Fusion-positive Rhabdomyosarcoma Research Articles

Abstract 1939: Loss of MST/Hippo signaling promotes tumorigenesis in a genetically engineered mouse model of fusion-positive alveolar rhabdomyosarcoma

Abstract While improvement in survival for pediatric cancer patients over the last 40 years has been encouraging, certain cancer types evade cure. One such example is fusion-positive alveolar rhabdomyosarcoma (aRMS), a pediatric soft tissue sarcoma of mesenchymal origin with skeletal muscle features and a 5-year survival rate of <50%. A hallmark of this aggressive malignancy is the t{2;13} translocation fusion gene PAX3-FOXO1 (PF). Primary cell-based modeling experiments in our laboratory and others have shown that PF is necessary but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously we showed that PF-positive aRMS tumorigenesis relies on alterations in Hippo pathway signaling, including upregulation of the YAP transcriptional co-activator, and downregulation of the MST/Hippo kinase, which occurs when the scaffold protein RASSF4 (itself a PF transcriptional target) binds and inhibits MST1/Hippo. We hypothesized that ablating MST/Hippo signaling in an existing genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis and provide insight into the role of this pathway in aRMS. To assess the role of MST/Hippo signaling in aRMS, MST1/2-floxed (Stk3F/F;Stk4F/F) mice were crossed with an established MSTWT aRMS GEMM driven by conditional expression of Pax3-Foxo1 from the endogenous Pax3 locus and conditional loss of Cdkn2a in Myf6-expressing cells. Statistical analysis revealed that compared to MSTWT aRMS control animals, MSTNull have significantly accelerated tumorigenesis (median survival 112 vs. 224 days, p< 0.0001) and increased tumor penetrance (76% vs. 27%). MSTNull animals developed tumors disproportionately in the head and neck as compared to control, and incurred multiple tumors per animal. Tumors were analyzed via immunohistochemistry for MyoD and myogenin, markers of RMS, as well as Myf5, an early marker of myogenesis. Tumors in both cohorts were for positive for aRMS markers. Tumor-derived cell lines were used for in vitro cell-based assays and molecular interrogation. We have identified the MST/Hippo signaling axis as an important tumor suppressor mechanism in aRMS. The rapid onset and increased penetrance of tumorigenesis in this GEMM provides a powerful tool for interrogating aRMS biology and screening novel therapeutics. Citation Format: Kristianne Oristian, Lisa E. Crose, Rex Bentley, Nina Kuprasertkul, David G. Kirsch, Corinne Linardic. Loss of MST/Hippo signaling promotes tumorigenesis in a genetically engineered mouse model of fusion-positive alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2017-1939

Abstract 3877: Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 [P3F] or PAX7-FOXO1 [P7F] resulting from 2;13 and 1;13 chromosomal translocations) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. To further examine the significance of DNA methylation, we generated genome-wide DNA methylation profiles for a new cohort of 48 RMS tumors for which we previously assessed mutation, copy number and expression status. Investigation of the RMS subsets defined by methylation clustering revealed a significant association of methylation with P3F versus P7F fusion status in the FP subset, and an association of methylation with RAS mutation status in the FN subset. Localization studies of differentially methylated probes showed these probes were not evenly distributed with respect to annotated genomic features. In particular, hypomethylated probes were enriched in FP tumors in the promoter region and in the intergenic region, whereas hypermethylated probes were enriched in these regions in FN tumors. In contrast, hypermethylated probes were enriched in FP tumors in the 3' UTR region whereas hypomethylated probes were enriched in FN tumors in these regions. In our new larger cohort of cases, there was a significant difference in the distribution of P3F binding sites between genes with and without differential methylation. Integrative analysis of P3F binding sites, promoter methylation and gene expression demonstrated that genes with P3F binding sites tended to be more highly expressed in FP tumors (compared to FN tumors) than genes without P3F binding sites regardless of promoter methylation status. Though promoter hypomethylation is most highly associated with enhanced expression among genes with P3F binding sites, the group of genes with P3F binding sites and promoter hypomethylation is small in number compared to the much larger group of genes with P3F binding sites but without promoter hypomethylation. In conclusion, these results demonstrate the interaction of these epigenetic changes with mutational alterations and transcriptional organization in RMS tumors and provide a direction for future studies of these epigenetic events. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Jack F. Shern, Sivasish Sindiri, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2017-3877

Abstract 693: Antibody-based targeting of the cell surface receptor tyrosine kinase FGFR4 in rhabdomyosarcoma and other cancers

