Gene fusions in vascular tumors and their underlying molecular mechanisms

Introduction: The group of vascular tumors contains many different entities, and is considered difficult by pathologists, as they often have overlapping histological characteristics. Chromosomal translocations have been identified in similar to 20% of mesenchymal tumors and are considered the drivers of tumor formation. Many translocations have been discovered over the past decade through next-generation sequencing. This technological advancement has also revealed several recurrent gene fusions in vascular tumors.Areas covered: This review will discuss the various vascular tumors for which recurrent gene fusions have been identified. The gene fusions and the presumed molecular mechanisms underlying tumorigenesis are shown, and potential implications for targeted therapies discussed. The identification of these gene fusions in vascular tumors has improved diagnostic accuracy, especially since several of these fusions can be easily detected using surrogate immunohistochemical markers.Expert opinion: The identification of gene fusions in a subset of vascular tumors over the past decade has improved diagnostic accuracy, and has provided the pathologists with novel diagnostic tools to accurately diagnose these often difficult tumors. Moreover, the increased understanding of the underlying molecular mechanisms can guide the development of targeted therapeutic strategies.Molecular tumour pathology - and tumour geneticsMTG

A remarkable response to pazopanib, despite recurrent liver toxicity, in a patient with a high grade endometrial stromal sarcoma, a case report

Experimentele farmacotherapi

Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma

Primary cilia are specialized cell surface projections found on most cell types. Involved in several signaling pathways, primary cilia have been reported to modulate cell and tissue organization. Although they have been implicated in regulating cartilage and bone growth, little is known about the organization of primary cilia in the growth plate cartilage and osteochondroma. Osteochondromas are bone tumors formed along the growth plate, and they are caused by mutations in EXT1 or EXT2 genes. In this study, we show the organization of primary cilia within and between the zones of the growth plate and osteochondroma. Using confocal and electron microscopy, we found that in both tissues, primary cilia have a similar formation but a distinct organization. The shortest ciliary length is associated with the proliferative state of the cells, as confirmed by Ki-67 immunostaining. Primary cilia organization in the growth plate showed that non-polarized chondrocytes (resting zone) are becoming polarized (proliferating and hypertrophic zones), orienting the primary cilia parallel to the longitudinal axis of the bone. The alignment of primary cilia forms one virtual axis that crosses the center of the columns of chondrocytes reflecting the polarity axis of the growth plate. We also show that primary cilia in osteochondromas are found randomly located on the cell surface. Strikingly, the growth plate-like polarity was retained in sub-populations of osteochondroma cells that were organized into small columns. Based on this, we propose the existence of a mixture ('mosaic') of normal lining (EXT+/- or EXTwt/wt) and EXT-/- cells in the cartilaginous cap of osteochondromas. Laboratory Investigation (2010) 90, 1091-1101; doi:10.1038/labinvest.2010.81; published online 26 April 2010Molecular tumour pathology - and tumour genetic

SUMOylation is associated with aggressive behavior in chondrosarcoma of bone

Simple Summary SUMO is a ubiquitin-like post-translational modification important for many cellular processes and is suggested to play a role in cancer cell cycle progression. The aim of our study is to understand the role of SUMOylation in tumor progression and aggressiveness. Chondrosarcoma of bone was employed as a model to investigate if SUMOylation contributes to its aggressiveness. We confirmed that SUMO expression levels correlate with aggressiveness of chondrosarcoma and disease outcome. Inhibition of SUMOylation showed promising effects on reduction of chondrosarcoma growth in vitro. Our study implies that SUMO expression could be used as a potential biomarker for disease outcome in chondrosarcoma. Multiple components of the SUMOylation machinery are deregulated in various cancers and could represent potential therapeutic targets. Understanding the role of SUMOylation in tumor progression and aggressiveness would increase our insight in the role of SUMO in cancer and clarify its potential as a therapeutic target. Here we investigate SUMO in relation to conventional chondrosarcomas, which are malignant cartilage forming tumors of the bone. Aggressiveness of chondrosarcoma increases with increasing histological grade, and a multistep progression model is assumed. High-grade chondrosarcomas have acquired an increased number of genetic alterations. Using immunohistochemistry on tissue microarrays (TMA) containing 137 chondrosarcomas, we showed that higher expression of SUMO1 and SUMO2/3 correlates with increased histological grade. In addition, high SUMO2/3 expression was associated with decreased overall survival chances (p = 0. 0312) in chondrosarcoma patients as determined by log-rank analysis and Cox regression. Various chondrosarcoma cell lines (n = 7), especially those derived from dedifferentiated chondrosarcoma, were sensitive to SUMO inhibition in vitro. Mechanistically, we found that SUMO E1 inhibition interferes with cell division and as a consequence DNA bridges are frequently formed between daughter cells. In conclusion, SUMO expression could potentially serve as a prognostic biomarker.MTG6Molecular tumour pathology - and tumour genetic

