Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip.

Alveolar soft-part sarcoma (ASPS) is a slow-growing soft tissue sarcoma with high mortality rates that affects adolescents and young adults. ASPS resists conventional chemotherapy; thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic capacities. Integrated blood vessels that are rich in pericytes (PCs) and metastatic potential are distinctive of ASPS. To mimic ASPS angiogenic microenvironment, a microfluidic coculture vasculature chip has been developed as a three-dimensional (3D) spheroid composed of mouse ASPS, a layer of PCs, and endothelial cells (ECs). This ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model to elucidate the cellular crosstalk within the tumor vasculature before metastasis. We successfully reproduce ASPS spheroid and leaky vessels representing the unique tumor vasculature to assess effective drug delivery into the core of a solid tumor. Furthermore, this ASPS angiogenesis model enabled us to investigate the role of proteins in the intracellular trafficking of bioactive signals from ASPS to PCs and ECs during angiogenesis, including Rab27a and Sytl2. The results can help to develop drugs targeting the crosstalk between ASPS and the adjacent cells in the tumoral microenvironment.

PEComa with ASPSCR1::TFE3 fusion: expanding the molecular genetic spectrum of TFE3-rearranged PEComa with an emphasis on overlap with alveolar soft part sarcoma.

AIMS: Mesenchymal neoplasms involving TFE3 gene fusions are diverse, mainly include alveolar soft part sarcoma (ASPS) that is characterised by ASPSCR1::TFE3 fusion, and a small subset of perivascular epithelioid cell tumours (PEComas) referred to as TFE3-rearranged PEComa, that most frequently harbours SFPQ::TFE3 fusion. Historically, ASPS and TFE3-rearranged PEComa are considered two distinctive entities despite their known morphological overlap. However, recent studies have suggested a potential histogenetic relationship between them, and several neoplasms that showed morphological features more closely fit PEComa rather than ASPS but harboured ASPSCR1::TFE3 fusion have been documented. In this study, we report three cases of PEComa with ASPSCR1::TFE3 fusion. METHODS AND RESULTS: Clinicopathological features were assessed and partner agnostic targeted next-generation sequencing on clinically validated platforms were performed. The patients are two females and one male with age at presentation ranging from 21 to 51 years. All three tumours were located in the viscera (rectum, kidney and cervix). On a relatively limited follow-up period (range = 9-15 months), all patients are alive without evidence of recurrent or metastatic disease. The neoplasms were composed of tight nested architecture of epithelioid clear cells separated by a delicate vascular network, two of which were associated with sheets of plump spindle cells, and none showed significant discohesive tumour morphology. Immunohistochemically, in addition to TFE3 protein, all three neoplasms demonstrated co-expression of melan-A and smooth muscle actin. RNA-sequencing identified ASPSCR1::TFE3 fusion in all three cases that were confirmed by subsequent fluorescence in-situ hybridisation analyses. CONCLUSIONS: Our study expands the molecular genetic spectrum of TFE3-rearranged PEComa and further indicates its close relationship to ASPS.

ASPSCR-1 and Sirt-5 alleviate Clonorchis liver fluke rCsNOSIP-induced oxidative stress, proliferation, and migration in cholangiocarcinoma cells.

BACKGROUND: Clonorchiasis, caused by the infection of Clonorchis sinensis (C. sinensis), is a kind of neglected tropical disease, but it is highly related to cholangiocarcinoma. It has been well known that NO from chronic inflammation responses are thought to be a major component of the damage and ultimate carcinogenesis ESPs such as nitric oxide synthase interacting protein (NOSIP) are thought to enhance the damage. The objective of this study was to identify the protein candidates interact with recombinant CsNOSIP (rCsNOSIP) and explore their role involved in CCA development or progression. METHODS: We applied HuProt microarray containing 21,000 probe sets for a systematic identification of rCsNOSIP-binding proteins and grouped binding hits by gene function. Pull-down assays were used to confirm the interaction of rCsNOSIP with alveolar soft part sarcoma (ASPSCR-1) and sirtuins 5 (Sirt-5). ASPSCR-1/Sirt-5 over-expression and siRNA knockdown experiments were employed for obtain of ASPSCR-1/Sirt-5 high or low expression (ASP-oe/Sirt5-oe or ASP-si/Sirt5-si) cholangiocarcinoma cell line (CCLP-1) cells. Nitric oxide (NO) and reactive oxygen species assay (ROS) as well as cell proliferation and wound-healing assays were performed to observe the effect of rCsNOSIP on ASP-oe/Sirt5-oe or ASP-si/Sirt5-si CCLP-1 cells. RESULTS: Seventy candidate proteins protein "hits" were detected as rCsNOSIP-binding proteins by HuProt microarray and bioinformatics analysis. Pull down assay showed that ASPSCR-1 and Sirt-5 could interact with rCsNOSIP. In addition, endotoxin-free-rCsNOSIP could increase the production of NO and ROS and promote the migration of CCLP-1 cells, while its effect on enhancing cell proliferation was not significant. Furthermore, ROS/NO production, proliferation, or migration were increased in ASP-si or Sirt5-si CCLP-1 cells but decreased in Asp-oe or Sirt5-oe CCLP-1 cells when stimulated with rCsNOSIP. CONCLUSIONS: Our findings suggest that CsNOSIP as a component of CsESPs might promote the development and invasion of CCA and Sirt5/ ASPSCR1 as host molecules might play a novel protective role against adverse stimulus during C. sinensis infection. This work supports the idea that CsESPs induce the occurrence and progression of CCA through ROS/RNS-induced oxidative and nitrative DNA damage.