Abstract Fibroblast Growth Factor Receptor 4 (FGFR4) is a receptor tyrosine kinase that is highly expressed in rhabdomyosarcoma (RMS), an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. FGFR4 is expressed during development in myoblasts, but not in terminally differentiated skeletal muscle. Of note, FGFR4 is directly induced by the chimeric fusion oncogene PAX3-FOXO1 found in the most aggressive fusion-positive RMS and is mutationally activated in approximately 10-13% of the remaining fusion-negative RMS. High FGFR4 expression is associated with adverse outcome and is a driver oncogene in RMS. Metastatic RMS has a dismal outcome in the clinic with <30% survival. Novel treatments are needed for this devastating disease of children and young adults. Given that FGFR4 is functionally important and differentially overexpressed in RMS, we therefore hypothesized that FGFR4 is an ideal target for antibody-based therapeutics. To test our hypothesis, we first generated 15 binders against FGFR4: 3 murine monoclonal antibodies (mAbs), 2 rabbit mAbs and 10 fully human mAbs. We then assessed the specificity and affinity of these binders for FGFR4 by ELISA and surface plasmon resonance. Using flow cytometry and confocal microscopy, we next evaluated whether the mAbs internalized upon binding FGFR4. We then selected the internalizing binders from our panel for development of antibody-drug conjugates (ADCs). To investigate the ability of internalizing anti-FGFR4 mAbs to deliver cytotoxic payloads to RMS cells, we used a rapid screening method which employs drug-conjugated secondary antibodies (2°ADC), and thus circumvents the need for direct conjugation of cytotoxic small molecules to mAbs. When the anti-FGFR4 primary antibody and 2°ADC are co-administered, target cells internalize both antibodies in complex with the antigen. In addition to rapid screening of binders, this method facilitates comparison of various cytotoxic payloads, such as DNA alkylating and tubulin polymerization inhibiting agents. From the internalization and cytotoxicity screening data, we identified two murine mAbs that bound FGFR4 with low nanomolar affinity, readily internalized and killed RMS cells when paired with an anti-murine 2°ADC carrying MMAF or DMDM. In summary, we have determined that our anti-FGFR4 ADCs can deliver cytotoxic payloads to target cells through receptor-mediated endocytosis. We will next convert the two internalizing murine mAbs into mouse/human chimeric mAbs, directly conjugate them with a cytotoxic payload and test them in vitro and in vivo against RMS cell lines and RMS patient derived cells. The ultimate goal of these studies is to demonstrate the potential for clinical utility of FGFR4 targeting and develop novel therapeutics that can make a clinical impact on RMS and other FGFR4 overexpressing cancers, such as hepatocellular carcinoma, breast and lung cancers. Citation Format: Martin Skarzynski, Nitya Shivaprasad, Baskar Subramanian, David Azorsa, Zhongyu Zhu, Dimiter Dimitrov, Javed Khan. Antibody-based targeting of the cell surface receptor tyrosine kinase FGFR4 in rhabdomyosarcoma and other cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2017-693

Histology, fusion status, and outcome in metastatic rhabdomyosarcoma: A report from the Children's Oncology Group.

Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) has historically been of prognostic and therapeutic importance. However, classification has been complicated by shifting histologic criteria required for an ARMS diagnosis. Children's Oncology Group (COG) studies after IRS-IV, which included the height of this diagnostic shift, showed both an increased number of ARMS and an increase in the proportion of fusion-negative ARMS. Following diagnostic standardization and histologic re-review of ARMS cases enrolled during this era, analysis of low-risk (D9602) and intermediate-risk (D9803) rhabdomyosarcoma (RMS) studies showed that fusion status rather than histology best predicts prognosis for patients with RMS. This analysis remains to be completed for patients with high-risk RMS. We re-reviewed cases on high-risk COG studies D9802 and ARST0431 with an enrollment diagnosis of ARMS. We compared the event-free survival (EFS) and overall survival by histology, PAX-FOXO1 fusion, and clinical risk factors (Oberlin score) for patients with metastatic RMS using the log-rank test. Histology re-review resulted in reclassification as ERMS for 12% of D9802 cases and 5% of ARST0431 cases. Fusion-negative RMS had a superior EFS to fusion-positive RMS; however, poorer outcome for metastatic RMS was most related to clinical risk factors including age, primary site, and number of metastatic sites. In contrast to low- or intermediate-risk RMS, in metastatic RMS, clinical risk factors have the most impact on patient outcome. PAX-FOXO1 fusion is more common in patients with a high Oberlin score, but fusion status is not an independent biomarker of prognosis.

The Role of PDGFR-β Activation in Acquired Resistance to IGF-1R Blockade in Preclinical Models of Rhabdomyosarcoma

To determine what alternative pathways may act as mechanisms of bypass resistance to type 1 insulin-like growth factor receptor (IGF-1R) blockade in rhabdomyosarcoma (RMS), we compared expression of receptor tyrosine kinase activity in a number of IGF-1R antibody-resistant and -sensitive RMS cell lines. We found that platelet-derived growth factor receptor β (PDGFR-β) activity was upregulated in three xenograft-derived IGF-1R antibody-resistant cell lines that arose from a highly sensitive fusion-positive RMS cell line (Rh41). Furthermore, we identified four additional fusion-negative RMS cell lines that similarly upregulated PDGFR-β activity when selected for IGF-1R antibody resistance in vitro. In the seven cell lines described, we observed enhanced growth inhibition when cells were treated with dual IGF-1R and PDGFR-β inhibition in vitro. In vivo studies have confirmed the enhanced effect of targeting IGF-1R and PDGFR-β in several mouse xenograft models of fusion-negative RMS. These findings suggest that PDGFR-β acts as a bypass resistance pathway to IGF-1R inhibition in a subset of RMS. Therapy co-targeting these receptors may be a promising new strategy in RMS care.

