A dynamic microRNA profile that tracks a chemotherapy resistance phenotype in osteosarcoma. Implications for novel therapeutics.

Osteosarcoma is a rare primary bone tumor for which no significant therapeutic advancement has been made since the late 1980s despite ongoing efforts. Overall, the five-year survival rate remains about 65%, and is much lower in patients with tumors unresponsive to methotrexate, doxorubicin, and cisplatin therapy. Genetic studies have not revealed actionable drug targets, but our group, and others, have reported that epigenomic biomarkers, including regulatory RNAs, may be useful prognostic tools for osteosarcoma. We tested if microRNA (miRNA) transcriptional patterns mark the transition from a chemotherapy sensitive to resistant tumor phenotype. Small RNA sequencing was performed using 14 patient matched pre-chemotherapy biopsy and post-chemotherapy resection high-grade osteosarcoma frozen tumor samples. Independently, small RNA sequencing was performed using 14 patient matched biopsy and resection samples from untreated tumors. Separately, miRNA specific Illumina DASL arrays were used to assay an independent cohort of 65 pre-chemotherapy biopsy and 26 patient matched post-chemotherapy resection formalin fixed paraffin embedded (FFPE) tumor samples. mRNA specific Illumina DASL arrays were used to profile 37 pre-chemotherapy biopsy and five post-chemotherapy resection FFPE samples, all of which were also used for Illumina DASL miRNA profiling. The National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatments dataset, including PCR based miRNA profiling and RNA-seq data for 86 and 93 pre-chemotherapy tumor samples, respectively, was also used. Paired differential expression testing revealed a profile of 17 miRNAs with significantly different transcriptional levels following chemotherapy. Genes targeted by the miRNAs were differentially expressed following chemotherapy, suggesting the miRNAs may regulate transcriptional networks. Finally, an in vitro pharmacogenomic screen using miRNAs and their target transcripts predicted response to a set of candidate small molecule therapeutics which potentially reverse the chemotherapy resistance phenotype and synergize with chemotherapy in otherwise treatment resistant tumors. Importantly, these novel therapeutic targets are distinct from targets identified by a similar pharmacogenomic analysis of previously published prognostic miRNA profiles from pre chemotherapy biopsy specimens.

Complete tumor necrosis after neoadjuvant chemotherapy defines good responders in patients with Ewing sarcoma.

BACKGROUND: Survival in patients who have Ewing sarcoma is correlated with postchemotherapy response (tumor necrosis). This treatment response has been categorized as the response rate, similar to what has been used in osteosarcoma. There is controversy regarding whether this is appropriate or whether it should be a dichotomy of complete versus incomplete response, given how important a complete response is for in overall survival of patients with Ewing sarcoma. The purpose of this study was to evaluate the impact that the amount of chemotherapy-induced necrosis has on (1) overall survival, (2) local recurrence-free survival, (3) metastasis-free survival, and (4) event-free survival in patients with Ewing sarcoma. METHODS: In total, 427 patients who had Ewing sarcoma or tumors in the Ewing sarcoma family and received treatment with preoperative chemotherapy and surgery at 10 international institutions were included. Multivariate Cox proportional-hazards analyses were used to assess the associations between tumor necrosis and all four outcomes while controlling for clinical factors identified in bivariate analysis, including age, tumor volume, location, surgical margins, metastatic disease at presentation, and preoperative radiotherapy. RESULTS: Patients who had a complete (100%) tumor response to chemotherapy had increased overall survival (hazard ratio [HR], 0.26; 95% CI, 0.14-0.48; p < .01), recurrence-free survival (HR, 0.40; 95% CI, 0.20-0.82; p = .01), metastasis-free survival (HR, 0.27; 95% CI, 0.15-0.46; p ≤ .01), and event-free survival (HR, 0.26; 95% CI, 0.16-0.41; p ≤ .01) compared with patients who had a partial (0%-99%) response. CONCLUSIONS: Complete tumor necrosis should be the index parameter to grade response to treatment as satisfactory in patients with Ewing sarcoma. Any viable tumor in these patients after neoadjuvant treatment should be of oncologic concern. These findings can affect the design of new clinical trials and the risk-stratified application of conventional or novel treatments.

Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

EWSR1-ATF1 dependent 3D connectivity regulates oncogenic and differentiation programs in Clear Cell Sarcoma.

Oncogenic fusion proteins generated by chromosomal translocations play major roles in cancer. Among them, fusions between EWSR1 and transcription factors generate oncogenes with powerful chromatin regulatory activities, capable of establishing complex gene expression programs in permissive precursor cells. Here we define the epigenetic and 3D connectivity landscape of Clear Cell Sarcoma, an aggressive cancer driven by the EWSR1-ATF1 fusion gene. We find that EWSR1-ATF1 displays a distinct DNA binding pattern that requires the EWSR1 domain and promotes ATF1 retargeting to new distal sites, leading to chromatin activation and the establishment of a 3D network that controls oncogenic and differentiation signatures observed in primary CCS tumors. Conversely, EWSR1-ATF1 depletion results in a marked reconfiguration of 3D connectivity, including the emergence of regulatory circuits that promote neural crest-related developmental programs. Taken together, our study elucidates the epigenetic mechanisms utilized by EWSR1-ATF1 to establish regulatory networks in CCS, and points to precursor cells in the neural crest lineage as candidate cells of origin for these tumors.

Genome-wide DNA methylation patterns reveal clinically relevant predictive and prognostic subtypes in human osteosarcoma.

Aberrant methylation of genomic DNA has been reported in many cancers. Specific DNA methylation patterns have been shown to provide clinically useful prognostic information and define molecular disease subtypes with different response to therapy and long-term outcome. Osteosarcoma is an aggressive malignancy for which approximately half of tumors recur following standard combined surgical resection and chemotherapy. No accepted prognostic factor save tumor necrosis in response to adjuvant therapy currently exists, and traditional genomic studies have thus far failed to identify meaningful clinical associations. We studied the genome-wide methylation state of primary tumors and tested how they predict patient outcomes. We discovered relative genomic hypomethylation to be strongly predictive of response to standard chemotherapy. Recurrence and survival were also associated with genomic methylation, but through more site-specific patterns. Furthermore, the methylation patterns were reproducible in three small independent clinical datasets. Downstream transcriptional, in vitro, and pharmacogenomic analysis provides insight into the clinical translation of the methylation patterns. Our findings suggest the assessment of genomic methylation may represent a strategy for stratifying patients for the application of alternative therapies.

Myc is a prognostic biomarker and potential therapeutic target in osteosarcoma.

BACKGROUND: Over the past four decades, outcomes for osteosarcoma patients have plateaued as there have been few emerging therapies showing clinical results. Thus, the identification of novel biomarkers and therapeutic strategies are urgently needed to address these primary obstacles in patient care. Although the Myc-oncogene has known roles in oncogenesis and cancer cell growth, its expression and function in osteosarcoma are largely unknown. METHODS: Expression of Myc was determined by Western blotting of osteosarcoma cell lines and patient tissues, and by immunohistochemistry of a unique osteosarcoma tissue microarray (TMA) constructed from 70 patient samples with extensive follow-up data. Myc specific siRNA and inhibitor 10058-F4 were applied to examine the effect of Myc inhibition on osteosarcoma cell proliferation. The clonogenicity and migration activity was determined by clonogenic and wound-healing assays. A mimic in vivo assay, three-dimensional (3D) cell culture model, was performed to further validate the effect of Myc inhibition on osteosarcoma cell tumorigenic markers. RESULTS: Myc was significantly overexpressed in human osteosarcoma cell lines compared with normal human osteoblasts, and also highly expressed in fresh osteosarcoma tissues. Higher Myc expression correlated significantly with metastasis and poor prognosis. Through the addition of Myc specific siRNA and inhibitor, we significantly reduced Myc protein expression, resulting in decreased osteosarcoma cell proliferation. Inhibition of Myc also suppressed the migration, clonogenicity, and spheroid growth of osteosarcoma cells. CONCLUSION: Our results support Myc as an emerging prognostic biomarker and therapeutic target in osteosarcoma therapy.

