Domizio Suva

Identification of Cancer Stem Cells in Ewing's Sarcoma

Cancer stem cells that display tumor-initiating properties have recently been identified in several distinct types of malignancies, holding promise for more effective therapeutic strategies. However, evidence of such cells in sarcomas, which include some of the most aggressive and therapy-resistant tumors, has not been shown to date. Here, we identify and characterize cancer stem cells in Ewings sarcoma family tumors (ESFT), a highly aggressive pediatric malignancy believed to be of mesenchymal stem cell (MSC) origin. Using magnetic bead cell separation of primary ESFT, we have isolated a subpopulation of CD133+ tumor cells that display the capacity to initiate and sustain tumor growth through serial transplantation in nonobese diabetic/severe combined immunodeficiency mice, re-establishing at each in vivo passage the parental tumor phenotype and hierarchical cell organization. Consistent with the plasticity of MSCs, in vitro differentiation assays showed that the CD133+ cell population retained the ability to differentiate along adipogenic, osteogenic, and chondrogenic lineages. Quantitative real-time PCR analysis of genes implicated in stem cell maintenance revealed that CD133+ ESFT cells express significantly higher levels of OCT4 and NANOG than their CD133- counterparts. Taken together, our observations provide the first identification of ESFT cancer stem cells and demonstration of their MSC properties, a critical step towards a better biological understanding and rational therapeutic targeting of these tumors.

EWS-FLI-1 Expression Triggers a Ewing's Sarcoma Initiation Program in Primary Human Mesenchymal Stem Cells

Ewings sarcoma family tumors (ESFT) express the EWS-FLI-1 fusion gene generated by the chromosomal translocation t(11;22)(q24;q12). Expression of the EWS-FLI-1 fusion protein in a permissive cellular environment is believed to play a key role in ESFT pathogenesis. However, EWS-FLI-1 induces growth arrest or apoptosis in differentiated primary cells, and the identity of permissive primary human cells that can support its expression and function has until now remained elusive. Here we show that expression of EWS-FLI-1 in human mesenchymal stem cells (hMSC) is not only stably maintained without inhibiting proliferation but also induces a gene expression profile bearing striking similarity to that of ESFT, including genes that are among the highest ESFT discriminators. Expression of EWS-FLI-1 in hMSCs may recapitulate the initial steps of Ewings sarcoma development, allowing identification of genes that play an important role early in its pathogenesis. Among relevant candidate transcripts induced by EWS-FLI-1 in hMSCs, we found the polycomb group gene EZH2, which we show to play a critical role in Ewings sarcoma growth. These observations are consistent with our recent findings using mouse mesenchymal progenitor cells and provide compelling evidence that hMSCs are candidate cells of origin of ESFT.

Low-energy femoral fractures associated with the long-term use of bisphosphonates: a case series from a Swiss university hospital.

AbstractBackground: Bisphosphonates are effective and well tolerated anti-resorptive drugs used for the treatment of osteoporosis. However, some concerns about their potential long-term negative effects are emerging. Objective: We report a series of patients with a history of bisphosphonate treatment admitted to our institution with a low-energy subtrochanteric fracture. Patients and methods: Eight patients fulfilling these two criteria within the last 2 years were included in our retrospective analysis. All cases were reported to the Swiss National Pharmacovigilance Centre. Results: All patients presented with a typical radiological pattern consisting of a cortical thickening at the lateral femoral subtrochanteric cortex with a horizontal fracture line originating precisely at this level. Four patients eventually developed a stress fracture or complete fracture of the contralateral femur. Two patients demonstrated delayed healing of their fracture. Five patients had been on alendronate therapy for a period ranging from 16 months to 8 years, two had been on ibandronate for 4 months and 1 year, respectively, after changing from alendronate, and one patient had been on pamidronate until 1 year before the fracture occurred. Seven patients were also receiving long-term proton pump inhibitor (PPI) treatment which could have contributed to the increased risk of fracture. Four patients were receiving both PPI and long-term corticosteroid treatment. The hypothesis of a negative pharmacodynamic interaction between bisphosphonates, PPIs and corticosteroids which could lead to a decrease in bone strength after long-term use needs further investigation. Conclusion: Prescribers should be aware of the possibility of these rare adverse reactions and the prolonged use of bisphosphonates should be reconsidered until long-term robust safety data are available.

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells.

