Munir R. Tanas

Identification of a Disease-Defining Gene Fusion in Epithelioid Hemangioendothelioma

A newly identified gene fusion defines the vascular cancer epithelioid hemangioendothelioma and encodes a chimeric transcription factor. FISHing for a Gene Fusion Mother was right: There is a time and place for everything. And at the molecular level, inappropriate behavior can have consequences much more severe than being grounded. Using an unbiased deep-sequencing approach coupled with traditional chromosomal karyotyping, Tanas et al. now describe the genes involved in a fusion event that defines epithelioid hemangioendothelioma (EHE), a rare vascular cancer. This genetic aberration may instigate the bad behavior—an improper transcriptional program in endothelial cells. A rare sarcoma, EHE is difficult to diagnose because it shares many characteristics with normal endothelial cells and resembles other abnormal vascular neoplasms, such as epithelioid hemangioma, a benign condition, and epithelioid angiosarcoma, an aggressive vascular cancer. Treatment for patients with localized EHE includes surgical removal, when possible, or liver transplantation in the case of hepatic involvement, and there is no treatment for metastatic disease. To aid in diagnosis and decipher the pathological processes behind this mysterious cancer, researchers and clinicians need a defining biomarker for EHE. Traditional cytogenetic techniques for identifying the genes involved in a genetic translocation are labor-intensive, especially for a rare cancer for which no cell lines are available. So, Tanas et al. took a shortcut; the authors combined cytogenetic methods with deep transcriptome sequencing, which they used to search in an unbiased way for the product of the t(1;3)(p36;q25) chromosomal translocation characteristic of EHE. The translocation involved two genes, WWTR1, which encodes a transcriptional coactivator that is highly expressed in endothelial cells, and CAMTA1, a DNA binding transcriptional regulatory protein that is normally expressed during brain development. The WWTR1/CAMTA1 gene fusion contains the strong endothelial cell promoter of WWTR1, which may drive the inappropriate expression of a protein-encoding fragment of CAMTA1 in endothelial cells. The authors suggest that this promoter switch initiates an ill-suited and ill-timed transcriptional program that may play a role in cancer biology. If this is the case, then the chimeric WWTR1/CAMTA1 transcription factor may represent a therapeutic target for EHE-specific drugs. To aid in disease diagnosis, Tanas et al. also devised a sensitive and specific fluorescence in situ hybridization assay to detect the EHE translocation. Together, these tools should teach researchers about the biology and prognosis of this rare cancer and eventually help bring the bad behavior under control. Integrating transcriptomic sequencing with conventional cytogenetics, we identified WWTR1 (WW domain–containing transcription regulator 1) (3q25) and CAMTA1 (calmodulin-binding transcription activator 1) (1p36) as the two genes involved in the t(1;3)(p36;q25) chromosomal translocation that is characteristic of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. This WWTR1/CAMTA1 gene fusion is under the transcriptional control of the WWTR1 promoter and encodes a putative chimeric transcription factor that joins the amino terminus of WWTR1, a protein that is highly expressed in endothelial cells, in-frame to the carboxyl terminus of CAMTA1, a protein that is normally expressed only in brain. Thus, CAMTA1 expression is activated inappropriately through a promoter-switch mechanism. The gene fusion is present in virtually all EHEs tested but is absent from all other vascular neoplasms, demonstrating it to be a disease-defining genetic alteration. A sensitive and specific break-apart fluorescence in situ hybridization assay was also developed to detect the translocation and will assist in the evaluation of this diagnostically challenging neoplasm. The chimeric WWTR1/CAMTA1 transcription factor may represent a therapeutic target for EHE and offers the opportunity to shed light on the functions of two poorly characterized proteins.

