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Corresponding author at: Dipartimento di Medicina Sperimentale, “La Sapienza”, Università di Roma, Viale Regina Elena 324, 00161 Roma, Italy. Tel.: +39 0649973011/335; fax: +39 064454820.
Malignant melanoma accounts for nearly 80% skin-cancer-related deaths; some 20% of
patients have occult nodal metastases on Sentinel Lymph Node (SLN) Biopsy, and in
most cases the SLN, is the only lymph node invaded by tumor cells. To detect the presence
of melanoma cells in SLNs, the reverse transcriptase-polymerase chain reaction (RT-PCR)
assay is used as a highly sensitive method to identify tyrosinase mRNA [
]. In the last years, many studies using RT-PCR have investigated the molecular pattern
of gene expression in melanoma patients, to identify a molecular panel of genes useful
to predict the outcome of disease. Much effort has been applied to the extracellular
matrix composition and its organisation, which plays a central role in normal cellular
homeostasis. It has long been recognised that the stroma surrounding a malignant tumor
differs from the normal extracellular matrix, and that different expression of molecules
as fibronectin, laminin and Tenascin C (TnC) has been associated with tumor progression
and/or outcome of patients [
Immunohistochemical expression of extracellular matrix components tenascin, fibronectin,
collagen type IV and laminin breast cancer: their prognostic value and role in tumor
invasion and progression.
]. TnC is an extracellular matrix glycoprotein up-regulated under conditions of tissue
remodelling in inflammatory processes, as well as during fetal and neoplastic growth
[
]; it consists of several isoforms derived from alternative splicing and the large
spliced variant is reported to regulate tumorigenesis in many tumor types [
]. Melanoma cells constitutively synthesise and secrete TnC in vitro. In this study
we investigated TnC expression in 69 SLNs obtained from 45 melanoma patients (25 males
and 20 females), mean age 58.15, enrolled between 1998 and 2003. Informed consent
was obtained from all patients. Tumor thickness, level of invasion, tumor type and
stage of disease, according to TNM classification were documented. The SLNs obtained
after lymphatic mapping were previously tested for the presence of capsular naevus
cells and the positive samples were excluded from the study. Half of each lymph node,
or more, was examined by Histology and IHC, and the remaining part was used for two
RT-PCR molecular assays. Total RNA extracted from the frozen tissues was reverse transcribed
and amplified in two different PCR assay with GAPDH as internal control and TnC primers
(Fig. 1, Panel a). The primers used for the experiments of RT-PCR recognised the large spliced
variant (between the domains A4 and B2) which is predominantly overexpressed in proliferative processes or tumorigenesis
in some tumors [
]. Nevertheless its expression has been never investigated in SLNs of melanoma. The
histologic examination gave a positive result for only one sample, which resulted
also positive for the IHC assay. The analysis performed by IHC showed a positive result
in six samples, whereas all the other samples were negative. The patient positive
for both histology and IHC died and among the patients with positive results by IHC
two died and three are disease free. All sample positive for histology and/or IHC
were also positive for RT-PCR assay. Patients were examined prospectively for recurrent
or metastatic disease at 3-month intervals, during a median follow-up of 50 months
(0.9–109.47 months). Twenty-four of 45 patients enrolled for the study are, at moment
disease free and of the 33 SLNs obtained from those patients 31/33 shown positivity
for TnC expression, whereas 2/33 resulted negative. We obtained 13 SLNs from eight
patients who showed progression of disease; of these SLNs 9/13 resulted positive for
TnC expression and 4/13 gave a negative result. Among the 23 SLNs from 13 patients
who died (13/45), 10/23 shown positive expressions while 13/23 were negative for TnC.
The obtained results from histologic, IHC and RT-PCR are shown in Table 1. To verify the correlation between TnC expression and the clinical features as Breslow
thickness, Clark's level, hystotype, lymph node site, melanoma site and gender, a
multivariate logistic regression analysis has been performed. TnC positivity was not
significantly correlated to any of the clinical features of patients. In order to
correlate the results obtained by RT-PCR with the outcome of patients, the Kaplan
Meyer exact test was applied; p < 0.005 was considered statistically significant. The statistical analysis demonstrated
as TnC mRNA positive expression is a good prognostic factor with a p = 0.0002 (Fig. 1, Panel b). The χ2-test performed to correlate the TnC expression with the outcome of patients, was
statistically significant (p = 0.015). The role of TnC as prognostic factor in cancer patients is still controversial:
in the same tumor type, TnC staining is associated with poor outcome in some studies
and with good prognosis in others, as described, for example, in breast and colon
cancer [
Immunohistochemical expression of extracellular matrix components tenascin, fibronectin,
collagen type IV and laminin breast cancer: their prognostic value and role in tumor
invasion and progression.
]. On one hand, cancer-produced TnC may help epithelial cell to detach from their substratum,
thus allowing metastatization; on the other hand it is now clear that TnC produced
from stromal cells has an opposite function. A favourable function of stromal TnC
would be that it helps to create a dense desmoplastic stroma which may act as a boundary
to contain tumor cells, thus hindering cancer invasion. Our results may suggest that
in the analysed SLNs, negative for the presence of tumoral cells as demonstrated by
histology and immunohistochemistry, TnC is mainly produced by stromal cells, probably
playing a biological defensive role. We believe that this preliminary study, as well
as other performed by our group at molecular level on the SLNs may be useful to outline
molecular prognostic factors in melanoma patients [
]. Furthermore, it would be crucial to evaluate if the positive expression of TnC in
SLNs is due to the stroma or to tumoral cells eventually present in the SLN. Secondary,
it should be appropriate to investigate the expression of TnC by IHC assay in an adequate
number of SLN in order to verify if the significance of the statistical analysis is
maintained using a technique more easily applied to prognostic purpose, even if less
sensitive than RT-PCR. Finally it is our future aim to investigate the role of different
spliced isoforms of TnC in SLNs to identify which isoform may better represent a molecular
marker for melanoma patients.
Fig. 1Molecular analysis of amplification product and Kaplan-Meyer disease-free survival
rate. Panel (a) Analysis of the RT-PCR products was performed by electrophoresis of
20 μl of the amplification products on a 2% agarose gel. Only samples that showed the
specific amplification product were considered positive. Each RT-PCR experiment included
a sample without RNA as a negative control and a sample with RNA extracted from M14
melanoma cell line as positive control. In A are shown some products of amplification
obtained with specific primers which recognize the large isoform of Tenascin C generating
an amplification of 328 bp. To assess the suitability of RNA extracted from SLNs an RT-PCR assay was performed
using GAPDH specific primers; all samples were found suitable. In B are shown some
products of amplification obtained with GAPDH specific primers which generate an amplification
of 168 bp. Lanes (1–4) Samples from sentinel lymph nodes analyzed for the expression of the
large isoform of Tenascin C. Lane (5) Positive control (RNA from M14 cell line). Lane
(6) Negative control (sample without RNA). M Molecular-weight marker. bp Base pair.
Panel (b) The probability of disease-free survival is shown for A, patients with SLN
positive for the large isoform of Tenascin C RNA expression; B, patients with SLN
negative for Large isoform of Tenascin C RNA expression.
Immunohistochemical expression of extracellular matrix components tenascin, fibronectin, collagen type IV and laminin breast cancer: their prognostic value and role in tumor invasion and progression.
