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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.

Epithelial marker expression and/or epithelial differentiation, as well as “anomalous” expression of keratins, are features of some soft tissue tumors. Recently, we have encountered an unusual mesenchymal tumor composed of bland, distinctly eosinophilic, keratin-positive epithelial cells, which were almost entirely obscured by xanthogranulomatous inflammation. Six cases were identified (5 F, 1 M; 16–62 years (median 21 years)) arising in soft tissue (n = 4) and bone (n = 2) and ranging in size from 2 to 7 cm. The tumors were generally circumscribed, with a fibrous capsule containing lymphoid aggregates, and consisted in large part of a sheet-like proliferation of foamy histiocytes, Touton-type and osteoclast-type giant cells, and chronic inflammatory cells. Closer inspection, however, disclosed a distinct population of uniform, cytologically bland mononuclear cells with brightly eosinophilic cytoplasm arranged singly and in small nests and cords. Overt squamous and/or glandular differentiation was absent. By immunohistochemistry, these cells were diffusely positive with the OSCAR and AE1/AE3 keratin antibodies, and focally positive for high-molecular weight keratins; endothelial and myoid markers were negative and SMARCB1 was retained. RNA-seq identified a PLEKHM1 variant of undetermined significance in one case, likely related to this patient’s underlying osteopetrosis. Follow-up to date has been benign. In summary, we have identified a novel tumor of soft tissue and bone with a predilection for young females, provisionally termed “xanthogranulomatous epithelial tumor”. These unusual lesions do not appear to arise from adnexa, or represent known keratin-positive soft tissue tumors, and the origin of their constituent epithelial cells is obscure. The natural history of this distinctive lesion appears indolent, although study of additional cases and longer term follow-up are needed.

ObjectivesSteatohepatitic 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.MethodsTranscriptomic 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.ResultsSteatohepatitic 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.ConclusionsSteatohepatitic 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.

Summary 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, PDGFRβ, and RET may be necessary for diagnostic confirmation.

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-NTRK1 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.

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.

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.

Gene fusions involving the anaplastic lymphoma kinase ( ALK) gene occurs in approximately 3-5% of lung adenocarcinomas. The most common fusion partner is EML4 but several other fusion partners to the ALK kinase domain have been identified. Herein we report a lung adenocarcinoma case harboring a fusion between ALK and Striatin ( STRN ), with extended responses to targeted therapy. Using CT-guided needle biopsy approach, we successfully generated a patient-derived xenograft model from this tumor and molecularly characterized the STRN-ALK gene fusion.

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., Understanding the molecular events controlling melanoma progression are necessary to find improved therapeutics. Here, the authors report oncogenic SOX2-GLI1 transcriptional complex to drive melanoma invasion through the induction of the sialyltransferase ST3GAL1, and report ST3GAL1-AXL axis as driver of melanoma metastasis.

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.

Objectives Mucinous cystic neoplasm of the liver is characterized by neoplastic mucinous and/or biliary epithelium surrounded by ovarian-type stroma. Immunohistochemical studies have shown that the ovarian-type stroma expresses estrogen receptor, suggesting potential hormonal responsiveness. The molecular biology of mucinous cystic neoplasm of the liver remains poorly studied. Methods Transcriptome sequencing and immunohistochemistry were performed on a series of mucinous cystic neoplasms. Results Mucinous cystic neoplasm of the liver exhibited significantly increased RNA expression of ovarian stromal markers WT1, PR, and ER2 and sex cord stromal markers SF-1, inhibin-α, and calretinin compared with nonneoplastic liver. Immunohistochemistry confirmed the RNA-level data. Evidence for sex hormone biosynthesis was identified by significant overexpression of multiple estrogen biosynthetic enzymes. Expression of 17β-hydroxysteroid dehydrogenase 1 was confirmed immunohistochemically. Pathway analysis also identified significant upregulation of the hedgehog and Wnt pathways and significant downregulation of T-helper 1 and T-helper 2 pathways. Conclusions Mucinous cystic neoplasm of the liver recapitulates ovarian stroma at the morphologic, DNA, RNA, and protein levels. These data support the concept that this tumor likely arises from ectopic primitive gonadal tissue and/or stromal cells with capacity to transdifferentiate to ovarian cortical cells.

Multiple isodicentric Y chromosome [idic(Y)] is a cytogenetic abnormality consisting of duplicative copies of the short-arm of Y chromosome. Several mechanisms have been postulated to explain the f...

