Transcriptomic and immunophenotypic characterization of two cases of adamantinoma-like Ewing sarcoma of the thyroid gland.

INTRODUCTION: Adamantinoma-like Ewing sarcoma (ALES) is a rare aggressive malignancy occasionally diagnosed in the thyroid gland. ALES shows basaloid cytomorphology, expresses keratins, p63, p40, frequently CD99, and harbours the t(11;22) EWSR1::FLI1 translocation. There is debate on whether ALES resembles more sarcoma or carcinoma. METHODS: We performed RNA sequencing from two ALES cases and compared findings with skeletal Ewing's sarcomas and nonneoplastic thyroid tissue. ALES was investigated by in situ hybridization (ISH) for high-risk human papillomavirus (HPV) DNA and immunohistochemistry for the following antigens: keratin 7, keratin 20, keratin 5, keratins (AE1/AE3 and CAM5.2), CD45, CD20, CD5, CD99, chromogranin, synaptophysin, calcitonin, thyroglobulin, PAX8, TTF1, S100, p40, p63, p16, NUT, desmin, ER, FLI1, INI1, and myogenin. RESULTS: An uncommon EWSR1::FLI transcript with retained EWSR1 exon 8 was detected in both ALES cases. Regulators of EWSR1::FLI1 splicing (HNRNPH1, SUPT6H, SF3B1) necessary for production of a functional fusion oncoprotein, as well as 53 genes (including TNNT1, NKX2.2) activated downstream to the EWSR1::FLI1 cascade, were overexpressed. Eighty-six genes were uniquely overexpressed in ALES, most of which were related to squamous differentiation. Immunohistochemically, ALES strongly expressed keratins 5, AE1/AE3 and CAM5.2, p63, p40, p16, and focally CD99. INI1 was retained. The remaining immunostains and HPV DNA ISH were negative. CONCLUSION: Comparative transcriptomic profiling reveals overlapping features of ALES with skeletal Ewing's sarcoma and an epithelial carcinoma, as evidenced by immunohistochemical expression of keratin 5, p63, p40, CD99, the transcriptome profile, and detection of EWSR1::FLI1 fusion transcript by RNA sequencing.

Excerno: Using Mutational Signatures in Sequencing Data to Filter False Variants Caused by Clinical Archival.

The accurate detection of point mutations from pathology slides using sequencing data is of great importance in cancer genomics and precision oncology. Formalin-fixation paraffin-embedding (FFPE) is a widely used technique to preserve pathology tissues. The FFPE process introduces artificial C > T mutations in next-generation sequencing, so we set out to develop excerno, a method to score and filter such spurious variants. FFPE mutational artifacts follow a mutational signature. By using the FFPE signature and Bayes' formula, we can calculate the probability of a mutation resulting from the FFPE process and use this probability to filter FFPE variants. We implement this method as the excerno R package. We tested excerno by simulating mutations across all 60-baseline mutational signatures from the Catalog of Somatic Mutations in Cancer (COSMIC) and combining them with mutations following the FFPE mutational signature. The sensitivity and specificity of excerno are adversely affected by the cosine similarity between the baseline and FFPE signatures (cosFFPE). Higher percentages of FFPE mutations (pctFFPE) result in increased sensitivity and reduced specificity. The specificity and sensitivity of excerno can be predicted as linear model with an interaction term using cosFFPE and pctFFPE, with an R2of0.84 and 0.79, respectively. Finally, we tested excerno using six RNA sequencing cancer samples and observed concordant trends of specificity and sensitivity with respect to our simulated data. The excerno R package can be used to annotate and filter FFPE-induced mutations in cancer genomics. Our method is adversely affected by cosFFPE and pctFFPE.

PO2RDF: representation of real-world data for precision oncology using resource description framework.