Pharmacological inhibition of Bcl-xL sensitizes osteosarcoma to doxorubicin

High-grade conventional osteosarcoma is the most common primary bone tumor. Prognosis for osteosarcoma patients is poor and resistance to chemotherapy is common. We performed an siRNA screen targeting members of the Bcl-2 family in human osteosarcoma cell lines to identify critical regulators of osteosarcoma cell survival. Silencing the anti-apoptotic family member Bcl-xL but also the pro-apoptotic member Bak using a SMARTpool of siRNAs as well as 4/4 individual siRNAs caused loss of viability. Loss of Bak impaired cell cycle progression and triggered autophagy. Instead, silencing Bcl-xL induced apoptotic cell death. Bcl-xL was expressed in clinical osteosarcoma samples but mRNA or protein levels did not significantly correlate with therapy response or survival. Nevertheless, pharmacological inhibition of a range of Bcl-2 family members showed that inhibitors targeting Bcl-xL synergistically enhanced the response to the chemotherapeutic agent, doxorubicin. Indeed, in osteosarcoma cells strongly expressing Bcl-xL, the Bcl-xL-selective BH3 mimetic, WEHI-539 potently enhanced apoptosis in the presence of low doses of doxorubicin. Our results identify Bcl-xL as a candidate drug target for sensitization to chemotherapy in patients with osteosarcoma

NTRK fusions are extremely rare in bone tumours

Aims Because of the efficacy of tropomyosin receptor kinase (Trk) inhibitor therapy in tumours with rearrangements of the neurotrophic tyrosine kinase receptor genes (NRTK genes), there has been a surge in demand for NTRK fusion screening. To date, most studies involving mesenchymal tumours have focused on soft tissue tumours, and data on bone tumours are sparse. Hence, we aimed to explore the frequency of NTRK fusions in a large series of primary bone tumours. Methods and results Immunohistochemical expression of pan-Trk was successfully assessed in 354 primary bone tumours by the use of tissue microarrays. In a selection of positive cases, additional molecular analysis for NTRK fusions was performed with anchored multiplex polymerase chain reaction-based targeted next-generation sequencing. Positivity was found in 19 cases (5%), which comprised Ewing sarcoma (n = 6, 33%), osteosarcoma (n = 11, 13%), and giant-cell tumour of bone (n = 2, 3%). In all except one case, cytoplasmic staining was observed. Weak staining was most often observed (n = 13), although five cases showed moderate staining and one case showed focal strong staining. Molecular analysis was successful in six cases, all of which were negative for NTRK fusions. Conclusion The likelihood of finding an NTRK fusion in bone tumours in clinical practice is extremely low. This may imply that, if more comprehensive large-scale molecular studies confirm this, routine predictive NTRK testing in bone tumour patients with advanced disease may be reconsidered.Molecular tumour pathology - and tumour geneticsMTG

Mismatch repair deficiency is rare in bone and soft tissue tumors

Introduction There has been an increased demand for mismatch repair (MMR) status testing in sarcoma patients after the success of immune checkpoint inhibition (ICI) in MMR deficient tumors. However, data on MMR deficiency in bone and soft tissue tumors is sparse, rendering it unclear if routine screening should be applied. Hence, we aimed to study the frequency of MMR deficiency in bone and soft tissue tumors after we were prompted by two (potential) Lynch syndrome patients developing sarcomas.Methods Immunohistochemical expression of MLH1, PMS2, MSH2 and MSH6 was assessed on tissue micro arrays (TMAs), and included 353 bone and 539 soft tissue tumors. Molecular data was either retrieved from reports or microsatellite instability (MSI) analysis was performed. In MLH1 negative cases, additional MLH1 promoter hypermethylation analysis followed. Furthermore, a systematic literature review on MMR deficiency in bone and soft tissue tumors was conducted.Results Eight MMR deficient tumors were identified (1%), which included four leiomyosarcoma, two rhabdomyosarcoma, one malignant peripheral nerve sheath tumor and one radiation-associated sarcoma. Three patients were suspected for Lynch syndrome. Literature review revealed 30 MMR deficient sarcomas, of which 33% were undifferentiated/unclassifiable sarcomas. 57% of the patients were genetically predisposed.Conclusion MMR deficiency is rare in bone and soft tissue tumors. Screening focusing on tumors with myogenic differentiation, undifferentiated/unclassifiable sarcomas and in patients with a genetic predisposition / co-occurrence of other malignancies can be helpful in identifying patients potentially eligible for ICI.Molecular tumour pathology - and tumour geneticsMTG

