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Research Corner Abstracts by Tom Swartz and Bruce Shriver
In This Issue
The division of retroperitoneal liposarcoma (RPLS) into well-differentiated (WD) and dedifferentiated (DD) subtypes is established. However, WD and DD are usually treated similarly. The investigators of this study hypothesized that WD and DD have distinct biological behaviors mandating different treatments. Their prospective sarcoma database identified all primary/recurrent RPLS treated between 1996 and 2007. 77 DD (52%) and 58 WD (39.2%) patients were analyzed for recurrence rate, recurrence free survival (RFS), and overall survival (OS). Their Results were as follows: At presentation, WD were mostly primary whereas DD were mostly recurrent (75.9% versus 58.4%; p = 0.04). A significant proportion of DD (37.7%) received chemotherapy compared to WD (1.7%; p < 0.0001). Multivisceral resection was more common in DD versus WD (45.5% versus 31%; p = 0.01). Gross total resection rates were equivalent (WD: 86.2%; DD: 85.7%). Overall and local recurrence were higher in DD versus WD (82.2% versus 50% and 71.2% versus 46.3%; p < 0.0001). Only 3.7% WD recurred as high grade metastatic disease. Median time to recurrence was 55.5 months in WD versus 13.5 months in DD (p < 0.0001). RFS and OS (1, 2, and 5 year) were higher in WD than DD (80.3% versus 55.9%; 65.1% versus 34.1%; 41.9% versus 7.8%; p < 0.0001) and (98% versus 88.1%; 95.6% versus 71.9%; 92.1% versus 36.5%; p < 0.0001) respectively. The investigators Conclude: WD and DD have distinct biological behaviors. Gross total resection is achievable in most WD; unlike DD, high-grade recurrence is uncommon. Treatment should therefore reflect these biologic differences by maximizing survivorship while avoiding unnecessarily extensive multivisceral resection.
Malignant peripheral nerve sheath tumors (MPNST) are chemoresistant sarcomas with a poor 5-year survival that arise in patients with neurofibromatosis type 1 (NF1) or sporadically. The investigators of this study tested three drugs for single and combinatorial effects on collected MPNST cell lines and in MPNST xenografts. The mammalian target of rapamycin complex 1 inhibitor RAD001 (Everolimus) decreased growth 19% to 60% after 4 days of treatment in NF1 and sporadic-derived MPNST cell lines. Treatment of subcutaneous sporadic MPNST cell xenografts with RAD001 significantly, but transiently, delayed tumor growth, and decreased vessel permeability within xenografts. RAD001 combined with the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib caused additional inhibitory effects on growth and apoptosis in vitro, and a small but significant additional inhibitory effect on MPNST growth in vivo that were larger than the effects of RAD001 with doxorubicin. RAD001 plus erlotinib, in vitro and in vivo, reduced phosphorylation of AKT and total AKT levels, possibly accounting for their additive effect. The investigators thus conclude that these results support the consideration of RAD001 therapy in NF1 patient and sporadic MPNST. The preclinical tests described allow rapid screening strata for drugs that block MPNST growth, prior to tests in more complex models, and should be useful to identify drugs that synergize with RAD001.
The role of radiation in improving survival for early-stage carcinosarcoma and leiomyosarcoma The investigators examined the effect of radiation on survival for early-stage uterine carcinosarcomas and leiomyosarcomas. A surveillance, epidemiology, and end results database was used to identify patients with stage I/II carcinosarcomas and leiomyosarcomas. Logistic regression and Cox models were developed to determine radiation use and survival. The RESULTS were as follows: Among 1819 women with carcinosarcomas and 1088 women with leiomyosarcomas, radiation was administered to 667 of the patients (37%) with carcinosarcomas and to 235 of the patients (22%) with leiomyosarcomas. In a multivariate model, adjuvant radiation reduced the risk of death by 21% in women with carcinosarcomas (hazard ratio, 0.79; 95% CI, 0.7-0.9). Radiation reduced mortality rates in patients with carcinosarcomas who had not undergone node dissection but had only a marginal effect on survival in node-negative women. Adjuvant radiation had no effect on survival for early-stage leiomyosarcomas (hazard ratio, 1.1; 95% CI, 0.9-1.4). The investigators CONCLUDE: Adjuvant radiotherapy improves survival for select patients with early-stage carcinosarcomas but is of limited value for leiomyosarcomas.