BACKGROUND: Next-generation sequencing provides comprehensive information about individuals' genetic makeup and is commonplace in precision oncology practice. Due to the heterogeneity of individual patient's disease conditions and treatment journeys, not all targeted therapies were initiated despite actionable mutations. To better understand and support the clinical decision-making process in precision oncology, there is a need to examine real-world associations between patients' genetic information and treatment choices. METHODS: To fill the gap of insufficient use of real-world data (RWD) in electronic health records (EHRs), we generated a single Resource Description Framework (RDF) resource, called PO2RDF (precision oncology to RDF), by integrating information regarding genes, variants, diseases, and drugs from genetic reports and EHRs. RESULTS: There are a total 2,309,014 triples contained in the PO2RDF. Among them, 32,815 triples are related to Gene, 34,695 triples are related to Variant, 8,787 triples are related to Disease, 26,154 triples are related to Drug. We performed two use case analyses to demonstrate the usability of the PO2RDF: (1) we examined real-world associations between EGFR mutations and targeted therapies to confirm existing knowledge and detect off-label use. (2) We examined differences in prognosis for lung cancer patients with/without TP53 mutations. CONCLUSIONS: In conclusion, our work proposed to use RDF to organize and distribute clinical RWD that is otherwise inaccessible externally. Our work serves as a pilot study that will lead to new clinical applications and could ultimately stimulate progress in the field of precision oncology.

Stage-Specific Non-Coding RNA Expression Patterns during In Vitro Human B Cell Differentiation into Antibody Secreting Plasma Cells.

The differentiation of B cells into antibody secreting plasma cells (PCs) is governed by a strict regulatory network that results in expression of specific transcriptomes along the activation continuum. In vitro models yielding significant numbers of PCs phenotypically identical to the in vivo state enable investigation of pathways, metabolomes, and non-coding (ncRNAs) not previously identified. The objective of our study was to characterize ncRNA expression during human B cell activation and differentiation. To achieve this, we used an in vitro system and performed RNA-seq on resting and activated B cells and PCs. Characterization of coding gene transcripts, including immunoglobulin (Ig), validated our system and also demonstrated that memory B cells preferentially differentiated into PCs. Importantly, we identified more than 980 ncRNA transcripts that are differentially expressed across the stages of activation and differentiation, some of which are known to target transcription, proliferation, cytoskeletal, autophagy and proteasome pathways. Interestingly, ncRNAs located within Ig loci may be targeting both Ig and non-Ig-related transcripts. ncRNAs associated with B cell malignancies were also identified. Taken together, this system provides a platform to study the role of specific ncRNAs in B cell differentiation and altered expression of those ncRNAs involved in B cell malignancies.

Frequent POLE-driven hypermutation in ovarian endometrioid cancer revealed by mutational signatures in RNA sequencing.

BACKGROUND: DNA polymerase epsilon (POLE) is encoded by the POLE gene, and POLE-driven tumors are characterized by high mutational rates. POLE-driven tumors are relatively common in endometrial and colorectal cancer, and their presence is increasingly recognized in ovarian cancer (OC) of endometrioid type. POLE-driven cases possess an abundance of TCT > TAT and TCG > TTG somatic mutations characterized by mutational signature 10 from the Catalog of Somatic Mutations in Cancer (COSMIC). By quantifying the contribution of COSMIC mutational signature 10 in RNA sequencing (RNA-seq) we set out to identify POLE-driven tumors in a set of unselected Mayo Clinic OC. METHODS: Mutational profiles were calculated using expressed single-nucleotide variants (eSNV) in the Mayo Clinic OC tumors (n = 195), The Cancer Genome Atlas (TCGA) OC tumors (n = 419), and the Genotype-Tissue Expression (GTEx) normal ovarian tissues (n = 84). Non-negative Matrix Factorization (NMF) of the mutational profiles inferred the contribution per sample of four distinct mutational signatures, one of which corresponds to COSMIC mutational signature 10. RESULTS: In the Mayo Clinic OC cohort we identified six tumors with a predicted contribution from COSMIC mutational signature 10 of over five mutations per megabase. These six cases harbored known POLE hotspot mutations (P286R, S297F, V411L, and A456P) and were of endometrioid histotype (P = 5e-04). These six tumors had an early onset (average age of patients at onset, 48.33 years) when compared to non-POLE endometrioid OC cohort (average age at onset, 60.13 years; P = .008). Samples from TCGA and GTEx had a low COSMIC signature 10 contribution (median 0.16 mutations per megabase; maximum 1.78 mutations per megabase) and carried no POLE hotspot mutations. CONCLUSIONS: From the largest cohort of RNA-seq from endometrioid OC to date (n = 53), we identified six hypermutated samples likely driven by POLE (frequency, 11%). Our result suggests the clinical need to screen for POLE driver mutations in endometrioid OC, which can guide enrollment in immunotherapy clinical trials.