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene‐targeting therapies since they differentiate into various cell‐lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non‐hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45+ leukocytes. After culture, leukocytes were replaced by a homogenous layer of adherent CD45− CD14− CD34− CD11b− CD90+ HLA‐ABC+ cells. Culture doubling time (mean = 4 days, range 1.6–6.7 days) was not correlated with patient age (27–81 years, n = 16). Amplified cultures supported long‐term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 106‐fold) were not age‐related. All 14 clones analyzed (from five patients, ages 27–78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM‐derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC‐based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC. J. Cell. Physiol. 198: 110–118, 2004.

IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells.

Background The EWS-FLI-1 fusion protein is associated with 85–90% of Ewings sarcoma family tumors (ESFT), the remaining 10–15% of cases expressing chimeric genes encoding EWS or FUS fused to one of several ets transcription factor family members, including ERG-1, FEV, ETV1 and ETV6. ESFT are dependent on insulin-like growth factor-1 (IGF-1) for growth and survival and recent evidence suggests that mesenchymal progenitor/stem cells constitute a candidate ESFT origin. Methodology/Principal Findings To address the functional relatedness between ESFT-associated fusion proteins, we compared mouse progenitor cell (MPC) permissiveness for EWS-FLI-1, EWS-ERG and FUS-ERG expression and assessed the corresponding expression profile changes. Whereas all MPC isolates tested could stably express EWS-FLI-1, only some sustained stable EWS-ERG expression and none could express FUS-ERG for more than 3–5 days. Only 14% and 4% of the total number of genes that were respectively induced and repressed in MPCs by the three fusion proteins were shared. However, all three fusion proteins, but neither FLI-1 nor ERG-1 alone, activated the IGF1 promoter and induced IGF1 expression. Conclusion/Significance Whereas expression of different ESFT-associated fusion proteins may require distinct cellular microenvironments and induce transcriptome changes of limited similarity, IGF1 induction may provide one common mechanism for their implication in ESFT pathogenesis.

Accuracy of Stress Radiography Techniques in Grading Isolated and Combined Posterior Knee Injuries: A Cadaveric Study

Background Stress radiography techniques have been shown to be superior to the arthrometer and clinical examination in evaluating the posterior cruciate ligament—deficient knee, but no precise relationship has been established between the extent of the lesion and the laxity measured by stress radiography. Hypothesis It is possible to establish a precise relation between posterior laxity and the anatomical lesions of the posterior cruciate ligament and posterior structures using stress radiography. Study Design Controlled laboratory study. Methods Measurements were performed on 15 fresh-frozen cadaveric knee specimens. A partial posterior cruciate ligament lesion was created by sectioning the anterolateral bundle, followed by a complete section. Then the lateral collateral ligament and the posterolateral corner were transected, and finally the medial collateral ligament and the posteromedial corner were sectioned. Stress radiography was performed first on the intact knee and again after each lesion was created using 4 techniques: Gravity Sag View, PCL-Press, Telos at 80°, and Telos at 30° of flexion. Results Telos 30 and Telos 80 revealed the best overall performance as a diagnostic test in terms of accuracy in discriminating between the different types of lesions. Using the Telos device, we determined the following cut-off points: for a partial lesion, less than 3 mm at 30° and less than 6 mm at 80°; for a complete lesion, between 4 mm and 9 mm at 30° and between 7 mm and 12 mm at 80°; for associated peripheral lesions, more than 9 mm at 30° and more than 12 mm at 80°. Conclusion The Telos 30° and 80° allow us to accurately distinguish between the different types of lesion and permit grading of posterior knee laxity. Clinical Relevance Stress radiography allows characterization of posterior knee injuries and helps to determine treatment strategy.

Influence of preoperative patient education on the risk of dislocation after primary total hip arthroplasty

OBJECTIVE Dislocation is a well-known complication after total hip arthroplasty (THA), and is the second-highest cause of revision surgery. Our objective was to assess the effect of preoperative patient education on the occurrence of hip dislocation within 6 months after primary THA. METHODS Between 1998 and 2007, we conducted a prospective cohort study at the Geneva University Hospital Department of Orthopaedic Surgery, including all primary THAs performed via an anterolateral transgluteal approach with the use of a 28-mm diameter head. The preoperative education session was introduced in June 2002 and included advice on muscle strengthening exercises and postoperative restrictions of range of motion as a means of preventing dislocation. The main outcome was the incidence of dislocation within 6 months of surgery. RESULTS A total of 597 patients who underwent 656 THAs between June 2002 and June 2007 participated in the education session, whereas 1,641 patients who underwent 1,945 procedures did not. Forty-six dislocations occurred over the study period, 5 (0.8%) in participants and 41 (2.1%) in nonparticipants (absolute risk reduction 1.3%; 95% confidence interval [95% CI] 0.4, 2.3), with the time interval between surgery and dislocation being significantly shorter among participants (0.2 versus 1.2 months). Nonparticipants had a 2.8 times higher risk of dislocation than participants (unadjusted odds ratio [OR] 2.80; 95% CI 1.10, 7.13). Adjustment for age, sex, comorbidities, and prior surgery did not change the results (adjusted OR 2.79; 95% CI 1.09, 7.15). CONCLUSION Our findings suggest that participation in a preoperative patient education session may reduce the risk of dislocation within 6 months after THA.

