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The hyalinizing trabecular adenoma/tumor is a rare and poorly characterized follicular-derived thyroid neoplasm recently shown to harbor recurrent PAX8–GLIS1or PAX8–GLIS3gene fusions. Here we sought to define the repertoire of genetic alterations of hyalinizing trabecular tumors, and whether PAX8–GLIS3fusions are pathognomonic for hyalinizing trabecular tumors. A discovery series of eight hyalinizing trabecular tumors was subjected to RNA-sequencing (n= 8), whole-exome sequencing (n= 3) or targeted massively parallel sequencing (n= 5). No recurrent somatic mutations or copy number alterations were identified in hyalinizing trabecular tumor, whereas RNA-sequencing revealed the presence of a recurrent genetic rearrangement involving PAX8(2q14.1) and GLIS3(9p24.2) genes in all cases. In this in-frame fusion gene, which comprised exons 1–2 of PAX8and exons 3–11 of GLIS3, GLIS3is likely placed under the regulation of PAX8. Reverse transcription RT-PCR and/or fluorescence in situ hybridization analyses of a validation series of 26 hyalinizing trabecular tumors revealed that the PAX8–GLIS3gene fusion was present in all hyalinizing trabecular tumors (100%). No GLIS1rearrangements were identified. Conversely, no PAX8–GLIS3gene fusions were detected in a cohort of 237 control thyroid neoplasms, including 15 trabecular thyroid lesions highly resembling hyalinizing trabecular tumor from a morphological standpoint, as well as trabecular/solid follicular adenomas, solid/trabecular variants of papillary carcinoma, and Hurthle cell adenomas or carcinomas. Our data provide evidence to suggest that the PAX8–GLIS3fusion is pathognomonic for hyalinizing trabecular tumors, and that the presence of the PAX8–GLIS3fusion in thyroid neoplasms may be used as an ancillary marker for the diagnosis of hyalinizing trabecular tumor, thereby avoiding overtreatment in case of misdiagnoses with apparently similar malignant tumors.

Integration of morphological, immunohistochemical, and molecular methods is often necessary for the precise diagnosis and optimal clinical management of sarcomas. We have validated and implemented a clinical molecular diagnostic assay, MSK- Fusion Solid, for detection of gene fusions in solid tumors, including sarcomas. Starting with RNA extracted from formalin-fixed paraffin-embedded tumor material, this targeted RNA sequencing assay utilizes anchored multiplex PCR to detect oncogenic fusion transcripts involving 62 genes known to be recurrently rearranged in solid tumors including sarcomas without prior knowledge of fusion partners. From 1/2016 to 1/2018, 192 bone and soft tissue tumors were submitted for MSK- Fusion Solid analysis and 96% (184/192) successfully passed all the pre-sequencing quality control parameters and sequencing steps. These sarcomas encompass 24 major tumor types, including 175 soft tissue tumors and 9 osteosarcomas. Ewing and Ewing-like sarcomas, rhabdomyosarcoma, and sarcoma-not otherwise specified were the three most common tumor types. Diagnostic in-frame fusion transcripts were detected in 43% of cases, including 3% (6/184) with novel fusion partners, specifically TRPS1-PLAG1, VCP-TFE3, MYLK-BRAF, FUS-TFCP2, and ACTB-FOSB, the latter in two cases of pseudomyogenic hemangioendothelioma, representing a novel observation in this sarcoma. Our experience shows that this targeted RNA sequencing assay performs in a robust and sensitive fashion on RNA extracted from most routine clinical specimens of sarcomas thereby facilitating precise diagnosis and providing opportunities for novel fusion partner discovery.

Several recent reports have described low-grade fibromyxoid sarcoma with sclerosing epithelioid fibrosarcoma-like areas. We evaluated cases of pure sclerosing epithelioid fibrosarcoma lacking areas of low-grade fibromyxoid sarcoma for FUSrearrangement to determine whether this entity could be related to low-grade fibromyxoid sarcoma. Available formalin-fixed paraffin-embedded tissue of 27 sclerosing epithelioid fibrosarcoma from 25 patients was retrieved and tabulated with clinical information. Unstained slides from formalin-fixed paraffin-embedded blocks were prepared and fluorescence in-situhybridization was performed using a commercial FUSbreak-apart probe. The median patient age at presentation was 50 (range, 14–78) years, with 14 males and 10 females. Sclerosing epithelioid fibrosarcoma most commonly involved the extremities (n=8) or chest (n=6). Sixteen patients had a median follow-up of 17 (range, 1–99) months; seven were alive and well at 12 (range, 5–30) months; three alive with disease at 28 (range, 9–99) months; five dead of disease at a median of 22 (range, 1–36) months and one was dead of unknown causes. Twelve patients were known to have metastases; the most common site was lung (n=7), followed by bone (n=3), lymph nodes (n=2) and peritoneum (n=1). Only 2 of 22 (9%) analyzable cases of sclerosing epithelioid fibrosarcoma showed rearrangement in the FUSlocus by fluorescence in-situhybridization. Although cytogenetically confirmed low-grade fibromyxoid sarcoma can have sclerosing epithelioid fibrosarcoma-like areas, FUSrearrangement, which is characteristic of low-grade fibromyxoid sarcoma, appears to be relatively rare in pure sclerosing epithelioid fibrosarcoma.

