Soft Tissue Sarcoma Cell Research Articles

Perivascular Epithelioid Cell Tumors (PEComas) Harboring TFE3 Gene Rearrangements Lack the TSC2 Alterations Characteristic of Conventional PEComas

Perivascular Epithelioid Cell Tumors (PEComas) Harboring TFE3 Gene Rearrangements Lack the TSC2 Alterations Characteristic of Conventional PEComas

Abstract 5651: p53-mediated apoptotic signaling overcomes the resistance to oncolytic adenovirus in human osteosarcoma cells

Abstract Despite major advances in the treatment of bone and soft tissue sarcomas, about one fourth of the patients show a poor response to conventional therapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required to cure patients with bone and soft tissue sarcomas. We previously developed an oncolytic adenovirus, OBP-301, in which human telomerase reverse transcriptase (hTERT) gene promoter drives viral E1 gene for replication. OBP-301 induces tumor-specific oncolytic cell death in a telomerase-dependent manner. Recently, we revealed that OBP-301 shows cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells were less sensitive to cytopathic activity of OBP-301. To enhance the cytopathic activity of OBP-301, we recently developed a novel telomerase-specific oncolytic adenovirus, OBP-702, which expresses tumor suppressor p53 gene. In this study, we investigated the antitumor effect of OBP-702 in OBP-301-sensitive (OST, HOS and U2OS) and OBP-301-resistant (MNNG/HOS and SaOS-2) human osteosarcoma cells. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved poly (ADP-ribose) polymerase (PARP) proteins using western blot analysis in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301 in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells. The ID50 value of OBP-702 was lower than that of OBP-301 in all cell lines. OBP-702 induced not only oncolysis, but also apoptotic cell death with the cleavage of PARP. OBP-702 infection induced more profound p53 expression than Ad-p53. However, p53-downstream target p21 proteins were not activated by OBP-702. Taken together, we demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 in human osteosarcoma cells. Oncolytic adenovirus-mediated p53 gene transduction would induce profound apoptosis through p53 upregulation without p21 activation, resulting in the enhancement of antitumor effect by OBP-301. OBP-702 would be a promising antitumor reagent for the treatment of OBP-301-resistant human osteosarcoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5651. doi:1538-7445.AM2012-5651

Abstract 3763: Treatment of soft tissue sarcoma cells by EGFR and/or STAT3 inhibitors

Abstract Background: We previously found Epidermal Growth Factor Receptor (EGFR) inhibitor can block liposarcoma cell EGFR (HER1) survival signalling. However, it did not interfere with HER2 regulated Janus Kinases (JAK), and Signal Transducer and Activator of Transcription (STAT) signal transduction. Our research suggested JAK/STAT may be one mechanism for EGFR inhibitor resistance. Targeting both EGFR and STAT3, which has never been tested in sarcoma may overcome this problem. The principal aim of this preliminary study was to investigate the effect and mechanism of both STAT3 inhibitor alone and in combination with EGFR inhibitor in the treatment of soft tissue sarcoma (STS) cell lines. Methods: STAT3 inhibitor (S3I-201) mono-therapy or in combination with EGFR inhibitor Gefitinib was investigated in 7 STS cell lines (449b, 778, SW872, SW684, SW982, GCT and HT1080). Crystal-violet colorimetric and clonogenic assays were used to measure drug effects. For assessing a potential role of treatment, we investigated expression and activity of STAT and EGFR by Western blot and immunohistochemistry staining before and after treatment. Data were analysed using Chou & Talalay method and CalcuSyn software. Results: All 7 STS cell lines expressed phosphorylated/total STAT3 and EGFR. Anti-proliferative and anti-clonogenic effects of S3I-201 monotherapy on STS were dose- and time-dependent. Sensitive cells to S3I-201 (IC50 Δ50μM) were HT1080, SW684, GCT and SW982 (90% HT1080 were inhibited at day 3 post-administration of 25μM drug), partially sensitive cells were 449B and 778, and non-sensitive cells (IC50 ≤ 200μM) were SW872 (only 29% SW872 were inhibited at day 5 with 200μM S3I-201). Western blot analysis of whole-cell lysates from HT1080 showed phosphorylated STAT3 (pSTAT3) levels were significantly diminished after S3I-201 treatment, whereas pSTAT3 were not down-regulated in resistant cells SW872. IC50 of Gefitinib monotherapy on STS were 20 - 40μM, and 778 had minor effect (33% cells were inhibited after 5 days treatment). Combination therapy with Gefitinib and S3I-201 achieved synergistic antiproliferative effect (mean CIs < 0.90) in 6/7 STS cell lines (CI: 449b (0.3-1), 778 (0.1-0.2), SW872 (0.6-0.7), SW684 (0.2-0.5), GCT (0.3-0.4) and HT1080 (0.5-0.6)), except SW982 (1-1.2). For the most synergistic 778 cells, the drug reduction index for Gefitinib and S3I-201 was 5.9 and 12.7. Although treatment with Gefitinib alone inhibited pSTAT3 expression on serum-starved 778, EGF stimulation reversed the inhibition function. However in combination therapy, STAT3 phosphorylation was not induced by EGF stimulation. Conclusion: Combination therapy targeting both EGFR and STAT3 is a worthwhile treatment to pursue in anti-sarcoma therapy. Further studies will focus on potential mechanisms as well as the anti-sarcoma effect in the animal models. These results will have a clinical implication in treatment of STS in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3763. doi:1538-7445.AM2012-3763

