Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

ABSTRACT: Prediction of outcome for melanoma patients with surgically-resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinicopathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage 4yr, 90%CI): presence of a nodular component in the primary melanoma (6.8, 0.6-76.0), and small cell size (11.1, 0.8-100.0) or low pigmentation (3.0, 0.8-100.0) in the nodal metastases. Absence of BRAF mutation (20.0, 1.0-1000.0) or NRAS mutation (16.7, 0.6-1000.0) were both favourable prognostic factors. A 46-gene expression signature with strong over-representation of immune response genes was predictive of better survival (10.9, 0.4-325.6); in the full cohort median survival was >100 months in those with the signature, but 10 months in those without. This relationship was validated in two previously published independent stage III melanoma datasets. We conclude that the presence of BRAF mutation, NRAS mutation and absence of an immune-related expressed gene profile predict poor outcome in melanoma patients with macroscopic stage III disease. Samples eligible for this study (n=79) were obtained from lymph node specimens (Melanoma Institute Australia (MIA) Biospecimen Bank) in which macroscopic tumor was observed, obtained from patients believed to be without distant metastases at the time of tumor banking based on clinical examination and computerised axial tomographic scanning of the brain, chest, abdomen and pelvis. Specimens were macro-dissected at time of banking and subsequently reviewed to meet minimum criteria for tumor cell content (>80%) and amount of necrosis (<30%). Linked clinical and pathologic data were obtained from the MIA research database. Total RNA was extracted from 20-30mg of fresh frozen tissue. Tissue samples were homogenized using a high-speed agitation polytron blender (Kinematica, Luzern, Switzerland) in the presence of Trizol. The RNA was isolated and purified with an RNeasy purification kit (Qiagen RNeasy purification kit- Qiagen Pty Ltd., Clifton Hill, Victoria, Australia) with DNAse I digestion on the column. The quality of the RNA preparations was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA, USA). RNA integrity scores were >8 for all samples analyzed. cRNA amplification and labelling with biotin were performed using Illumina TotalPrep RNA amplification kit according to the manufacturer’s directions (Ambion, Inc., Austin, TX, USA) with 250ng total RNA as input material. Gene expression analysis was performed using the Sentrix Human-6 v3 Expression BeadChips (Illumina, Inc., San Diego, CA, USA), and BeadStation system from Illumina as per manufacturer’s instructions. Expression BeadChip using array annotation based on R-2.11.0 and illuminaHumanv3.db. Quality control was performed on all chips using R/Bioconductor and the lumi package (www.bioconductor.org). Data normalization was performed using a variance-stabilizing transform (VST) and quantile normalization as implemented in the lumi package for R/Bioconductor.

ORGANISM(S): Homo sapiens

SUBMITTER: Graham Mann

PROVIDER: E-GEOD-53118 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in patients with stage III melanoma.

Mann Graham J GJ Pupo Gulietta M GM Campain Anna E AE Carter Candace D CD Schramm Sarah-Jane SJ Pianova Svetlana S Gerega Sebastien K SK De Silva Chitra C Lai Kenneth K Wilmott James S JS Synnott Maria M Hersey Peter P Kefford Richard F RF Thompson John F JF Yang Yee Hwa YH Scolyer Richard A RA

The Journal of investigative dermatology 20120830 2

Prediction of outcome for melanoma patients with surgically resected macroscopic nodal metastases is very imprecise. We performed a comprehensive clinico-pathologic assessment of fresh-frozen macroscopic nodal metastases and the preceding primary melanoma, somatic mutation profiling, and gene expression profiling to identify determinants of outcome in 79 melanoma patients. In addition to disease stage <II at initial presentation, the following clinical and pathologic factors were independent pre ...[more]

PMID: 22931913

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Project description:In patients with metastatic melanoma, the identification and validation of accurate prognostic biomarkers will assist rational treatment planning. Studies based on "-omics" technologies have focussed on a single high-throughput data type such as gene or microRNA transcripts. Occasionally, these features were evaluated in conjunction with limited clinico-pathologic data. With the increased availability of multiple data types, there is a pressing need to tease apart which of these sources contain the most valuable prognostic information. We evaluated and integrated several data types derived from the same tumor specimens in AJCC stage III melanoma patients - gene, protein, and microRNA expression as well as clinical, pathologic and mutation information - to determine their relative impact on prognosis. We used classification frameworks based on pre-validation and bootstrap multiple imputation classification to compare the prognostic power of each data source, both individually as well as integratively. We found that the prognostic utility of clinico-pathologic information was not out-performed by various "-omics" platforms. Rather, a combination of clinico-pathologic variables and mRNA expression data performed best. Furthermore, a patient-based classification analysis revealed that the prognostic accuracy of various data types was not the same for different patients, providing useful insights for ongoing developments in the individualized treatment of melanomas patients. SPECIAL NOTE: In this study, survival data were re-extracted from the MIA research database for all patients and brought up to date, revealing discrepancies affecting survival class in the case of four patients compared with the previous dataset (GSE53118: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE53118). The current survival data are considered to be more accurate although the expression information has not changed. In addition, there were 5 samples (122, 144, 195, 264, and 358) for which gene expression information were not available at the time of analysis. However, the associated clinical information for these samples is provided since it was analysed elsewhere in the accompanying publication. Samples eligible for this study (n=84) were obtained from lymph node specimens (Melanoma Institute Australia (MIA) Biospecimen Bank) in which macroscopic tumor was observed, obtained from patients believed to be without distant metastases at the time of tumor banking based on clinical examination and computerised axial tomographic scanning of the brain, chest, abdomen and pelvis. Specimens were macro-dissected at time of banking and subsequently reviewed to meet minimum criteria for tumor cell content (>80%) and amount of necrosis (<30%). Linked clinical and pathologic data were obtained from the MIA research database. We previously analyzed the distribution of survival times in these samples and identified more favorable and less favorable groups as patients having time from surgery to death from melanoma greater than 4 years with no sign of relapse (n=25) or less than 1 year (n=22), respectively (Mann et al. 2013, PMID: 22931913). Since that publication, survival data have been re-extracted from the MIA research database for all patients and brought up to date, revealing discrepancies affecting survival class in the case of four patients compared with the previous dataset. The current survival data are considered to be more accurate. MRNA expression profiling and somatic mutation profiling, were performed as previously described in Mann et al. 2013 (PMID: 22931913).