Inflammatory liposarcoma is a variant of well-differentiated liposarcoma/atypical lipomatous tumor that consists of a mixture of lymphocytes, histiocytes, scattered atypical stromal cells, mature adipocytes, and rarely lipoblasts. When the inflammatory infiltrate predominates, the morphological features overlap with various fibroinflammatory disorders including sclerosing mesenteritis and retroperitoneal fibrosis, making the diagnosis difficult. Well-differentiated liposarcoma/atypical lipomatous tumor and dedifferentiated liposarcoma have characteristic molecular markers in the form of giant marker and ring chromosomes consisting of amplicons of 12q13-15, which includes MDM2. MDM2 immunohistochemistry (IHC) (Zymed; clone IF2) and dual color fluorescence in situhybridization utilizing MDM2(12q15) and chromosome 12 centromeric probes were performed on formalin-fixed and paraffin-embedded specimens from inflammatory well-differentiated liposarcoma (17 cases), sclerosing mesenteritis (14 cases), and idiopathic retroperitoneal fibrosis (10 cases). MDM2 expression as detected by IHC is a very sensitive tool in recognizing inflammatory well-differentiated liposarcoma (17 of 17); however, 21% (3 of 14) and 10% (1 of 10) of sclerosing mesenteritis and retroperitoneal fibrosis, respectively, displayed weak MDM2 immunoexpression. The MDM2fluorescence in situhybridization assay was very specific for inflammatory well-differentiated liposarcoma as 15 of 17 (88%) cases showed MDM2amplification, whereas none of the cases of sclerosing mesenteritis or idiopathic retroperitoneal fibrosis showed amplification. Five cases of retroperitoneal fibrosis were noncontributory secondary to autofluorescence, potentially limiting the usefulness of the assay in certain situations such as inappropriate fixation. Increased MDM2 expression and/or MDM2amplification can be employed to aid discrimination of inflammatory well-differentiated liposarcoma from fibroinflammatory mimics. MDM2fluorescence in situhybridization is a very specific method (100%), but less sensitive (88%), whereas MDM2 expression by IHC is very sensitive (100%), but less specific (83%). Therefore, a positive screen of difficult cases with MDM2 IHC would require confirmation by the fluorescence in situhybridization. However, lack of MDM2 immunoexpression would rule out the possibility of inflammatory well-differentiated liposarcoma.

Well-differentiated liposarcoma/atypical lipomatous tumor and dedifferentiated liposarcoma can be difficult to distinguish from benign lipomatous neoplasms and other high-grade sarcomas, respectively. Cytogenetics in these tumors has identified ring and giant chromosomes composed of 12q13-15 amplicons including the MDM2gene. Identifying MDM2amplification by fluorescence in situhybridization may prove an adjunctive tool in the diagnosis of lipomatous neoplasms. Dual color fluorescence in situhybridization employing a laboratory-developed BAC label probe cocktail specific for MDM2(12q15) and a probe for the centromeric region of chromosome 12 (Abbott Molecular, DesPlaines, IL) was performed on formalin-fixed and paraffin-embedded tissue including whole sections from atypical lipomatous tumors (n=13), dedifferentiated liposarcomas (n=14), benign lipomatous tumors (n=30), and pleomorphic sarcoma, not otherwise specified (n=10), and a tissue microarray containing a variety of high-grade sarcomas (n=63). An MDM2/chromosome 12 ratio ≥2.0 was considered amplified, <2.0 nonamplified, and cases displaying >2 signals of both probes and an MDM2ratio <2.0 polysomic for chromosome 12. Of the well-differentiated and dedifferentiated liposarcomas, 100% showed amplification of MDM2. Chromosome 12 polysomy was noted in 89% of spindle cell/pleomorphic lipomas, while all angiolipomas and lipomas were nonamplified and eusomic. MDM2amplification was observed in 40% of pleomorphic sarcomas and a small subset of high-grade sarcomas (3/63). MDM2/chromosome 12 fluorescence in situhybridization is a sensitive and specific tool (both 100%) in evaluating low-grade lipomatous neoplasms. The specificity decreases in high-grade sarcomas, as MDM2amplification was observed in a small portion of pleomorphic sarcomas and high-grade sarcomas other than dedifferentiated liposarcomas. Importantly, none of the benign lipomatous lesions were MDM2amplified and even cells in areas of well-differentiated liposarcomas with minimal cytologic atypia were amplified, making the probe a valuable tool in the diagnosis of even limited biopsy samples of well-differentiated lipomatous neoplasms.