Robert E. Turcotte

Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape

An oncohistone deranges inhibitory chromatin Missense mutations (that change one amino acid for another) in histone H3 can produce a so-called oncohistone and are found in a number of pediatric cancers. For example, the lysine-36–to-methionine (K36M) mutation is seen in almost all chondroblastomas. Lu et al. show that K36M mutant histones are oncogenic, and they inhibit the normal methylation of this same residue in wild-type H3 histones. The mutant histones also interfere with the normal development of bone-related cells and the deposition of inhibitory chromatin marks. Science, this issue p. 844 The lysine-36–to–methionine mutation in histone H3 is oncogenic and interferes with inhibitory chromatin marks. Several types of pediatric cancers reportedly contain high-frequency missense mutations in histone H3, yet the underlying oncogenic mechanism remains poorly characterized. Here we report that the H3 lysine 36–to–methionine (H3K36M) mutation impairs the differentiation of mesenchymal progenitor cells and generates undifferentiated sarcoma in vivo. H3K36M mutant nucleosomes inhibit the enzymatic activities of several H3K36 methyltransferases. Depleting H3K36 methyltransferases, or expressing an H3K36I mutant that similarly inhibits H3K36 methylation, is sufficient to phenocopy the H3K36M mutation. After the loss of H3K36 methylation, a genome-wide gain in H3K27 methylation leads to a redistribution of polycomb repressive complex 1 and de-repression of its target genes known to block mesenchymal differentiation. Our findings are mirrored in human undifferentiated sarcomas in which novel K36M/I mutations in H3.1 are identified.

Endothelin-1 (ET-1) promotes MMP-2 and MMP-9 induction involving the transcription factor NF-κB in human osteosarcoma

In the present study, we have investigated the effect of (i) ET-1 (endothelin-1) and its precursor, big ET-1, on MMP (matrix metalloproteinase)-2 and MMP-9 synthesis and activity in osteosarcoma tissue, and (ii) ET-1 receptor antagonists on cell invasion. Using Western blotting, zymography, RT-PCR (reverse transcription-PCR), immunohistochemistry, immunofluorescence and Northern blotting, we have shown that ET-1 and ET-1 receptors (ET(A) and ET(B)) were expressed in these cells. Additionally, we have demonstrated that ET-1 markedly induced the synthesis and activity of MMP-2, which was significantly increased when compared with MMP-9. Furthermore, inhibition of NF-kappaB (nuclear factor kappaB) activation blocked MMP-2 production and activity, indicating the involvement of NF-kappaB, a ubiquitous transcription factor playing a central role in the differentiation, proliferation and malignant transformation. Since ET-1 acts as an autocrine mediator through gelatinase induction and because inhibition of ET(A) receptor is beneficial for reducing both basal and ET-1-induced osteosarcoma cell invasion, targeting this receptor could be an attractive therapeutic alternative for the successful treatment of osteosarcoma.

Giant cell tumor of bone express p63

p63 contributes to skeletal development and tumor formation; however, little is known regarding its activity in the context of bone and soft tissue neoplasms. The purpose of this study was to investigate p63 expression in giant cell tumor of bone and to determine whether it can be used to discriminate between other giant cell-rich tumors. Seventeen cases of giant cell tumor of bone were examined to determine the cell type expressing p63 and identify the isoforms present. Total RNA or cell protein was extracted from mononuclear- or giant cell-enriched fractions or intact giant cell tumor of bone and examined by RT-PCR or western blot, respectively. Immunohistochemistry was used to evaluate p63 expression in paraffin embedded sections of giant cell tumor of bone and in tumors containing multinucleated giant cells, including: giant cell tumor of tendon sheath, pigmented villonodular synovitis, aneurysmal bone cyst, chondroblastoma, and central giant cell granuloma. The mononuclear cell component in all cases of giant cell tumor of bone was found to express all forms of TAp63 (α, β, and γ), whereas only low levels of the TAp63 α and β isoforms were detected in multinucleated cells; ΔNp63 was not detected in these tumors. Western blot analysis identified p63 protein as being predominately localized to mononuclear cells compared to giant cells. This was confirmed by immunohistochemical staining of paraffin-embedded tumor sections, with expression identified in all cases of giant cell tumor of bone. Only a proportion of cases of aneurysmal bone cyst and chondroblastoma showed p63 immunoreactivity whereas it was not detected in central giant cell granuloma, giant cell tumor of tendon sheath, or pigmented villonodular synovitis. The differential expression of p63 in giant cell tumor of bone and central giant cell granuloma suggest that these two tumors may have a different pathogenesis. Moreover, p63 may be a useful biomarker to differentiate giant cell tumor of bone from central giant cell granuloma and other giant cell-rich tumors, such as giant cell tumor of tendon sheath and pigmented villonodular synovitis.