Revisiting cytomorphology, including unusual features and clinical scenarios of 8 cases of alveolar soft part sarcoma with TFE3 immunohistochemical staining in 7 cases.

OBJECTIVES: To present a comprehensive analysis of cytomorphological features, including clinical scenarios, for 8 cases (4 males, 4 females, aged 17-39 years, average = 28.5) of, retrospectively diagnosed alveolar soft part sarcoma (ASPS), with TFE3 immunostaining in 7 cases. METHODS: Conventional Papanicolaou and May Grunwald-Giemsa (MGG) stained smears and corresponding tissue sections were critically reviewed. Fine needle aspiration cytology was performed for primary diagnosis in 6 cases and for metastatic lesions in 2 cases. TFE3 and other immunohistochemical stains were tested using polymer detection technique. RESULTS: Tumour sites were thigh (n = 6), shoulder (1) and neck (1). Tumour size (n = 6) varied from 5 to 14.5 cm (average = 7.2). Seven out of 8 cases were correctly diagnosed on cytosmears. The smears were mostly hypercellular (5), composed of cohesive clusters (8), including cell balls and pseudopapillae (3) and singly scattered cells (8). Tumour cells were round to oval, containing central to eccentric nuclei (8), abundant granular (8) to finely vacuolated (7) cytoplasm that was ill- to well-defined, intracytoplasmic rod-like or needle-shaped crystals (3) and prominent nucleoli (8), Additionally, there were binucleated cells (7), multinucleation (2), intracytoplasmic inclusions (3), intranuclear inclusions (2), intercellular stroma (5) and bare nuclei (8). Immunohistochemically, 7/8 tumours were positive for TFE3. CONCLUSIONS: This constitutes the largest series describing cytomorphological spectrum of ASPS with TFE3 immunostaining results. Frequently observed features and rod-like/needle-shaped crystals on MGG smears, can help to differentiate ASPS from its mimics. TFE3 immunostaining aids in substantiating diagnoses, in an appropriate clinicoradiological context.

Genomics, Morphoproteomics, and Treatment Patterns of Patients with Alveolar Soft Part Sarcoma and Response to Multiple Experimental Therapies.

Overexpression of transcription factor 3 in alveolar soft part sarcoma(ASPS) results in upregulation of cell proliferation pathways. No standard treatment algorithm exists for ASPS; multikinase inhibitors[tyrosine kinase inhibitor (TKI)] and immune checkpoint inhibitors (ICI) have shown clinical benefit. To date, no studies have reported on management strategies or sequencing of therapy. We evaluated ASPS treatment patterns and responses in an experimental therapeutics clinic. Genomic and morphoproteomic analysis was performed to further elucidate novel targets. We retrospectively reviewed patients with ASPS treated on clinical trials. Demographic and clinical next-generation sequencing (NGS) profiles were collected. AACR GENIE database was queried to further evaluate aberrations in ASPS. Morphoproteomic analysis was carried out to better define the biology of ASPS with integration of genomic and proteomic findings. Eleven patients with ASPS were identified; 7 received NGS testing and mutations in CDKN2A (n = 1) and hepatocyte growth factor (n = 1) were present. Ten patients were treated with TKIs with stable disease as best response and 4 patients with ICI (three partial responses). Within GENIE, 20 patients were identified harboring 3 called pathogenic mutations. Tumor mutation burden was low in all samples. Morphoproteomic analysis confirmed the expression of phosphorylated c-Met. In addition, fatty acid synthase and phosphorylated-STAT3 were detected in tumor cell cytoplasm and nuclei. Patients with ASPS have a quiescent genome and derive clinical benefit from VEGF-targeting TKIs. Morphoproteomic analysis has provided both additional correlative pathways and angiogenic mechanisms that are targetable for patients with ASPS. Our study suggests that sequential therapy with TKIs and immune checkpoint inhibitors is a reasonable management strategy.