A Knock-In Mouse Model of Gastrointestinal Stromal Tumor Harboring Kit K641E

A mouse model of gastrointestinal stromal tumor (GIST) has been developed by a knock-in gene targeting strategy, which introduced a Kit gene K641E mutation, originally identified in sporadic human GISTs and in the germ line of familial GIST syndrome patients. Homozygous and heterozygous Kit K641E mice develop gastrointestinal pathology with complete penetrance and all Kit K641E homozygotes die by age 30 weeks due to gastrointestinal obstruction by hyperplastic interstitial cells of Cajal (ICC) or GISTs. Heterozygous mice have less extensive ICC hyperplasia and smaller GISTs, suggesting a dose-response relationship between oncogenically activated Kit and ICC proliferation. Immunoprecipitation and Western blotting reveal GISTs to contain abundant phosphorylated/activated Kit. In addition to ICC hyperplasia and GISTs, homozygous Kit K641E mice exhibit loss-of-function Kit phenotypes, including white coat color, decreased numbers of dermal mast cells, and sterility, indicating that despite its oncogenic activity the mutant form cannot accomplish many activities of the wild-type gene. Kit K641E reproduces the pathology associated with the familial GIST syndrome and thus is an excellent model to study Kit pathway activation, ICC biology, GIST pathogenesis, and preclinical validations of GIST therapies and mechanisms of drug resistance.

Fluorescence in situ hybridization in the diagnosis of soft tissue neoplasms: a review.

This paper presents an overview of the role of fluorescence in situ hybridization (FISH) in the diagnosis of soft tissue neoplasms. Many soft tissue neoplasms harbor characteristic translocations or amplification of gene regions, which can be assessed by FISH, and can be used to assist in their diagnosis. We discuss the major morphologic categories in which FISH has come to be used including high-grade round cell sarcomas, spindle cell sarcomas, low-grade myxoid neoplasms, adipocytic neoplasms, and malignant melanocytic neoplasms on the basis of a recent review of soft tissue neoplasms which were analyzed by FISH. We also review the molecular alterations (translocations and amplification of gene regions), which have come to define many of these diagnostic entities and the most effective way to evaluate them with FISH with attention to potential pitfalls. Finally, we discuss the advantages and disadvantages of FISH as a technique when appraising soft tissue neoplasms.

Utility of FISH in the diagnosis of angiomatoid fibrous histiocytoma: a series of 18 cases

Angiomatoid fibrous histiocytoma is a mesenchymal neoplasm of intermediate malignancy and uncertain histogenesis/line of differentiation, which occurs most commonly in the extremities of children to young adults. It has a characteristic appearance characterized by a proliferation of histiocytoid cells with a lymphoid cuff and fibrous pseudocapsule, simulating the appearance of a neoplasm occurring within a lymph node. However, these classic histological features are not always present. Given the variable appearance of the neoplastic cells and the lack of consistently positive immunohistochemical markers, diagnosis can be problematic. Angiomatoid fibrous histiocytoma has been found to harbor three related translocations, a t(12;16)(q13;p11) resulting in a FUS/ATF1 fusion gene, t(12;22)(q13;q12) resulting in a EWSR1/ATF1 fusion, and t(2;22)(q33;q12) resulting in a EWSR1/CREB1 fusion. Fluorescence in situ hybridization (FISH) probes to EWSR1 and FUS, in theory, should detect all three translocations/gene fusions. We evaluated 18 cases of angiomatoid fibrous histiocytoma for rearrangements of EWSR1 and FUS by FISH, the largest series to date. We found that 13 of 17 (76%) cases of angiomatoid fibrous histiocytoma harbored rearrangements of EWSR1; rearrangements of FUS were not detected in any of the cases. This study affirms that the rearrangement of EWSR1 is a common genetic event in angiomatoid fibrous histiocytoma, and is thus useful diagnostically. This study supports the fact that the rearrangement of FUS is present in only a small minority of angiomatoid fibrous histiocytomas. Interestingly, 24% of the cases were translocation negative, and did not contain rearrangements of EWSR1 or FUS by FISH. Although it is possible that these cases contained cryptic rearrangements of EWSR1 or FUS that were not detectable by our FISH probes, it also raises the possibility that another translocation/gene fusion may be present in angiomatoid fibrous histiocytoma. Finally, we discuss some of the potential pitfalls of this technique, including confusion with other mesenchymal neoplasms containing rearrangement of EWSR1, in particular Ewings sarcoma/PNET.

