The risk of developing cutaneous squamous cell carcinoma (SCC) is markedly increased in organ transplant recipients (OTRs) compared to the normal population.,Next to sun exposure, the immunosuppressive regimen is an important risk factor for the development of SCC in OTRs.,Various gene mutations (e.g.,TP53) and genetic alterations (e.g. loss of CDKN2A, amplification of RAS) have been found in SCCs.,The aim of this genome-wide study was to identify pathways and genomic alterations that are consistently involved in the formation of SCCs and their precursor lesions, actinic keratoses (AKs).,To perform the analysis in an isogenic background, RNA and DNA were isolated from SCC, AK and normal (unexposed) epidermis (NS) from each of 13 OTRs.,Samples were subjected to genome-wide expression analysis and genome SNP analysis using Illumina’s HumanWG-6 BeadChips and Infinium II HumanHap550 Genotyping BeadChips, respectively. mRNA expression results were verified by quantitative PCR.,Hierarchical cluster analysis of mRNA expression profiles showed SCC, AK and NS samples to separate into three distinct groups.,Several thousand genes were differentially expressed between epidermis, AK and SCC; most upregulated in SCCs were hyperproliferation related genes and stress markers, such as keratin 6 (KRT6), KRT16 and KRT17.,Matching to oncogenic pathways revealed activation of downstream targets of RAS and cMYC in SCCs and of NFκB and TNF already in AKs.,In contrast to what has been reported previously, genome-wide SNP analysis showed very few copy number variations in AKs and SCCs, and these variations had no apparent relationship with observed changes in mRNA expression profiles.,Vast differences in gene expression profiles exist between SCC, AK and NS from immunosuppressed OTRs.,Moreover, several pathways activated in SCCs were already activated in AKs, confirming the assumption that AKs are the precursor lesions of SCCs.,Since the drastic changes in gene expression appeared unlinked to specific genomic gains or losses, the causal events driving SCC development require further investigation.,Other molecular mechanisms, such as DNA methylation or miRNA alterations, may affect gene expression in SCCs of OTRs.,Further study is required to identify the mechanisms of early activation of NFκB and TNF, and to establish whether these pathways offer a feasible target for preventive intervention among OTRs.
The rationale for using small molecule inhibitors of oncogenic proteins as cancer therapies depends, at least in part, on the assumption that metastatic tumors are primarily clonal with respect to mutant oncogene.,With the emergence of BRAFV600E as a therapeutic target, we investigated intra- and inter-tumor heterogeneity in melanoma using detection of the BRAFV600E mutation as a marker of clonality.,BRAF mutant-specific PCR (MS-PCR) and conventional sequencing were performed on 112 tumors from 73 patients, including patients with matched primary and metastatic specimens (n = 18).,Nineteen patients had tissues available from multiple metastatic sites.,Mutations were detected in 36/112 (32%) melanomas using conventional sequencing, and 85/112 (76%) using MS-PCR.,The better sensitivity of the MS-PCR to detect the mutant BRAFV600E allele was not due to the presence of contaminating normal tissue, suggesting that the tumor was comprised of subclones of differing BRAF genotypes.,To determine if tumor subclones were present in individual primary melanomas, we performed laser microdissection and mutation detection via sequencing and BRAFV600E-specific SNaPshot analysis in 9 cases.,Six of these cases demonstrated differing proportions of BRAFV600Eand BRAFwild-type cells in distinct microdissected regions within individual tumors.,Additional analyses of multiple metastatic samples from individual patients using the highly sensitive MS-PCR without microdissection revealed that 5/19 (26%) patients had metastases that were discordant for the BRAFV600E mutation.,In conclusion, we used highly sensitive BRAF mutation detection methods and observed substantial evidence for heterogeneity of the BRAFV600E mutation within individual melanoma tumor specimens, and among multiple specimens from individual patients.,Given the varied clinical responses of patients to BRAF inhibitor therapy, these data suggest that additional studies to determine possible associations between clinical outcomes and intra- and inter-tumor heterogeneity could prove fruitful.