Molecular Landscape in Alveolar Soft Part Sarcoma: Implications for Molecular Targeted Therapy.

Alveolar soft part sarcoma (ASPS) is a highly aggressive rare soft tissue sarcoma (STS) with poor prognosis especially in the metastatic form. ASPS is resistant to standard chemotherapy. Although, early diagnosis and surgical resection of operable tumor could lead to improved patient survival but novel treatment options are needed for advanced (metastatic) ASPS. This malignancy exhibits highly angiogenic behavior which reflects hyper-activation and over expression of angiogenic factors. Understanding the molecular events in this type of sarcoma is important in finding novel molecular based targeted therapies. We aim to review molecular aspects of ASPS growth and treatment.

Alveolar soft part sarcoma: A case report with emphasis on some unusual cytological features.

Alveolar soft part sarcoma is a very rare, slow growing highly angiogenic tumor with poor prognosis. Most common site in children and infants is head and neck region and in adults it most commonly occurs in extremities especially thigh. In our case study, an 8 years old female patient presented with a gradually progressive left shoulder lump. FNAC from the lesion showed cellular smears with polyhedral and spindly cells showing abundant finely vacuolated cytoplasm, nuclear pleomorphism, intranuclear pseudoinclusions, and few bare nuclei. Perivascular arrangement of cells was peculiar in addition to the presence of intracytoplasmic metachromatic PAS positive diastase resistant granules. A presumptive diagnosis of alveolar soft part sarcoma with differentials of granular cell tumor and PEComa was considered and the lesion was excised. Although the histopathological features were not characteristic (ie, showing mainly solid pattern without classic alveolar pattern), immunohistochemistry were diagnostic (negative for S 100, Desmin, Cytokeratin, EMA, and moderate to strong nuclear positivity for TFE3). Thus, the diagnosis of alveolar soft part sarcoma was established. This case is being presented for its rarity and unique cytological and histopathological features.

Exploring the Histogenesis and Diagnostic Strategy Using Immunoassay and RT-PCR in Alveolar Soft Part Sarcoma.

Alveolar soft part sarcoma (ASPS) is a rare soft tissue sarcoma, but it's easily misdiagnosed in rare locations. The derivation of ASPS is still uncertain, therefore we conducted this study to explore the histogenesis of ASPS by analyzing stem cell markers (ALDH1, CD29, CD133 and Nestin). Protein TFE3 and fusion gene ASPS-TFE3 were tested in paraffin to explore diagnostic strategy and molecular pathological features. In this study, nine cases of ASPS were immunostained with stem cell surface markers (ALDH1, CD29, CD133 and Nestin) and protein TFE3. Seven cases of ASPS mRNA were successfully extracted from nine paraffin-embedded tissues. The expression of fusion gene ASPL-TFE3 was examined by reverse transcriptase-polymerase chain reaction. The immunohistochemical staining of nine patients showed that CD29 and Nestin were negative in all nine cases (0/9). CD133 was weakly positive in one cases (1/9) and ALDH1 was weakly positive in one cases (1/9). TFE3 was positive in nine cases (9/9). Seven paraffin tissues could be successfully extracted with mRNA in nine cases. The results of Reverse Transcription Polymerase Chain Reaction (RT-PCR) showed that ASPL-TFE3 fusion transcripts could be tested in the seven cases (four cases being type 2 and three cases being type 1). The positive rate of CD133 and ALDH1 were less than 1% and the expression of CD29 and Nestin were negative in ASPS. Immunohistochemistry results indicated that the histogenesis of ASPS maybe not derive from mesenchymal stem cells. Immunohistochemistry staining showed that TFE3 protein expression was highly sensitive in ASPS. Furthermore, RT-PCR results showed that fusion gene ASPL-TFE3 (ASPL-TFE3 type 1 and ASPL-TFE3 type 2) was expressed in ASPS, which could provide information for clinical molecular pathological diagnosis and improve the diagnosis rate of rare atypical ASPS.