Abstract 2434: DNA methylation characterization of fusion-positive and fusion-negative rhabdomyosarcoma primary tumors and cell lines

Abstract BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 or PAX7-FOXO1) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. The current study will explore these issues in a larger, separate cohort of RMS tumors and compare DNA methylation in RMS cell lines and primary tumors. METHODS: DNA methylation was examined in 48 RMS tumors (21 FP and 27 FN) as well as 10 RMS cell lines (5 FP and 5 FN) on the Illumina HumanMethylation450 BeadChip platform. RESULTS: Unsupervised clustering analysis using the most variable probes (top 1%) in the 48 RMS tumors revealed that patterns of DNA methylation segregated these tumors into two distinct subgroups; one subgroup contains all 21 FP cases along with 2 FN cases and a second subgroup contains 25 of the 27 FN cases. A principal component analysis confirmed this close association of methylation pattern and fusion status, and showed that the two “discordant” FN cases map in a region between the FP and FN clusters. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Application of an 11-gene methylation signature developed in our earlier study classified these 48 cases into FP and FN categories with >95% accuracy. In contrast to our previous findings, there was a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Though unsupervised clustering analysis indicated that FP tumors and cell lines cluster as do the FN tumors and cell lines, a principal component analysis clarified these relationships by showing that the two groups of cell lines are located at a considerable distance from the two tumor subtypes. Analysis of the most varied probes in the tumors indicated that the vast majority of these probes are hypermethylated in all RMS cell lines. In a complementary analysis with the most variable probes (top 1%) in the cell lines, most of these probes are hypomethylated in all RMS tumors, though two smaller groups of probes show differential methylation between both groups of FP and FN samples. CONCLUSIONS: This study provides an independent validation that FP and FN RMS tumors possess distinct and characteristic methylation profiles. The enrichment of PAX3-FOXO1 binding sites in genes that are differentially methylated between these FP and FN tumors suggests that the PAX3-FOXO1 fusion protein may contribute to this methylation pattern. These analyses also indicate that RMS cell lines do not faithfully recapitulate the DNA methylation patterns that characterize primary tumors. Citation Format: Wenyue Sun, Bishwanath Chatterjee, John F. Shern, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. DNA methylation characterization of fusion-positive and fusion-negative rhabdomyosarcoma primary tumors and cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2434.

Abstract 4457: Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy

Abstract Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric tumor driven by an oncogenic fusion protein, PAX3-FOXO1, which acts as a transcription factor. Conventional chemotherapy is effective for low risk patients who have a 5-year overall survival greater than 65%, while high risk patients, including those with metastatic disease, have less than 40% survival. Consequently, we hypothesize that targeting the fusion protein or its collaborators in transcription regulation will provide novel therapies for this aggressive subtype of RMS. To identify new druggable PAX3-FOXO1 interactors, we performed a combined proteomic and genetic screen which led to the discovery of the NuRD complex (Nucleosome Remodelling and Deacetylase) as a major PAX3-FOXO1 co-regulator. The NuRD complex is unique among the chromatin remodelling complexes due to its dual enzymatic activity. It can act by histone deacetylation through HDAC1/2 (histone deacetylases) or influence nucleosome positioning through CHD4 (chromodomain-DNA-binding protein 4). Intriguingly, it has been associated with both activating and repressive activities in gene expression and its role in cancer development is not fully understood yet. We found that in FP-RMS, silencing of CHD4 affected the expression of approximately 50% of PAX3-FOXO1 regulated target genes. These were mainly genes which are usually upregulated, suggesting an activating role for NuRD. Consistent with CHD4 activation activity, ChIP-seq experiments demonstrated that CHD4 and HDAC2 co-localize with the fusion protein in cis-regulatory sites of a subset of its target genes. Interestingly, gene expression analysis showed that both CHD4 and HDAC2 are highly expressed in tumor tissue and myoblasts when compared to normal skeletal muscle, inferring a potential role of the NuRD complex in maintaining the undifferentiated phenotype observed in FP-RMS. Importantly, CHD4 silencing had no effect on myoblasts proliferation whereas a profound growth reduction was seen in FP-RMS cell lines, suggesting a unique tumour dependency on this chromatin remodeler. In addition, depletion of CHD4 caused a complete regression of xenograft tumours in mice.In summary, we have identified the NuRD complex as an essential positive co-regulator of PAX3-FOXO1 transcriptional activity. Our data propose a critival role of one of the NuRD core component CHD4 in FP-RMS cell viability, making CHD4 an attractive new target for therapy. To our knowledge, CHD4 is the first chromatin remodeler identified to associate with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy.Ongoing work is currently underway to identify first-in-class small molecules to inhibit CHD4 protein. Citation Format: Joana G. Marques, Berkley Gryder, Maria Boehm, Marco Wachtel, Young Song, Hsien-Chao Chou, Rajesh Patidar, Hongling Liao, Javed Khan, Beat W. Schaefer. Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4457.