Fas Death Pathway in Sarcomas Correlates with Epidermal Growth Factor Transcription Modulation of apoptosis may influence sarcoma pathogenesis and/or aggressiveness. The Fas death pathway, mediated by FasL or TGFbeta, is one of two apoptotic pathways. Recent studies report that EGF can modulate TGFbeta and/or FasL expression/activity; thus, EGF has the potential to influence activation of the Fas pathway. EGF is not always detectable in mesenchymal tumors. Thus, the investigators hypothesized EGF would define which Fas ligand predominates. They assayed 57 surgically removed human sarcomas for 10 genes involved in the Fas pathway. Skeletal muscle biopsies from eight patients served as controls. Sample transcripts were detected by real-time RT-PCR. They attempted to identify relevant predictor variables. The 57 sarcomas were segregated into two categories defined by EGF mRNA content: (1) 23 tumors with EGF concentrations that approximated muscle EGF transcript levels (high-EGF tumors); and (2) 34 tumors that either lacked EGF mRNA, or whose mRNA levels were very low and frequently undetected by PCR (low-EGF tumors). TGFbeta1 expression best predicted Fas transcript concentrations in the 34 low-EGF sarcomas, while FasL predicted Fas mRNA levels in the remaining 23 high-EGF sarcomas. The results suggest ligand activity in the Fas death pathway correlates with EGF transcription in sarcomas.
Synovial sarcoma is a soft tissue malignancy characterized by the fusion of SS18 to either SSX1, SSX2, or SSX4 genes. SS18 and SSX are transcriptional cofactors involved in activation and repression of gene transcription, respectively. SS18 interacts with SWI/SNF, whereas SSX associates with the polycomb chromatin remodeling complex. Thus, fusion of these two proteins brings together two opposing effects on gene expression and chromatin structure. Recent studies have shown that a significant number of genes are down-regulated by the SS18-SSX fusion protein and that the clinically applicable histone deacetylase (HDAC) inhibitor romidepsin inhibits synovial sarcoma growth. Therefore, the investigators of this study set out to identify direct targets of SS18-SSX among genes down-regulated in synovial sarcoma and investigated if romidepsin can specifically counteract SS18-SSX–mediated transcriptional dysregulation. They report that the tumor suppressor early growth response 1 (EGR1) is repressed by the SS18-SSX protein through a direct association with the EGR1 promoter. This SS18-SSX binding correlates with trimethylation of Lys27 of histone H3 (H3K27-M3) and recruitment of polycomb group proteins to this promoter. In addition, they found that romidepsin treatment reverts these modifications and reactivates EGR1 expression in synovial sarcoma cell models. Their data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma. Furthermore, their work highlights a possible mechanism behind the efficacy of a clinically applicable HDAC inhibitor in synovial sarcoma treatment.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Highly malignant, RMS frequently fails to respond to conventional aggressive multimodal radiation, surgery, and chemotherapy treatment protocols. Other tumors of mesenchymal origin, such as locally aggressive fibromatoses and desmoid tumors, have been successfully treated with a selective estrogen receptor (ER) modulator, tamoxifen. In an effort to identify new targets for RMS therapy, the investigators of this study investigated the previously uncharacterized ER pathway in RMS cell culture and primary tumors. They detected ER isoform β (ERβ), but not isoform α, RNA, and protein in five RMS cell lines. Immunohistochemical staining of primary RMS tumor sections confirmed high levels of ERβ but not ERα protein. RMS cell growth was dramatically inhibited in steroid-free conditions, and this growth inhibition was rescued with 17-β-estradiol (E2) supplementation. Exposure of RMS cells to 4'OH-tamoxifen (4OHT) decreased cell viability and inhibited colony formation as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony-forming assays. 4OHT also induced apoptotic signaling in RMS cells as detected by cleavage of caspase-3 and poly(ADP)ribose polymerase. This effect increased 3- to 8-fold in steroid-deprived conditions but was rescued by supplementation with E2. Immunofluorescence studies detected a change in the subcellular localization of ERβ in response to 4OHT. Thus the investigators believe together, these data suggest an active ERβ-mediated signal transduction pathway in RMS. And the ability of 4OHT to induce apoptotic signaling and disrupt estradiol-mediated proliferation provides a rationale to explore a role for selective ER modulators in the treatment of RMS.