In vitro activated human T lymphocytes very efficiently attach to allogenic multipotent mesenchymal stromal cells and transmigrate under them

The regulatory effect of human multipotent mesenchymal stromal cells (MSC) on allogenic T lymphocytes is extremely powerful and of important clinical relevance, but the mechanisms underlying this process are not fully elucidated. We report here that T lymphocytes activated with a sub‐mitogenic stimulus such as phytohemaglutinin alone (PHA), or with mitogenic stimuli such as PHA + interleukin‐2 (P‐IL2), or immobilized anti‐CD3 + anti‐CD28 mAb (a3‐28), tightly bound allogenic MSC and transmigrated within 4 h under them, where they remained for approximately 60 h. Allogenic MSC induced T cell proliferation in cultures containing sub‐mitogenic PHA concentrations, and inhibited the mitogenic effect of P‐IL2 or a3‐28. Anti‐γ‐IFN mAb or L‐tryptophan complementation partially restored proliferation in P‐IL2 and a3‐28 cultures, whereby γ‐IFN‐synthesizing CD3+ cells were detectable. MSC‐lymphocyte contact hindrance using transwells abrogated proliferation in PHA cultures, restored it integrally in P‐IL2 cultures, and partially in a3‐28 cultures. These data suggest that MSC‐induced T lymphocyte regulation results from the combination of various processes. Allogenic cell–cell contact, as demonstrated by the PHA co‐cultures is per se stimulatory, whereas γ‐IFN synthesized by activated T lymphocytes, which activates indolamine 2,3‐dioxygenase in MSC, and L‐tryptophan depletion, which is induced by this enzyme, are inhibitory. Transmigration is nevertheless pivotal for the establishment of the inhibition by these mediators because it targets lymphocytes under the stroma in small extracellular spaces surrounded by MSC, where L‐tryptophan is efficiently destroyed, leading to T lymphocyte proliferation arrest. In conclusion lymphocyte transmigration under allogenic MSC potentiates the inhibitory effect of soluble mediators generated by these cells. J. Cell. Physiol. 214: 588–594, 2008.

Short duration of antibiotic prophylaxis in open fractures does not enhance risk of subsequent infection

We undertook a retrospective case-control study to assess the clinical variables associated with infections in open fractures. A total of 1492 open fractures were retrieved; these were Gustilo and Anderson grade I in 663 (44.4%), grade II in 370 (24.8%), grade III in 310 (20.8%) and unclassifiable in 149 (10.0%). The median duration of prophylaxis was three days (interquartile range (IQR) 1 to 3), and the median number of surgical interventions was two (1 to 9). We identified 54 infections (3.6%) occurring at a median of ten days (IQR 5 to 20) after trauma. Pathogens intrinsically resistant to the empirical antibiotic regimen used (enterococci, Enterobacter spp, Pseudomonas spp) were documented in 35 of 49 cases (71%). In multivariable regression analyses, grade III fractures and vascular injury or compartment syndrome were significantly associated with infection. Overall, compared with one day of antibiotic treatment, two to three days (odds ratio (OR) 0.6 (95% confidence interval (CI) 0.2 to 2.0)), four to five days (OR 1.2 (95% CI 0.3 to 4.9)), or > five days (OR 1.4 (95% CI 0.4 to 4.4)) did not show any significant differences in the infection risk. These results were similar when multivariable analysis was performed for grade III fractures only (OR 0.3 (95% CI 0.1 to 3.4); OR 0.6 (95% CI 0.2 to 2.1); and OR 1.7 (95% CI 0.5 to 6.2), respectively). Infection in open fractures is related to the extent of tissue damage but not to the duration of prophylactic antibiotic therapy. Even for grade III fractures, a one-day course of prophylactic antibiotics might be as effective as prolonged prophylaxis.

Epigenetic Features of Human Mesenchymal Stem Cells Determine Their Permissiveness for Induction of Relevant Transcriptional Changes by SYT-SSX1

Background A characteristic SYT–SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism. Methodology/Principal Findings We addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression. Conclusions/Significance Our observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.