Gene Expression Profiling of Paraffin-Embedded Primary Melanoma Using the DASL Assay Identifies Increased Osteopontin Expression as Predictive of Reduced Relapse-Free Survival

Abstract

Purpose Gene expression studies in melanoma have been few because tumors are small and cryopreservation is rarely possible. The purpose of this study was to evaluate the Illumina DASL Array Human Cancer Panel for gene expression studies in formalin-fixed melanoma primary tumors, and to identify prognostic biomarkers. Experimental Design Primary tumors from two studies were sampled using a tissue microarray needle. Study 1: 254 tumors from a melanoma cohort recruited 2000-2006. Study 2: 218 tumors from a case-control study of patients undergoing sentinel node biopsy. Results RNA was obtained from 76% of blocks. 1.4% of samples failed analysis (transcripts from less than 250 of the 502 genes on the DASL chip detected). Increasing age of the block and increased melanin in the tumor were associated with reduced number of genes detected. The gene whose expression was most differentially expressed in association with relapse free survival in study 1 was osteopontin (SPP1, p=2.11×10−6) and supportive evidence for this was obtained in study 2 used as a validation set (p=0.006) (unadjusted data). Osteopontin level in study 1 remained a significant predictor of relapse free survival when data were adjusted for age, sex, tumor site and histological predictors of relapse. Genes whose expression correlated most strongly with osteopontin were PBX1, BIRC5 (Survivin) and HLF. Conclusion Expression data were obtained from 74% of primary melanomas and provided confirmatory evidence that osteopontin expression is a prognostic biomarker. These results suggest that predictive biomarker studies may be possible using stored blocks from mature clinical trials.

The role of osteopontin expression in melanoma progression

“…Previous studies have reported that increased OPN mRNA expression is associated with unfavourable patient prognosis in human cancers, including melanoma [9,29]. In addition, we extended our studies to OPN protein levels and therefore fabricated tissue microarray platforms for 93 primary melanomas.…”

“…In human malignant melanoma, OPN expression is significantly associated with reduced relapse-free survival and additional histological parameters that are associated with poor clinical outcome [7,24,29,33]. OPN is a prognostic marker in a variety of cancers [30,[39][40][41].…”

“…Several studies indicate that OPN protein is overexpressed in the majority of human cancers, including malignant melanoma, and is associated with tumor progression [14,[23][24][25][26][27][28][29][30]. Extensive microarray analyses of melanomas demonstrated that OPN also plays a role in melanoma progression [14,28,31,32].…”

Differential gene expression profiling of primary cutaneous melanoma and sentinel lymph node metastases

“…Highly pigmented specimens were avoided due to previously published technical issues that reported low RNA yield in the presence of abundant melanin. 19 A total of 22 samples were screened; however, only 19 samples were used, 3 being excluded on the basis of poor RNA quality. In all, 10 primary melanomas and 9 sentinel lymph node metastases were evaluated, including 4 autologous pairs of primary melanoma and sentinel lymph node metastasis (from 4 patients).…”

“…Others, using cell lines or tissue samples, have studied DNA expression profiling of normal melanocytes, nevi, primary melanomas, and regional and distant metastases. [7][8][9][10][11][12][13][14][15][16][17][18][19] Conway et al 19 used the cDNA-mediated annealing, selection, extension, and ligation assay to evaluate formalin-fixed, paraffin-embedded archival primary melanoma specimens, and reported osteopontin overexpression as a potential prognostic biomarker. Rangel et al, 20 using immunohistochemistry, had previously shown that overexpression of osteopontin, correlated with the likelihood of sentinel lymph node metastasis.…”