Alveolar Soft Part Sarcoma Tumor Research Articles

ASPSCR1::TFE3 Drives Alveolar Soft Part Sarcoma by Inducing Targetable Transcriptional Programs.

Alveolar soft part sarcoma (ASPS) is a rare mesenchymal malignancy driven by the ASPSCR1::TFE3 fusion. A better understanding of the mechanisms by which this oncogenic transcriptional regulator drives cancer growth is needed to help identify potential therapeutic targets. In this study, we characterized the transcriptional and chromatin landscapes of ASPS tumors and preclinical models, identifying the essential role of ASPSCR1::TFE3 in tumor cell viability by regulating core transcriptional programs involved in cell proliferation, angiogenesis, and mitochondrial biology. ASPSCR1::TFE3 directly interacted with key epigenetic regulators at enhancers and promoters to support ASPS-associated transcription. Among the effector programs driven by ASPSCR1::TFE3, cell proliferation was driven by high levels of cyclin D1 expression. Disruption of cyclin D1/CDK4 signaling led to a loss of ASPS proliferative capacity, and combined inhibition of CDK4/6 and angiogenesis halted tumor growth in xenografts. These results define the ASPS oncogenic program, reveal mechanisms by which ASPSCR1::TFE3 controls tumor biology, and identify a strategy for therapeutically targeting tumor cell-intrinsic vulnerabilities. Significance: The ASPSCR1::TFE3 fusion propels the growth of alveolar soft part sarcoma by activating transcriptional programs that regulate proliferation, angiogenesis, mitochondrial biogenesis, and differentiation and can be therapeutically targeted to improve treatment.

Tivantinib (ARQ 197), a selective inhibitor of MET, in patients with microphthalmia transcription factor–associated tumors

Microphthalmia transcription factor (MITF)-associated (MiT) tumors are a family of rare malignancies, including alveolar soft part sarcoma (ASPS), clear cell sarcoma (CCS), and translocation-associated renal cell carcinoma (tRCC) that have dysregulated expression of oncogenic MITF family proteins. The MET receptor tyrosine kinase gene is transcriptionally activated by MITF family proteins, making MET a potential therapeutic target for MiT tumors. This study assessed the activity of tivantinib (ARQ 197), a selective MET inhibitor, in patients with MiT-associated tumors. This multicenter, single-arm, phase 2 trial enrolled patients with advanced MiT tumors. Patients initially received tivantinib 120 mg orally twice daily, then 360 mg twice daily per protocol amendment. The primary endpoint was overall response rate. Secondary endpoints included safety, progression-free survival, pharmacokinetics, and correlative studies. A total of 47 patients (median age, 25 years; range, 11-73 years) with ASPS (n = 27), CCS (n = 11), tRCC (n = 6), or other tumor types (n = 3) were enrolled. Common grade 3/4 drug-related adverse events included anemia (4%) and neutropenia (4%). Three patients (6.4%) experienced 4 treatment-related serious adverse events (grade 3 febrile neutropenia, thrombocytopenia, and deep vein thrombosis, and grade 4 thrombocytopenia). Best response was partial response in 1 CCS patient (2%) and stable disease in 28 patients (60%). Median progression-free survival was 3.6 months (overall), 5.5 months (ASPS), and 1.9 months (CCS and tRCC). Baseline MET expression was strongly or focally positive in tumor samples from 14 of 19 patients (74%). Tivantinib was safe and tolerable in patients with MiT tumors, but antitumor activity was modest.

Technique for differentiating alveolar soft part sarcoma from other tumors in paraffin-embedded tissue: comparison of immunohistochemistry for TFE3 and CD147 and of reverse transcription polymerase chain reaction for ASPSCR1-TFE3 fusion transcript

