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Andrew D Kelly,
Benjamin Haibe-Kains,
Katherine A Janeway,
Katherine E Hill,
Eleanor Howe,
Jeffrey Goldsmith,
Kyle Kurek,
Antonio R Perez-Atayde, Nancy Francoeur,
Jian-Bing Fan,
Craig April,
Hal Schneider,
Mark C Gebhardt,
Aedin Culhane,
John Quackenbush,
Dimitrios Spentzos
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ABSTRACT: BACKGROUND: Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly since prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded (FFPE) tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability. METHODS: We performed miRNA and mRNA microarray FFPE assays in 65 osteosarcoma biopsy and 26 paired post-chemotherapy resection specimens and used the only publicly available miRNA dataset, generated independently by another group, to externally validate our strongest findings (n=29). We used supervised principal components analysis and logistic regression for survival and chemoresponse, and miRNA activity and target gene set analysis to study miRNA regulatory activity. RESULTS: Several miRNA-based models with as few as 5 miRNAs were prognostic independently of pathologically assessed chemoresponse (median RFS: 59 months vs. not-yet-reached; adjusted HR=2.90; p=0.036). The independent dataset supported the reproducibility of recurrence and survival findings. The prognostic value of the profile was independent of confounding by known prognostic variables, including chemoresponse, tumor location, and metastasis at diagnosis. Model performance improved when chemoresponse was added as a covariate (median RFS: 59 vs. not-yet-reached; HR=3.91; p=0.002). Most prognostic miRNAs are located at 14q32 - a locus already linked to osteosarcoma - and their gene targets display deregulation patterns associated with outcome. We also identified miRNA profiles predictive of chemoresponse (75-80% accuracy), which did not overlap with prognostic profiles. CONCLUSIONS: FFPE tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA/mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for FFPE-based miRNA biomarker studies in cancer.
Genome Medicine 01/2013; 5(1):2.
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Elena Fountzilas,
Andrew D Kelly,
Antonio R Perez-Atayde,
Jeffrey Goldsmith,
Panagiotis A Konstantinopoulos, Nancy Francoeur,
Mick Correll,
Renee Rubio,
Lan Hu,
Mark C Gebhardt,
John Quackenbush,
Dimitrios Spentzos
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ABSTRACT: MicroRNAs (miRNAs) are nucleic acid regulators of many human mRNAs, and are associated with many tumorigenic processes. miRNA expression levels have been used in profiling studies, but some evidence suggests that expression levels do not fully capture miRNA regulatory activity. In this study we integrate multiple gene expression datasets to determine miRNA activity patterns associated with cancer phenotypes and oncogenic pathways in mesenchymal tumors - a very heterogeneous class of malignancies.
Using a computational method, we identified differentially activated miRNAs between 77 normal tissue specimens and 135 sarcomas and we validated many of these findings with microarray interrogation of an independent, paraffin-based cohort of 18 tumors. We also showed that miRNA activity is imperfectly correlated with miRNA expression levels. Using next-generation miRNA sequencing we identified potential base sequence alterations which may explain differential activity. We then analyzed miRNA activity changes related to the RAS-pathway and found 21 miRNAs that switch from silenced to activated status in parallel with RAS activation. Importantly, nearly half of these 21 miRNAs were predicted to regulate integral parts of the miRNA processing machinery, and our gene expression analysis revealed significant reductions of these transcripts in RAS-active tumors. These results suggest an association between RAS signaling and miRNA processing in which miRNAs may attenuate their own biogenesis.
Our study represents the first gene expression-based investigation of miRNA regulatory activity in human sarcomas, and our findings indicate that miRNA activity patterns derived from integrated transcriptomic data are reproducible and biologically informative in cancer. We identified an association between RAS signaling and miRNA processing, and demonstrated sequence alterations as plausible causes for differential miRNA activity. Finally, our study highlights the value of systems level integrative miRNA/mRNA assessment with high-throughput genomic data, and the applicability of paraffin-tissue-derived RNA for validation of novel findings.