RNA-Seq Reveals Differences in Expressed Tumor Mutation Burden in Colorectal and Endometrial Cancers with and without Defective DNA-Mismatch Repair.

Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.

Transcriptomic and Proteomic Analysis of Steatohepatitic Hepatocellular Carcinoma Reveals Novel Distinct Biologic Features.

OBJECTIVES: Steatohepatitic hepatocellular carcinoma is a distinct variant of hepatocellular carcinoma strongly associated with underlying nonalcoholic steatohepatitis. The molecular biology of steatohepatitic hepatocellular carcinoma is not fully elucidated, and thus we aimed to investigate the molecular underpinnings of this entity. METHODS: Transcriptomic analysis using RNAseq was performed on eight tumor-nonneoplastic pairs of steatohepatitic hepatocellular carcinoma with comparison to conventional hepatocellular carcinoma transcriptomes curated in The Cancer Genome Atlas. Immunohistochemistry was used to validate key RNA-level findings. RESULTS: Steatohepatitic hepatocellular carcinoma demonstrated a distinctive differential gene expression profile compared with The Cancer Genome Atlas curated conventional hepatocellular carcinomas (n = 360 cases), indicating the distinctive steatohepatitic hepatocellular carcinoma morphology is associated with a unique gene expression profile. Pathway analysis comparing tumor-nonneoplastic pairs revealed significant upregulation of the hedgehog pathway based on GLI1 overexpression and significant downregulation of carnitine palmitoyltransferase 2 transcript. Glutamine synthetase transcript was significantly upregulated, and fatty acid binding protein 1 transcript was significantly downregulated and immunohistochemically confirmed, indicating steatohepatitic hepatocellular carcinoma tumor cells display a zone 3 phenotype. CONCLUSIONS: Steatohepatitic hepatocellular carcinoma demonstrates a distinctive morphology and gene expression profile, phenotype of zone 3 hepatocytes, and activation of the hedgehog pathway and repression of carnitine palmitoyltransferase 2, which may be important in tumorigenesis.

Semi-Supervised Topological Analysis for Elucidating Hidden Structures in High-Dimensional Transcriptome Datasets.

Topological data analysis (TDA) is a powerful method for reducing data dimensionality, mining underlying data relationships, and intuitively representing the data structure. The Mapper algorithm is one such tool that projects high-dimensional data to 1-dimensional space by using a filter function that is subsequently used to reconstruct the data topology relationships. However, domain context information and prior knowledge have not been considered in current TDA modeling frameworks. Here, we report the development and evaluation of a semi-supervised topological analysis (STA) framework that incorporates discrete or continuously labeled data points and selects the most relevant filter functions accordingly. We validate the proposed STA framework with simulation data and then apply it to samples from Genotype-Tissue Expression data and ovarian cancer transcriptome datasets. The graphs generated by STA for these 2 datasets, based on gene expression profiles, are consistent with prior knowledge, thereby supporting the effectiveness of the proposed framework.

Malignant Peritoneal Mesothelioma Arising in Young Adults With Long-standing Indwelling Intra-abdominal Shunt Catheters.

Only 50% to 70% of patients with mesothelioma report asbestos exposure. Other exposures (eg, radiation) play a role in some cases, but some patients have no obvious cause. We describe a series of patients with long-standing indwelling intra-abdominal shunt catheters who developed malignant peritoneal mesothelioma, suggesting a novel association. We identified 7 patients who had shunts and subsequently developed mesothelioma (5 women; median age: 31 y, range: 18 to 45 y). Clinical history and pathology materials were reviewed, and RNA sequencing was performed. Clinical presentations varied; 6 patients had hydrocephalus and a ventriculoperitoneal shunt, and 1 patient had portal hypertension and a portoatrial shunt. The median duration of shunt therapy in 5 cases was 29 years (range: 12 to 35 y); the remaining 2 patients also had shunts for many years, but specific details were unavailable. Two patients had radiotherapy for malignancies in childhood. One had an alleged exposure to asbestos and 1 had prior exposure to talc. The rest had no known risk factors. Histologically, all tumors were purely epithelioid. Treatments included surgical debulking, chemotherapy, and palliative care. All 7 died of disease (median survival: 7 mo, range: 1 to 18 mo). Molecular testing showed loss of NF2 and CDKN2A/B and a BAP1 mutation in 1 case, and no genomic alterations associated with mesothelioma in 2 cases. Peritoneal mesothelioma may represent a complication of long-standing indwelling shunt catheters. The mechanism is unknown, but chronic peritoneal irritation may play a role. Albeit rare, mesothelioma should be considered in patients with a shunt who present with new ascites.

