An Empirical Evaluation of Normalization Methods for MicroRNA Arrays in a Liposarcoma Study.

BACKGROUND: Methods for array normalization, such as median and quantile normalization, were developed for mRNA expression arrays. These methods assume few or symmetric differential expression of genes on the array. However, these assumptions are not necessarily appropriate for microRNA expression arrays because they consist of only a few hundred genes and a reasonable fraction of them are anticipated to have disease relevance. METHODS: We collected microRNA expression profiles for human tissue samples from a liposarcoma study using the Agilent microRNA arrays. For a subset of the samples, we also profiled their microRNA expression using deep sequencing. We empirically evaluated methods for normalization of microRNA arrays using deep sequencing data derived from the same tissue samples as the benchmark. RESULTS: In this study, we demonstrated array effects in microRNA arrays using data from a liposarcoma study. We found moderately high correlation between Agilent data and sequence data on the same tumors, with the Pearson correlation coefficients ranging from 0.6 to 0.9. Array normalization resulted in some improvement in the accuracy of the differential expression analysis. However, even with normalization, there is still a significant number of false positive and false negative microRNAs, many of which are expressed at moderate to high levels. CONCLUSIONS: Our study demonstrated the need to develop more efficient normalization methods for microRNA arrays to further improve the detection of genes with disease relevance. Until better methods are developed, an existing normalization method such as quantile normalization should be applied when analyzing microRNA array data.

The miR-17-92 cluster and its target THBS1 are differentially expressed in angiosarcomas dependent on MYC amplification.

Angiosarcomas (ASs) represent a heterogeneous group of malignant vascular tumors that may occur spontaneously as primary tumors or secondarily after radiation therapy or in the context of chronic lymphedema. Most secondary ASs have been associated with MYC oncogene amplification, whereas the role of MYC abnormalities in primary AS is not well defined. Twenty-two primary and secondary ASs were analyzed by array-comparative genomic hybridization (aCGH) and by deep sequencing of small RNA libraries. By aCGH and subsequently confirmed by fluorescence in situ hybridization, MYC amplification was identified in three out of six primary tumors and in 8 out of 12 secondary AS. We have also found MAML1 as a new potential oncogene in MYC-amplified AS. Significant upregulation of the miR-17-92 cluster was observed in MYC-amplified AS compared to AS lacking MYC amplification and the control group (other vascular tumors, nonvascular sarcomas). Moreover, MYC-amplified ASs were associated with a significantly lower expression of thrombospondin-1 (THBS1) than AS without MYC amplification or controls. Altogether, our study implicates MYC amplification not only in the pathogenesis of secondary AS but also in a subset of primary AS. Thus, MYC amplification may play a crucial role in the angiogenic phenotype of AS through upregulation of the miR-17-92 cluster, which subsequently downregulates THBS1, a potent endogenous inhibitor of angiogenesis.

mRNA and protein levels of FUS, EWSR1, and TAF15 are upregulated in liposarcoma.

Translocations or mutations of FUS, EWSR1, and TAF15 (FET) result in distinct genetic diseases. N-terminal translocations of any FET protein to a series of transcription factors yields chimeric proteins that contribute to sarcomagenesis, whereas mutations in the conserved COOH-terminal domain of wild-type FUS were recently shown to cause familial amyotrophic lateral sclerosis. We thus investigated whether the loss of one FUS allele by translocation in liposarcoma may be followed by mutations in either the remaining FUS allele or the paralogous EWSR1. Furthermore, we investigated the strength of the FET promoters and their contributions to sarcomagenesis given the proteins' frequent involvement in oncogenic translocations. We sequenced the respective genomic regions of both FUS and EWSR1 in 96 liposarcoma samples. Additionally, we determined FET transcript and protein levels in several liposarcoma cell lines. We did not observe sequence variations in either FUS or EWSR1. However, protein copy numbers reached an impressive 0.9 and 5.5 Mio of FUS and EWSR1 per tumor cell, respectively. Compared with adipose-derived stem cells, FUS and EWSR1 protein expression levels were elevated on average 28.6-fold and 7.3-fold, respectively. TAF15 mRNA levels were elevated on average 3.9-fold, although with a larger variation between samples. Interestingly, elevated TAF15 mRNA levels did not translate to strongly elevated protein levels, consistent with its infrequent occurrence as translocation partner in tumors. These results suggest that the powerful promoters of FET genes are predominantly responsible for the oncogenic effect of transcription factor translocations in sarcomas.