Development of curative therapies for Ewing sarcomas by interdisciplinary cooperative groups in Europe.

Curative therapies for Ewing sarcoma have been developed within cooperative groups. Consecutive clinical trials have systematically assessed the impact and timing of local therapy and the activity of cytotoxic drugs and their combinations. They have led to an increase of long-term disease-free survival to around 70% in patients with localized disease. Translational research in ES remains an area in which interdisciplinary and international cooperation is essential for future progress. This article reviews current state-of-the art therapy, with a focus on trials performed in Europe, and summarizes novel strategies to further advance both the cure rates and quality of survival.

An orbital perivascular epithelioid cell tumor (PEComa) in a 9-year-old boy: Case report and review of the literature.

Perivascular epithelioid cell tumors (PEComas) are a family of benign neoplasms characterized by smooth muscle and melanocytic differentiation. Orbital cases are rare. A 9-year-old male presented with a slowly growing orbital mass. Magnetic resonance imaging (MRI) revealed a well-defined orbital mass without intracranial extension. The microscopic appearance of the complete resection specimen showed large nests of epithelioid cells with wide cytoplasm containing melanin pigment and round to oval nuclei with mild cytonuclear atypia and low mitotic activity. Immunohistochemistry was positive for HMB45 and negative for melanA, smooth muscle actin, desmin and S-100 protein. Pangenomic RNA-sequencing identified an in-frame NONO-TFE3 rearrangement, and clustering data showed that the tumor's gene expression profile was grouped with other previously studied PEComas. A diagnosis of orbital pigmented PEComa with uncertain malignant potential associated with a NONO-TFE3 rearrangement was made. There was no recurrence after 1 year of follow-up.

Natural history and treatment efficacy in an ambispective case series of NTRK-rearranged mesenchymal tumors.

BACKGROUND: Apart for infantile fibrosarcoma (IFS), very little is known about NTRK-rearranged mesenchymal tumors (NMTs). The objective of this study is to describe the distribution, characteristics, natural history, and prognosis of NMT. PATIENTS AND METHODS: This study was carried out as a translational research program, retrospectively from a cohort of 500 soft tissue sarcoma (STS; excluding IFS) and prospectively both in routine practice and from the RNASARC molecular screening program (N = 188; NCT03375437). RESULTS: Using RNA-sequencing, NTRK fusion was detected in 16 patient tumors diagnosed as STS: 8 samples of sarcoma with simple genomics (4 NTRK-rearranged spindle cell neoplasm, 3 ALK/ROS wild-type inflammatory myofibroblastic tumor, and 1 quadruple Wild-type gastrointestinal stromal tumor) and 8 samples of sarcomas with complex genomics (dedifferentiated liposarcoma, intimal sarcoma, leiomyosarcoma, undifferentiated pleomorphic sarcoma, high-grade uterine sarcoma, malignant peripheral nerve sheath tumor). Among the eight patients with simple genomics, four were treated with tyrosine receptor kinase inhibitor (TRKi) at different stages of the disease and all benefited from the treatment, including one complete response. Among the eight other patients, six evolved with metastatic spreading and the median metastatic survival was 21.9 months, as classically reported in these tumor types. Two of them received a first-generation TRKi without objective response. CONCLUSIONS: Our study confirms low frequency and histotype diversity of NTRK fusion in STS. While the activity of TRKi in simple genomics NMT is confirmed, our clinical data encourage subsequent studies focusing on the biological relevance of NTRK fusions in sarcomas with complex genomics together with the efficacy of TRKi in this population.

Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7.

To understand the initiation of the transcription of protein-coding genes, we have dissected the role of the basal transcription/DNA repair factor TFIIH. Having succeeded in reconstituting a functionally active TFIIH from baculovirus recombinant polypeptides, we were able to analyze the role of XPB, XPD, and cdk7 subunits in the transcription reaction. Designing mutated recombinant subunits, we show that the XPB helicase is absolutely required for transcription to open the promoter around the start site whereas the XPD helicase, which is dispensable, stimulates transcription and allows the CAK complex to be anchored to TFIIH. In addition, we also show that cdk7 may phosphorylate the carboxy-terminal domain (CTD) of RNA pol II in the absence of promoter opening.

