Comparison between three molecular methods for detection of blood melanoma tyrosinase mRNA. Correlation with melanoma stages and S100B, LDH, NSE biochemical markers

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Abstract

Background

The molecular monitoring of circulating tumor cells by reverse transcriptase-PCR (RT-PCR) for patients with melanoma, is still under debate. It may be affected by: a) pre-analytical variability, b) frequency of melanoma-associated gene transcripts and c) the reliability of the methods employed. Few commercial methods are available for the detection of tyrosinase mRNA in blood.

Objective

Comparison between two RT-PCR-nested methods with a third one based on real-time methodology, for detection and quantitation of tyrosinase transcripts, respectively.

Methods

Sixty-two melanoma patients with different AJCC stages and 20 healthy subjects were enrolled. All blood samples were extracted in duplicate with two different methods. Two nested-PCR methods (one commercial and one in house) plus a real time commercial kit were employed.

Results

The two nested PCR methods employed were overimposable, specific and sensitive, at least in the stage III, where there was a concordance between sentinel lymph nodes detection and blood tyrosinase positivity. The different extraction methods did not affect the quality of results, while the commercial real-time kit cannot be used.

Conclusion

Tyrosinase mRNA detection may be therefore employed to monitor the melanoma patients over time in function of response to therapy.

Introduction

In the last years, the molecular monitoring of circulating RNA markers of tumor cells by reverse transcriptase-PCR (RT-PCR) has been employed routinely to evaluate both treatment and outcome in patients with haematological diseases [1], [2], [3]. Moreover, the application of this laboratory approach for patients with solid malignancies, in particular melanoma, is still under debate [4], [5], [6].

Histological analyses on lymph nodes is generally employed to evaluate the stage of disease, while tyrosinase transcripts, detected on lymph nodes and blood, may represent a specific marker of melanocyte presence [4], [5], [6], [7], [8].

Some reports also showed the presence of tyrosinase mRNA in the peripheral blood is associated with the stage of disease and may predict disease progression in patients with advanced melanomas [4], [5], [6], [7], [8].

Qualitative and semi-quantitative techniques have been performed by some researchers to assay the presence of specific melanoma markers (Tyrosinase, Mart-1, gp100, MelanA/Mart-1) in the peripheral blood in association with micro-metastase load [5], [9], [10], [11], [12], [13], [14], [15]. However, investigations by different groups led to conflicting reports on both: (a) the frequency of melanoma-associated gene transcripts in patients with early or advanced disease [7]; and (b) the reliability of the methods employed [16]. The majority of these studies investigated tyrosinase as a single marker, whereas other studies used multiple marker PCR assays, including tyrosinase, MelanA/MART1, gp100, and other ones [17], [18], [19], [20]. There is no uniform consensus that multimarker PCRs may differ in diagnostic relevance as compared with the single tyrosinase mRNA detection.

To investigate the reliability of RT-PCR assays, two interlaboratory quality assurance studies have been performed recently. A multicenter study performed by the European Organisation for Research and Treatment of Cancer (EORTC) Melanoma Group compared the results of various in-house assays on a series of samples, consisting of spiked whole blood, peripheral blood mononuclear cells, and cDNAs [21], [22] with largely homogenous results.

The second quality assurance study evaluated a commercially available method on blood samples of melanoma patients with very heterogeneous results [23]. One of the principle reasons for such discordance is the heterogeneity of cDNA integrity, generated from blood samples, as showed in detail in the EORTC quality assurance study.

Rigorous quality control on a per-sample basis when applying RT-PCR assays in clinical trials is therefore mandatory, following the recommendations of the EORTC Melanoma Group [22].

With conventional or semiquantitative RT-PCR, however, the determination of cDNA quality by means of housekeeping genes is far to be optimal. Quality control by using detection of small amounts of plasmid or foreign cellular standards [16] mixed with the sample is also criticized, because potential damages before mixture cannot be controlled.

Generally, only few commercial methods are available for the detection of tyrosinase mRNA in blood: some of these use the classical RT-PCR technique [16], and few utilize the real-time approach [19].

The aim of the present study is the comparison of results between two RT-PCR-nested (one commercial and one home-made) methods and a third based on real-time technique, for the detection and quantitation of tyrosinase transcripts, respectively. These data will be correlated with the different stages of melanoma and with the S100B, LDH, NSE positivities over cut-off. In fact, these molecules have been considered in the past as possible melanoma markers, more in the disease follow up than in primary diagnosis [11], [12]. These markers, in fact, lack both of sensitivity and specificity.

Section snippets

Patient selection

Sixty-two patients who had given informed consent before the analysis were employed. Clinical disease status was determined according to the recent American Joint Committee on Cancer guidelines [24]. The patients consisted of two groups: (a) 62 subjects with primary cutaneous melanoma (group 1); (b) 20 healthy volunteers as negative controls (group 2) sharing sex and age with the group 1.

Patients' enrolment

Sixty-two consecutive melanoma patients attending to the Dermatological Department of Catholic University

Results

Table 2 reports the results obtained using two nested (one commercial and one in house) and a commercially available real-time PCR, to detect tyrosinase in 62 blood samples of melanoma patients (subdivided by stages AJCC) and 20 normal subjects. In addition a comparison with LDH, S100B and NSE positives over cut-off was made. As shown in Table 2, all patients were histologically classified and, in dependence of Breslow thickness and lymph node status, were also histologically and IHC

Discussion

Controversial data are reported in the literature concerning the role of peripheral blood tyrosinase mRNA reverse transcription PCR analysis in melanoma patient management. In fact, some papers underlined the clinical relationship between tyrosinase mRNA expression and disease outcome, while others evidenced the high degree of variability in the positive rates due to a transient shedding of melanoma cells in the bloodstream. In addition, tyrosinase mRNA expression should not be considered as a

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