MicroRNA-mRNA networks define translatable molecular outcome phenotypes in osteosarcoma.

There is a lack of well validated prognostic biomarkers in osteosarcoma, a rare, recalcitrant disease for which treatment standards have not changed in over 20 years. We performed microRNA sequencing in 74 frozen osteosarcoma biopsy samples, constituting the largest single center translationally analyzed osteosarcoma cohort to date, and we separately analyzed a multi-omic dataset from a large NCI supported national cooperative group cohort. We validated the prognostic value of candidate microRNA signatures and contextualized them in relevant transcriptomic and epigenomic networks. Our results reveal the existence of molecularly defined phenotypes associated with outcome independent of clinicopathologic features. Through machine learning based integrative pharmacogenomic analysis, the microRNA biomarkers identify novel therapeutics for stratified application in osteosarcoma. The previously unrecognized osteosarcoma subtypes with distinct clinical courses and response to therapy could be translatable for discerning patients appropriate for more intensified, less intensified, or alternate therapeutic regimens.

Author Correction: Intragenic antagonistic roles of protein and circRNA in tumorigenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Clinical and Molecular Analysis of Pathologic Fracture-associated Osteosarcoma: MicroRNA profile Is Different and Correlates with Prognosis.

BACKGROUND: MicroRNAs are small, noncoding RNAs that regulate the expression of posttranslational genes. The presence of some specific microRNAs has been associated with increased risk of both local recurrence and metastasis and worse survival in patients with osteosarcoma. Pathologic fractures in osteosarcoma are considered to be more the manifestation of a neoplasm with a more aggressive biological behavior than the cause itself of worse prognosis. However, this has not been proved at the biological or molecular level. Currently, there has not been a microRNA profiling study of patients who have osteosarcoma with and without pathologic fractures that has described differences in terms of microRNA profiling between these two groups and their correlation with biologic behavior. QUESTIONS/PURPOSES: (1) In patients with osteosarcoma of the extremities, how do the microRNA profiles of those with and without pathologic fractures compare? (2) What relationship do microRNAs have with local recurrence, risk of metastasis, disease-specific survival, and overall survival in osteosarcoma patients with pathologic fractures? METHODS: Between 1994 and 2013, 217 patients were diagnosed and treated at our institution for osteosarcoma of the extremities. Patients were excluded if (1) they underwent oncologic resection of the osteosarcoma at an outside institution (two patients) or (2) they were diagnosed with an extraskeletal osteosarcoma (29 patients) or (3) they had less than 1 year of clinical follow-up and no oncologic outcome (local recurrence, metastasis, or death) (four patients). A total of 182 patients were eligible. Of those, 143 were high-grade osteosarcomas. After evaluation of tumor samples before chemotherapy treatment, a total of 80 consecutive samples were selected for sequencing. Demographic and clinical comparison between the sequenced and non-sequenced patients did not demonstrate any differences, confirming that both groups were comparable. Diagnostic samples from the extremities of 80 patients with high-grade extremity osteosarcomas who had not yet received chemotherapy underwent microRNA sequencing for an ongoing large-scale osteosarcoma genome profiling project at our institution. Six samples were removed after a second look by a musculoskeletal pathologist who verified cellularity and quality of samples to be sequenced, leaving a total of 74 patients. Of these, two samples were removed as they were confirmed to be pelvic tumors in a second check after sequencing. The final study sample was 72 patients (11 patients with pathologic fractures and 61 without). Sequencing data were correlated with fractures and local recurrence, risk of metastasis, disease-specific survival, and overall survival through Kaplan-Meier analyses. RESULTS: Several microRNAs were expressed differently between the two groups. Among the markers with the highest differential expression (edgeR and DESeq algorithms), Hsa-mIR 656-3p, hsa-miR 493-5p, and hsa-miR 381-3p were upregulated in patients with pathologic fractures, whereas hsa-miR 363, hsa-miR 885-5p, and has-miR 20b-5p were downregulated. The highest differential expression fracture and nonfracture-associated microRNA markers also distinguished groups of patients with different metastasis risk, a well as different disease-specific and overall survival. Furthermore, the profile of pathologic fractures demonstrated a higher differential expression for microRNA markers that were previously associated with a higher risk of metastasis and lower survival rates in patients with osteosarcoma. CONCLUSIONS: In patients who have osteosarcoma, the microRNA profiles of those with pathologic fractures are different than of patients without pathologic fractures. The highest differential expression mircroRNA molecules in patients with pathologic fractures predict also higher risk of metastatic disease as well as worse disease-specific survival and overall survival. Furthermore, we found higher differential expression of microRNAs in the pathologic fracture group previously associated with poor prognosis. The higher risk of metastasis and poorer overall survival in patients with pathologic fractures is inherent to tumor aggressive biologic behavior. It is plausible that the fracture itself is not the direct cause of worse prognosis but another manifestation of tumor biologic aggressiveness. Identification of these molecules through liquid biopsies may help to determine which patients may benefit from surgery before fractures occur. The same technology can be applied to identify patterns of response to conventional chemotherapy, assisting in more specific and accurate systemic therapy. LEVEL OF EVIDENCE LEVEL: III, prognostic study.

