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.

Spindle cell rhabdomyosarcoma of bone with FUS-TFCP2 fusion: confirmation of a very recently described rhabdomyosarcoma subtype.

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.

Fluorescence in-situ hybridization identifies Mastermind-like 2 (MAML2) rearrangement in odontogenic cysts with mucous prosoplasia: a pilot study.

AIMS: The pathogenesis of intraosseous mucoepidermoid carcinoma (IMEC) remains unknown. Coexistence with odontogenic cysts (ODC) has been reported in 32-48% of IMEC. Furthermore, prosoplastic mucous cells are often seen in the epithelial lining of ODCs. MECT1-MAML2 fusion transcripts have been identified in >66% of salivary gland MEC cases. The aim of this study was to investigate the presence of MAML2 rearrangement in ODCs featuring mucous prosoplasia. METHODS AND RESULTS: Ten cases of ODC with a mucous cell component and three cases of IMEC were evaluated using fluorescence in-situ hybridization. All cases occurred in the mandible. The ODCs exhibited a M:F ratio of 4:1 (mean age 49.2 years), while all IMECs occurred in women (mean age 68.3 years). All three IMECs demonstrated MAML2 rearrangement, in 26-61% of tumour cells. Successful hybridization was observed in nine of 10 cases of ODC. In two of these nine, there was MAML2 rearrangement in 12% and 24% of the lining epithelial cells, while three of the nine showed rearrangement in 7-8% of cells; the remaining four cases were negative. CONCLUSIONS: We identified MAML2 rearrangements in five of nine ODCs lined by mucus-secreting cells. This suggests that at least a subset of ODCs with mucous prosoplasia are characterized by molecular events considered diagnostic for intraosseous and extraosseous MEC.

Histopathologic, immunophenotypic and cytogenetic features of pulmonary mucoepidermoid carcinoma.

Pulmonary mucoepidermoid carcinoma is an uncommon but distinctive manifestation of mucoepidermoid carcinoma. Pulmonary mucoepidermoid carcinoma occurs in adults and children and can cause diagnostic problems, especially in small biopsies. Few studies have characterized the histologic and immunophenotypic features of pulmonary mucoepidermoid carcinoma. t(11;19)(q21;p13) is considered disease-defining for mucoepidermoid carcinoma; its significance in pulmonary mucoepidermoid carcinoma warrants further study. Forty three pulmonary mucoepidermoid carcinomas were re-reviewed and graded according to the Brandwein grading system for mucoepidermoid carcinoma. Four cases were excluded because of a split opinion between pathology report and re-review. These cases were negative for MAML2 rearrangement by FISH. TTF-1, napsin A, p40 and p63 immunostains were scored: 0 (negative), 1 (1-25% tumor cells), 2 (26-50%), 3 (51-75%) or 4 (>75%). FISH to detect MAML2 rearrangement used a MAML2-11q21 break-apart probe. Thirty nine pulmonary mucoepidermoid carcinoma (4 low, 30 intermediate, 5 high grade) contained mucous, epidermoid and intermediate cells and lacked keratinization and in situ carcinoma of the overlying epithelium. All cases with available gross description (n=22) had a central/endo- or peribronchial location. All 25 cases tested for immunohistochemistry were positive (scores 1-4) for p63; 23 also expressed p40. In six cases, the p63 score was higher than p40. TTF-1 and napsin were uniformly negative in all 25 cases. MAML2 rearrangement was identified by FISH in each of the 24 cases tested (3 low, 19 intermediate, 2 high grade). Clinical history was available in 29 patients (15 men) (median age, 48 years) with follow-up in 24 (median, 8.4 years). Five patients died of unrelated causes; one developed metastatic pulmonary mucoepidermoid carcinoma. In conclusion, features helpful in distinguishing pulmonary mucoepidermoid carcinoma from other lung cancers include its central/endo- or peribronchial location together with the presence of mucous cells, p63 expression, lack of keratinization and MAML2 rearrangement. TTF-1 and napsin are typically not expressed.

Ewing sarcoma of the head and neck: The Mayo Clinic experience.

BACKGROUND: Treatment options of Ewing sarcoma of the head and neck include surgery, radiotherapy (RT), and chemoradiotherapy. However, local control can be challenging. METHODS: We conducted a retrospective review of all patients with head and neck Ewing sarcoma treated from 1972 to 2015 at a single tertiary care hospital. RESULTS: Seventeen patients met criteria (median 21 years, range 5-58 years; 5 women). Mean follow-up was 10.4 years (range 2.2-39 years). Tumors occurred commonly in the cervical spine (5/17), the skull (3/17), and the paranasal sinuses (3/17). A total of 14 of 17 patients underwent surgical resection, 9 with gross total resection. After multimodality therapy, the 5-year overall survival (OS) and recurrence-free survival (RFS) was 87% and 75%, respectively. CONCLUSION: Combined multimodal treatment resulted in a 5-year OS and RFS of 87% and 75%, respectively. Aggressive surgical resection with adjuvant chemoradiotherapy should be considered. Although negative margin surgery is the goal, subtotal resection may be acceptable in the setting of adjuvant treatment.

Development and Verification of an RNA Sequencing (RNA-Seq) Assay for the Detection of Gene Fusions in Tumors.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.

Clinical utility of myb rearrangement detection and p63/p40 immunophenotyping in the diagnosis of adenoid cystic carcinoma of minor salivary glands: a pilot study.

OBJECTIVES: MYB rearrangement is observed in approximately 28% to 86% of adenoid cystic carcinomas (ACCs). Also, ACC features a p63+/p40+ immunophenotype in greater than 90% of cases, compared with p63+/p40- polymorphous low-grade adenocarcinoma (PLGA). Our aim was to investigate the incidence of (1) MYB rearrangement and (2) p63/p40 immunoreactivity in ACC and PLGA of minor salivary glands (MSGs). STUDY DESIGN: Seven cases of ACC as well as five of PLGA were evaluated by using a MYB (6 q23.3) break-apart fluorescence in situ hybridization (FISH) probe. In addition, all cases were immunohistochemically stained with p63 and p40 antibodies. RESULTS: All five successfully hybridized ACCs featured MYB rearrangement, whereas PLGAs did not show MYB rearrangement. Interestingly, one case of PLGA demonstrated a single intact copy of MYB in greater than 88% of the neoplastic cells. All ACCs exhibited consistent p63+/p40+ staining, whereas PLGAs demonstrated a p63+/p40- immunophenotype. CONCLUSIONS: (1) MYB rearrangement is encountered in ACCs but not PLGAs of MSGs; (2) MYB aberrations, for example, monosomy or deletion, can be seen in PLGAs; (3) combined p63/p40 immunostaining can be used to differentiate ACC from PLGA in incisionally biopsied specimens; and (4) performance of either FISH or p63/p40 immunohistochemistry is expected to be able to confirm the diagnosis of ACC or PLGA in small intraoral biopsies, since both techniques appeared to be diagnostically accurate in this pilot study.