Aims Rhabdomyosarcomas of bone are extremely rare, with fewer than 10 reported cases. A very rare subtype of spindle cell/sclerosing rhabdomyosarcoma harbouring a FUS-TFCP2 fusion and involving both soft tissue and bone locations has been reported very recently. We report only the fourth case of this unusual, clinically aggressive rhabdomyosarcoma. Material and results A previously well 72-year-old male presented with a destructive lesion of the mandible. Morphological and immunohistochemical study of a needle biopsy and the subsequent resection showed a spindle cell rhabdomyosarcoma. RNA-seq, RT-PCR and FISH confirmed the presence of the FUS-TFCP2 fusion. Conclusions Spindle cell rhabdomyosarcomas carrying the FUS-TFCP2 fusion are very rare rhabdomyosarcoma variants with osseous predilection. The classification and differential diagnosis of this unusual molecular variant of spindle cell/sclerosing rhabdomyosarcoma are discussed.

TO THE EDITOR: Indolent T-cell lymphoproliferative disorder of the gastrointestinal tract (GI TLPD) is a newly recognized entity in the World Health Organization (WHO) classification of lymphoid neoplasms.[1][1] GI TLPD is defined as a clonal T-cell proliferation occurring in the GI tract, most

Aberrant expression of neuroendocrine markers is extremely rare in endothelial neoplasms, with only a single report describing three cases. Although originally classified as conventional angiosarcoma, further assessment of these tumors revealed a strikingly composite morphology composed of retiform and epithelioid elements reminiscent of composite hemangioendothelioma, a rare subtype of hemangioendothelioma. To further investigate these findings, available materials from 11 morphologically distinctive endothelial tumors showing neuroendocrine marker expression were retrieved from our archives. Immunohistochemistry for CD31, CD34, FLI-1, synaptophysin, chromogranin, D2-40, ERG, keratin (OSCAR), and CAMTA1 was performed. Total RNA from five cases were extracted and subjected to whole transcriptome sequencing. Clinical follow-up was obtained. These tumors were found to arise in five males and six females in patients from 9 to 55 years in age (median 47 years). They arose both in superficial (wrist, ankle, scalp, hip, and foot) and deep (periaortic tissues, C5 vertebra, pulmonary vein, and liver) locations. All contained elongated, retiform vascular channels lined by hyperchromatic 'hobnail' endothelial cells and a solid growth of uniform epithelioid cells reminiscent of epithelioid hemangioendothelioma. Hemangioma-like foci also lined by hobnail endothelial cells were frequently present. Mitotic activity was typically

Gastroblastoma is a rare distinctive biphasic tumor of the stomach. The molecular biology of gastroblastoma has not been studied, and no affirmative diagnostic markers have been developed. We retrieved two gastroblastomas from the consultation practices of the authors and performed transcriptome sequencing on formalin-fixed paraffin-embedded tissue. Recurrent predicted fusion genes were validated at genomic and RNA levels. The presence of the fusion gene was confirmed on two additional paraffin-embedded cases of gastroblastoma. Control cases of histologic mimics (biphasic synovial sarcoma, leiomyoma, leiomyosarcoma, desmoid-type fibromatosis, EWSR1-FLI1-positive Ewing sarcoma, Wilms' tumor, gastrointestinal stromal tumor, plexiform fibromyxoma, Sonic hedgehog-type medulloblastomas, and normal gastric mucosa and muscularis propria were also analyzed. The gastroblastomas affected two males and two females aged 9-56 years. Transcriptome sequencing identified recurrent somatic MALAT1-GLI1 fusion genes, which were predicted to retain the key domains of GLI1. The MALAT1-GLI1 fusion gene was validated by break-apart and dual-fusion FISH and RT-PCR. The additional two gastroblastomas were also positive for the MALAT1-GLI1 fusion gene. None of the other control cases harbored MALAT1-GLI1. Overexpression of GLI1 in the cases of gastroblastomas was confirmed at RNA and protein levels. Pathway analysis revealed activation of the Sonic hedgehog pathway in gastroblastoma and gene expression profiling showed that gastroblastomas grouped together and were most similar to Sonic hedgehog-type medulloblastomas. In summary, we have identified an oncogenic MALAT1-GLI1 fusion gene in all cases of gastroblastoma that may serve as a diagnostic biomarker. The fusion gene is predicted to encode a protein that includes the zinc finger domains of GLI1 and results in overexpression of GLI1 protein and activation of the Sonic hedgehog pathway.