PAX7 is a required target for microRNA-206-induced differentiation of fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS can be parsed based on clinical outcome into two subtypes, fusion-positive RMS (FP-RMS) or fusion-negative RMS (FN-RMS) based on the presence or absence of either PAX3-FOXO1 or PAX7-FOXO1 gene fusions. In both RMS subtypes, tumor cells show histology and a gene expression pattern resembling that of developmentally arrested skeletal muscle. Differentiation therapy is an attractive approach to embryonal tumors of childhood including RMS; however, agents to drive RMS differentiation have not entered the clinic and their mechanisms remain unclear. MicroRNA-206 (miR-206) expression increases through normal muscle development and has decreased levels in RMS compared with normal skeletal muscle. Increasing miR-206 expression drives differentiation of RMS, but the target genes responsible for the relief of the development arrest are largely unknown. Using a combinatorial approach with gene and proteomic profiling coupled with genetic rescue, we identified key miR-206 targets responsible for the FN-RMS differentiation blockade, PAX7, PAX3, NOTCH3, and CCND2. Specifically, we determined that PAX7 downregulation is necessary for miR-206-induced cell cycle exit and myogenic differentiation in FN-RMS but not in FP-RMS. Gene knockdown of targets necessary for miR-206-induced differentiation alone or in combination was not sufficient to phenocopy the differentiation phenotype from miR-206, thus illustrating that miR-206 replacement offers the ability to modulate a complex network of genes responsible for the developmental arrest in FN-RMS. Genetic deletion of miR-206 in a mouse model of FN-RMS accelerated and exacerbated tumor development, indicating that both in vitro and in vivo miR-206 acts as a tumor suppressor in FN-RMS at least partially through downregulation of PAX7. Collectively, our results illustrate that miR-206 relieves the differentiation arrest in FN-RMS and suggests that miR-206 replacement could be a potential therapeutic differentiation strategy.

Histology, Fusion Status, and Outcome in Alveolar Rhabdomyosarcoma With Low-Risk Clinical Features: A Report From the Children's Oncology Group.

Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) is of prognostic and therapeutic importance. Criteria for classifying these entities evolved significantly from 1995 to 2013. ARMS is associated with inferior outcome; therefore, patients with alveolar histology have generally been excluded from low-risk therapy. However, patients with ARMS and low-risk stage and group (Stage 1, Group I/II/orbit III; or Stage 2/3, Group I/II) were eligible for the Children's Oncology Group (COG) low-risk rhabdomyosarcoma (RMS) study D9602 from 1997 to 1999. The characteristics and outcomes of these patients have not been previously reported, and the histology of these cases has not been reviewed using current criteria. We re-reviewed cases that were classified as ARMS on D9602 using current histologic criteria, determined PAX3/PAX7-FOXO1 fusion status, and compared these data with outcome for this unique group of patients. Thirty-eight patients with ARMS were enrolled onto D9602. Only one-third of cases with slides available for re-review (11/33) remained classified as ARMS by current histologic criteria. Most cases were reclassified as ERMS (17/33, 51.5%). Cases that remained classified as ARMS were typically fusion-positive (8/11, 73%), therefore current classification results in a similar rate of fusion-positive ARMS for all clinical risk groups. In conjunction with data from COG intermediate-risk treatment protocol D9803, our data demonstrate excellent outcomes for fusion-negative ARMS with otherwise low-risk clinical features. Patients with fusion-positive RMS with low-risk clinical features should be classified and treated as intermediate risk, while patients with fusion-negative ARMS could be appropriately treated with reduced intensity therapy.

CDK4 Amplification Reduces Sensitivity to CDK4/6 Inhibition in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS. We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS. Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3–FOXO1 or PAX7–FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from ‘aberrant’ hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3–FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3–FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2′-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

Abstract 3278: Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma

Abstract BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Based on the presence or absence of a characteristic fusion gene (either PAX3-FOXO1 or PAX7-FOXO1), RMS comprises two major subtypes: fusion-positive (FP) and fusion-negative (FN). The goal of this study is to analyze DNA methylation differences between FP and FN tumors and to determine how these methylation differences contribute to biological differences between FP and FN RMS. METHODS: DNA methylation profiles of 37 RMS tumors (20 FP and 17 FN) and 10 RMS cell lines (5 FP and 5 FN) as well as 8 normal tissues (4 muscles and 4 bone marrows) were assessed using DNA methylation arrays. Expression array analysis was performed on 58 RMS tumors (33 FP and 25 FN, including 16 with matched methylation data). Bioinformatic methods including classification, differential methylation, differential expression, methylation-expression correlation and gene set analysis were carried out. RESULTS: Unsupervised clustering clearly distinguished the FP and FN subsets in 37 RMS primary tumors and in 10 RMS cell lines. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Integrative methylation and gene expression analysis revealed that methylation differences between FP and FN tumors could either be positively or negatively associated with mRNA expression. Though there was no significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation, PAX3-FOXO1 binding sites were enriched among genes that were both differentially methylated and differentially expressed. An 11-gene DNA methylation signature, classifying the RMS tumors into FP and FN subsets, was established in 37 RMS primary tumors and validated in 10 RMS cell lines. Pyrosequencing studies of 6 of the methylation signature genes independently confirmed differential methylation between FP and FN cases for the CpG site queried on the methylation array as well as other nearby CpG sites. To further investigate the relationship between DNA methylation and expression, we focused on the EMILIN1 gene, which shows higher methylation and lower mRNA expression in FP tumors compared to FN tumors. After treating RMS cell lines with 5-Aza-2′-deoxycytidine, the EMILIN1 gene demonstrated decreased DNA methylation and increased mRNA expression in multiple FP cell lines. CONCLUSION: FP and FN RMS tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important role of epigenetics in RMS tumorigenesis, suggest novel approaches for identifying fusion status, and may find new therapeutic targets in RMS. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Frederic G. Barr. Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2015-3278

