A. M. Aglianò

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.

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.

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.

Fibronectin and laminin expression in sentinel lymph nodes of patients with malignant melanoma

SIR, Malignant melanoma (MM) has shown an alarming increase in incidence over the last few decades; currently it represents roughly 5% of skin cancers and 1% of all malignant tumours, with an annual increase in incidence and mortality of 2–3% during the last 30 years. Sentinel lymph node (SLN) biopsy has greatly improved the staging and prognostic evaluation of primary cutaneous MM; in particular, the Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR), which is able to detect melanoma cells expressing tyrosinase mRNA, offers an additional technique to the histopathological examination. The remodelling of cell adhesion molecules during the process of tumour progression is known to play a key functional role in cancer progression and metastasis. To date, although there have been several attempts to identify molecular markers among cell adhesion molecules in melanoma lesions and in peripheral blood from patients with MM, few studies have focused on the involvement of cell adhesion molecules in SLNs of such patients. Fibronectin and laminin are two adhesion molecules that have been shown to be involved in melanoma progression even though their role in SLNs has been poorly investigated. Recently, our group investigated the molecular profile of SLNs, suggesting a role as prognostic indicators for survivin, tyrosinase, MIA and Mart-1 expression. In this study we investigated the role of fibronectin and laminin mRNA expression as prognostic indicators in SLNs of patients with MM and correlated the results to progression of disease in a median follow-up of 51Æ5 months. The presence of capsular naevus cells in SLNs was tested and the positive samples were excluded from the study. We analysed 72 SLNs from 48 patients (mean age 58Æ3 years) with MM; informed consent was obtained from each patient. There were 22, 20 and six patients with stage I, II and III disease, respectively. Sections of each lymph node were stained with haematoxylin and eosin, and immunohistochemistry was performed by using antibodies to HMB-45 antigen and S100 protein that were detected with the avidinbiotin-peroxidase technique. Negative controls were obtained by using normal animal serum instead of specific primary antibodies. The results are shown in Table 1. After surgical excision the SLNs were frozen in liquid nitrogen and stored at –80 C until use in the RT-PCR assay. One microgram of total RNA extracted from the frozen tissues was reverse transcribed in a final volume of 20 lL, then 5 lL of cDNA was amplified in PCR buffer containing 25 pmol each of upstream and downstream specific primers. All the recommended precautions were taken to avoid the possibility of false-positive results. Each RT-PCR experiment included a sample without RNA as negative control and RNA extracted from the SK-MEL 5 melanoma cell line as positive control. The amplification products were separated by electrophoresis on 2% agarose gel: those showing specific amplification products were considered positive. The suitability of all samples was investigated by an RT-PCR with b-actin-specific primers. Laminin expression was found to be positive in 45 of 72 and negative in 27 of 72 samples examined. Positive expression was associated with death or disease progression in 20 of 45 cases, whereas negativity was associated with disease progression in 16 of 27 cases; 11 of 27 remain disease free. Fibronectin gene expression was found in 61 of 72 specimens: positivity was associated with disease progression in 26 of 61 positive samples, while 35 of 61 were disease free. Eleven of 72 samples did not have fibronectin gene expression: negativity was associated with death or disease progression in 10 of 11 cases, whereas one of 11 is currently disease free. The features of patients and the results obtained by the RT-PCR assay are listed in Table 1. Agarose gel electrophoresis of RT-PCR products is shown in Figure 1. In order to verify the statistical significance of our results we first determined the correlation between overall survival, Clark level and fibronectin and laminin expression by the Kaplan– Meier method. We failed to find a statistically significant correlation for the expression of laminin (P = 0Æ57); in contrast, a significant correlation was found between the Clark level and overall survival (P = 0Æ0036) and between negativity for fibronectin and overall survival (P = 0Æ0024). The log-rank test showed a strong correlation between absence of fibronectin expression and Clark level (Spearman correlation P = 0Æ35). The Cox proportional hazards model was applied to the multivariate survival analysis and demonstrated that absence of fibronectin expression and Clark level are statistically significant negative prognostic factors (P = 0Æ012 and P = 0Æ030, respectively).

High levels of transforming growth factor-alpha (TGF-α) 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.