Phase I trial of the HDAC inhibitor LBH589 in combination with sorafenib in patients with renal cell carcinoma, non-small cell lung cancer and soft tissue sarcomas.

440 Background: LBH589 is a novel histone deacetylase inhibitor (HDACi) that induces apoptosis of tumor cells. In renal cell carcinoma (RCC) and non-small-cell lung cancer (NSCLC) cell lines, the combination of sorafenib and HDACi was found to have synergistic inhibition, which correlated with the induction of an ER stress response. In this phase I study, we evaluated the combination of LBH589 and sorafenib in previously treated patients with RCC (9pts), soft tissue sarcomas (1pt), and NSCLC (6pts). The trial was designed to determine the safety profile and maximum tolerated dose of LBH589 and sorafenib when administered concurrently. Methods: Patients were dosed with either i.v. or oral LBH 589 (three times per week, continuously) every twenty eight days in combination with standard daily dose sorafenib (400 mg bid). The dose escalation was based on a “3+3” algorithmic design. LBH was initially administered at an i.v. dose of 5 mg/m2 with escalation to 10 mg/m2. Due to the manufacturer’s phase-out of the i.v. formulation, this was then changed to an oral formulation administered three times a week (doses 15 mg, 20 mg, and 25 mg). Results: Sixteen patients, median age 57 years, have been treated. Dose limiting toxicities were observed with grade 4 thrombocytopenia in two patients at the oral dose of 25 mg. There were no other grade 4 events. Grade 3 events included fatigue (2 pts), hypophosphatemia (2 pts), hypertension (1 pts), anemia (1 pt), rash (1 pt) and hand-foot erythroderma (1 pt). Common toxicities for the combination were fatigue (81%), weight loss (62%), loss of appetite (56%), diarrhea (56%), rash (50%), thrombocytopenia (31%), and hand-foot erythroderma (25%). No patients had significant QT prolongation. There was 1 partial response in a patient with lung cancer (31 weeks). Stable disease was noted in seven patients with RCC (78+, 48, 47, 31, 21, 17, and 10+ weeks). Seven patients had progressive disease Conclusions: The administration of oral LBH589 at a dose of 20 mg was found to be well tolerated and will be used in the expansion phase of the trial. Prolonged stable disease was observed in patients previously treated with sorafenib alone, sunitinib and axitinib.