Abstract 3093: CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes an aggressive, PAX3-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in a subset of fusion-positive RMS. Other tumor types with CDK4 amplification or overexpression, including liposarcoma and neuroblastoma, are sensitive to CDK4/6 inhibition, suggesting that CDK4/6-targeted therapies may provide a new treatment strategy in fusion-positive RMS. To evaluate the role of CDK4 in chromosomal region 12q13-14 amplification in fusion-positive RMS and the potential clinical utility of CDK4/6 inhibition in this disease setting, we examined the biological consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition in fusion-positive RMS in vitro and in vivo. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and non-amplified fusion-positive RMS cells via G1-phase cell cycle arrest. This arrest was associated with reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, however, were not observed in RMS cells overexpressing CDK4 or in fusion-positive tumors harboring 12q13-14 amplification relative to control cells or tumors lacking amplification, respectively. Treatment with the small molecule CDK4/6 inhibitor LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell cycle arrest. All fusion-positive RMS cell lines showed sensitivity to CDK4/6 inhibition, with evidence of differential antitumor activity resulting from an inverse relationship between CDK4 expression and inhibitor vulnerability. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and non-amplified fusion-positive RMS. Our findings demonstrate that CDK4 is necessary for RB-E2F-mediated G1-phase cell cycle progression, proliferation, and transformation in fusion-positive RMS regardless of CDK4 amplification status. Our studies further indicate that single-agent LEE011 is active in the setting of fusion-positive RMS and suggest that CDK4 amplification may be a marker of reduced sensitivity whereas low CDK4 expression may be associated with higher susceptibility to CDK4/6 inhibition. Collectively, our data provide preclinical evidence supporting further investigation of CDK4/6-targeted therapies in treatment regimens for fusion-positive RMS. Citation Format: Mary E. Olanich, Wenyue Sun, Stephen M. Hewitt, Zied Abdullaev, Svetlana D. Pack, Frederic G. Barr. CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2015-3093

Abstract 597: Coding and noncoding gene targets in pediatric soft tissue sarcomas

Abstract BACKGROUND: Genomic studies on pediatric soft tissue sarcomas (STS) such as rhabdomyosarcoma (RMS) and Ewing family of tumors (EFT) have shown that the overall burden of somatic mutations in these tumors is relatively low when compared to most adult tumors. This observation along with recent studies suggest that other features such as chromosomal translocations that appear predominantly in these tumors may play an important role in their biology. Genes resulting from these translocations are known to regulate protein-coding genes that are required for tumor formation. Our studies on these tumors show that these chimeric fusion genes can hijack the cellular machinery that comprises both protein-coding and non-coding networks for tumorigenesis. RESULTS: Gene expression signature of the more aggressive RMS subset that comprises of PAX-FKHR-positive alveolar RMS suggested that the protein-coding NOS1 is most strongly associated and prognostically significant gene in this group. Functional assays confirmed that PAX-FKHR directly regulated its expression. Decreasing NOS1 expression using lentiviral vectors resulted in a decrease in cell survival and reduced tumor growth in vivo. Similar expression analyses comparing EFT, which express the fusion gene EWS-FLI1, with other tumors and normal tissues showed that the non-coding features were more strongly associated with EFT than the coding genes. EWS-FLI1 directly regulates a long intergenic non-coding RNA (lincRNA), FEZF1-AS1. Matrigel assays and tail vein injection models suggested that the lincRNA promotes cell invasion and migration. Chromatin-RNA immunoprecipitation assays revealed interaction of the lincRNA with DNA regions, both coding and non-coding, which are involved in invasion and metastasis. CONCLUSIONS: Fusion genes in pediatric STS activate genomic regions that facilitate tumor pathogenesis by regulating the interactive network between coding and non-coding genes. While fusion-positive RMS are dependent on NOS1 for survival, EFT need lincRNA FEZF1-AS1 for cell migration and tumor propagation. EFT are known to be highly metastatic tumors and our results underscore the complex regulatory networks that are required for cellular migration in these tumors. Understanding the importance of these gene interactions will help development of effective tumor-specific drug therapies that improve treatment while decreasing unwanted side-effects. Citation Format: Sheetal A. Mitra, Anirban P. Mitra, Jonathan D. Buckley, Timothy J. Triche. Coding and noncoding gene targets in pediatric soft tissue sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 597. doi:10.1158/1538-7445.AM2015-597