// Nicholas B. Larson 1 , Shannon K. McDonnell 1 , Zach Fogarty 1 , Melissa C. Larson 1 , John Cheville 2 , Shaun Riska 1 , Saurabh Baheti 1 , Alexandra M. Weber 3 , Asha A. Nair 1 , Liang Wang 4 , Daniel O’Brien 1 , Jaime Davila 1 , Daniel J. Schaid 1 and Stephen N. Thibodeau 5 1 Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA 2 Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 3 Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI, USA 4 Department of Pathology, Medical College of Wisconsin, Milwaukee, WI, USA 5 Division of Laboratory Genetics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA Correspondence to: Nicholas B. Larson, email: Larson.nicholas@mayo.edu Keywords: prostate cancer, genetic risk, expression quantitative trait loci, mediation Received: April 26, 2017 Accepted: July 29, 2017 Published: September 08, 2017 ABSTRACT Large-scale genome-wide association studies have identified multiple single-nucleotide polymorphisms associated with risk of prostate cancer. Many of these genetic variants are presumed to be regulatory in nature; however, follow-up expression quantitative trait loci (eQTL) association studies have to-date been restricted largely to cis -acting associations due to study limitations. While trans -eQTL scans suffer from high testing dimensionality, recent evidence indicates most trans -eQTL associations are mediated by cis -regulated genes, such as transcription factors. Leveraging a data-driven gene co-expression network, we conducted a comprehensive cis -mediator analysis using RNA-Seq data from 471 normal prostate tissue samples to identify downstream regulatory associations of previously identified prostate cancer risk variants. We discovered multiple trans -eQTL associations that were significantly mediated by cis -regulated transcripts, four of which involved risk locus 17q12, proximal transcription factor HNF1B , and target trans -genes with known HNF response elements ( MIA2 , SRC , SEMA6A , KIF12 ). We additionally identified evidence of cis -acting down-regulation of MSMB via rs10993994 corresponding to reduced co-expression of NDRG1 . The majority of these cis -mediator relationships demonstrated trans -eQTL replicability in 87 prostate tissue samples from the Gene-Tissue Expression Project. These findings provide further biological context to known risk loci and outline new hypotheses for investigation into the etiology of prostate cancer.

Background Uterine leiomyomas, in contrast to sarcomas, tend to cease growth following menopause. In the setting of a rapidly enlarging uterine mass in a postmenopausal patient, clinical distinction of uterine leiomyoma from sarcoma is difficult and requires pathologic examination. Case presentation A 74-year-old woman presented with postmenopausal bleeding and acute blood loss requiring transfusion. She was found to have a rapidly enlarging uterine mass clinically suspicious for sarcoma. An abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. A 15.5 cm partially necrotic intramural mass was identified in the uterine corpus. The tumor was classified as a cellular leiomyoma. RNA sequencing identified a KAT6B-KANSL1 fusion that was confirmed by RT-PCR and Sanger sequencing. After 6 months of follow-up, the patient remains asymptomatic without evidence of disease. Conclusion Prior studies of uterine leiomyomas have identified KAT6B (previously MORF) rearrangements in uterine leiomyomas, but this case is the first to identify a KAT6B-KANSL1 gene fusion in a uterine leiomyoma. While alterations of MED12 and HMGA2 are most common in uterine leiomyomas, a range of other genetic pathways have been described. Our case contributes to the evolving molecular landscape of uterine leiomyomas.

To determine early somatic changes in high-grade serous ovarian cancer (HGSOC), we performed whole genome sequencing on a rare collection of 16 low stage HGSOCs. The majority showed extensive structural alterations (one had an ultramutated profile), exhibited high levels of p53 immunoreactivity, and harboured a TP53 mutation, deletion or inactivation. BRCA1 and BRCA2 mutations were observed in two tumors, with nine showing evidence of a homologous recombination (HR) defect. Combined Analysis with The Cancer Genome Atlas (TCGA) indicated that low and late stage HGSOCs have similar mutation and copy number profiles. We also found evidence that deleterious TP53 mutations are the earliest events, followed by deletions or loss of heterozygosity (LOH) of chromosomes carrying TP53, BRCA1 or BRCA2. Inactivation of HR appears to be an early event, as 62.5% of tumours showed a LOH pattern suggestive of HR defects. Three tumours with the highest ploidy had little genome-wide LOH, yet one of these had a homozygous somatic frame-shift BRCA2 mutation, suggesting that some carcinomas begin as tetraploid then descend into diploidy accompanied by genome-wide LOH. Lastly, we found evidence that structural variants (SV) cluster in HGSOC, but are absent in one ultramutated tumor, providing insights into the pathogenesis of low stage HGSOC.

