A 44-year old woman with recurrent solitary fibrous tumor (SFT)/hemangiopericytoma was enrolled in a clinical sequencing program including whole exome and transcriptome sequencing. A gene fusion of the transcriptional repressor NAB2 with the transcriptional activator STAT6 was detected. Transcriptome sequencing of 27 additional SFTs all revealed the presence of a NAB2-STAT6 gene fusion. Using RT-PCR and sequencing, we detected this fusion in 51 of 51 SFTs, indicating high levels of recurrence. Expression of NAB2-STAT6 fusion proteins was confirmed in SFT, and the predicted fusion products harbor the early growth response (EGR)-binding domain of NAB2 fused to the activation domain of STAT6. Overexpression of the NAB2-STAT6 gene fusion induced proliferation in cultured cells and activated EGR-responsive genes. These studies establish NAB2-STAT6 as the defining driver mutation of SFT and provide an example of how neoplasia can be initiated by converting a transcriptional repressor of mitogenic pathways into a transcriptional activator.

Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong TFE3 immunoreactivity, FISH analysis for TFE3 gene rearrangement was applied to the index case as well as to 9 additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, 9 pseudomyogenic HE and 3 epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in 6 cases, lung in 3 and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and RT-PCR. YAP1 gene rearrangements were then confirmed in 7 of the remaining 9 TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions.

The family of pediatric fibroblastic and myofibroblastic proliferations encompasses a wide spectrum of pathologic entities with overlapping morphologies and ill-defined genetic abnormalities. Among the superficial lesions, lipofibromatosis (LPF), composed of an admixture of adipose tissue and fibroblastic elements, in the past has been variously classified as infantile fibromatosis or fibrous hamartoma of infancy. In this regard, we have recently encountered a group of superficial soft tissue tumors occurring in children and young adults, with a notably infiltrative growth pattern reminiscent of LPF, variable cytologic atypia and a distinct immunoprofile of S100 protein and CD34 reactivity, suggestive of neural differentiation. SOX10 and melanocytic markers were negative in all cases tested. In contrast, a control group of classic LPF displayed bland, monomorphic histology and lacked S100 protein immunoreactivity. In order to define the pathogenetic abnormalities in these seemingly distinctive groups, we performed RNA sequencing for fusion gene discovery in 2 cases each, followed by screening for any novel alterations identified in a larger cohort representing both entities. The 2 index LPF-like neural tumors (LPF-NT) showed TPR-NTRK1 and TPM3-NTRK1 gene fusions, which were further validated by FISH and RT-PCR. Subsequent FISH screening of 14 LPF-NT identified recurrent NTRK1 gene rearrangements in 10 (71%) cases. Of the NTRK1 negative LPF-NT cases, one case each showed ROS1 and ALK gene rearrangements. In contrast none of the 25 classic LPF showed NTRK1 gene rearrangements, although regional abnormalities were noted in the 1q21–22 region by FISH in a majority of cases. Furthermore, NTRK1 immunostaining was positive only in NTRK1-rearranged S100 positive LPF-NT, but negative in classic LPF. These results suggest that NTRK1 oncogenic activation through gene fusion defines a novel and distinct subset of soft tissue tumors resembling LPF, but displaying cytologic atypia and a neural immunophenotype, provisionally named LPF-like neural tumors.