The diagnosis of alveolar soft part sarcoma is commonly based on characteristic histology and distinctive periodic acid-Schiff-positive crystals; however, the characteristic crystals may not always be observed, rendering the diagnosis difficult. Three important characteristics of alveolar soft part sarcoma, the presence of ASPSCR1-TFE3 fusion transcript, nuclear immunoreactivity for TFE3, and immunoreactivity for monocarboxylate transporter 1 and CD147, have recently been reported. To identify the best marker for alveolar soft part sarcoma in formalin-fixed, paraffin-embedded tissues, we evaluated the sensitivity and specificity of the detection of the ASPSCR1-TFE3 fusion transcript along with the immunoreactivity for TFE3 and CD147 in 24 alveolar soft part sarcomas and 23 non-alveolar soft part sarcoma tumors, including 5 granular cell tumors, 5 paragangliomas, 3 clear cell sarcomas, and 10 clear cell renal cell carcinomas. The ASPSCR1-TFE3 fusion transcript was detected in 24 of 24 alveolar soft part sarcomas (7 type 1, 17 type 2), and TFE3 immunoreactivity was observed in 22 of 24 alveolar soft part sarcomas. In non-alveolar soft part sarcoma tumors, the ASPSCR1-TFE3 fusion transcript was not detected; however, the TFE3 immunoreactivity was observed in 2 of 5 granular cell tumors. CD147 immunoreactivity was demonstrated in 20 of 24 alveolar soft part sarcomas, 3 of 5 granular cell tumors, and 8 of 10 clear cell renal cell carcinomas. Our results demonstrate that the most sensitive marker of alveolar soft part sarcoma was the presence of the ASPSCR1-TFE3 fusion transcript. Thus, detection of the ASPSCR1-TFE3 fusion transcript was considered applicable for formalin-fixed, paraffin-embedded tissues with superior sensitivity as compared with TFE3 immunohistochemical staining. In alveolar soft part sarcomas with unusual locations or histology, we consider that the detection of the ASPSCR1-TFE3 fusion transcript would be the highly effective diagnostic technique.

ASPS-1, A Novel Cell Line Manifesting Key Features of Alveolar Soft Part Sarcoma

In vitro growth of alveolar soft part sarcoma (ASPS) and establishment of an ASPS cell line, ASPS-1, are described in this study. Using a recently developed xenograft model of ASPS derived from a lymph node metastasis, organoid nests consisting of 15 to 25 ASPS cells were isolated from ASPS xenograft tumors by capture on 70 μm filters and plated in vitro. After attachment to the substratum, these nests deposited small aggregates of ASPS cells. These cells grew slowly and were expanded over a period of 3 years and have maintained characteristics consistent with those of both the original ASPS tumor from the patient and the xenograft tumor including (1) presence of the alveolar soft part locus-transcription factor E3 type 1 fusion transcript and nuclear expression of the alveolar soft part locus-transcription factor E3 type 1 fusion protein; (2) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS; and (3) expression of upregulated ASPS transcripts involved in angiogenesis (ANGPTL2, HIF-1-α, MDK, c-MET, VEGF, and TIMP-2), cell proliferation (PRL, PCSK1), metastasis (ADAM9), as well as the transcription factor BHLHB3 and the muscle-specific transcripts TRIM63 and ITGβ1BP3. This ASPS cell line forms colonies in soft agar and retains the ability to produce highly vascularized ASPS tumors in NOD.SCID/NCr mice. Immunohistochemistry of selected ASPS markers on these tumors indicated similarity to those of the original patient tumor as well as to the xenografted ASPS tumor. We anticipate that this ASPS cell line will accelerate investigations into the biology of ASPS including identification of new therapeutic approaches for treatment of this slow growing soft tissue sarcoma.

Abstract 1621: The proprotein convertase PCSK1 is a novel drug target in alveolar soft part sarcoma (ASPS)

Abstract Proprotein convertases (PCs) are a family of calcium-dependent serine endoproteases which mediate the proteolytic cleavage, at pairs of dibasic amino acids, of many precursor proteins, including growth factors and pro-hormones, into their functionally active forms. One family member, PCSK1, was identified as a potential therapeutic target in ASPS. This was based on gene expression profiling of 4 primary and 3 metastatic ASPS tumors (BMC Cancer 9:22, 2009). Affymetrix 133 Plus 2 arrays revealed that PCSK1, but not PCSK2 or furin, was significantly up-regulated (range 18.6 to 131 fold) relative to universal RNA, a multi-organ pool of non-tumor RNA's. PCSK1 expression was confirmed by immunohistochemistry and Western Blot analysis in patient tumors, in an ASPS xenograft model, and in a cell line developed from the xenograft tumor (ASPS-1; J Pediatr. Hematol. Oncol., in press 2010) as well as in tumors produced by ASPS-1 in immunocompromised mice. ASPS-1 was utilized, in-vitro, to evaluate a panel of PCSK1 siRNA's, the most potent of these produced a 75% reduction in the PCSK1 RNA transcript, reduced expression of PCSK1 protein and produced a 50% reduction in ASPS-1 growth. As part of an effort to develop new therapeutic approaches toward the treatment of ASPS we have implemented a high-throughput screening effort to identify small molecule inhibitors of PCSK1. Utilizing recombinant PCSK1 and the fluorescent substrate (Pyr-Arg-Thr-Lys-Arg-MCA) a high-throughput screen of a small molecule library (∼15 K compounds) was initiated. Results of a pilot screen of ∼2400 compounds at concentrations ranging from 0.1 to 100 uM yielded several inhibitors of PCSK1 which were retested in a 20 concentration assay to confirm activity. Preliminary analysis of structure-activity relationships identified several active inhibitor chemotypes, comparable in potency to the known peptide PCSK1 inhibitor, Ac-Leu-Leu-Arg-Val-Lys-Arg-NH2, containing aryl groups linked by aliphatic chains containing peptide bonds. Such compounds may act as substrate mimetic inhibitors and will be further evaluated for inhibition of growth of ASPS-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1621. doi:10.1158/1538-7445.AM2011-1621

