Fernanda Amary

Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non‐neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U‐CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence‐like phenotype. The U‐CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]

Frequent mutation of the major cartilage collagen gene COL2A1 in chondrosarcoma

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H3f3a (histone 3.3) G34w Immunohistochemistry: A Reliable Marker Defining Benign and Malignant Giant Cell Tumor of Bone

mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro. Copyright

EGFR inhibitors identified as a potential treatment for chordoma in a focused compound screen

Chondrosarcoma is a heterogeneous collection of malignant bone tumors and is the second most common primary malignancy of bone after osteosarcoma. Recent work has identified frequent, recurrent mutations in IDH1 or IDH2 in nearly half of central chondrosarcomas. However, there has been little systematic genomic analysis of this tumor type, and, thus, the contribution of other genes is unclear. Here we report comprehensive genomic analyses of 49 individuals with chondrosarcoma (cases). We identified hypermutability of the major cartilage collagen gene COL2A1, with insertions, deletions and rearrangements identified in 37% of cases. The patterns of mutation were consistent with selection for variants likely to impair normal collagen biosynthesis. In addition, we identified mutations in IDH1 or IDH2 (59%), TP53 (20%), the RB1 pathway (33%) and Hedgehog signaling (18%).

In-depth genetic analysis of sclerosing epithelioid fibrosarcoma reveals recurrent genomic alterations and potential treatment targets

Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component.

The Chemical Form of Metal Species Released from Corroded Taper Junctions of Hip Implants: Synchrotron Analysis of Patient Tissue

Chordoma is a rare malignant bone tumour with a poor prognosis and limited therapeutic options. We undertook a focused compound screen (FCS) against 1097 compounds on three well‐characterized chordoma cell lines; 154 compounds were selected from the single concentration screen (1 µm), based on their growth‐inhibitory effect. Their half‐maximal effective concentration (EC50) values were determined in chordoma cells and normal fibroblasts. Twenty‐seven of these compounds displayed chordoma selective cell kill and 21/27 (78%) were found to be EGFR/ERBB family inhibitors. EGFR inhibitors in clinical development were then studied on an extended cell line panel of seven chordoma cell lines, four of which were sensitive to EGFR inhibition. Sapitinib (AstraZeneca) emerged as the lead compound, followed by gefitinib (AstraZeneca) and erlotinib (Roche/Genentech). The compounds were shown to induce apoptosis in the sensitive cell lines and suppressed phospho‐EGFR and its downstream pathways in a dose‐dependent manner. Analysis of substituent patterns suggested that EGFR‐inhibitors with small aniline substituents in the 4‐position of the quinazoline ring were more effective than inhibitors with large substituents in that position. Sapitinib showed significantly reduced tumour growth in two xenograft mouse models (U‐CH1 xenograft and a patient‐derived xenograft, SF8894). One of the resistant cell lines (U‐CH2) was shown to express high levels of phospho‐MET, a known bypass signalling pathway to EGFR. Neither amplifications (EGFR, ERBB2, MET) nor mutations in EGFR, ERBB2, ERBB4, PIK3CA, BRAF, NRAS, KRAS, PTEN, MET or other cancer gene hotspots were detected in the cell lines. Our findings are consistent with the reported (p‐)EGFR expression in the majority of clinical samples, and provide evidence for exploring the efficacy of EGFR inhibitors in the treatment of patients with chordoma and studying possible resistance mechanisms to these compounds in vitro and in vivo.

Digital PCR analysis of circulating tumor DNA: a biomarker for chondrosarcoma diagnosis, prognostication, and residual disease detection

Purpose: Sclerosing epithelioid fibrosarcoma (SEF) is a highly aggressive soft tissue sarcoma closely related to low-grade fibromyxoid sarcoma (LGFMS). Some tumors display morphologic characteristics of both SEF and LGFMS, hence they are known as hybrid SEF/LGFMS. Despite the overlap of gene fusion variants between these two tumor types, SEF is much more aggressive. The current study aimed to further characterize SEF and hybrid SEF/LGFMS genetically to better understand the role of the characteristic fusion genes and possible additional genetic alterations in tumorigenesis. Experimental Design: We performed whole-exome sequencing, SNP array analysis, RNA sequencing (RNA-seq), global gene expression analyses, and/or IHC on a series of 13 SEFs and 6 hybrid SEF/LGFMS. We also expressed the FUS-CREB3L2 and EWSR1-CREB3L1 fusion genes conditionally in a fibroblast cell line; these cells were subsequently analyzed by RNA-seq, and expression of the CD24 protein was assessed by FACS analysis. Results: The SNP array analysis detected a large number of structural aberrations in SEF and SEF/LGFMS, many of which were recurrent, notably DMD microdeletions. RNA-seq identified FUS-CREM and PAX5-CREB3L1 as alternative fusion genes in one SEF each. CD24 was strongly upregulated, presumably a direct target of the fusion proteins. This was further confirmed by the gene expression analysis and FACS analysis on Tet-On 3G cells expressing EWSR1-CREB3L1. Conclusions: Although gene fusions are the primary tumorigenic events in both SEF and LGFMS, additional genomic changes explain the differences in aggressiveness and clinical outcome between the two types. CD24 and DMD constitute potential therapeutic targets. Clin Cancer Res; 23(23); 7426–34. ©2017 AACR.

