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.

Tyrosinase expression as a molecular marker for investigating the presence of circulating tumor cells in melanoma patients.

In 1991, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was introduced to assess the expression of Tyrosinase in the peripheral blood of melanoma patients, in order to identify the presence of Circulating Melanoma Cells. To date, hundreds of studies, some of which are reviewed here, were performed to assess the clinical value of tyrosinase expression alone, and/or, in addition to other molecular markers. Unfortunately no consensus on the utility of tyrosinase detection exists. In this paper, we underline the presence of too many variables that may interfere with the detection of circulating melanoma cells: from withdrawal and RNA extraction, to Reverse Transcriptase-Polymerase Chain Reaction and the assays used for the analysis of amplification products.

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.

Erythropoietin expression in primary rat Sertoli and peritubular myoid cells.

Kidney and liver are the major organs of erythropoietin (Epo) synthesis. However, Epo messenger RNA (mRNA) has been detected in several organs, such as brain, lung, and testis. Furthermore, functional Epo receptors have been demonstrated on different cell types, including rat Leydig cells. The aim of the study was to identify testicular cells expressing Epo mRNA and to quantitate its levels by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). Besides whole testis, Epo transcripts were found in Sertoli and peritubular myoid cells, while no signal was detected in Leydig cells. Exposure of Sertoli cells to CoCl(2) led to an increase of Epo mRNA level. Semiquantitative competitive RT-PCR presented an increase in the level of Epo mRNA in Sertoli cells stimulated by follicle-stimulating hormone, while exposure of peritubular myoid cells cultures to testosterone reduced Epo mRNA expression. Due to the blood-testis barrier, basal expression of Epo suggests a not yet defined function of this hormone in testis.

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.

Follicle-stimulating hormone, testosterone, and hypoxia differentially regulate UDP-glucuronosyltransferase 1 isoforms expression in rat sertoli and peritubular myoid cells.

Uridine diphosphoglucuronosyltransferases (UGTs) are detoxifying enzymes responsible for the metabolism of endogenous and xenobiotics compounds. UGT isoforms are widely distributed in rat tissues showing a constitutive and inducible gene expression. However, little information is available concerning UGTs expression in testis. The UGT1A1, UGT1A2, and UGT1B1 mRNAs expression in whole rat testis, in Sertoli and peritubular myoid cells in basal conditions, and after hormonal and hypoxic stimulation were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). Constitutive expression of each UGT1 isoform was present in rat testis with higher levels of UGT1A2. UGT transcripts were also detected in Sertoli and peritubular myoid cells. After FSH stimulation, Sertoli cells showed an increase in UGT1B1 mRNA expression, whereas the levels of UGT1A1 and UGT1A2 resulted unmodified. The main effect induced by testosterone was a decrease of UGT1B1 mRNA expression in peritubular myoid cells, whereas in Sertoli cells an increase in UGT1A1 and UGT1B1 was observed. In hypoxic conditions, a reduction in UGTs mRNA levels was detected in both cell types. These findings suggest that rat UGT1 isoforms are regulated in testis by hormonal and environmental factors. Thus, it was speculated that alterations in UGTs expression and/or activity may be involved in the pathogenesis of testis injury.

Detection of basic fibroblast growth factor mRNA in urinary bladder cancer: correlation with local relapses.

Natural history of bladder cancer is characterized by high risk of disease progression even for patients with a clinical diagnosis of superficial disease; in these tumors, the occurrence of local relapse is known to be dependent on the angiogenesis rate. Basic fibroblast growth factor (bFGF), has been described to be elevated in urine and serum of patients with bladder cancer. We investigated the expression of bFGF at mRNA level in a panel of 32 transitional cell tumors of the urinary bladder and in normal bladder tissues used as controls. Expression of bFGF was found elevated in most tumors of high stage, where its presence was found correlated with the occurrence of early local relapses. Furthermore, bFGF was found highly expressed in the majority of tumors showing a high bcl-2 expression rate. Our data suggest that bFGF expression could contribute to the progression of disease; it may provide a prognostic indicator in the identification of patients with high risk for occurrence of local relapses.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

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.

Prevalence of papillomavirus, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus type 2 in urinary bladder cancer.

Recent epidemiological studies suggest that the risk for urological malignancies may be related to the exposure to infectious agents. Human Papillomaviruses type 16 and 18 (HPV 16, HPV 18), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and herpes simplex virus type 2 (HSV-2) have been suggested previously as cofactors in the pathogenesis of some malignancies in humans. The present paper, the presence of HPV 16, HPV 18, EBV, CMV and HSV-2 genomes was investigated in a panel of 35 biopsies from urinary bladder carcinomas using the polymerase chain reaction (PCR). Sequences of EBV, HPV, CMV and HSV-2 genomes were detected in 34%, 31%, 11% and 9% of tissue samples respectively, while in 20% of patients we found more than one viral infection. Absence of viral genomes was found in normal bladder. To our knowledge, this is the first report concerning the association of EBV, CMV and HSV-2 with bladder cancer. This finding may raise the question whether such viral infection may contribute to development and progression of some types of urological malignancies in humans.