Molecular Genetic Landscape of Sclerosing Pneumocytomas.

OBJECTIVES: Sclerosing pneumocytomas are rare pulmonary neoplasms that are typically benign. However, rare patients experience progressive disease, and therapy targeting specific genetic underpinnings could be an attractive therapeutic option. Recent studies have found recurrent AKT 1 mutations in sclerosing pneumocytoma, but little is known about whether oncogenic fusion genes may also be present. METHODS: To better understand the genetic background, 10 sclerosing pneumocytomas were subjected to next-generation sequencing cancer mutation panel testing (n = 9) and/or RNA sequencing (n = 3). The patients were all women (average age, 47 years; range, 17-74 years). RESULTS: Eight patients had solitary sclerosing pneumocytomas, while one had two tumors, and one had many bilateral tumors. Recurrent mutations were noted in genes involved in the mTOR pathway, including AKT1, PIK3R1, and PTEN. AKT1 alterations were particularly common, present in 78%. No recurrent genetic fusions were identified. The patient in our study with multiple bilateral lesions was treated with the mammalian target of rapamycin (mTOR) inhibitor everolimus, with no objective radiographic evidence of treatment response after 4 months. CONCLUSIONS: Our data further support that abnormal activation of the mTOR pathway is a consistent genetic event in sclerosing pneumocytoma. This warrants further exploration to determine if mTOR pathway inhibitors may be effective in patients with metastatic or recurrent disease.

ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL.

Understanding the molecular events controlling melanoma progression is of paramount importance for the development of alternative treatment options for this devastating disease. Here we report a mechanism regulated by the oncogenic SOX2-GLI1 transcriptional complex driving melanoma invasion through the induction of the sialyltransferase ST3GAL1. Using in vitro and in vivo studies, we demonstrate that ST3GAL1 drives melanoma metastasis. Silencing of this enzyme suppresses melanoma invasion and significantly reduces the ability of aggressive melanoma cells to enter the blood stream, colonize distal organs, seed and survive in the metastatic environment. Analysis of glycosylated proteins reveals that the receptor tyrosine kinase AXL is a major effector of ST3GAL1 pro-invasive function. ST3GAL1 induces AXL dimerization and activation that, in turn, promotes melanoma invasion. Our data support a key role of the ST3GAL1-AXL axis as driver of melanoma metastasis, and highlight the therapeutic potential of targeting this axis to treat metastatic melanoma.

Data-driven Sublanguage Analysis for Cancer Genomics Knowledge Modeling: Applications in Mining Oncological Genetics Information from Patients' Genetic Reports.

Despite an abundance of information in clinical genetic testing reports, information is oftentimes not well documented/utilized for decision making. Unstructured information in genetic reports can contribute to long-term patient management and future translational research. Thus, we proposed a knowledge model that could manage unstructured information in medical genetic reports and facilitate knowledge extraction, curation and updating. For this pilot study, we used a dataset including 1,565 cancer genetics reports of Mayo Clinic patients. We used a previously developed, data-driven discovery pipeline that involves both semantic annotation and co-occurrence association analysis to establish a knowledge model. We showed that compared to genetic reports, around 56% of testing results are missing or incomplete in the clinical notes. We built a genetic report knowledge model and highlighted four key semantic groups including "Genes and Gene Products" and "Treatments". Coverage of term annotation was 99.5%. Accuracies of term annotation and relationship extraction were 98.9% and 92.9% respectively.

Developing a FHIR-based Framework for Phenome Wide Association Studies: A Case Study with A Pan-Cancer Cohort.

Phenome Wide Association Studies (PheWAS) enables phenome-wide scans to discover novel associations between genotype and clinical phenotypes via linking available genomic reports and large-scale Electronic Health Record (EHR). Data heterogeneity from different EHR systems and genetic reports has been a critical challenge that hinders meaningful validation. To address this, we propose an FHIR-based framework to model the PheWAS study in a standard manner. We developed an FHIR-based data model profile to enable the standard representation of data elements from genetic reports and EHR data that are used in the PheWAS study. As a proof-of-concept, we implemented the proposed method using a cohort of 1,595 pan-cancer patients with genetic reports from Foundation Medicine as well as the corresponding lab tests and diagnosis from Mayo EHRs. A PheWAS study is conducted and 81 significant genotype-phenotype associations are identified, in which 36 significant associations for cancers are validated based on a literature review.