Cabozantinib and dastinib exert anti-tumor activity in alveolar soft part sarcoma.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is an extremely rare metastatic soft tissue tumor with a poor prognosis for which no effective systemic therapies have yet been established. Therefore, the development of novel effective treatment approaches is required. Tyrosine kinases (TKs) are being increasingly used as therapeutic targets in a variety of cancers. The purpose of this study was to identify novel therapeutic target TKs and to clarify the efficacy of TK inhibitors (TKIs) in the treatment of ASPS. EXPERIMENTAL DESIGN: To identify novel therapeutic target TKs in ASPS, we evaluated the antitumor effects and kinase activity of three TKIs (pazopanib, dasatinib, and cabozantinib) against ASPS cells using an in vitro assay. Based on these results, we then investigated the phosphorylation activities of the identified targets using western blotting, in addition to examining antitumor activity through in vivo assays of several TKIs to determine both the efficacy of these substances and accurate targets. RESULTS: In cell proliferation and invasion assays using pazopanib, cabozantinib, and dasatinib, all three TKIs inhibited the cell growth in ASPS cells. Statistical analyses of the cell proliferation and invasion assays revealed that dasatinib had a significant inhibitory effect in cell proliferation assays, and cabozantinib exhibited marked inhibitory effects on cellular functions in both assays. Through western blotting, we also confirmed that cabozantinib inhibited c-MET phosphorylation and dasatinib inhibited SRC phosphorylation in dose-dependent fashion. Mice that received cabozantinib and dasatinib had significantly smaller tumor volumes than control animals, demonstrating the in vivo antitumor activity of, these substances. CONCLUSIONS: Our findings suggest that cabozantinib and dasatinib may be more effective than pazopanib against ASPS cells. These in vitro and in vivo data suggest that c-MET may be a potential therapeutic target in ASPS, and cabozantinib may be a particularly useful therapeutic option for patients with ASPS, including those with pazopanib-resistant ASPS.

Metastatic Alveolar Soft Part Sarcoma of the Spinal Cord: A Case Report and Review of Literature.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare, malignant soft-tissue neoplasm typically seen in young adults that possesses an unusual tendency to metastasize. Metastases to the intramedullary compartment of the spinal cord, however, are exceptionally rare and have not been described in the literature. CASE DESCRIPTION: We report the case of a 23-year-old woman with disseminated ASPS to the lung and brain who presented with progressive lower-extremity weakness and loss of sensation after radiation and chemotherapy. Magnetic resonance imaging revealed a 1.3-cm avidly enhancing lesion within the central thoracic spinal cord at T3. A T2-T4 laminectomy was undertaken and resulted in a gross total resection. Histopathologically, the mass was composed of organoid nests containing epithelioid cells with eosinophilic, granular cytoplasm separated by sinusoidal spaces. Immunohistochemistry demonstrated convincing positive TFE3 staining. Postoperative imaging confirmed the complete resection of the mass, and her examination was notable for intact sensation and impaired motor function that gradually improved. CONCLUSIONS: A review of the literature found that the reported case represents the first instance of primary or metastatic ASPS in the spinal cord. Metastatic ASPS should thus be included in the differential diagnosis in patients with known disease and neurologic impairment or back pain. Imaging of the spine should then be considered.

Primary alveolar soft part sarcoma of uterine corpus: a case report with immunohistochemical, ultrastructural study and review of literature.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare mesenchymal malignancy. ASPS usually occurs most commonly in the deep soft tissues of the thigh and buttock or the head and neck regions. ASPS that originate from the uterine corpus are even more rare, with only 10 previous cases reported in the English literature. CASE PRESENTATION: In our case, the alveolar features were completely lost and the tumour shows a solid, non-alveolar pattern and the nuclei have marked variation in nuclear size, and multinucleation. The correct pathological diagnosis has been made by immuno- histochemical and ultrastructural features, which rvealed overexpression of TFE3 and peculiar cytoplasmic crystalline inclusions. In this paper, an additional case of primary ASPS of uterine corpus is reported with immunohistochemical, ultrastructural study and review of literature in the effort to delineate its clinical and pathological features. In this unusual site, the diagnosis can be problematic because ASPS can mimic other primary or metastatic uterine neoplasms. CONCLUSIONS: Thus, in this unusual presentation an essential diagnostic marker is the nuclear over-expression of TFE3 as well as ultrastructural study, which reveals the presence of peculiar cytoplasmic crystalline inclusions.

Alveolar Soft Part Sarcoma.

