Abstract 5070: Validation of a fresh-tissue based prognostic gene signature in formalin-fixed, paraffin-embedded melanomas
Authors: Brunner Georg , Suter Ludwig , Schulze Hans-Joachim , Berking Carola , Heinecke Achim , Atzpodien Jens 
Published: June 2012

Abstract
Melanoma incidence is rapidly increasing – with a doubling rate of 10-20 years. Precision and reliability of conventional histological and clinical staging, however, remain limited in predicting clinical outcome. On the other hand, complementary molecular prognostic markers are not yet available.
We have recently identified, for the first time, a prognostic gene signature expressed in fresh-frozen primary melanomas (n = 135), which is associated with overall survival (multivariate Cox regression analysis: p = 0.0004, hazard ratio 3.83). The clinical value of a signature-based risk score is its ability to identify patients at low risk, not identified by conventional AJCC staging, and to define risk patients in need of adjuvant therapy. The purpose of the present study was to establish analysis of the signature genes in formalin-fixed, paraffin-embedded (FFPE) melanoma tissue and to validate prognostic significance.
We developed a sensitive and robust methodology to analyze and normalize gene expression in FFPE tissue samples (some of them more than 20 years old): Total RNA was prepared from FFPE sections matching the above fresh-frozen primary melanomas (131 out of 135), quality-controled, and transcribed into cDNA. Human reference RNA was included as an internal standard. Following pre-amplification of the cDNA, expression of the nine signature genes (KRT9, KBTBD10, DCD, ECG2/SPINK7, PIP, SCGB1D2, SCGB2A2, COL6A6, HES6) and of four house-keeping genes (18S rRNA, GAPDH, GUSB, BPNT1) was quantified by real-time PCR using TaqMan assays specific for short amplicons. Gene expression data were normalized, in two steps, to correct for inter-assay technical variability (based on the reference RNA data) and inter-sample variability of RNA quality (based on the data for the house-keeping genes). Significance of correlation of FFPE gene expression data (CT values or estimated mRNA copy numbers) with data from matched fresh-frozen tissue samples (two-sided t-test) or with patient overall survival (univariate Cox regression analysis; clinical follow-up data up to 273 months) was evaluated.
The majority of FFPE primary melanomas (125 out of 131) yielded mRNA of sufficient quality. Expression of all nine signature genes in FFPE melanomas correlated with that in matched fresh-frozen samples. Significance of correlation was higher with CT values (r = 0.58 – 0.19; p = 0.001 – 0.05) than with estimated mRNA copy numbers. Expression of 7 out of the 9 genes (dichotomized CT values) in FFPE melanomas was significantly associated with patient overall survival (p = 0.0001 – 0.0335).
Thus, our prognostic melanoma gene signature was successfully transfered from fresh-frozen onto FFPE tissue samples. This facilitates clinical use of a gene-signature based prognostic risk score and, in addition, allows the retrospective prognostic analysis of primary melanomas.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5070. doi:10.1158/1538-7445.AM2011-5070