長期経過で軟部転移を繰り返した軟部淡明細胞肉腫の1例

長期経過で軟部転移を繰り返した軟部淡明細胞肉腫の1例

The Vascularised Groin Chamber: A Novel Model for Growing Primary Human Liposarcoma in Nude Mice.

BackgroundThe preclinical development of anti-sarcoma drugs has been primarily based on the subcutaneous transplantation of xenografts. Transplant survival remains an obstacle of current models which has been attributed to the period of hypoxia after transplantation. We hypothesized that primary soft tissue sarcoma models with an intrinsic tissue engineered vascular supply would be easily reproducible. The aim of this study was to establish a model of primary human soft tissue sarcoma with an intrinsic vascular supply.MethodsPrimary soft tissue sarcoma cells from resected human liposarcomas isolated and divided into tumour fragments were transplanted into a silicon chamber, placed around the superficial epigastric vessels in mice. Sarcoma xenograft samples were analysed histomorphologically (light/electron microscopy and immunohistochemistry).ResultsAll primary soft tissue sarcoma transplants engrafted, leading to solid tumours within 3 weeks. Histological and immunohistochemical staining confirmed the mouse xenografts as identical high grade liposarcomas compared to original tumour tissue.ConclusionThis study established a reproducible xenograft model of primary human liposarcoma. This animal model could be of high value for studying human soft tissue sarcomas and their therapy.

Pediatric head and neck malignancies in sub-Saharan Africa

Introduction Cancers are relatively rare in children however recent reports suggest that malignancies are becoming a major source of pediatric deaths. Method Using the database of the cancer registry of the University College Hospital, Ibadan we reviewed all newly diagnosed cases of head and neck cancers in children under 19years old at the hospital between 1981 and 2008. Results A total of 1,021 cases of Head and Neck cancers were seen in children. The hospital based incidence of pediatric head and neck cancers is 36 cases per year. There were 627 males and 394 females [M:F ratio of 1.6:1] with mean ages of 8.21 and 7.70 years respectively. Boys were more affected than girls in all years of life while the peak age of onset for both sexes is the third year of life. The commonest anatomical site involved is the eye/orbit; other common sites were the nasopharynx, paranasal sinuses, nasal cavity and thyroid gland. Neural malignancies constitute the commonest malignancies seen (35.3%), other are lymphomas (33.1%), squamous cell carcinoma (9.1%) and soft tissue sarcoma (8.6%). Retinoblastoma is the commonest lesion seen among the patients with a slight male preponderance [M:F ratio of 1.2:1] Burkitt lymphoma (BL) is seen in all age groups but there is greater frequency in the older ages. The incidence of carcinomas is higher in the older age groups, relatively rare lesions like Hodgkins lymphoma and thyroid malignancies are almost exclusive to older children. Conclusion The pattern of head and neck malignancies in children in sub-Saharan Africa is changing; dominant lesions like lymphomas are being gradually replaced by other malignancies such as neural malignancies, soft tissue sarcomas and squamous cell carcinoma.

Clinicopathological prognostic factors of superficial non‐small round cell soft tissue sarcomas

We undertook this study to assess the therapeutic results of superficial soft tissue sarcomas as related to the presence of microscopic invasion, and to identify prognostic factors so as to optimize the therapeutic strategy. From 1995 to 2008, 105 patients who were treated surgically for superficial non-small round cell soft tissue sarcoma were investigated with regard to clinical results and microscopic invasion, and the influence exerted on prognosis was analyzed. We analyzed overall, metastasis-free, and local recurrence-free survival rates and determined the difference in survivorship between with and without fascia invasion. The 5-year overall survival rate and 5-year disease-free survival rate were 95.3% and 81.8%. For overall survival, age (P < 0.05), grade (P < 0.05), tumor size (P < 0.05), and fascial invasion (P < 0.0001) were significant unfavorable prognostic factors, while for metastasis-free survival, grade (P < 0.01) and fascial invasion (P < 0.001) were significant unfavorable prognostic factors. For local recurrence-free survival, fascial invasion alone (P < 0.01) was a significant unfavorable prognostic factor. Fascial invasion on pathological examination of resected specimens was identified as a significant unfavorable prognostic factor. Selecting the postoperative adjuvant therapy based on a detailed evaluation of any fascial invasion is an important part of the therapeutic strategy.