Utilization of Fluorescence In Situ Hybridization in the Diagnosis of 230 Mesenchymal Neoplasms: An Institutional Experience

CONTEXT Mesenchymal neoplasms harbor characteristic translocations and amplification of gene regions amenable to evaluation by fluorescence in situ hybridization (FISH). OBJECTIVE To determine the utility of FISH in the diagnosis of mesenchymal neoplasms. DESIGN Two hundred thirty soft tissue cases analyzed by FISH were reviewed retrospectively. RESULTS Morphologic patterns where FISH was used included high-grade round cell sarcomas (n  =  67), nonmyogenic spindle cell sarcomas (n  =  40), low-grade myxoid neoplasms (n  =  34), adipocytic neoplasms (n  =  20), and melanocytic neoplasms (n  =  19). Fifty cases did not fit into the previously mentioned categories. SYT FISH (96% of monophasic synovial sarcomas were positive; 0% of malignant peripheral nerve sheath tumor were positive) and DDIT3 FISH (100% of myxoid/round cell liposarcomas; no other neoplasm positive) were very sensitive and specific. EWSR1 FISH was very sensitive and specific in the differential diagnosis of melanocytic neoplasms (88% of clear cell sarcomas were positive; all melanomas were negative). EWSR1 FISH was sensitive among high-grade round cell sarcomas (positive in 100% of desmoplastic small round cell tumors and 96% of Ewing sarcoma/primitive neuroectodermal tumors) but not specific because clear cell sarcoma, extraskeletal myxoid chondrosarcoma, and a subset of round cell liposarcomas also harbor rearrangements of EWSR1. FUS FISH was very sensitive in detecting low-grade fibromyxoid sarcomas (91% positive) but not specific because most myxoid/round cell liposarcomas also contain rearrangements of FUS. All atypical lipomatous tumors were positive for amplification of MDM2, whereas all lipomas were negative. FOXO1A FISH was positive in ∼70% of cases of alveolar rhabdomyosarcoma. CONCLUSION FISH is a useful adjunct in the diagnosis of mesenchymal neoplasms.

Epithelioid sarcoma: the University of Washington experience.

BACKGROUND Epithelioid sarcoma is a rare sarcoma with a high local recurrence rate that frequently metastasizes to lymph nodes. We reviewed our experience with adjuvant therapy in patients with this disease. METHODS Between 1990 and 2003, we treated 11 patients with epithelioid sarcoma. Patient, tumor, and treatment characteristics were analyzed, and effect of treatment on survival was evaluated by the Kaplan-Meier method. RESULTS Nine men and 2 women were treated. Tumors presented on the trunk, the upper extremities, and the lower extremities. Five patients developed nodal disease. All patients underwent surgery for the primary tumor, and 7 patients had nodal evaluation. Ten patients underwent adjuvant chemotherapy, and 9 underwent radiotherapy. Recurrence developed in 9 patients. Five-year disease-free and overall survival rates were 46% and 65%, respectively. Chemotherapy and radiation therapy did not impact disease-free survival. CONCLUSIONS Although surgery remains the primary treatment modality, multi-institutional trials are needed to develop more effective adjuvant therapy for patients with epithelioid sarcoma.

Angiomatoid fibrous histiocytoma: an expansion of the clinical and histological spectrum

Summary Angiomatoid fibrous histiocytoma, a fibrohistiocytic tumour of intermediate malignancy that usually presents on extremities of young patients, has a broader clinical/histological spectrum than is widely appreciated. We summarise our experience with an emphasis on unusual features. Twenty-seven cases were analysed for clinical and histological features, including immunohistochemistry and FISH for rearrangements of EWSR1 or FUS. Five (19%) occurred in patients >40 years old, and ten (37%) occurred outside the extremities. Three that occurred in patients >40 years old arose in atypical locations. Evaluation for classical histological features (lymphocytic cuff, fibrous pseudocapsule, pseudovascular spaces, haemorrhage, haemosiderin, and histiocytoid morphology) showed that all had two or more classical features. Unusual features were noted in many cases. Ten (37%) displayed significant areas of sclerosis; three of these ten had areas with a perineurioma-like pattern. Nine displayed at least moderate pleomorphism, with two exhibiting striking pleomorphism. Eight had eosinophils in the stroma, one with numerous eosinophils. One had a reticulated pattern of cells in a myxoid stroma. Mitotic rates were low [average 0.67/10 high power fields (HPFs)]. Three had atypical mitotic figures. Thirteen of 20 (65%) were CD68 positive, 11 of 17 (65%) were EMA positive, and 10 of 18 (56%) were desmin positive. Thirteen of 16 (81%) had a rearrangement of EWSR1; none had a FUS rearrangement. This series expands the spectrum of angiomatoid fibrous histiocytoma.