Myxoid\Round Cell Liposarcoma (MRCLS) Revisited: An Analysis of 418 Primarily Managed Cases

BackgroundObjectives of this study were to evaluate oncologic outcomes and to provide guidelines for the management of primary myxoid (MLS) and round cell liposarcoma (RCLS).MethodsA multicenter, retrospective study of 418 cases of MRCLS primarily managed by Canadian multidisciplinary sarcoma teams.ResultsStudy included 418 cases (MLS: 311 patients and RCLS: 107;xa0>xa05% round cell) with a median age of 45xa0years and a median follow-up of 5.2xa0years. Median tumor size was 10xa0cm, and 81% were deep and 90% were in lower limb. The majority of patients underwent surgical resection and radiotherapy, with a small percentage (6%) receiving chemotherapy. The overall 10-year local control rate was 93% with no differences between MLS and RCLS. Radiotherapy was significant in preventing local relapse and reducing tumor diameter (medianxa0=xa018%) and improving microscopic margin status, but did not impact survival. Radiotherapy and the margin status were independent predictors of local recurrence. The 5- and 10-year metastatic-free survivals were 84 and 77% respectively for MLS and 69 and 46% for RCLS. The initial site of metastasis was found in multiple locations (34%) and bone involvement was frequent (40%) with predilection for spine (79%). Round cell percent (>xa05%) and tumor diameter (>xa010xa0cm) correlated with increased risk for metastasis and death.ConclusionsMLS and RCLS showed different metastatic risk but equally good local control. Radiotherapy was effective in preventing local recurrence and should be delivered as neoadjuvant. New staging strategies are to be defined to account for the unusual metastatic pattern.

High-Speed Burring with and without the Use of Surgical Adjuvants in the Intralesional Management of Giant Cell Tumor of Bone: A Systematic Review and Meta-Analysis.

Local control rates for Giant Cell Tumor of Bone (GCT) have been reported in a large number of retrospective series. However, there remains a lack of consensus with respect to the need for a surgical adjuvant when intralesional curettage is performed. We have systematically reviewed the literature and identified six studies in which two groups from the same patient cohort were treated with intralesional curettage and high-speed burring with or without a chemical or thermal adjuvant. Studies were evaluated for quality and pooled data was analyzed using the fixed effects model. Data from 387 patients did not indicate improved local control with the use of surgical adjuvants. Given the available data, we conclude that surgical adjuvants are not required when meticulous tumor removal is performed.

A systematic review and meta-analysis of intralesional versus wide resection for intramedullary grade I chondrosarcoma of the extremities.

BackgroundThe surgical management of grade I intramedullary chondrosarcoma of bone remains controversial. The purpose of this study was to perform a systematic review and meta-analysis of published data to determine the oncologic outcomes of intralesional versus wide resection for grade I intramedullary chondrosarcoma.MethodsLiterature searches were performed through Medline, EMBASE, and the Cochrane Database. Cohort studies in which one patient group with grade I chondrosarcoma underwent wide resection and one underwent intralesional curettage were included. Two reviewers independently assessed all eligible papers with the Newcastle-Ottawa Quality Assessment Scale for Cohort Studies. The outcome measures were the pooled odds ratio and 95% confidence intervals for the risk of local recurrence and metastasis calculated through the random-effects method.ResultsFive eligible studies were identified including a total of 190 patients, 78 of whom underwent intralesional resection and 112 of whom underwent wide resection. Only one pelvic lesion was identified, which underwent wide resection. There were a total of five local recurrences and three metastases. The risk for local recurrence and metastasis did not differ significantly between the two groups, with an odds ratio for intralesional resection of 2.26 (95% confidence interval, 0.41–12.62) and 0.44 (95% confidence interval, 0.04–5.21) respectively.ConclusionsIntralesional curettage as an alternative to wide resection for extrapelvic grade I chondrosarcoma of bone does not greatly increase the risk for local recurrence or metastasis. Overall effect estimates, however, should be interpreted with caution as a result of the relatively small number of events.

The efficacy of chemical adjuvants on giant-cell tumour of bone: AN IN VITRO STUDY

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H(2)O(2)) and 50% zinc chloride (ZnCI(2)) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions. Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H(2)O(2) and 50% ZnCI(2). The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures. These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.