Therapeutic Vulnerability of an In Vivo Model of Alveolar Soft Part Sarcoma (ASPS) to Antiangiogenic Therapy

In vivo growth of alveolar soft part sarcoma (ASPS) was achieved using subcutaneous xenografts in sex-matched nonobese diabetic severe combined immunodeficiency mice. One tumor, currently at passage 6, has been maintained in vivo for 32 months and has maintained characteristics consistent with those of the original ASPS tumor including (1) tumor histology and staining with periodic acid Schiff/diastase, (2) the presence of the ASPL-TFE3 type 1 fusion transcript, (3) nuclear staining with antibodies to the ASPL-TFE3 type 1 fusion protein, (4) maintenance of the t(X;17)(p11;q25) translocation characteristic of ASPS, (5) stable expression of signature ASPS gene transcripts and finally, the development and maintenance of a functional vascular network, a hallmark of ASPS. The ASPS xenograft tumor vasculature encompassing nests of ASPS cells is highly reactive to antibodies against the endothelial antigen CD34 and is readily accessible to intravenously administered fluorescein isothiocyanate-dextran. The therapeutic vulnerability of this tumor model to antiangiogenic therapy, targeting vascular endothelial growth factor and hypoxia-inducible factor-1 alpha, was examined using bevacizumab and topotecan alone and in combination. Together, the 2 drugs produced a 70% growth delay accompanied by a 0.7 net log cell kill that was superior to the antitumor effect produced by either drug alone. In summary, this study describes a preclinical in vivo model for ASPS which will facilitate investigation into the biology of this slow growing soft tissue sarcoma and demonstrates the feasibility of using an antiangiogenic approach in the treatment of ASPS.

Molecular analyses of cell origin and detection of circulating tumor cells in the peripheral blood in alveolar soft part sarcoma

Alveolar soft part sarcoma (ASPS) is a distinct, rare soft tissue tumor with an unknown histogenesis and a tendency for late widespread metastases to lung, bone, and brain. It is now clear that they are caused by a specific unbalanced translocation, der(17)t(X;17)(p11;q25), which results in the formation of an ASPSCR1–TFE3 (alias ASPL–TFE3) fusion gene;. The rearrangement results in the expression of chimeric transcripts, which can be identified by means of reverse transcriptase–polymerase chain reaction (RT-PCR). We investigated the histogenesis of ASPS and attempted to detect circulating ASPS tumor cells in peripheral blood. The immunohistochemical and genetic details of four cases and one cell line of ASPS were examined. An immunohistochemical analysis and RT-PCR did not detect myogenic differentiation gene MYOD1. The sensitivity of nested RT-PCR for detection of circulating ASPS cells was assessed by demonstrating that the tumor cell–associated gene translocation could be detected in 50 tumor cells/2 mL of blood. Clinically, it was detectable in a peripheral blood sample (2 mL) of ASPS patient with distant metastases. The findings suggest that ASPS is not of skeletal muscle origin. ASPS tumor cells in the peripheral blood could be monitored by RT-PCR.

Alveolar Soft Part Sarcoma: A Bimarker Diagnostic Strategy Using TFE3 Immunoassay and ASPL-TFE3 Fusion Transcripts in Paraffin-Embedded Tumor Tissues