A Collision Tumor Involving a Primary Leiomyosarcoma of the Lower Extremity and a Metastatic Medullary Thyroid Carcinoma: A Case Report.

CASE: A 49-year-old man presented with a rapidly growing thigh mass. Histologic analyses demonstrated separate regions that were consistent with a collision tumor composed of a primary leiomyosarcoma and a metastatic medullary thyroid carcinoma. After responding to chemotherapy, the patient underwent resection of the tumor and a total thyroidectomy; he was disease-free 9 years after the diagnosis. CONCLUSION: A wide diagnostic differential and thorough histologic analysis are necessary in patients presenting with neoplasms of the extremities. A leiomyosarcoma may be a hospitable location for metastatic disease, and the presence of a collision tumor should be considered when pathology findings are equivocal.

Expression and Therapeutic Potential of SOX9 in Chordoma.

Purpose: Conventional chemotherapeutic agents are ineffective in the treatment of chordoma. We investigated the functional roles and therapeutic relevance of the sex-determining region Y (SRY)-box 9 (SOX9) in chordoma.Experimental Design: SOX9 expression was examined by immunohistochemistry (IHC) using 50 chordoma tissue samples. SOX9 expression in chordoma cell lines was examined by Western blot and immunofluorescent assays. We used synthetic human SOX9 siRNA to inhibit the expression of SOX9. Cell proliferation ability and cytotoxicity of inhibiting SOX9 were assessed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) and clonogenic assays. The effect of SOX9 knockdown on chordoma cell motility was evaluated by a wound-healing assay and a Transwell invasion chamber assay. Knockdown of SOX9 induced apoptosis, cell-cycle arrest, as well as decreased expression of cancer stem cell markers were determined by Western blot and flow cytometric assays. The effect of the combination of SOX9 siRNA and the chemotherapeutic drug doxorubicin/cisplatin on chordoma cells was assessed by an MTT assay.Results: Tissue microarray and IHC analysis showed that SOX9 is broadly expressed in chordomas and that higher expression levels of SOX9 correlated with a poor prognosis. RNA interference (RNAi)-mediated knockdown of SOX9 inhibited chordoma cell growth, decreased cell motility, and induced apoptosis as well as cell-cycle arrest. Moreover, the combination of SOX9 inhibition and chemotherapeutic drugs had an enhanced anti-cancer effect on chordoma cells.Conclusions: Our results demonstrate that SOX9 plays a crucial role in chordoma. Targeting SOX9 provides a new rationale for treatment of chordoma. Clin Cancer Res; 23(17); 5176-86. ©2017 AACR.