Perivascular epithelioid cell neoplasms (PEComa) are a family of rare mesenchymal tumors with hybrid myo-melanocytic differentiation. Although most PEComas harbor loss of function TSC1/TSC2 mutations, a small subset were reported to carry TFE3 gene rearrangements. As no comprehensive genomic study has addressed the molecular classification of PEComa, we sought to investigate by multiple methodologies the incidence and spectrum of genetic abnormalities and their potential genotype-phenotype correlations in a large group of 38 PEComas. The tumors were located in soft tissue (11 cases) and visceral sites (27) including uterus, kidney, liver, lung and urinary bladder. Combined RNA sequencing and Fluorescence In Situ Hybridization (FISH) analysis identified 9 (23%) TFE3 gene rearranged tumors, with 3 cases showing a SFPQ/PSF-TFE3 fusion and one case a novel DVL2-TFE3 gene fusion. The TFE3-positive lesions showed a distinctive nested/alveolar morphology and were equally distributed between soft tissue and visceral sites. Additionally, novel RAD51B gene rearrangements were identified in 3 (8%) uterine PEComas, which showed a complex fusion pattern and were fused to RRAGB/OPHN1 genes in two cases. Other non-recurrent gene fusions, HTR4-ST3GAL1 and RASSF1-PDZRN3, were identified in 2 cases. Targeted exome sequencing using the IMPACT assay was used to address if the presence of gene fusions are mutually exclusive from TSC gene abnormalities. TSC2 mutations were identified in 80% of the TFE3 fusion-negative cases tested. Co-existent TP53 mutations were identified in 63% of the TSC2 mutated PEComas. Our results showed that TFE3-rearranged PEComas lacked co-existing TSC2 mutations, indicating alternative pathways of tumorigenesis. In summary, this comprehensive genetic analysis significantly expands our understanding of molecular alterations in PEComas and brings forth the genetic heterogeneity of these tumors.

Spindle cell rhabdomyosarcoma (RMS) is a rare form of RMS with different clinical characteristics and behavior between children and adult patients. Its genetic hallmark remains unknown and it remains debatable if there is pathogenetic relationship between the spindle cell and the so-called sclerosing RMS. We studied two pediatric and one adult spindle cell RMS by next generation RNA sequencing and used FusionSeq for data analysis to detect novel fusions. An SRF-NCOA2 gene fusion was detected in a spindle cell RMS from the posterior neck in a 7 month-old child. The fusion matched the tumor karyotype and was further confirmed by fluorescence in situ hybridization (FISH) and by RT-PCR, which showed fusion of SRF exon 6 to NCOA2 exon 12. Additional 14 spindle cell (from 8 children and 6 adults) and 4 sclerosing (from 2 children and 2 adults) RMS were tested by FISH for the presence of abnormalities in NCOA2, SRF, as well as for PAX3 and NCOA1, identifying NCOA2 rearrangements in two additional spindle cell RMS from a 3 month-old and a 4 week-old child, both arising in the chest wall. In the latter tumor, TEAD1 was identified by rapid amplification of cDNA ends (RACE) to be the NCOA2 gene fusion partner. None of the adult tumors were positive for NCOA2 rearrangement. Despite similar histomorphology in adults and young children, these results suggest that spindle cell RMS is a heterogeneous disease genetically as well as clinically. Our findings also support a relationship between NCOA2-rearranged spindle cell RMS occurring in young childhood and the so-called congenital RMS, which often displays rearrangements at 8q13 locus (NCOA2).

Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

BACKGROUND Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer. CONCLUSIONS The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.

PHF1 gene rearrangements have been recently described in around 50% of ossifying fibromyxoid tumors (OFMT) including benign and malignant cases, with a small subset showing EP400-PHF1 fusions. In the remaining cases no alternative gene fusions have been identified. PHF1-negative OFTs, especially if lacking S100 protein staining or peripheral ossification, are difficult to diagnose and distinguish from other soft tissue mimics. In seeking more comprehensive molecular characterization, we investigated a large cohort of 39 OFMT of various anatomic sites, immunoprofiles and grades of malignancy. Tumors were screened for PHF1 and EP400 rearrangements by FISH. RNA sequencing was performed in two index cases (OFMT1, OFMT3), negative for EP400-PHF1 fusions, followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. Two novel fusions were identified ZC3H7B-BCOR in OFMT1 and MEAF6-PHF1 in OFMT3. After being validated by FISH and RT-PCR, these abnormalities were screened on the remaining cases. With these additional gene fusions, 33/39 (85%) of OFMTs demonstrated recurrent gene rearrangements, which can be used as molecular markers in challenging cases. The most common abnormality is PHF1 gene rearrangement (80%), being present in benign, atypical and malignant lesions, with fusion to EP400 in 44% of cases. ZC3H7B-BCOR and MEAF6-PHF1 fusions occurred predominantly in S100 protein-negative and malignant OFMT. As similar gene fusions were reported in endometrial stromal sarcomas, we screened for potential gene abnormalities in JAZF1 and EPC1 by FISH and found two additional cases with EPC1-PHF1 fusions.