Mechanism of action of a WWTR1(TAZ)-CAMTA1 fusion oncoprotein

The WWTR1 (protein is known as TAZ)-CAMTA1 (WC) fusion gene defines epithelioid hemangioendothelioma, a malignant vascular cancer. TAZ (transcriptional coactivator with PDZ binding motif) is a transcriptional coactivator and end effector of the Hippo tumor suppressor pathway. It is inhibited by phosphorylation by the Hippo kinases LATS1 and LATS2. Such phosphorylation causes cytoplasmic localization, 14-3-3 protein binding and the phorphorylation of a terminal phosphodegron promotes ubiquitin-dependent degradation (the phosphorylation of the different motifs has several effects). CAMTA1 is a putative tumor suppressive transcription factor. Here we demonstrate that TAZ-CAMTA1 (TC) fusion results in its nuclear localization and constitutive activation. Consequently, cells expressing TC display a TAZ-like transcriptional program that causes resistance to anoikis and oncogenic transformation. Our findings elucidate the mechanistic basis of TC oncogenic properties, highlight that TC is an important model to understand how the Hippo pathway can be inhibited in cancer, and provide approaches for targeting this chimeric protein.

A metabolic synthetic lethal strategy with arginine deprivation and chloroquine leads to cell death in ASS1-deficient sarcomas.

Sarcomas comprise a large heterogeneous group of mesenchymal cancers with limited therapeutic options. When treated with standard cytotoxic chemotherapies, many sarcomas fail to respond completely and rapidly become treatment resistant. A major problem in the investigation and treatment of sarcomas is the fact that no single gene mutation or alteration has been identified among the diverse histologic subtypes. We searched for therapeutically druggable targets that are common to a wide range of histologies and hence could provide alternatives to the conventional chemotherapy. Seven hundred samples comprising 45 separate histologies were examined. We found that almost 90% were arginine auxotrophs, as the expression of argininosuccinate synthetase 1 was lost or significantly reduced. Arginine auxotrophy confers sensitivity to arginine deprivation, leading temporarily to starvation and ultimately to cell survival or death under different circumstances. We showed that, in sarcoma, arginine deprivation therapy with pegylated arginine deiminase (ADI-PEG20) maintains a prolonged state of arginine starvation without causing cell death. However, when starvation was simultaneously prolonged by ADI-PEG20 while inhibited by the clinically available drug chloroquine, sarcoma cells died via necroptosis and apoptosis. These results have revealed a novel metabolic vulnerability in sarcomas and provided the basis for a well-tolerated alternative treatment strategy, potentially applicable to up to 90% of the tumors, regardless of histology.

TAZ and YAP are frequently activated oncoproteins in sarcomas

TAZ (WWTR1) and YAP are transcriptional coactivators and oncoproteins inhibited by the Hippo pathway. Herein we evaluate 159 sarcomas representing the most prevalent sarcoma types by immunohistochemistry for expression and activation (nuclear localization) of TAZ and YAP. We show that 50% of sarcomas demonstrate activation of YAP while 66% of sarcomas demonstrate activated TAZ. Differential activation of TAZ and YAP are identified in various sarcoma types. At an RNA level, expression of WWTR1 or YAP1 predicts overall survival in undifferentiated pleomorphic sarcoma and dedifferentiated liposarcoma. Immunohistochemistry demonstrates that TAZ and YAP expression and activation are positively correlated with grade in the well-differentiated liposarcoma to dedifferentiated liposarcoma tumor progression sequence as well as conventional chondrosarcomas. TAZ and YAP are constitutively activated oncoproteins in sarcoma cell lines. Knock-down of TAZ and YAP demonstrate differential activity for the two proteins. Verteporfin decreases colony formation in soft agar as well as CTGF expression in sarcoma cell lines harboring activated TAZ and YAP.