An imprinted non-coding genomic cluster at 14q32 defines clinically relevant molecular subtypes in osteosarcoma across multiple independent datasets.

BACKGROUND: A microRNA (miRNA) collection on the imprinted 14q32 MEG3 region has been associated with outcome in osteosarcoma. We assessed the clinical utility of this miRNA set and their association with methylation status. METHODS: We integrated coding and non-coding RNA data from three independent annotated clinical osteosarcoma cohorts (n = 65, n = 27, and n = 25) and miRNA and methylation data from one in vitro (19 cell lines) and one clinical (NCI Therapeutically Applicable Research to Generate Effective Treatments (TARGET) osteosarcoma dataset, n = 80) dataset. We used time-dependent receiver operating characteristic (tdROC) analysis to evaluate the clinical value of candidate miRNA profiles and machine learning approaches to compare the coding and non-coding transcriptional programs of high- and low-risk osteosarcoma tumors and high- versus low-aggressiveness cell lines. In the cell line and TARGET datasets, we also studied the methylation patterns of the MEG3 imprinting control region on 14q32 and their association with miRNA expression and tumor aggressiveness. RESULTS: In the tdROC analysis, miRNA sets on 14q32 showed strong discriminatory power for recurrence and survival in the three clinical datasets. High- or low-risk tumor classification was robust to using different microRNA sets or classification methods. Machine learning approaches showed that genome-wide miRNA profiles and miRNA regulatory networks were quite different between the two outcome groups and mRNA profiles categorized the samples in a manner concordant with the miRNAs, suggesting potential molecular subtypes. Further, miRNA expression patterns were reproducible in comparing high-aggressiveness versus low-aggressiveness cell lines. Methylation patterns in the MEG3 differentially methylated region (DMR) also distinguished high-aggressiveness from low-aggressiveness cell lines and were associated with expression of several 14q32 miRNAs in both the cell lines and the large TARGET clinical dataset. Within the limits of available CpG array coverage, we observed a potential methylation-sensitive regulation of the non-coding RNA cluster by CTCF, a known enhancer-blocking factor. CONCLUSIONS: Loss of imprinting/methylation changes in the 14q32 non-coding region defines reproducible previously unrecognized osteosarcoma subtypes with distinct transcriptional programs and biologic and clinical behavior. Future studies will define the precise relationship between 14q32 imprinting, non-coding RNA expression, genomic enhancer binding, and tumor aggressiveness, with possible therapeutic implications for both early- and advanced-stage patients.

MicroRNA-155 expression is independently predictive of outcome in chordoma.

BACKGROUND: Chordoma pathogenesis remains poorly understood. In this study, we aimed to evaluate the relationships between microRNA-155 (miR-155) expression and the clinicopathological features of chordoma patients, and to evaluate the functional role of miR-155 in chordoma. METHODS: The miRNA expression profiles were analyzed using miRNA microarray assays. Regulatory activity of miR-155 was assessed using bioinformatic tools. miR-155 expression levels were validated by reverse transcription-polymerase chain reaction. The relationships between miR-155 expression and the clinicopathological features of chordoma patients were analyzed. Proliferative, migratory and invasive activities were assessed by MTT, wound healing, and Matrigel invasion assays, respectively. RESULTS: The miRNA microarray assay revealed miR-155 to be highly expressed and biologically active in chordoma. miR-155 expression in chordoma tissues was significantly elevated, and this expression correlated significantly with disease stage (p = 0.036) and the presence of metastasis (p = 0.035). miR-155 expression also correlated significantly with poor outcomes for chordoma patients (hazard ratio, 5.32; p = 0.045). Inhibition of miR-155 expression suppressed proliferation, and the migratory and invasive activities of chordoma cells. CONCLUSIONS: We have shown miR-155 expression to independently affect prognosis in chordoma. These results collectively indicate that miR-155 expression may serve not only as a prognostic marker, but also as a potential therapeutic target in chordoma.