A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three-hybrid system.

We describe a three-hybrid system that involves three polypeptides that allow or prevent the formation of the transcriptional activator. Beside the two-hybrid fusion proteins, the third partner is under the control of the Met25 promoter, which is positively regulated in medium lacking methionine. We document a situation where such a third partner promotes interaction between two proteins, one fused to a DNA-binding domain and the other fused to an activator domain. This is demonstrated for cdk7-MAT1 interaction stabilized by the presence of cyclin H; these three polypeptides are found either free or associated with the transcription/DNA repair factor TFIIH. We also document the capacity of our system to conditionally inhibit the interaction between two polypeptides that otherwise elicit a positive two-hybrid response. This is demonstrated for Ras-Raf interaction precluded by an excess of Raf. The presence of a methionine-regulated promoter provides an "on" or "off" switch for the formation of the transcriptional activator, thus also providing an excellent control to evaluate the activation or inhibition properties of the third partner.

A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II.

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.

Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK.

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

Bisphosphonate effects in rat unloaded hindlimb bone loss model: three-dimensional microcomputed tomographic, histomorphometric, and densitometric analyses.

The effects of antiresorptive drugs on bone loss remain unclear. Using three-dimensional microtomography, dual X-ray/densitometry, and histomorphometry, we evaluated tiludronate effects in the bone loss model of immobilization in tail-suspended rats after 7, 13, and 23 days. Seventy-eight 12-week-old Wistar male rats were assigned to 13 groups: 1 baseline group, and for each time point, 1 control group treated with vehicle and three tail-suspended groups treated with either tiludronate (0.5 or 5 mg/kg) or vehicle, administered s. c. every other day, during the last week before sacrifice. In primary spongiosa (ISP), immobilization-induced bone loss plateaued after day 7 and was prevented by tiludronate. In secondary spongiosa (IISP), bone loss appeared at day 13 with a decrease in trabecular thickness and trabecular number (Tb.N) as assessed by three-dimensional microtomography. Osteoclastic parameters did not differ in tail-suspended rats versus control rats, whereas bone formation showed a biphasic pattern: after a marked decrease at day 7, osteoblastic activity and recruitment normalized at days 13 and 23, respectively. At day 23, the 80% decrease in bone mass was fully prevented by high-dose tiludronate with an increase in Tb.N without preventing trabecular thinning. In summary, at day 7, tiludronate prevented bone loss in ISP. After day 13, tiludronate prevented bone loss in ISP and IISP despite a further decrease in bone formation. Thus, the preventive effects of tiludronate in this model may be related to the alteration in bone modeling with an increase in Tb.N in ISP and subsequently in IISP.

The visualization and evaluation of bone architecture in the rat using three-dimensional X-ray microcomputed tomography.

Microcomputed tomography allows the true three-dimensional structure of bone to be assessed by a nondestructive analysis. This article describes how this technique has for the first time been applied to rat bone to determine the effects of aging, ovariectomy, and antiresorptive drugs on bone structure and how these results compare with those determined by histological and histomorphometric techniques. During the procedure, a micro X-ray source is directed toward the bone sample. Modifications in the X-ray beam induced by bone crystals are determined for a range of acquisitions before three-dimensional reconstruction of bone architecture is performed. Morphometric parameters determined were trabecular bone volume/tissue volume, trabecular number, and trabecular thickness. The results show that ovariectomy has a dramatic effect on rat bone structure. Following treatment with the bone resorption inhibitor tiludronate, the morphometric parameters were significantly improved. The results obtained with three-dimensional microcomputed tomography were in agreement with observations made using classical techniques. Microcomputed tomography should prove useful for evaluating the antiresorptive effects of bisphosphonates on bone architecture and in allowing between-drug comparisons.