Neoadjuvant and adjuvant chemotherapy with modified mesna, adriamycin, ifosfamide, and dacarbazine (MAID) regimen for adult high-grade non-small round cell soft tissue sarcomas

Good local control of high-grade non-small round cell soft tissue sarcomas (NSRCSTSs) has been achieved with significant advances in surgical techniques and radiotherapy. However, the role of chemotherapy remains controversial. Our aim was to investigate the efficacy, feasibility and adverse effects of neoadjuvant and adjuvant chemotherapy with modified mesna, adriamycin, ifosfamide and dacarbazine (MAID) regimen for NSRCSTSs. We conducted a retrospective review of 40 consecutive patients (29 men, 11 women; median age 47 years) with high-grade NSRCSTSs treated in two referral centers between 2004 and 2009 (median follow-up 38.5 months). Patients with distant or nodal metastases at diagnosis were excluded. The regimen consisted of ifosfamide 2,500 mg/m(2)/6 h (days 1-3), mesna 2,500 mg/m(2)/6 h (days 1-3), tetrahydropyranyl adriamycin 20 mg/m(2)/0.5 h (days 1-3), and dacarbazine 300 mg/m(2)/1 h (days 1-3). Among the 26 evaluable patients, there were 8 with a partial response, 15 with stable disease, and 3 with progressive disease. Two- and 5-year overall survival rates were 92 and 86%, respectively, and corresponding disease-free survival rates were 80 and 77%. All relapses were metastases without local recurrence. Grade 3-4 neutropenia, anemia and thrombocytopenia were observed in 38, 18, and 21 patients, respectively. No serious infectious complications occurred due to the administration of granulocyte colony-stimulating factor and prophylactic antibiotics. No other life-threatening serious adverse events were observed. The modified MAID regimen achieved a better outcome with less serious adverse events than previously reported and is a potential option in the management of NSRCSTSs. Further evaluation with long-term follow-up is required.

Effective Oncolytic Vaccinia Therapy for Human Sarcomas

Approximately one fourth of bone and soft-tissue sarcomas recur after prior treatment. GLV-1h68 is a recombinant, replication-competent vaccinia virus that has been shown to have oncolytic effects against many human cancer types. We sought to determine whether GLV-1h68 could selectively target and lyse a panel of human bone and soft-tissue sarcoma cell lines invitro and invivo. GLV-1h68 was tested in a panel of four cell lines including: fibrosarcoma HT-1080, osteosarcoma U-2OS, fibrohistiocytoma M-805, and rhabdomyosarcoma HTB-82. Gene expression, infectivity, viral proliferation, and cytotoxicity were characterized invitro. HT-1080 xenograft flank tumors grown invivo were injected intratumorally with a single dose of GLV-1h68. All four cell lines supported robust viral transgene expression invitro. At a multiplicity of infection (MOI) of five, GLV-1h68 was cytotoxic to three cell lines, resulting in >80% cytotoxicity over 7 d. Invivo, a single injection of GLV-1h68 into HT-1080 xenografts exhibited localized intratumoral luciferase activity peaking at d 2-4, with gradual resolution over 8 d and no evidence of spread to normal tissues. Treated animals exhibited near-complete tumor regression over a 28-d period without observed toxicity. GLV-1h68 has potent direct oncolytic effects against human sarcoma invitro and invivo. Recombinant vaccinia oncolytic virotherapy could provide a new platform for the treatment of patients with bone and soft tissue sarcomas. Future clinical trials investigating oncolytic vaccinia as a therapy for sarcomas are warranted.