Uterine inflammatory myofibroblastic tumors in pregnant women with and without involvement of the placenta: a study of 6 cases with identification of a novel TIMP3-RET fusion.

Uterine inflammatory myofibroblastic tumors (IMTs) have been reported in association with pregnancy and, in some instances, secondarily involve the placenta. The clinicopathological spectrum of these tumors in the setting of pregnancy is not well defined. We investigated the clinical, morphologic, immunohistochemical, molecular cytogenetic, and genetic features of 6 uterine IMTs occurring in pregnant women. Each tumor was discovered at parturition, and none was identified by prenatal ultrasound. Patient age ranged from 25 to 41 years (mean 31.5). Tumor size ranged from 1.5 to 9 cm (mean 4.7). Four of 6 had usual IMT features, with at least focal deciduoid change in 3. Necrosis was identified in 3 tumors; and multinucleated cells, in 3 tumors. Sex hormone receptor expression was consistent with estrogen receptor negative or focally weakly positive and progesterone receptor diffusely moderately or moderately to strongly positive in all 6 tumors. ALK immunohistochemistry was strongly positive in 5 tumors, and all of these had an ALK rearrangement detected by break-apart fluorescence in situ hybridization. Subsequent RNA sequencing of these 5 tumors identified a TIMP3-ALK fusion in 4 and a THBS1-ALK in 1. In the ALK-negative tumor, RNA sequencing detected a novel TIMP3-RET fusion that was confirmed by RET break-apart fluorescence in situ hybridization. Follow-up was available for 2 of 6 patients 5 and 19 months after diagnosis. Neither patient developed recurrence. ALK immunohistochemistry will distinguish most uterine IMTs, but if ALK expression and gene studies are negative, in the appropriate morphologic context, evaluation of other tyrosine kinase genes known to be more commonly altered in extrauterine IMTs such as ROS1, NTRK3, PDGFRß, and RET may be necessary for diagnostic confirmation.

Bioinformatics and DNA-extraction strategies to reliably detect genetic variants from FFPE breast tissue samples.

BACKGROUND: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples. RESULTS: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates. CONCLUSIONS: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

Desmoplastic Infantile Ganglioglioma: A MAPK Pathway-Driven and Microglia/Macrophage-Rich Neuroepithelial Tumor.

MAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%-27% variant allele frequency) and 1 showed a TPM3-NRTK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19-139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.

Gene expression differences between matched pairs of ovarian cancer patient tumors and patient-derived xenografts.

As patient derived xenograft (PDX) models are increasingly used for preclinical drug development, strategies to account for the nonhuman component of PDX RNA expression data are critical to its interpretation. A bioinformatics pipeline to separate donor tumor and mouse stroma transcriptome profiles was devised and tested. To examine the molecular fidelity of PDX versus donor tumors, we compared mRNA differences between paired PDX-donor tumors from nine ovarian cancer patients. 1,935 differentially expressed genes were identified between PDX and donor tumors. Over 90% (n = 1767) of these genes were down-regulated in PDX models and enriched in stroma-specific functions. Several protein kinases were also differentially expressed in PDX tumors, e.g. PDGFRA, PDGFRB and CSF1R. Upon in silico removal of these PDX-donor tumor differentially expressed genes, a stronger transcriptional resemblance between PDX-donor tumor pairs was seen (average correlation coefficient increases from 0.91 to 0.95). We devised and validated an effective bioinformatics strategy to separate mouse stroma expression from human tumor expression for PDX RNAseq. In addition, we showed most of the PDX-donor differentially expressed genes were implicated in stromal components. The molecular similarities and differences between PDX and donor tumors have implications in future therapeutic trial designs and treatment response evaluations using PDX models.

RNA sequencing identifies a novel USP9X-USP6 promoter swap gene fusion in a primary aneurysmal bone cyst.

Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full-length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell-rich primary bone tumors. In this report, we present a case of a 16-year-old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X-USP6 promoter swap fusion. This result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin-specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate-specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.