Abstract 481: Rationale for targeting the protein chaperone network in rhabdomyosarcoma

Abstract Cancer cells face unique proteotoxic stresses by virtue of genomic instability, increased translational output, and hypoxic environments. Coordinately, these stresses enhance dependence on cellular chaperones in order to preserve functional proteins and enable cancer cell survival. We hypothesized that perturbing specific nodes in this chaperone network may uncover unexpected vulnerabilities in certain cancer cells that can be exploited for therapeutic benefit. We found that amongst a large panel of patient-derived cancer cell lines, rhabdomyosarcomas exhibit exquisite sensitivity to the selective HSP70 inhibitor MAL3-101. Rhabdomyosarcoma is the most common soft tissue sarcoma affecting children and adolescents, and survival is exceedingly poor in patients with high risk disease. We observed that MAL3-101, at low micromolar concentrations, rapidly induces apoptosis in both fusion-positive and fusion-negative rhabdomyosarcoma cells. Unexpectedly, this was not accompanied by degradation of previously described obligate HSP70 clients or of the alveolar rhabdomyosarcoma driver oncogene PAX3-FOXO1. Instead, transcriptome profiling revealed that the predominant effector of HSP70 inhibition was activation of the unfolded protein response (UPR). We observed that combined knockdown of the constitutive HSP70 family member HSPA8 as well as the inducible members HSPA1A/B is required to phenocopy the effects of MAL3-101 treatment, confirming HSP70 as the critical target of MAL3-101. Through genetic experiments, we further showed that HSP70 inhibition with MAL3-101 induces cell death via the UPR effector CHOP, a pro-apoptotic transcription factor. Surprisingly, we found that the ER-resident UPR sensor IRE1 exhibits the greatest control on CHOP levels after MAL3-101 treatment, and appeared to do so independently of its endonuclease activity. Rhabdomyosarcoma cells grown under continuous drug exposure to generate isogenic models of resistance demonstrated a novel disconnect between HSP70 inhibition and UPR activation, which appears to be mediated in part by upregulation of another HSP70 family member, HSPA12A. Together, these data highlight a notable dependence of rhabdomyosarcoma cells on cytosolic HSP70 chaperones to silence a lethal unfolded protein response. Our data also suggest a previously underexplored link between the kinase activity of IRE1 and transcriptional control of CHOP, and identify CHOP as a potential biomarker of HSP70 inhibitor efficacy. The poor prognosis of alveolar rhabdomyosarcoma patients harboring a recurrent amplification at 12q13-14, which includes the locus encoding for CHOP, suggests an ideal target population for trials of early phase therapies that might target this vulnerability. Altogether, our findings provide a promising novel mechanism-based therapeutic strategy to improve outcomes in patients suffering from high risk rhabdomyosarcoma. Citation Format: Amit J. Sabnis, Christopher J. Guerriero, Jeffrey L. Brodsky, Trever Bivona. Rationale for targeting the protein chaperone network in rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2015-481

Abstract 3273: PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis

Abstract The PAX3-FOXO1 fusion gene, which is generated by a 2; 13 chromosomal translocation, is a characteristic feature of fusion-positive rhabdomyosarcoma (RMS), a major RMS subtype associated with aggressive behavior and poor prognosis. This study utilizes a novel inducible expression system in human myoblasts to dissect the molecular mechanism and contribution of PAX3-FOXO1 in RMS tumorigenesis.A human myoblast cell line (immortalized by BMI1 and TERT expression) was transduced with a retroviral construct that constitutively expresses MYCN and a lentiviral based- doxycycline inducible construct that variably and reversibly expresses PAX3-FOXO1. Focus formation and animal xenograft experiments were performed to study transformation in vitro and tumorigenesis in vivo, respectively. Myogenic differentiation was assessed by light microscopy and by western blot or immunohistochemical assays of muscle-specific protein expression.PAX3-FOXO1-transduced myoblasts treated with doxycycline demonstrated a time- and dose-dependent increase in expression of PAX3-FOXO1 mRNA and protein and its downstream targets genes; doxycycline withdrawal led to cessation of fusion protein expression. Though myoblasts expressing PAX3-FOXO1 or MYCN alone did not show any evidence of transformation in culture, combined PAX3-FOXO1 and MYCN expression resulted in myoblast transformation. Under differentiation-promoting culture conditions, combined PAX3-FOXO1 and MYCN expression inhibited myogenic differentiation. Intramuscular injection of myoblasts with MYCN and PAX3-FOXO1 resulted in rapid RMS tumor formation in NOD-SCID mice when fusion protein expression was induced by feeding mice a doxycycline-containing diet. Myoblasts with MYCN expression alone did not form any tumors while PAX3-FOXO1 induction without MYCN expression resulted in RMS tumors only after a much longer latency period. After tumors formed from myoblasts expressing PAX3-FOXO1 with or without MYCN, down-regulation of PAX3-FOXO1 expression by doxycycline withdrawal resulted in tumor regression associated with widespread myogenic differentiation. However, the regressed tumors slowly grew back in the absence of doxycycline induction demonstrating a PAX3-FOXO1-independent oncogenic mechanism for recurrence.The PAX3-FOXO1 fusion protein collaborated with MYCN in the initial stage of RMS tumorigenesis to promote dysregulated cell proliferation and inhibit myogenic differentiation. Though most cells in the initial tumor were dependent on the fusion protein, recurrent tumors formed in which the fusion protein was not required to maintain the tumorigenic phenotype. Citation Format: Puspa R. Pandey, Stephen M. Hewitt, Markku M. Miettinen, Frederic G. Barr. PAX3-FOXO1 is essential for initiation but not for recurrence during rhabdomyosarcoma tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3273. doi:10.1158/1538-7445.AM2015-3273