We previously reported an extremely rare case of follicular dendritic cell sarcoma (FDCS) presented as a thyroid mass. Given the rarity of this disease, there are no personalized and molecularly targeted treatment options due to the lack of knowledge in the genomic makeup of the tumor. A 44-year-old white woman was diagnosed with an extranodal FDCS in thyroid. The patient underwent a total thyroidectomy, central compartment dissection, parathyroid re-implantation, and adjuvant radiation therapy. Tumor DNA sequencing of 236 genes by FoundationOne panel found truncating mutations in PTEN and missense mutations in RET and TP53. However, patient-matched germline DNA was not sequenced which is critical for identification of true somatic mutations. Furthermore, the FoundationOne panel doesn't measure genomic rearrangements which have been shown to be abundant in sarcomas and are associated with sarcoma tumorigenesis and progression. In the current study, we carried out comprehensive genomic sequencing of the tumor, adjacent normal tissues, and patient-matched blood, in an effort to understand the genomic makeup of this rare extranodal FDCS and to identify potential therapeutic targets. Eighty-one somatic point mutations were identified in tumor but not in adjacent normal tissues or blood. A clonal truncating mutation in the CLTCL1 gene, which stabilizes the mitotic spindle, was likely a driver mutation of tumorigenesis and could explain the extensive copy number aberrations (CNAs) and genomic rearrangements in the tumor including a chr15/chr17 local chromothripsis resulted in 6 expressed fusion genes. The fusion gene HDGFRP3→SHC4 led to a 200-fold increase in the expression of oncogene SHC4 which is a potential target of the commercial drug Dasatinib. Missense mutations in ATM and splice-site mutation in VEGFR1 were also detected in addition to the TP53 missense mutation reported by FoundationOne.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Motivation: RNA-seq has become the method of choice to quantify genes and exons, discover novel transcripts and detect fusion genes. However, reliable variant identification from RNA-seq data remains challenging because of the complexities of the transcriptome, the challenges of accurately mapping exon boundary spanning reads and the bias introduced during the sequencing library preparation. Method: We developed RVboost, a novel method specific for RNA variant prioritization. RVboost uses several attributes unique in the process of RNA library preparation, sequencing and RNA-seq data analyses. It uses a boosting method to train a model of ‘good quality’ variants using common variants from HapMap, and prioritizes and calls the RNA variants based on the trained model. We packaged RVboost in a comprehensive workflow, which integrates tools of variant calling, annotation and filtering. Results: RVboost consistently outperforms the variant quality score recalibration from the Genome Analysis Tool Kit and the RNA-seq variant-calling pipeline SNPiR in 12 RNA-seq samples using ground-truth variants from paired exome sequencing data. Several RNA-seq–specific attributes were identified as critical to differentiate true and false variants, including the distance of the variant positions to exon boundaries, and the percent of the reads supporting the variant in the first six base pairs. The latter identifies false variants introduced by the random hexamer priming during the library construction. Availability and implementation: The RVboost package is implemented to readily run in Mac or Linux environments. The software and user manual are available at http://bioinformaticstools.mayo.edu/research/rvboost/. Supplementary information: Supplementary data are available at Bioinformatics online.

Detection of rare events happening in a set of DNA/protein sequences could lead to new biological discoveries. One kind of such rare events is the presence of patterns called motifs in DNA/protein sequences. Finding motifs is a challenging problem since the general version of motif search has been proven to be intractable. Motifs discovery is an important problem in biology. For example, it is useful in the detection of transcription factor binding sites and transcriptional regulatory elements that are very crucial in understanding gene function, human disease, drug design, etc. Many versions of the motif search problem have been proposed in the literature. One such is the (ℓ, d)-motif search (or Planted Motif Search (PMS)). A generalized version of the PMS problem, namely, Quorum Planted Motif Search (qPMS), is shown to accurately model motifs in real data. However, solving the qPMS problem is an extremely difficult task because a special case of it, the PMS Problem, is already NP-hard, which means that any algorithm solving it can be expected to take exponential time in the worse case scenario. In this paper, we propose a novel algorithm named qPMS7 that tackles the qPMS problem on real data as well as challenging instances. Experimental results show that our Algorithm qPMS7 is on an average 5 times faster than the state-of-art algorithm. The executable program of Algorithm qPMS7 is freely available on the web at http://pms.engr.uconn.edu/downloads/qPMS7.zip. Our online motif discovery tools that use Algorithm qPMS7 are freely available at http://pms.engr.uconn.edu or http://motifsearch.com.