Alveolar soft part sarcoma is a rare neoplasm usually arising in the soft tissues of the lower limbs in adults and in the head and neck region in children. It presents primarily as a slowly growing mass or as metastatic disease. It is characterized by a specific chromosomal alteration, der(17)t(X:17)(p11:q25), resulting in fusion of the transcription factor E3 (TFE3) with alveolar soft part sarcoma critical region 1 (ASPSCR1) at 17q25. This translocation is diagnostically useful because the tumor nuclei are positive for TFE3 by immunohistochemistry. Real-time polymerase chain reaction to detect the ASPSCR1-TFE3 fusion transcript on paraffin-embedded tissue blocks has been shown to be more sensitive and specific than detection of TFE3 by immunohistochemical stain. Cathepsin K is a relatively recent immunohistochemical stain that can aid in the diagnosis. The recent discovery of the role of the ASPSCR1-TFE3 fusion protein in the MET proto-oncogene signaling pathway promoting angiogenesis and cell proliferation offers a promising targeted molecular therapy.

Modeling alveolar soft part sarcomagenesis in the mouse: a role for lactate in the tumor microenvironment.

Alveolar soft part sarcoma (ASPS), a deadly soft tissue malignancy with a predilection for adolescents and young adults, associates consistently with t(X;17) translocations that generate the fusion gene ASPSCR1-TFE3. We proved the oncogenic capacity of this fusion gene by driving sarcomagenesis in mice from conditional ASPSCR1-TFE3 expression. The completely penetrant tumors were indistinguishable from human ASPS by histology and gene expression. They formed preferentially in the anatomic environment highest in lactate, the cranial vault, expressed high levels of lactate importers, harbored abundant mitochondria, metabolized lactate as a metabolic substrate, and responded to the administration of exogenous lactate with tumor cell proliferation and angiogenesis. These data demonstrate lactate's role as a driver of alveolar soft part sarcomagenesis.

Proteomics identified overexpression of SET oncogene product and possible therapeutic utility of protein phosphatase 2A in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is an exceedingly rare sarcoma refractory to standard chemotherapy. Although several molecular targeting drugs have been applied for ASPS, their clinical significance has not yet been established, and novel therapeutic strategies have long been required. The aim of this study was to identify proteins aberrantly regulated in ASPS and to clarify their clinical significance. Protein expression profiling of tumor and nontumor tissues from 12 ASPS patients was performed by 2-D difference gel electrophoresis and mass spectrometry. We found that the expression of 145 proteins differed significantly. Among them, further investigation was focused on the SET protein, which has multifunctional roles in cancers. Immunohistochemistry confirmed overexpression of SET in all 15 ASPS cases examined. Gene silencing of SET significantly decreased cell proliferation, invasion, and migration against a background of induced apoptosis. SET is known to be an inhibitor of phosphatase 2A (PP2A), which functions as a tumor suppressor by inhibiting the signal transduction pathway and inducing apoptosis. We found that a PP2A activator, FTY720, decreased cell proliferation through apoptosis. Together, our findings may suggest the possible contribution of SET to the tumor progression and the utility of FTY720 for treatment of ASPS.

Mammalian target of rapamycin pathway activity in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a distinct type of soft tissue sarcoma holding a specific ASPL-TFE3 fusion transcript. Curative therapy is based on surgical removal, whereas lately, antiangiogenic targeted therapy regimens have proven effective. In ASPS, analysis of small series additionally display mTOR (mammalian target of rapamycin) pathway activity, thus making mTOR a possible additive target in ASPS, because it is in other tumor entities. Therefore, we systematically evaluated mTOR pathway activity in a large series of ASPS in comparison with soft tissue sarcomas of other differentiation (non-ASPS). Upstream and downstream factors of mTOR signaling and ancillary targets were analyzed in 103 cases (22 ASPS, 81 non-ASPS) by immunohistochemistry mostly using phospho-specific antibodies. TFE3 (transcription factor for immunoglobulin heavy-chain enhancer 3) translocation status was determined by FISH and RT-PCR. All ASPS were positive in TFE3 break-apart FISH and exhibited specific fusion products when RNA was available (type 1: 9x, type 2: 11x), whereas TFE3-immunoreactive non-ASPS did not. In ASPS, TFE3-, cMET-, pAKT T308- (all P < .0001), pp70S6K- (P = .002), and p4EBP1 (P = .087) expression levels were elevated, whereas pAKT S473 was decreased (P < .0001). In addition, ASPS exhibited higher TFE3-, cMET-, pAKT T308-, and pp70S6K- expression levels compared with TFE3-immunopositive non-ASPS sarcomas (all P < .001). We demonstrate elevated mTOR complex 1 (mTORC1) activity in ASPS independent of mTOR complex 2 (mTORC2) activation. mTORC1 activity seems to be related to the existence of ASPL-TFE3 fusion transcripts because TFE3-immunoreactive non-ASPS without ASPL-TFE3 fusion transcripts exhibit significantly lower mTORC1 activation status. Small molecule-based targeting of mTOR might therefore represent a potential mechanism in ASPS alone or in combination with contemporary upstream approaches.