Abstract A5: microRNA response to hypoxic stress in soft tissue sarcoma cells

Abstract Many solid tumors encounter hypoxia; a state in which the tumor tissue is deprived of oxygen. Hypoxia is of great clinical importance since it promotes malignant progression by increasing genetic instability, angiogenesis, local invasion and distant metastases. Furthermore hypoxia may lower the susceptibility to apoptosis, and induce chemo/radiation therapy resistance. These alterations are in part mediated by hypoxia inducible transcription factors like HIF1 that affect gene expression. Less well studied in hypoxia is the relatively new level of post-transcriptional regulation by microRNAs. MiRNAs are small non-protein coding RNA molecules that regulate gene expression by binding to the 3′UTRs of target mRNAs, thereby causing translation inhibition and/or mRNA degradation. Several studies have identified hypoxia-regulated miRNAs (HRMs). However, the lack of a general HRM profile suggests cell type specific miRNA responses to hypoxic stress. In a pilot study with three cancer cell lines of different histogenetic origin we observed, in line with the current literature, strong hypoxic miRNA-responses in each cell line, but little overlap in HRMs among the cell lines. Therefore we decided to focus on the modulation of the hypoxic response by miRNAs in a single class of rare mesenchymal tumors, soft tissue sarcomas. We cultured 13 sarcoma cell lines (derived from fibrosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma and synovial sarcoma) under standard conditions in 20% oxygen (normoxia) or in 1 % oxygen (hypoxia) for 0h, 6h, 24h and 48h. Hypoxic conditions were verified by Hypoxia Inducible Factor 1α (HIF1α) upregulation at the protein level and by the upregulation of transcripts of HIF1 target genes CA-IX and VEGFA. MiRNA expression profiling by microarray technology using LNA™ modified oligonucleotides identified several soft tissue sarcoma specific HRMs. There was considerably more overlap in differentially expressed miRNAs among the various soft tissue sarcoma subtypes than among tumor types from more diverse origins. Although miR-210 expression was below the detection level on our array platform, RT-qPCR indicated a 2 to 24 fold upregulation of this commonly found HRM after 24h of hypoxia. The top three (p&amp;lt;0.0002) differentially and overexpressed miRNAs among all sarcoma subtypes are miR-185*, miR-485-5p and miR-216a. Potential target genes of these miRNAs were identified by online target prediction algorithms (e.g. TargetScan). HIF1AN (predicted target of miR-216a and mir-485-5p) and HIF3α (11 predicted target sites for mir-485-5p) were selected as they both appear to play modulatory roles in the hypoxic response. We confirmed the downregulation of HIF1AN (a HIF1-inhibitor) under hypoxic conditions, which would fit the hypothesis that it is regulated by our HRMs. For HIF3α several transcripts with different 3′UTRs exist; only the longest 3′UTRs contain all eleven potential binding sites for miR-485-5p. The presence of these long transcripts is confirmed in all but one cell line. Therefore it is plausible that HIF3α is targeted by miR-485-5p. Luciferase reporter assays and transfection experiments, transiently over-expressing and down-regulating miR-485-5p and miR-216a, are being carried out for functional studies and target gene validation. In Conclusion: We are the first to describe the miRNA response to hypoxia in soft tissue sarcomas and identified new HRMs. Furthermore we identified the hypoxia related genes HIF1 AN and HIF3α as potential targets of our HRMs. This research will further elucidate the role of miRNAs in the hypoxic response in soft tissue sarcomas and may ultimately lead to novel treatment strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A5.

Immunoglobulin G Locus Events in Soft Tissue Sarcoma Cell Lines

Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes.