A network model for angiogenesis in ovarian cancer.

BACKGROUND: We recently identified two robust ovarian cancer subtypes, defined by the expression of genes involved in angiogenesis, with significant differences in clinical outcome. To identify potential regulatory mechanisms that distinguish the subtypes we applied PANDA, a method that uses an integrative approach to model information flow in gene regulatory networks. RESULTS: We find distinct differences between networks that are active in the angiogenic and non-angiogenic subtypes, largely defined by a set of key transcription factors that, although previously reported to play a role in angiogenesis, are not strongly differentially-expressed between the subtypes. Our network analysis indicates that these factors are involved in the activation (or repression) of different genes in the two subtypes, resulting in differential expression of their network targets. Mechanisms mediating differences between subtypes include a previously unrecognized pro-angiogenic role for increased genome-wide DNA methylation and complex patterns of combinatorial regulation. CONCLUSIONS: The models we develop require a shift in our interpretation of the driving factors in biological networks away from the genes themselves and toward their interactions. The observed regulatory changes between subtypes suggest therapeutic interventions that may help in the treatment of ovarian cancer.

Prognostic significance of miRNA-1 (miR-1) expression in patients with chordoma.

Reliable prognostic biomarkers for chordoma have not yet been established. Recent studies revealed that expression of miRNA-1 (miR-1) is frequently downregulated in several cancer types including chordoma. The goal of this follow-up study is to investigate the expression of miR-1 as a prognostic biomarker and further confirm the functional role of miR-1 in chordoma cell growth and proliferation. We determined the relative expression levels of miR-1 and Met in chordoma tissue samples and correlated those to clinical variables. The results showed that miR-1 was downregulated in 93.7% of chordoma tissues and expression was inversely correlated with Met expression. miR-1 expression levels also correlated with clinical prognosis. To characterize and confirm the functional role of miR-1 in the growth and proliferation of chordoma cells, miR-1 precursors were stably transfected into chordoma cell lines UCH-1 and CH-22. Cell Proliferation Assay and MTT were used to evaluate cell growth and proliferation. Restoring expression of miR-1 precursor decreased cell growth and proliferation in UCH-1 and CH-22 cells. These results indicate that suppressed miR-1 expression in chordoma may in part be a driver for tumor growth, and that miR-1 has potential to serve as prognostic biomarker and therapeutic target for chordoma patients.

Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.

Next-generation sequencing is increasingly employed in biomedical investigations. Strong concordance between microarray and mRNA-seq levels has been reported in high quality specimens but information is lacking on formalin-fixed, paraffin-embedded (FFPE) tissues, and particularly for microRNA (miRNA) analysis. We conducted a preliminary examination of the concordance between miRNA-seq and cDNA-mediated annealing, selection, extension, and ligation (DASL) miRNA assays. Quantitative agreement between platforms is moderate (Spearman correlation 0.514-0.596) and there is discordance of detection calls on a subset of miRNAs. Quantitative PCR (q-RT-PCR) performed for several discordant miRNAs confirmed the presence of most sequences detected by miRNA-seq but not by DASL but also that miRNA-seq did not detect some sequences, which DASL confidently detected. Our results suggest that miRNA-seq is specific, with few false positive calls, but it may not detect certain abundant miRNAs in FFPE tissue. Further work is necessary to fully address these issues that are pertinent for translational research.

Stem cell-like gene expression in ovarian cancer predicts type II subtype and prognosis.