Project description:Circulating tumor cells (CTCs) are critical in the development of distant organ tumor metastasis, and are associated with advanced cancer stage and poor patient outcome. Here, we present the first genome-wide nucleotide-level characterization of CTCs. Our single-nucleotide polymorphism (SNP) analysis in patients with melanoma involved: 1) global comparative genomic analysis of CTCs and matched regional metastases, 2) identification of key genomic aberrations in CTCs, 3) verification of these target genes in aggressive distant tumor metastases, and 4) evidence of selective expression and functional consequence of CTC-associated genes in melanomas. We report 131 aberrant loci in CTCs that are potentially pro-metastatic, and show that such expression of a 5-marker gene panel (CSMD2, CNTNAP5, FLJ14051, ADAM6, TRPM2) in melanomas confers prognostic utility. Successful treatment of melanoma requires understanding of the metastatic process and identification of patients with tumors most likely to develop aggressive metastatic disease. Melanomas are heterogeneous, and CTCs have long been recognized as vehicles for cancer spread, representing particularly aggressive tumor clones that can evolve into successful clinical metastases. Elucidation of genomic aberrations in CTCs will aid in the development of prognostic biomarkers and therapeutic strategies to target CTCs to prevent or control distant cancer spread. This study provides the first detailed genomic confirmation of the close relation between CTCs and tumor metastases, and illustrates how CTCs can be utilized as a novel approach and rational source for identification of pro-metastatic genes in cancer research. Three individual patient cohorts were utilized in the study. CNV and LOH loci were evaluated initially in metastatic melanoma patients (n=13) in a discovery cohort. SNP loci that harbored CNV/LOH in CTCs were then separately verified for: (a) presence in distant organ metastasis (AJCC Stage IV melanoma) (n=27), and (b) relevance to prognosis in regional melanoma metastasis (AJCC Stage III melanoma) (n=35). The first discovery patient cohort group was utilized for capture of CTCs, and consisted of peripheral blood mononuclear cell (PBMC) and tumor specimens from metastatic melanoma patients (n=13). CTC-related loci were verified in a second cohort of patients with Stage IV distant organ metastases (n=27), including 15 brain, 4 lung, and 8 gastrointestinal (bowel, liver) metastases. The third patient cohort consisted of early passage (<12) established melanoma cell lines derived from 35 regional melanoma metastases (Stage III) for evaluation of the prognostic utility of the CTC-associated aberrant loci.

Project description:The biology underlying nodal metastasis is poorly understood. Transcriptome profiling has helped to characterize both primary tumors seeding nodal metastasis and the metastasis themselves. The interpretation of these data, however, is not without ambiguities. Here we profiled the transcriptomes of 17 papillary thyroid cancer (PTC) nodal metastases, associated primary tumors and primary tumors from N0 patients. We also included patient-matched normal thyroid and lymph node samples as controls to address some limits of previous studies. We found that the transcriptomes of patient-matched primary tumors and metastases were more similar than of unrelated metastases/primary pairs, a result also reported in other organ systems, and that part of this similarity reflected patient background. We found that the comparison of patient-matched primary tumors and metastases was heavily confounded by the presence of lymphoid tissues in the metastasis samples. An original data adjustment procedure was developed to circumvent this problem. It revealed a differential expression of stroma-related gene expression signatures also regulated in other organ systems. The comparison of N0 vs. N+ primary tumors uncovered a signal irreproducible across independent PTC datasets. This signal was also detectable when comparing the normal thyroid tissues adjacent to N0 and N+ tumors, suggesting a cohort specific bias also likely to be present in previous studies with similar statistical power. Classification of N0 vs. N+ yielded an accuracy of 63%, but additional statistical controls not presented in previous studies, revealed that this is likely to occur by chance alone. To address this issue, we used large datasets from The Cancer Genome Atlas and showed that N0 vs. N+ classification rates could not be reached randomly for most cancers. Yet, it was significant, but of limited accuracy (<70%) for thyroid, breast and head and neck cancers. We profiled the transcriptomes of 11 primary PTCs with no detectable nodal invasion, 17 primary PTCs with nodal invasion and 17 patient-matched nodal metastases. We also profiled a number of control samples. These included 24 patient-matched normal thyroid tissues (11 from N0, 13 from N+ patients), and 4 normal lymph nodes; technical and biological replicates including additional nodal metastasis for 3 patients, adjacent blocks for 5 primary tumors, 4 normal thyroid tissues and 1 nodal metastasis.

Dataset Information

BRAF Mutation, NRAS Mutation, and the Absence of an Immune-Related Expressed Gene Profile Predict Poor Outcome in Patients with Stage III Melanoma

Publications

BRAF mutation, NRAS mutation, and the absence of an immune-related expressed gene profile predict poor outcome in patients with stage III melanoma.

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