Up-regulation of the DR5 Expression by Proteasome Inhibitor MG132 Augments TRAIL-Induced Apoptosis in Soft Tissue Sarcoma Cell Lines

PurposeCurrent chemotherapeutics for treating locally advanced or metastatic soft tissue sarcomas (STS) are limited. Accordingly, the present in vitro study was conducted to evaluate the effects of treatment of STS cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) applied as a single agent or in combination with a proteasome inhibitor, MG132.Materials and MethodsSensitivity to TRAIL and activity of TRAIL-induced apoptotic pathways were analyzed in four STS cell lines: HTB-82 (rhabdomyosarcoma), HT-1080 (fibrosarcoma), HTB-93 (synovial sarcoma), and HTB-94 (chondrosarcoma). Reduction of the dye dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) was used to evaluate cytotoxic activity; western blots were used to evaluate TRAIL-induced apoptosis.ResultsTRAIL induced apoptosis in HTB-93 cells, but had little effect in HTB-82, HT-1080, or HTB-94 cells. Expression of TRAIL receptor-1 and -2 did not correlate with sensitivity to TRAIL. Co-incubation of cells with TRAIL and a proteasome inhibitor, MG132, augmented the apoptotic effect of TRAIL in both TRAIL-sensitive and TRAIL-resistant cells. This effect was due to up-regulation of TRAIL receptors and members of the pro-apoptotic BCL-2 family by MG132.ConclusionThese data show that combining TRAIL with MG132 enhances apoptosis and overcomes TRAIL resistance. This restoration of TRAIL sensitivity occurs through an increase in the expression of death receptor 5 and of pro-apoptotic BCL-2 family members such as BAX.

Abstract 5415: Preclinical evaluation of telomerase-specific oncolytic virotherapy for human bone and soft tissue sarcomas

Abstract Purpose: Bone and soft tissue sarcomas frequently occur in young children and show aggressive progression, resistance to conventional chemotherapy and poor prognosis, indicating a requirement for novel antitumor therapy to improve the clinical outcome. Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor, but not of normal, cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that non-epithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear if OBP-301 is cytopathic for non-epithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bones (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1 and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1 and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified-OBP-301, (OBP-405), was used to confirm an antitumor effect on OBP-301-resistant sarcomas. Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non-CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression, not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301-resistant OUMS-27 and NMFH-1 cells. Conclusions: We showed that OBP-301 has antitumor effects on both non-ALT-type and ALT-type sarcoma cells through upregulation of human telomerase reverse transcriptase mRNA. Furthermore, coxsackie and adenovirus receptor-negative sarcoma cells were efficiently killed by fiber-modified OBP-301-derived-OBP-405 through virus-integrin binding. A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5415. doi:10.1158/1538-7445.AM2011-5415

Abstract 4350: Translating diagnostic gene expression profiles for pediatric solid tumors