Alveolar soft part sarcoma (ASPS) is a malignancy with low incidence, but with poor prognosis if misdiagnosed. Immunohistochemical assay using TFE3 antibody has been shown to be a sensitive technique for ASPS diagnosis. A specific chromosomal translocation, t(X;17)(p11.2;q25), results in the ASPL-TFE3 fusion gene: it is detectable using reverse-transcription polymerase chain reaction (RT-PCR) in frozen tumor tissues of ASPS. However, the diagnostic usefulness of these markers has not been investigated in Chinese ASPS patients. Here, we report the first systematic study applying TFE3 immunoassay and ASPL-TFE3 fusion transcript detection to archival paraffin-embedded tissues in a large Chinese ASPS patient population. Sixteen patients had been diagnosed with ASPS (age, 3 to 58 y; 3 male patients and 13 female patients). Their tumors presented predominantly in the extremities (8/16), and were often located in the region of the orbit when affecting infants and children (3/16). Others had tumors in the chest wall, breast, and right pubis, respectively. One patient exhibited a tumor in the renal hilum, a location that had not been previously reported. Two patients had tumor metastases in the lung and the brain. ASPS tumors showed the best immunoreactivity to the TFE3 antibody (16/16). However, their immunoreactivity to other antibodies, including myoglobin (13/16), actin (10/16), desmin (2/16), and vimentin (2/16), was of various degrees. Positive staining was observed for the neural markers, NSE (9/16) and CgA (7/16), respectively. Using a strategy of RT-PCR, followed by a nested PCR with a different primer set, we were able to detect the expression of the chimeric ASPL-TFE3 mRNA in 11 of the 16 ASPS tumors. Of these 11, 7 were type 1 ASPL-TFE3 and 4 were type 2 ASPL-TFE3, including the tumor located in the renal hilum. No expression of ASPL-TFE3 fusion transcripts was detectable in all 38 control tumors. Our results demonstrate that the "bimarker strategy," a combination of TFE3 immunostaining and ASPL-TFE3 chimeric transcript detection, might have sufficient sensitivity and specificity in diagnosing most of the ASPSs. As both diagnostic techniques can be applied to widely available archival paraffin-embedded tissues, the usefulness of the strategy is largely implicated in routine pathology laboratories.

Immunohistochemical Discrimination Between the ASPL-TFE3 Fusion Proteins of Alveolar Soft Part Sarcoma

Alveolar soft part sarcoma (ASPS), a rare soft tissue sarcoma, is characterized by a chromosomal translocation der(17)t(X;17)(p11;q25) resulting in the production of 2 fusion proteins encoded by regions of the genes for alveolar soft part locus (ASPL) and the transcription factor E3 (TFE3). In this study, polyclonal antibodies were generated to 25 mer peptides encompassing the junctional regions of ASPL-TFE3 type 1 and ASPL-TFE3 type 2. The specificity of the affinity purified antibodies for the synthetic peptides and recombinant expressed ASPL-TFE3 type 1 and ASPL-TFE3 type 2 proteins was evaluated by enzyme-linked immunosorbent assay and was highly fusion type specific. Immunohistochemical staining of formalin-fixed, paraffin-embedded ASPS tumors with the fusion-specific antibodies resulted in intense nuclear staining and differentiation between tumors that express the type 1 protein and tumors that express the type 2 protein. These antibodies will be useful for the differential diagnosis of type 1 and type 2 ASPS and also in the detection of the fusion proteins in biochemical and cell biologic investigations.

Angiogenesis-Promoting Gene Patterns in Alveolar Soft Part Sarcoma

We examined a cohort of patients with alveolar soft part sarcoma (ASPS) treated at our institution and showed the characteristic ASPSCR1-TFE3 fusion transcript in their tumors. Investigation of potential angiogenesis-modulating molecular determinants provided mechanistic and potentially therapeutically relevant insight into the enhanced vascularity characteristic of this unusual tumor. Medical records of 71 patients with ASPS presenting at the University of Texas M.D. Anderson Cancer Center (1986-2005) were reviewed to isolate 33 patients with formalin-fixed paraffin-embedded material available for study. RNA extracted from available fresh-frozen and formalin-fixed paraffin-embedded human ASPS tumors were analyzed for ASPSCR1-TFE3 fusion transcript expression using reverse transcription-PCR and by angiogenesis oligomicroarrays with immunohistochemical confirmation. Similar to previous studies, actuarial 5- and 10-year survival rates were 74% and 51%, respectively, despite frequent metastasis. ASPSCR1-TFE3 fusion transcripts were identified in 16 of 18 ASPS samples. In the three frozen samples subjected to an angiogenesis oligoarray, 18 angiogenesis-related genes were up-regulated in tumor over adjacent normal tissue. Immunohistochemistry for jag-1, midkine, and angiogenin in 33 human ASPS samples confirmed these results. Comparison with other sarcomas indicates that the ASPS angiogenic signature is unique. ASPS is a highly vascular and metastatic tumor with a surprisingly favorable outcome; therapeutically resistant metastases drive mortality. Future molecular therapies targeting overexpressed angiogenesis-promoting proteins (such as those identified here) could benefit patients with ASPS.