BMC Genomics 07/2012; 13:332. · 4.07 Impact Factor
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ABSTRACT: Resistance to platinum-based chemotherapy develops in the majority of patients with epithelial ovarian cancer (EOC). Platinum compounds form electrophilic intermediates that mediate DNA cross-linking and induce double-strand DNA breaks. Because the cellular response to electrophilic xenobiotics is partly mediated by Keap1-Nrf2 pathway, we evaluated the presence of Kelch-like ECH-associated protein 1 (Keap1) mutations and NF-E2-related factor 2 (Nrf2) pathway activation in EOC and correlated these with platinum resistance and clinical outcome. Nrf2 immunohistochemistry revealed nuclear localization (a surrogate of pathway activation) in over half of EOC patient specimens examined, with more common occurrence in the clear cell EOC subtype. Quantitative real-time PCR revealed that Nrf2 target genes were upregulated in tumors with nuclear positivity for Nrf2. Microarray analysis also showed upregulation of Nrf2 target genes in clear cell EOCs compared with other EOC subtypes. In addition, Keap1 sequence analysis revealed genetic mutations in 29% of clear cell samples and 8% of nonclear cell tumors. RNAi-mediated knockdown of Keap1 was associated with Nrf2 pathway activation and resistance to carboplatin in vitro. Importantly, patients with evidence of Nrf2 pathway activation had fewer complete clinical responses to platinum-based therapy, were enriched for platinum resistance, and had shorter median overall survival compared with those who did not show evidence of Nrf2 pathway activation. Our findings identify Keap1 mutations in EOC and they suggest a previously unrecognized role for the Keap1-Nrf2 pathway in mediating chemotherapeutic responses in this disease.
Cancer Research 06/2011; 71(15):5081-9. · 7.86 Impact Factor
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ABSTRACT: To define a gene expression profile of BRCAness that correlates with chemotherapy response and outcome in epithelial ovarian cancer (EOC).
A publicly available microarray data set including 61 patients with EOC with either sporadic disease or BRCA(1/2) germline mutations was used for development of the BRCAness profile. Correlation with platinum responsiveness was assessed in platinum-sensitive and platinum-resistant tumor biopsy specimens from six patients with BRCA germline mutations. Association with poly-ADP ribose polymerase (PARP) inhibitor responsiveness and with radiation-induced RAD51 foci formation (a surrogate of homologous recombination) was assessed in Capan-1 cell line clones. The BRCAness profile was validated in 70 patients enriched for sporadic disease to assess its association with outcome.
The BRCAness profile accurately predicted platinum responsiveness in eight out of 10 patient-derived tumor specimens, and between PARP-inhibitor sensitivity and resistance in four out of four Capan-1 clones. [corrected] When applied to the 70 patients with sporadic disease, patients with the BRCA-like (BL) profile had improved disease-free survival (34 months v 15 months; log-rank P = .013) and overall survival (72 months v 41 months; log-rank P = .006) compared with patients with a non-BRCA-like (NBL) profile, respectively. The BRCAness profile maintained independent prognostic value in multivariate analysis, which controlled for other known clinical prognostic factors.
The BRCAness profile correlates with responsiveness to platinum and PARP inhibitors and identifies a subset of sporadic patients with improved outcome. Additional evaluation of this profile as a predictive tool in patients with sporadic EOC is warranted.
Journal of Clinical Oncology 08/2010; 28(22):3555-61. · 18.37 Impact Factor
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ABSTRACT: Diagnosis of soft tissue sarcomas (STS) is challenging. Many remain unclassified (not-otherwise-specified, NOS) or grouped in controversial categories such as malignant fibrous histiocytoma (MFH), with unclear therapeutic value. We analyzed several independent microarray datasets, to identify a predictor, use it to classify unclassifiable sarcomas, and assess oncogenic pathway activation and chemotherapy response.
We analyzed 5 independent datasets (325 tumor arrays). We developed and validated a predictor, which was used to reclassify MFH and NOS sarcomas. The molecular "match" between MFH and their predicted subtypes was assessed using genome-wide hierarchical clustering and Subclass-Mapping. Findings were validated in 15 paraffin samples profiled on the DASL platform. Bayesian models of oncogenic pathway activation and chemotherapy response were applied to individual STS samples. A 170-gene predictor was developed and independently validated (80-85% accuracy in all datasets). Most MFH and NOS tumors were reclassified as leiomyosarcomas, liposarcomas and fibrosarcomas. "Molecular match" between MFH and their predicted STS subtypes was confirmed both within and across datasets. This classification revealed previously unrecognized tissue differentiation lines (adipocyte, fibroblastic, smooth-muscle) and was reproduced in paraffin specimens. Different sarcoma subtypes demonstrated distinct oncogenic pathway activation patterns, and reclassified MFH tumors shared oncogenic pathway activation patterns with their predicted subtypes. These patterns were associated with predicted resistance to chemotherapeutic agents commonly used in sarcomas.
STS profiling can aid in diagnosis through a predictor tracking distinct tissue differentiation in unclassified tumors, and in therapeutic management via oncogenic pathway activation and chemotherapy response assessment.
PLoS ONE 01/2010; 5(4):e9747. · 4.09 Impact Factor