Abstract BACKGROUND: Features of cancer genomics including gene expression levels can be employed to create biomarker profiles that can predict prior to therapy the diagnosis and prognosis of an individual patient. We aim to translate robust and reproducible diagnostic and prognostic profiles for pediatric solid tumors into clinical tools applicable to all tumor specimens, fresh frozen (FF) or formalin fixed (FFPE). RESULTS: We used Affymetrix GeneChip Human Exon 1.0 ST (HuEx) arrays on 24 cell lines or tumors (4 fusion-positive rhabdomyosarcoma [RMSpos], 5 fusion-negative RMS [RMSneg], 6 Ewing sarcoma family of tumors [ESFT], 5 neuroblastoma [NB], and 4 osteosarcoma [OS]) to identify a 41-feature diagnostic metagene that clearly distinguished both the original test samples and a set of validation samples. We then incorporated the 41-feature metagene into a 48-gene panel. To translate this diagnostic signature, we compared the performance of HuEx arrays with several mid-plex assay platforms including Fluidigm's quantitative reverse-transcriptase PCR (q-RT-PCR) and NanoString's nCounter™ Digital Analyzer by measuring gene expression on an 18-cell line panel (4 RMSpos, 4 RMSneg, 5 ESFT, 1 NB, and 4 OS). With q-RT-PCR, we observed r2 values of 0.703 ± 0.085 (RMSpos), 0.558 ± 0.138 (RMSneg), 0.689 ± 0.070 (ESFT), and 0.607 ± 0.121 (OS). nCounter reported r2 values of 0.672 ± 0.171 (RMSpos), 0.620 ± 0.143 (RMSneg), 0.675 ± 0.094 (ESFT), and 0.583 ± 0.129 (OS). Hierarchical clustering with all platforms was able to distinguish the RMSpos, ESFT, and NB cell lines; however, the RMSneg and OS cell lines were less clearly distinguished because some of the OS cell lines lacked high expression of genes characteristic of OS tumors. Cross-platform comparisons yielded good correlation between nCounter and Q-RT-PCR (average r2 = 0.710 ± 0.183). An 18-sample dilution series revealed that EWS-FLI1 type I or PAX3-FKHR transcripts were consistently detected by q-RT-PCR or nCounter even at 1:1000 RNA dilution. Among biological duplicates, q-RT-PCR reported an average r2 = 0.916 ± 0.039 while nCounter obtained average r2 = 0.983 ± 0.011 including raw cell lysates. In addition, we also compared Fluidigm's q-RT-PCR with Applied Biosystems’ Taqman Low-Density Arrays (TLDA) on five NB patient bone marrow samples to detect residual tumor cells. We observed an average r2 = 0.929 ± 0.119 across five NB genes and one housekeeping gene. Finally, we have also successfully profiled FF vs. FFPE tumors on HuEx and are currently applying these mid-plex technologies to paired FF and FFPE samples in order to create diagnostic profiles applicable to routine FFPE material. CONCLUSIONS: Mid-plex platform can reliably distinguish bone and soft tissue sarcoma cell lines and can be used to translate the application of diagnostic signatures. These data warrant further studies to analyze FFPE samples, to compare additional mid-plex platforms, and to test potential prognostic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2011-4350

Abstract 5047: Survival signal pathways as potential therapeutic targets in soft tissue sarcoma cell lines

Abstract Introduction: Soft tissue sarcomas (STS) are rare malignancies but have poor survival rate. Surgery with standard chemotherapy has been the mainstay of treatment but newer strategies are necessary to improve survival. The investigations for targeted therapy are underway but little is known about potential targets until now. We previously presented potential molecular targets in osteosarcoma (OS) cell lines (101st AACR annual meeting 2010, abstract No.2451). We tried to compare the protein and mRNA profilings between the OS and STS cell lines to suggest the feasibility for developing molecular targeted agents in STS. Methods: We examined the protein expression of c-Met, Akt, mTOR, and their phosphorylated status, and PTEN in seven OS (MG-63, HOS, KHOS/NP, SK-ES-1, U-2OS, Saos-2, and G-292) and five STS cell lines (SK-UT-1, A-673, SW-871, SW-982, HT-1080) using western blotting. The gene expression profilings of the cell lines were performed by 44K microarray (Agilent Inc.). Results: Microarray data showed that whole genome expression pattern of STS was different from OS cell lines. But, the expressions of 94 mRNAs within the signal transduction pathways, which represent the currently utilized drug targets, revealed similar pattern between two groups of cell lines. To verify the possibility of developing molecular targeted agents in STS, we examined the activation patterns of signal transduction pathway proteins in each STS cell line. All STS cell lines except A-673 with low c-Met, but high p-Met, expressed moderate level of c-Met and p-Met. PI3K was expressed in all cell lines, with higher p-PI3K in SK-UT-1 and A-673 than in other STS cell lines. All but SW-872 cell line expressed Akt, and SK-UT-1, SW-872 and HT-1080 had higher levels of p-Akt. SW872 showed negligible PTEN and high level of p-Akt. SW982 generally repressed level of p-Met, p-PI3K, p-Akt, p-mTOR comparing with other STS cell lines. Meanwhile, mTOR and p-mTOR were expressed relatively even in all STS cell lines. Conclusion: We presented that STS cell lines had variously activated signal pathway status as in OS cell lines. Common potential targets such as mTOR was revealed, and the specific targets including PI3K or Akt were also presented. It suggests the possibility of developing specific molecular targeted agents in each type of STSs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5047. doi:10.1158/1538-7445.AM2011-5047