P63 does not regulate brachyury expression in human chordomas and osteosarcomas

The mechanisms of metal release from the articulation at the head cup bearing and the tapered junctions of orthopaedic hip implants are known to differ and the debris generated varies in size, shape and volume. Significantly less metal is lost from the taper junction between Cobalt-Chromium-Molybdenum (CoCrMo) and Titanium (Ti) components (fretting-corrosion dominant mechanism), when compared to the CoCrMo bearing surfaces (wear-corrosion dominant mechanism). Corrosion particles from the taper junction can lead to Adverse Reactions to Metal Debris (ARMD) similar to those seen with CoCrMo bearings. We used synchrotron methods to understand the modes underlying clinically significant tissue reactions to Co, Cr and Ti by analysing viable peri-prosthetic tissue. Cr was present as Cr2O3 in the corroded group in addition to CrPO4 found in the metal-on-metal (MoM) group. Interestingly, Ti was present as TiO2 in an amorphous rather than rutile or anatase physical form. The metal species were co-localized in the same micron-scale particles as result of corrosion processes and in one cell type, the phagocytes. This work gives new insights into the degradation products from metal devices as well as guidance for toxicological studies in humans.

Recurrent rearrangements of FOS and FOSB define osteoblastoma

Conventional chondrosarcoma is the most common primary bone tumor in adults. Prognosis corresponds with tumor grade but remains variable, especially for individuals with grade (G) II disease. There are currently no biomarkers available for monitoring or prognostication of chondrosarcoma. Circulating tumor DNA (ctDNA) has recently emerged as a promising biomarker for a broad range of tumor types. To date, little has been done to study the presence of ctDNA and its potential utility in the management of sarcomas, including chondrosarcoma. In this study, we have assessed ctDNA levels in a cohort of 71 patients, 32 with sarcoma, including 29 individuals with central chondrosarcoma (CS) and 39 with locally aggressive and benign bone and soft tissue tumors, using digital PCR. In patients with CS, ctDNA was detected in pretreatment samples in 14/29 patients, which showed clear correlation with tumor grade as demonstrated by the detection of ctDNA in all patients with GIII and dedifferentiated disease (n = 6) and in 8/17 patients with GII disease, but never associated with GI CS. Notably detection of ctDNA preoperatively in GII disease was associated with a poor outcome. A total of 14 patients with CS had ctDNA levels assessed at multiple time points and in most patients there was a clear reduction following surgical removal. This research lays the foundation for larger studies to assess the utility of ctDNA for chondrosarcoma diagnosis, prognostication, early detection of residual disease and monitoring disease progression.

Meningioma metastasis to the bone — review of a rare finding with emphasis on distribution and imaging appearances

In case 2, at low magnification the overall configuration of the gland was disrupted, but on higher magnification it was clear that this was due to a foreign body-type reaction to the keratin liberated from Hassall’s corpuscles (Figure 1A). Another finding pointing to a reactive process was the presence of marked cytological atypia in the absence of mitotic activity. Moreover, as radiation injury affects also the stroma and vessels, it can be useful to look for radiation-induced changes in these components. Obviously, awareness of a previous history of mediastinal radiation is an important clue for the correct diagnosis. The mediastinum is a frequent site of radiotherapy because of the variety of neoplasms that can involve the organs located within and nearby. Moreover, the ever-increasing number of cardiac operations being carried out nowadays (with excision of the pre-aortic fat and its subsequent microscopic examination) is bound to increase the number of incidental lesions of the type here reported. In summary, radiation-induced atypia of vestigial thymic epithelium can occur many years after the exposure, and can be so pronounced as to simulate a malignant process. The purpose of this short communication is to document this occurrence and to underscore the importance of identifying this lesion, as the two cases we came across resulted in considerable diagnostic challenge.