Myogenin, AP2β, NOS-1, and HMGA2 Are Surrogate Markers of Fusion Status in Rhabdomyosarcoma

Pediatric rhabdomyosarcoma (RMS) is traditionally classified on the basis of the histologic appearance into alveolar (ARMS) and embryonal (ERMS) subtypes. The majority of ARMS contain a PAX3-FOXO1 or PAX7-FOXO1 gene fusion, but about 20% do not. Intergroup Rhabdomyosarcoma Study stage-matched and group-matched ARMS typically behaves more aggressively than ERMS, but recent studies have shown that it is, in fact, the fusion status that drives the outcome for RMS. Gene expression microarray data indicate that several genes discriminate between fusion-positive and fusion-negative RMS with high specificity. Using tissue microarrays containing a series of both ARMS and ERMS, we identified a panel of 4 immunohistochemical markers-myogenin, AP2β, NOS-1, and HMGA2-which can be used as surrogate markers of fusion status in RMS. These antibodies provide an alternative to molecular methods for identification of fusion-positive RMS, particularly in cases in which there is scant or poor-quality material. In addition, these antibodies may be useful in fusion-negative ARMS as an indicator that a variant gene fusion may be present.

Abstract 24: Diagnosing small round cell tumors using whole transcriptome expression profiling.

Abstract INTRODUCTION: Differential diagnosis of malignant small round cell tumors (SRCTs) can be challenging due to their undifferentiated character. Their primitive histopathological features often render the identification of morphological features difficult and therefore, no definitive diagnosis may be possible. This is crucial in pediatric SRCTs as disparate approaches to therapy are used for distinct tumor types based on a patient's risk. This study sought to discover and implement diagnostic signatures for a variety of SRCTs using whole transcriptome profiling. METHODS: RNAs from 115 prospectively-obtained pediatric SRCTs were profiled on Affymetrix Human Exon microarrays. These comprised of Ewing sarcoma (n=10), neuroblastoma (n=10), PAX-FKHR fusion-negative rhabdomyosarcoma (F-RMS, n=20), PAX-FKHR fusion-positive rhabdomyosarcoma (F+RMS, n=19), lymphoma (n=10), Wilms tumor (n=10), clear cell sarcoma (n=7), rhabdoid tumor (n=9), medulloblastoma (n=10), and osteosarcoma (n=10). Expressions of 1,393,765 probe selection regions (PSRs) were analyzed. Binary screens were performed for each tumor type to identify diagnostic PSRs. Selected PSRs were combined as a signature, and its diagnostic potential was assessed by k-nearest neighbor algorithms. Primers designed to interrogate loci corresponding to the diagnostic PSRs were tested on cell lines and cases representing the various tumor types using the WaferGen SmartChip MyDesign platform. RESULTS: Binary screens identified hundreds of diagnostic PSRs corresponding to annotated and unannotated transcripts for each tumor type. To limit signature size, the top 6 PSRs were chosen for each tumor type. 17%-83% of features in each signature represented non-exonic content. Hierarchical clustering demonstrated the ability of each signature to diagnose the corresponding tumor by binary and multiplex analyses. Discrimination analysis showed that signatures for Ewing sarcoma, neuroblastoma, F+RMS, lymphoma and osteosarcoma were the best performing and most exclusive. Overall, each tumor signature had robust diagnostic potential (binary discrimination, all p<0.0001). To explore the possibility of translating the signatures to a quantitative PCR platform, RNA from 8 cell lines corresponding to 5 tumor types were used: TC32, CHLA10, TC252 (Ewing sarcoma); RD (F-RMS); Rh30, JR (F+RMS); REH (surrogate for lymphoma); and Saos-2 (osteosarcoma). Diagnostic primers sets showed substantial efficacy in distinguishing cell lines and cases corresponding to the tumor type of interest when compared with other samples. CONCLUSIONS: Using pediatric SRCTs as an example, this study illustrates that whole transcriptome analysis can reveal potential transcripts that can robustly diagnose malignancies that pose a histological challenge. Such concise genomic signatures can be translated to quantitative PCR-based assays for routine clinical implementation. Citation Format: Anirban P. Mitra, Dana Haydel, Sheetal A. Mitra, Jude Dunne, Scott Silveria, Larry Wang, Timothy J. Triche. Diagnosing small round cell tumors using whole transcriptome expression profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 24. doi:10.1158/1538-7445.AM2013-24

