Rosalyn Jewell

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

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 from 2000 to 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 <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 = 2.11 106) 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 histologic 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. (Clin Cancer Res 2009;15(22):693946)

Patterns of Expression of DNA Repair Genes and Relapse From Melanoma

Purpose: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. Experimental Design: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. Results: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10−6). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. Conclusions: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies. Clin Cancer Res; 16(21); 5211–21. ©2010 AACR.

MGMT promoter methylation is associated with temozolomide response and prolonged progression‐free survival in disseminated cutaneous melanoma

To investigate the predictive and prognostic value of O6‐methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single‐agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow‐up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation‐related transcriptional silencing of MGMT mRNA expression was assessed by real‐time RT‐PCR. Response to chemotherapy and progression‐free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p < 0.005). DTIC/TMZ therapy response rate was found to be significantly associated with MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter‐methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single‐agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma.

Inherited variation in the PARP1 gene and survival from melanoma

We report the association of an inherited variant located upstream of the poly(adenosine diphosphate‐ribose) polymerase 1 (PARP1) gene (rs2249844), with survival in 11 BioGenoMEL melanoma cohorts. The gene encodes a protein involved in a number of cellular processes including single‐strand DNA repair. Survival analysis was conducted for each cohort using proportional hazards regression adjusting for factors known to be associated with survival. Survival was measured as overall survival (OS) and, where available, melanoma‐specific survival (MSS). Results were combined using random effects meta‐analysis. Evidence for a role of the PARP1 protein in melanoma ulceration and survival was investigated by testing gene expression levels taken from formalin‐fixed paraffin‐embedded tumors. A significant association was seen for inheritance of the rarer variant of PARP1, rs2249844 with OS (hazard ratio (HR) = 1.16 per allele, 95% confidence interval (CI) 1.04–1.28, p = 0.005, eleven cohorts) and MSS (HR = 1.20 per allele, 95% CI 1.01–1.39, p = 0.03, eight cohorts). We report bioinformatic data supportive of a functional effect for rs2249844. Higher levels of PARP1 gene expression in tumors were shown to be associated with tumor ulceration and poorer OS.

The clinicopathological and gene expression patterns associated with ulceration of primary melanoma

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502‐gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up‐regulation of pro‐inflammatory cytokines suggests that ulceration is associated with tumor‐related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation.

Clinicopathologic features of V600E and V600K melanoma--letter.

The presence of a BRAF mutation in a melanoma tumor predicts response to BRAF inhibitors; however, the biological characteristics of tumors with different BRAF mutations have not been investigated until recently. Menzies and colleagues reported that the rarer V600K BRAF mutations were found more

Laryngo-Onycho-Cutaneous Syndrome

Laryngo-onycho-cutaneous syndrome (LOC) is a rare autosomal recessive genodermatosis characterized by altered cry at birth, skin erosions, nail abnormalities, and excessive granulation tissue in the conjunctivae and larynx. Shabbir first described this condition in 1986 in several affected individuals born to consanguineous families from the Punjabi regions of India and Pakistan. In 2003, the molecular basis of LOC syndrome was elucidated with the discovery of a unique mutation affecting the N-terminus of the α3 chain of laminin-332. In 2008, LOC syndrome was reclassified as a variant of junctional epidermolysis bullosa-other (JEB-O) and termed JEB-LOC. In this chapter, we provide an overview of the clinical features, pathogenesis, and management of LOC syndrome.

Identification of differentially expressed genes in matched formalin‐fixed paraffin‐embedded primary and metastatic melanoma tumor pairs

To take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Identification of differentially expressed genes in matched formalin-fixed paraffin-embedded primary and metastatic melanoma tumor pairs Rosalyn Jewell, Angana Mitra, Caroline Conway, James Iremonger, Christy Walker, Floor de Kort, Martin Cook, Andy Boon, Valerie Speirs and Julia Newton-Bishop

β-Catenin–mediated immune evasion pathway frequently operates in primary cutaneous melanomas

Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with &bgr;-catenin–mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where &bgr;-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a &bgr;-catenin–mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.