Although ovarian cancer is often initially chemotherapy-sensitive, the vast majority of tumors eventually relapse and patients die of increasingly aggressive disease. Cancer stem cells are believed to have properties that allow them to survive therapy and may drive recurrent tumor growth. Cancer stem cells or cancer-initiating cells are a rare cell population and difficult to isolate experimentally. Genes that are expressed by stem cells may characterize a subset of less differentiated tumors and aid in prognostic classification of ovarian cancer. The purpose of this study was the genomic identification and characterization of a subtype of ovarian cancer that has stem cell-like gene expression. Using human and mouse gene signatures of embryonic, adult, or cancer stem cells, we performed an unsupervised bipartition class discovery on expression profiles from 145 serous ovarian tumors to identify a stem-like and more differentiated subgroup. Subtypes were reproducible and were further characterized in four independent, heterogeneous ovarian cancer datasets. We identified a stem-like subtype characterized by a 51-gene signature, which is significantly enriched in tumors with properties of Type II ovarian cancer; high grade, serous tumors, and poor survival. Conversely, the differentiated tumors share properties with Type I, including lower grade and mixed histological subtypes. The stem cell-like signature was prognostic within high-stage serous ovarian cancer, classifying a small subset of high-stage tumors with better prognosis, in the differentiated subtype. In multivariate models that adjusted for common clinical factors (including grade, stage, age), the subtype classification was still a significant predictor of relapse. The prognostic stem-like gene signature yields new insights into prognostic differences in ovarian cancer, provides a genomic context for defining Type I/II subtypes, and potential gene targets which following further validation may be valuable in the clinical management or treatment of ovarian cancer.

MicroRNA paraffin-based studies in osteosarcoma reveal reproducible independent prognostic profiles at 14q32.

BACKGROUND: Although microRNAs (miRNAs) are implicated in osteosarcoma biology and chemoresponse, miRNA prognostic models are still needed, particularly because prognosis is imperfectly correlated with chemoresponse. Formalin-fixed, paraffin-embedded tissue is a necessary resource for biomarker studies in this malignancy with limited frozen tissue availability. METHODS: We performed miRNA and mRNA microarray formalin-fixed, paraffin-embedded 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 five miRNAs were prognostic independently of pathologically assessed chemoresponse (median recurrence-free survival: 59 months versus not-yet-reached; adjusted hazards ratio = 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 recurrence-free survival: 59 months versus not-yet-reached; hazard ratio = 3.91; P = 0.002). Most prognostic miRNAs were 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% to 80% accuracy), which did not overlap with prognostic profiles. CONCLUSIONS: Formalin-fixed, paraffin-embedded tissue-derived miRNA patterns are a powerful prognostic tool for risk-stratified osteosarcoma management strategies. Combined miRNA and mRNA analysis supports a possible role of the 14q32 locus in osteosarcoma progression and outcome. Our study creates a paradigm for formalin-fixed, paraffin-embedded-based miRNA biomarker studies in cancer.

A microRNA activity map of human mesenchymal tumors: connections to oncogenic pathways; an integrative transcriptomic study.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Metabolomic profiling from formalin-fixed, paraffin-embedded tumor tissue using targeted LC/MS/MS: application in sarcoma.

The relatively new field of onco-metabolomics attempts to identify relationships between various cancer phenotypes and global metabolite content. Previous metabolomics studies utilized either nuclear magnetic resonance spectroscopy or gas chromatography/mass spectrometry, and analyzed metabolites present in urine and serum. However, direct metabolomic assessment of tumor tissues is important for determining altered metabolism in cancers. In this respect, the ability to obtain reliable data from archival specimens is desirable and has not been reported to date. In this feasibility study, we demonstrate the analysis of polar metabolites extracted directly from ten formalin-fixed, paraffin-embedded (FFPE) specimens, including five soft tissue sarcomas and five paired normal samples. Using targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) via selected reaction monitoring (SRM), we detect an average of 106 metabolites across the samples with excellent reproducibility and correlation between different sections of the same specimen. Unsupervised hierarchical clustering and principal components analysis reliably recovers a priori known tumor and normal tissue phenotypes, and supervised analysis identifies candidate metabolic markers supported by the literature. In addition, we find that diverse biochemical processes are well-represented in the list of detected metabolites. Our study supports the notion that reliable and broadly informative metabolomic data may be acquired from FFPE soft tissue sarcoma specimens, a finding that is likely to be extended to other malignancies.