Genomic and clinical analysis of fusion gene amplification in rhabdomyosarcoma: A report from the Children's Oncology Group

Alveolar rhabdomyosarcoma (RMS) is an aggressive pediatric cancer of the myogenic lineage with frequent chromosomal translocations involving the PAX3 or PAX7 and FOXO1 genes. Based on previous studies indicating that the fusion genes are amplified in a subset of these cancers, we conducted a comprehensive molecular and clinical investigation of these amplification events. Using oligonucleotide arrays to localize amplicons, we found that the minimal 1p36 amplicon measured 0.13 Mb and only contained PAX7 whereas the minimal 13q14 amplicon measured 0.53 Mb and contained FOXO1 and the poorly characterized LOC646982 gene. Application of a fluorescence in situ hybridization assay to over 100 fusion-positive cases revealed that the fusion gene is amplified in 93% of PAX7-FOXO1-positive and 9% of PAX3-FOXO1-positive cases. While most cells in amplified PAX7-FOXO1-positive cases contained the amplicon, only a fraction of cells in the amplified PAX3-FOXO1-positive cases contained the amplicon. Expression studies demonstrated that the fusion transcripts were generally expressed at higher levels in amplified cases, and that the PAX7-FOXO1 fusion transcript was expressed at higher levels than the PAX3-FOXO1 fusion transcript. Finally, fusion gene amplification and PAX7-FOXO1 fusion status were each associated with significantly improved outcome; a multivariate analysis demonstrated that this predictive value was independent of other standard prognostic parameters. These findings therefore provide further evidence for a novel good prognosis subset of fusion-positive RMS.

Abstract 4350: Translating diagnostic gene expression profiles for pediatric solid tumors

Abstract BACKGROUND: Features of cancer genomics including gene expression levels can be employed to create biomarker profiles that can predict prior to therapy the diagnosis and prognosis of an individual patient. We aim to translate robust and reproducible diagnostic and prognostic profiles for pediatric solid tumors into clinical tools applicable to all tumor specimens, fresh frozen (FF) or formalin fixed (FFPE). RESULTS: We used Affymetrix GeneChip Human Exon 1.0 ST (HuEx) arrays on 24 cell lines or tumors (4 fusion-positive rhabdomyosarcoma [RMSpos], 5 fusion-negative RMS [RMSneg], 6 Ewing sarcoma family of tumors [ESFT], 5 neuroblastoma [NB], and 4 osteosarcoma [OS]) to identify a 41-feature diagnostic metagene that clearly distinguished both the original test samples and a set of validation samples. We then incorporated the 41-feature metagene into a 48-gene panel. To translate this diagnostic signature, we compared the performance of HuEx arrays with several mid-plex assay platforms including Fluidigm's quantitative reverse-transcriptase PCR (q-RT-PCR) and NanoString's nCounter™ Digital Analyzer by measuring gene expression on an 18-cell line panel (4 RMSpos, 4 RMSneg, 5 ESFT, 1 NB, and 4 OS). With q-RT-PCR, we observed r2 values of 0.703 ± 0.085 (RMSpos), 0.558 ± 0.138 (RMSneg), 0.689 ± 0.070 (ESFT), and 0.607 ± 0.121 (OS). nCounter reported r2 values of 0.672 ± 0.171 (RMSpos), 0.620 ± 0.143 (RMSneg), 0.675 ± 0.094 (ESFT), and 0.583 ± 0.129 (OS). Hierarchical clustering with all platforms was able to distinguish the RMSpos, ESFT, and NB cell lines; however, the RMSneg and OS cell lines were less clearly distinguished because some of the OS cell lines lacked high expression of genes characteristic of OS tumors. Cross-platform comparisons yielded good correlation between nCounter and Q-RT-PCR (average r2 = 0.710 ± 0.183). An 18-sample dilution series revealed that EWS-FLI1 type I or PAX3-FKHR transcripts were consistently detected by q-RT-PCR or nCounter even at 1:1000 RNA dilution. Among biological duplicates, q-RT-PCR reported an average r2 = 0.916 ± 0.039 while nCounter obtained average r2 = 0.983 ± 0.011 including raw cell lysates. In addition, we also compared Fluidigm's q-RT-PCR with Applied Biosystems’ Taqman Low-Density Arrays (TLDA) on five NB patient bone marrow samples to detect residual tumor cells. We observed an average r2 = 0.929 ± 0.119 across five NB genes and one housekeeping gene. Finally, we have also successfully profiled FF vs. FFPE tumors on HuEx and are currently applying these mid-plex technologies to paired FF and FFPE samples in order to create diagnostic profiles applicable to routine FFPE material. CONCLUSIONS: Mid-plex platform can reliably distinguish bone and soft tissue sarcoma cell lines and can be used to translate the application of diagnostic signatures. These data warrant further studies to analyze FFPE samples, to compare additional mid-plex platforms, and to test potential prognostic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2011-4350