patients undergoing ILP with TNF had severe systemic toxicity. No association was found between systemic TNF levels and the clinical outcome, whereas elevated TNF perfusion levels as well as systemic IL-6 and IL-8 levels were constantly elevated in patients with severe toxicity. These results are suggestive of an important role of TNFalpha levels in the perfusion system (more than leakage of perfusate) in causing postoperative toxicity, although other ILP-related factors should not be excluded.

Involvement of the glycoproteic Ib-V-IX complex in nickel-induced platelet activation.

We studied the effect of nickel ions on platelet function because hypernickelemia has been found in patients with acute myocardial infarction. We previously demonstrated that nickel can activate an intracellular pathway leading to cytoskeleton reorganization consequent to tyrosine phosphorylation of p60(src) in human platelets independently of integrin alpha-IIb-beta(3). Moreover, in von Willebrand factor-stimulated platelets, the tyrosine phosphorylation of pp60(c-src) is closely associated with the activation of phosphatidylinositol 3-kinase (PIK), and two adhesion receptors, glycoprotein (Gp)Ib and GpIIb/IIIa(alpha-IIb-beta(3)), are involved. In our study, 1 and 5 mM nickel in the presence of fibrinogen induced platelet aggregation (independently of protein kinase C activation) and secretion. The pretreatment with a PIK inhibitor, wortmannin, strongly decreased nickel-induced platelet aggregation. Platelet treatment with mocarhagin, a cobra venom metalloproteinase that cleaves GpIba, significantly reduced aggregation induced by 5 mM without affecting the response to other agonists such as adenosine diphosphate (ADP). Moreover, nickel caused PIK translocation to the cytoskeleton. Taken together, these observations suggest a partial involvement of both integrins alpha-IIb-beta(3) and GpIb-V-IX complex in Ni(2+)-induced platelet activation.

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.