Epidermal Growth Factor Receptor Blockade in Combination with Conventional Chemotherapy Inhibits Soft Tissue Sarcoma Cell Growth In vitro and In vivo The epidermal growth factor receptor (EGFR) is highly expressed in many human soft tissue sarcomas (STS). However, EGFR blockade has not apparently been used for human STS therapy. Thus, the investigators of this study examined the in vitro and in vivo effects and the underlying mechanisms before considering EGFR blockade as a therapy for STS patients.
Human STS tissues and cell lines were used to study EGFR expression and activation. Western blot analysis was used to evaluate effects of EGFR activation on downstream signaling. Cell culture assays were used to assess the effect of EGF stimulation as well as EGFR blockade (using an EGFR tyrosine kinase inhibitor, Iressa; AstraZeneca) on STS cell growth, apoptosis, and chemosensitivity. An in vivo study (HT1080 human fibrosarcoma cell line in nude/nude mice: Iressa, doxorubicin, Iressa + doxorubicin, vehicle) was used to examine tumor growth; pEGFR, proliferating cell nuclear antigen, and terminal deoxyribonucleotide transferase–mediated nick-end labeling staining helped assess the effect of therapy in vivo on STS EGFR activation, proliferation, and apoptosis.
The Results were as follows: EGFR was expressed and activated in STS cell lines and tumors, probably due to ligand binding rather than EGFR mutation. Stimulation caused activation of AKT and mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G0-G1 cell cycle arrest, and decreased cyclin D1 expression. In vivo, Iressa + doxorubicin had markedly synergistic anti-STS effects.
The investigators Conclude: EGFR blockade combined with conventional chemotherapy results in anti-human STS activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve anti-STS therapy.
Prognostic Significance of Macrophage Infiltration in Leiomyosarcomas Macrophages are migratory cells that are frequently recruited to the site of tumors. Their presence is associated with poor clinical outcome in a variety of epithelial malignancies. The investigators of this study sought to examine the prognostic significance of tumor-associated macrophages in sarcomas. Global gene expression profiling data of a series of soft tissue tumors were analyzed for macrophage-associated gene expression. Immunohistochemistry on tissue microarrays containing leiomyosarcoma cases with known clinical outcome was used to verify the presence of macrophages and to examine the relationship between tumor-associated macrophages and clinical outcome. The Results were as follows: Gene expression profiling revealed high-level expression of several macrophage-associated genes such as CD163 and CD68 in a subset of leiomyosarcomas, indicating the presence of variable numbers of tumor-infiltrating macrophages. This was confirmed by CD68 and CD163 immunostaining of a tissue microarray containing 149 primary leiomyosarcomas. Kaplan-Meier survival analysis showed that high density of tumor-infiltrating macrophages as identified by CD163 or CD68 staining is associated with a significantly worse disease-specific survival in nongynecologic leiomyosarcomas, whereas leiomyosarcomas arising from the gynecologic tract showed no significant association between macrophage infiltration and survival. The presence of tumor necrosis did not correlate significantly with outcome. The investigators Conclude: An increased density of CD163- or CD68-positive tumor-infiltrating macrophages is associated with poor outcome in nongynecologic leiomyosarcomas. This may help the clinical management of patients with leiomyosarcomas.