Cediranib for metastatic alveolar soft part sarcoma.

PURPOSE: Alveolar soft part sarcoma (ASPS) is a rare, highly vascular tumor, for which no effective standard systemic treatment exists for patients with unresectable disease. Cediranib is a potent, oral small-molecule inhibitor of all three vascular endothelial growth factor receptors (VEGFRs). PATIENTS AND METHODS: We conducted a phase II trial of once-daily cediranib (30 mg) given in 28-day cycles for patients with metastatic, unresectable ASPS to determine the objective response rate (ORR). We also compared gene expression profiles in pre- and post-treatment tumor biopsies and evaluated the effect of cediranib on tumor proliferation and angiogenesis using positron emission tomography and dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Of 46 patients enrolled, 43 were evaluable for response at the time of analysis. The ORR was 35%, with 15 of 43 patients achieving a partial response. Twenty-six patients (60%) had stable disease as the best response, with a disease control rate (partial response + stable disease) at 24 weeks of 84%. Microarray analysis with validation by quantitative real-time polymerase chain reaction on paired tumor biopsies from eight patients demonstrated downregulation of genes related to vasculogenesis. CONCLUSION: In this largest prospective trial to date of systemic therapy for metastatic ASPS, we observed that cediranib has substantial single-agent activity, producing an ORR of 35% and a disease control rate of 84% at 24 weeks. On the basis of these results, an open-label, multicenter, randomized phase II registration trial is currently being conducted for patients with metastatic ASPS comparing cediranib with another VEGFR inhibitor, sunitinib.

A broad survey of cathepsin K immunoreactivity in human neoplasms.

Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.

Combining integrated genomics and functional genomics to dissect the biology of a cancer-associated, aberrant transcription factor, the ASPSCR1-TFE3 fusion oncoprotein.

Oncogenic rearrangements of the TFE3 transcription factor gene are found in two distinct human cancers. These include ASPSCR1-TFE3 in all cases of alveolar soft part sarcoma (ASPS) and ASPSCR1-TFE3, PRCC-TFE3, SFPQ-TFE3 and others in a subset of paediatric and adult RCCs. Here we examined the functional properties of the ASPSCR1-TFE3 fusion oncoprotein, defined its target promoters on a genome-wide basis and performed a high-throughput RNA interference screen to identify which of its transcriptional targets contribute to cancer cell proliferation. We first confirmed that ASPSCR1-TFE3 has a predominantly nuclear localization and functions as a stronger transactivator than native TFE3. Genome-wide location analysis performed on the FU-UR-1 cell line, which expresses endogenous ASPSCR1-TFE3, identified 2193 genes bound by ASPSCR1-TFE3. Integration of these data with expression profiles of ASPS tumour samples and inducible cell lines expressing ASPSCR1-TFE3 defined a subset of 332 genes as putative up-regulated direct targets of ASPSCR1-TFE3, including MET (a previously known target gene) and 64 genes as down-regulated targets of ASPSCR1-TFE3. As validation of this approach to identify genuine ASPSCR1-TFE3 target genes, two up-regulated genes bound by ASPSCR1-TFE3, CYP17A1 and UPP1, were shown by multiple lines of evidence to be direct, endogenous targets of transactivation by ASPSCR1-TFE3. As the results indicated that ASPSCR1-TFE3 functions predominantly as a strong transcriptional activator, we hypothesized that a subset of its up-regulated direct targets mediate its oncogenic properties. We therefore chose 130 of these up-regulated direct target genes to study in high-throughput RNAi screens, using FU-UR-1 cells. In addition to MET, we provide evidence that 11 other ASPSCR1-TFE3 target genes contribute to the growth of ASPSCR1-TFE3-positive cells. Our data suggest new therapeutic possibilities for cancers driven by TFE3 fusions. More generally, this work establishes a combined integrated genomics/functional genomics strategy to dissect the biology of oncogenic, chimeric transcription factors.