A murine mesenchymal stem cell model for initiating events in osteosarcomagenesis points to CDK4/CDK6 inhibition as a therapeutic target

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16(Ink4a) is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15(Ink4b), p16(Ink4a), or p19(Arf) transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15(Ink4b), p16(Ink4a), or p19(Arf) were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16(INK4A) are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.Osteosarcoma is a tumour with a highly complex genome, which hampers the identification of driver genes. Using a model of murine mesenchymal stem cells (MSCs) with deficient p15(Ink4b), p16(Ink4a), or p19(Arf) that transform earlier compared to wild-type MSCs, the authors demonstrated that loss of p16(Ink4a) is a driver of osteosarcomagenesis. This can be exploited with a CDK4/CDK6 inhibitor, as osteosarcoma cells showed sensitivity to palbociclib which might be used as a novel therapeutic option.Molecular tumour pathology - and tumour geneticsMTG

FOS rearrangement and expression in cementoblastoma

Cementoblastomas are rare odontogenic tumors developing in close proximity to the roots of teeth. Due to their striking morphologic resemblance to osteoblastomas of the peripheral skeleton, we set out to determine whether cementoblastomas harbor the same FOS rearrangements with overexpression of c-FOS as has recently been described for osteoblastomas. In total, 16 cementoblastomas were analyzed for FOS expression by immunohistochemistry and for FOS rearrangements by fluorescence in situ hybridization (FISH). We observed strong and diffuse staining of c-FOS in 71% of cementoblastomas and identified a FOS rearrangement in all cases (n=3) applicable for FISH. In the remaining cases, FISH failed due to decalcification. Cementoblastomas harbor similar FOS rearrangements and show overexpression of c-FOS like osteoblastomas, suggesting that both entities might represent parts of the spectrum of the same disease.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

A phase ib clinical trial of metformin and chloroquine in patients with IDH1-mutated solid tumors

Simple SummaryMutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in high-grade chondrosarcoma, high-grade glioma and intrahepatic cholangiocarcinoma. Due to the lack of effective treatment options, these aggressive types of cancer have a dismal outcome. The metabolism of IDH1-mutated cancer cells is reprogrammed in order to produce the oncometabolite D-2-hydroxyglutarate (D-2HG). In this clinical trial, we used the oral antidiabetic drug metformin and the oral antimalarial drug chloroquine to disrupt the vulnerable metabolism of IDH1-mutated solid tumors. We found that the combination regimen of metformin and chloroquine is well tolerated, but the combination did not induce a clinical response in this patient population. Secondly, we confirmed the clinical usefulness of D/L-2HG ratios in serum as a biomarker and the ddPCR-facilitated detection of an IDH1 mutation in circulating DNA from peripheral blood.Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in 60% of chondrosarcoma, 80% of WHO grade II-IV glioma and 20% of intrahepatic cholangiocarcinoma. These solid IDH1-mutated tumors produce the oncometabolite D-2-hydroxyglutarate (D-2HG) and are more vulnerable to disruption of their metabolism. Methods: Patients with IDH1-mutated chondrosarcoma, glioma and intrahepatic cholangiocarcinoma received oral combinational treatment with the antidiabetic drug metformin and the antimalarial drug chloroquine. The primary objective was to determine the occurrence of dose-limiting toxicities (DLTs) and the maximum tolerated dose (MTD). Radiological and biochemical tumor responses to metformin and chloroquine were investigated using CT/MRI scans and magnetic resonance spectroscopy (MRS) measurements of D-2HG levels in serum. Results: Seventeen patients received study treatment for a median duration of 43 days (range: 7-74 days). Of twelve evaluable patients, 10 patients discontinued study medication because of progressive disease and two patients due to toxicity. None of the patients experienced a DLT. The MTD was determined to be 1500 mg of metformin two times a day and 200 mg of chloroquine once a day. A serum D/L-2HG ratio of >= 4.5 predicted the presence of an IDH1 mutation with a sensitivity of 90% and a specificity of 100%. By utilization of digital droplet PCR on plasma samples, we were able to detect tumor-specific IDH1 hotspot mutations in circulating tumor DNA (ctDNA) in investigated patients. Conclusion: Treatment of advanced IDH1-mutated solid tumors with metformin and chloroquine was well tolerated but did not induce a clinical response in this phase Ib clinical trial.Molecular tumour pathology - and tumour geneticsMTG