Critical Role of Notch Signaling in Osteosarcoma Invasion and Metastasis Notch signaling is an important mediator of growth and survival in several cancer types, with Notch pathway genes functioning as oncogenes or tumor suppressors in different cancers. However, the role of Notch in osteosarcoma is unknown. The investigators of this study assessed the expression of Notch pathway genes in human osteosarcoma cell lines and patient samples. They then used pharmacologic and retroviral manipulation of the Notch pathway and studied the effect on osteosarcoma cell proliferation, survival, anchorage-independent growth, invasion, and metastasis in vitro and in vivo. The Results were as follows: Notch pathway genes, including Notch ligand DLL1, Notch1 and Notch2, and the Notch target gene HES1, were expressed in osteosarcoma cells, and expression of HES1 was associated with invasive and metastatic potential. Blockade of Notch pathway signaling with a small molecule inhibitor of secretase eliminated invasion in Matrigel without affecting cell proliferation, survival, or anchorage-independent growth. Manipulation of Notch and HES1 signaling showed a crucial role for HES1 in osteosarcoma invasiveness and metastasis in vivo. The investigators thus Conclude: These studies identify a new invasion and metastasis-regulating pathway in osteosarcoma and define a novel function for the Notch pathway: regulation of metastasis. Because the Notch pathway can be inhibited pharmacologically, these findings point toward possible new treatments to reduce invasion and metastasis in osteosarcoma.
Preliminary Data Presented on CP-751,871 in Sarcoma On June 2, Pfizer announced preliminary results from clinical trials of its investigational compound CP-751,871 in sarcoma. CP-751,871, a fully human IgG2 monoclonal antibody, is a highly specific inhibitor of the IGF-1R pathway. It is believed that through this inhibition, CP-751,871 blocks one of the key signaling pathways in cancer cells that lead to uncontrolled growth and survival of tumor cells. The insulin-like growth factor receptor (IGF-1R) is increasingly recognized by the medical community as a relevant target for investigation in cancer research. The results were presented at the 44th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago, IL.
Phase I data presented at ASCO showed single agent CP-751,871 was generally well-tolerated in patients with relapsed or refractory sarcoma (n=22), including Ewing's sarcoma (n=9). A response of stable disease or better was seen in 12 out of 20 evaluable patients, including one confirmed partial response in a 12-year-old patient with Ewing's sarcoma. CP-751,871 was generally well tolerated in patients with relapsed or refractory sarcoma. Grade 3 or 4 treatment-related side effects reported included Grade 4 uric acid increase (n=1) and Grade 3 bilateral deep-vein thrombosis (n=1).
On May 29th, Epeius Biotechnologies announced the results of an on-going Phase I/II study of Rexin-G for metastatic bone and soft tissue sarcoma. Rexin-G is the first targeted gene therapy vector that has been tested in the clinic. In this study, cohorts of 6 to 7 patients with all types of sarcoma, including osteosarcoma, Ewing's sarcoma, leiomyosarcoma, malignant fibrous histiocytoma, chondrosarcoma, fibrosarcoma, liposarcoma, angiosarcoma, spindle cell sarcoma, and malignant mixed Mullerian tumor of ovary, were treated with 1 x 10e11 cfu Rexin-G, administered by IV over 5 minutes, 2 times a week for 4 weeks (Cumulative Dose = 8 x 10e11 cfu) followed by a 2-week rest period.
Patients with Grade 1 or less toxicity were given progressive intra-patient dose-escalations consisting of additional treatment cycles of 1 to 2 x 10e11 cfu three times a week for 4 weeks (Cumulative Dose per cycle: 1.2-2.4 x 10e12 cfu).
The higher dosing regimens were associated with prolonged disease stabilization and a median overall survival of greater than 6 months, which was three times longer than that observed in the low-dose group. Further, histologic examination of resected tumors showed 50-90% necrosis. No dose-limiting toxicity was observed, even at the higher doses of Rexin-G.
V5N3 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative. |