A Phase II Study of Flavopiridol in Patients With Previously Untreated Advanced Soft Tissue Sarcoma

Purpose. Flavopiridol is a potent cyclin-dependent kinase (CDK) inhibitor that has preclinical activity in many tumours. This synthetic flavonoid was tested in a phase II nonrandomized, nonblinded multicentre clinical trial to determine its activity and toxicity in patients with previously untreated metastatic or locally advanced soft tissue sarcoma. Methods. A total of 18 patients with histologically confirmed nonoperable soft tissue was treated with flavopiridol administered at a dose of 50 mg/m 2 IV over 1 hour daily ×3 days every 3 weeks. Results. Eighteen patients were accrued to the study over a period of 6 months. No objective responses were noted in the seventeen evaluable patients. Eight patients (47%) exhibited stable disease after 2 cycles (median duration of 4.3 months (range 1.4–6.9 months). Kaplan-Meier estimates for 3- and 6-month progression-free survivial rates were 44 percent and 22 percent, respectively. The only grade 3 toxicities were diarrhea (N = 2), nausea (N = 2), gastritis (N = 1), and fatigue (N = 1). Ninety-four percent of patients received ≥ 90% of the planned dose intensity, during 55 treatment cycles. Conclusions. Flavopiridol was well tolerated at the dose and schedule used in this study, however, no objective treatment responses were seen and thus our results do not support further exploration of flavopiridol as a monotherapy at this dose and schedule in soft tissue sarcomas.

Evidence for the role of matrix metalloproteinase-13 in bone resorption by giant cell tumor of bone☆☆☆

Giant cell tumor of bone (GCT) is an aggressively osteolytic primary bone tumor that is characterized by the presence of abundant multinucleated osteoclast-like giant cells, hematopoietic monocytes, and a distinct mesenchymal stromal cell component. Previous work in our laboratory has shown that matrix metalloproteinase (MMP)-13 is the principal proteinase expressed by the stromal cells of GCT. The release of cytokines, particularly interleukin-1beta, by the giant cells of GCT acts on stromal cells to stimulate a surge in MMP-13 secretion. The purpose of this study was to determine the bone resorption capabilities of the cellular elements of GCT and the significance of the MMP-13 expression involved in GCT bone resorption. We present a 3-dimensional histomorphometric technique developed to analyze resorption pit depth and yield an accurate measurement of bone resorption with a direct physical view of lacunae on bone slices. In this study, we demonstrate that the mesenchymal stromal cells and the multinucleated giant cells of GCT are independently capable of bone resorption. However, coculture of these 2 cell fractions shows a synergistic increase in bone resorption. In addition, inhibition of MMP-13 reduces resorptive activity of the cells indicating that MMP-13 likely plays an important role in this tumor. This cell-cell cooperation involves giant cell-derived cytokine up-regulation of MMP-13 in the stromal cells, which in turn assists the giant cells in bone resorption. Future research will involve elucidation of the role of cell-cell/matrix communication pathways in bone resorption and tumorigenesis in GCT.

The epigenetic regulation of SOX9 by miR-145 in human chondrosarcoma.

Chondrosarcoma is the most common primary bone malignancy in the adult population with a high rate of pulmonary metastasis. Chondrosarcoma is managed with surgical excision as the tumors do not respond well to conventional chemotherapy or radiation therapy. Thus, there exists a dire need to develop systemic treatment options to target chondrosarcoma cells for metastatic spread. We hypothesized that the expression of miR‐145 is low in chondrosarcoma, leading to decreased transcriptional control of SOX9 (the master regulator of chondrogenesis), and downstream activation of the transcription factor ETV5. We have previously shown that ETV5 activates MMP‐2 expression in chondrosarcoma, which in turn increases local bone matrix resorption. In this study, we confirm high expression of SOX9 in human chondrosarcoma using real‐time PCR, Western blotting, and immunofluorescence. An ETV5 promoter–reporter plasmid was transfected into chondrosarcoma cells to determine if SOX9 directly regulates the expression of ETV5. Co‐transfection of the ETV5 promoter‐plasmid with SOX9 lentivirus significantly increased the luciferase activity derived from the ETV5 promoter, from which the regulatory relationship between SOX9 and ETV5 is established. MiR‐145 was found to be down‐regulated in chondrosarcoma cell lines, patient samples, and further confirmed with a public sarcoma database. After stable miR‐145 lentiviral transfection, the subsequent mRNA expression levels of SOX9, ETV5, and MMP‐2 were significantly decreased in chondrosarcoma cells. The results generated by this study may have important clinical significance in the treatment of patients with chondrosarcoma in that targeted miRNA may have the potential to downregulate the upstream activators of proteases such as MMP‐2. J. Cell. Biochem. 116: 37–44, 2015.