Abstract 2665: NBTXR3 hafnium oxide nanoparticle activated by ionizing radiation demonstrates marked radio-enhancement and antitumor effect via high energy deposit in human soft tissue sarcoma

Abstract Local and systemic control of Soft Tissue Sarcoma (STS) remains a clinical challenge. Radiation therapy is part of the standard of care of STS. The narrowness of its therapeutic window represents the main concern for different clinical settings. Thus, local delivery of radiation doses is critical to ensure optimal benefit-risk ratio. NBTXR3, biocompatible hafnium oxide nanoparticles were designed as therapeutics to be activated by ionizing radiation to achieve tumor control by enhancement of local energy deposition. A global non clinical program was implemented in mesenchymal tumor models. In vitro clonogenic survival assays were performed in two human fibrosarcoma cell lines (HT1080 and Hs913t) and a human liposarcoma cell line (SW872) to evaluate the cellular response to radiation-activated NBTXR3 at increasing concentrations. A marked radio-enhancement was observed in human STS cell lines with dose enhancement factors estimated at 4 Gy ranging from 1.48 up to 5.36 according to NBTXR3 concentrations. Further, in human immortalized MRC5V1 fibroblasts, no significant decrease in clonogenic surviving fraction was observed upon activation of NBTXR3 under similar conditions suggesting a differential radiation response between fibroblasts and fibrosarcoma, liposarcoma cell lines. This differential radiation response was further confirmed between HT1080 fibrosarcoma cell line and MRC5V1 fibroblasts upon activation of NBTXR3 at increasing concentration using high energy gamma-rays. Mechanistic studies have demonstrated in HT1080 cells that NBTXR3 activation induces an increase of 53BP1 foci associated with more “complex” DNA damages than ionizing radiation alone. The level of unrepaired DNA damages was also estimated by flow cytometry analysis showing a higher level of gamma-H2AX at 48h following NBTXR3 activation than after radiotherapy alone. DNA fragmentation and Annexin V staining have demonstrated that these unrepaired DNA damages upon NBTXR3 activation induces apoptosis in HT1080 fibrosarcoma cells. In vivo, tolerance and tumor growth delay were investigated in two human xenografted tumor models, HT1080 fibrosarcoma and a patient derived liposarcoma LPS80T3 (poorly differentiated grade 3). No toxicity related to NBTXR3 was reported. In both models, a significant advantage was demonstrated in terms of tumor growth inhibition and survival when compared to radiation therapy alone. These findings establish a novel approach of treatment, which may be applied to a broad range of tumors. Therefore, incrementing the amount of energy deposited within the tumor through the activation of NBTXR3 crystalline nanoparticles constitutes a revolutionary approach for future clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2011-2665

Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy for Human Bone and Soft Tissue Sarcomas

Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor cells but not of normal cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that nonepithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear whether OBP-301 is cytopathic for nonepithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bone (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1, and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1, and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified OBP-301 (termed OBP-405) was used to confirm an antitumor effect on OBP-301-resistant sarcomas. OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non-CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression and not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301-resistant OUMS-27 and NMFH-1 cells. A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas.

Suppression of Soft Tissue Sarcoma Growth by a Host Defense-Like Lytic Peptide

BackgroundSoft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K3H3L9 on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum.Methods In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K3H3L9, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K3H3L9 was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed.ResultsThe peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K3H3L9 in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group.ConclusionThese findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K3H3L9 in athymic and syngeneic mouse models.

Clear Cell Sarcoma of the Knee. A Report of Two Rare Cases

Clear cell sarcoma of soft tissue is a rare type of soft tissue sarcoma. It is derived from melanoblast-like cells located within subcutaneous tissue, tendon and aponeuroses. The tumour is also known as malignant melanoma of soft parts because it has similar morphology to malignant melanoma. Unlike malignant melanoma, however, it is not associated with a cutaneous lesion. We report here two cases of this tumour occurring in young adults.