Local recurrence and assessment of sentinel lymph node biopsy in deep soft tissue leiomyosarcoma of the extremities

<p>Abstract</p> <p>Background</p> <p>Leiomyosarcoma of deep soft tissues of the extremities is a rare malignant tumour treated primarily by surgery. The incidence of local recurrence and lymph node metastasis is uncertain and it is not known whether a sentinel lymph node biopsy is indicated in these tumours.</p> <p>Methods</p> <p>A retrospective review of patients treated for extremity deep soft tissue leiomyosarcoma at our institution over a 10-year period was conducted. Patients developing local recurrence or lymph node metastasis were identified. The presence or absence of lymphatics in the primary tumours was assessed by immunohistochemical expression of LYVE-1 and podoplanin.</p> <p>Results</p> <p>27 patients (mean age 62 years) were included in the study. 15 were female and 12 male. Lymph node metastasis was seen in only two cases (7%); intratumoural lymphatics were identified in the primary tumours of both these cases. Local recurrence occurred in 25.9% of cases despite complete excision and post-operative radiotherapy; the mean time to recurrence was 10.1 months.</p> <p>Conclusion</p> <p>On the basis of this study, we do not advocate sentinel lymph node biopsy in this group of patients except in those cases in which intratumoural lymphatics can be demonstrated. Close follow up is important especially for high grade leiomyosarcomas, particularly in the first year, as these tumours have a high incidence of local recurrence.</p

Concurrent Oral 9 - Rheumatoid Arthritis: Aetiopathogenesis [OP59-OP64]: OP59. The Value of Interleukin-17 Serum Level in Rheumatoid Arthritis Immunopathogenesis

Background: Interleukin (IL)-17 is the main Th-1 cytokine, produced by activated T-lymphocytes. The potential IL-17 value in rheumatoid arthritis (RA) pathogenesis consists of its independent inflammatory response induction and mediated stimulation of proinflammatory factors synthesis resulting in joint destruction. The aim of study was to determine the role of IL-17 in immuno-inflammatory/autoimmune reactions development and to reveal IL-17 serum level associations with clinical and immunological characteristics of RA. Methods: 50 patients with early RA (disease duration >, Russia), anti-CCP antibodies (Axies-Shield Diagnostic, UK) were revealed using ELISA immunoassay. Results: On the base of IL-17 serum level patients were divided in two groups: group1 (n = 28) were patients with normal IL-17 serum level and group2 (n = 22) were those with high IL-17 serum level. In the group2, the rate of patients' pain assessment by visual analogue scale (67.3 ± 7.2 vs 32.8 ± 4.6; P < 0.001), tender (16.7 ± 2.0 vs 8.4 ± 1.1; P < 0.01) and swollen (12.3 ± 2.3 vs 3.9 ± 0.8; P < 0.01) joint count, DAS28 (5.0 ± 0.4 vs 2.8 ± 0.2 P < 0.01) were significantly higher compare to group1. It was found that in group2 the higher T-lymphocyte amount (CD3) was due to CD4 higher quantity, at the same time CD8 amount was significantly lower (22.2 ± 1.5% vs 28.4 ± 1.7%, P < 0.05) compare to group1. This caused the immunoregulative index increasing and indicated in the lost of autoimmune process regulation, including B-lymphocytes (CD19) activation. The CD154 expression was significantly lower in the group2 (3.4 ± 0.4% vs 10.8 ± 2.8%, P < 0.05) compare to group1. The difference in autoimmune reaction indices wasn't significant between groups except antibody-producing B-lymphocytes (13.7 ± 1.5% vs 8.5 ± 1.0%, P < 0.05) and IgM RF serum level (2.9 ± 0.3 U/ml vs 1.6 ± 0.5 U/ml, P < 0.05), which were significantly higher in group1. The IL-17 level had a positive correlative connections with DAS28 (r = 0.7; P < 0.05), circulative immune complex level (r = 0.38; P < 0.05), anti-CCP antibodies (r = 0.4; P < 0.05), IgM RF (r = 0.41; P < 0.05), CD4 (r = 0.38; P < 0.05) and negative correlative connection with CD8 (r = -0.39; P < 0.05). Conclusions: The importance of IL-17 value in immuno-inflammatory and autoimmune reactions development through T-lymphocytes activation in RA pathogenesis was confirmed. Thus the influence on T-depended immuno-inflammatory reaction products synthesis could be a new therapeutic target of RA patients' management. Disclosure statement: All authors have declared no conflicts of interes

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

<p>Abstract</p> <p>Background</p> <p>Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.</p> <p>Methods</p> <p>HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.</p> <p>Results</p> <p>17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.</p> <p>Conclusions</p> <p>Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in <it>in vivo </it>induction of HIF. <it>In vitro </it>data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.</p

Prognostic impact of reduced connexin43 expression and gap junction coupling of neoplastic stromal cells in giant cell tumor of bone

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in alpha-smooth muscle actin positive than alpha-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB

Serum and synovial fluid ANGPTL4 concentrations are elevated in RA.

<p>(A) Synovial fluid from RA patients contains more ANGPTL4 (203.3±264.8 ng/ml, range 68.7–847.3 ng/ml) than that from patients with non-inflammatory OA (64.4±13.6 ng/ml, range 50.4–82.2 ng/ml). *, p<0.05. (B) Serum from RA patients contains more ANGPTL4 (363.4±138.7 ng/ml, range 24.0–2235.0) than that from OA patients (39.0±3.4 ng/ml, range 11.9–100.5 ng/ml) or normal controls (45.8±6.7 ng/ml, range 6.5–154.7 ng/ml). **, p<0.01. (C) Serum from ‘high ANGPTL4’ RA patients is more likely to have detectable RANKL (black shading; RANKL-positive) than either serum from ‘low ANGPTL4’ RA patients or controls. White shading; RANKL-negative. *, p<0.05.</p

HIF-1α is expressed by osteoclasts in RA.

<p>(A) HIF-1α expression by osteoclasts in the RA synovium (arrows). Representative image from n = 3 cases. (B) HIF-1α (green) and ANGPTL4 (red) expression co-localises in 2 bone-apposing osteoclasts (arrows). All scale bars represent 100 µM. (C) ANGPTL4 (solid lines) and SLC2A1 (Glut-1; dashed lines) mRNA expression in monocyte-derived osteoclasts from RA patients following 24 h exposure to normoxia or hypoxia (2% O₂). Hypoxic fold-change in mRNA expression; *, p<0.05.</p

ANGPTL4 is expressed by osteoclasts in RA.

<p>(A) ANGPTL4 is strongly expressed by osteoclasts (arrows) in the RA synovium. Inset: Representative isotype control image. (B) Resorbing osteoclasts adjacent to bone (starred) express ANGPTL4. (C) ANGPTL4 (red) expression co-localises with cathepsin K-positive (green) osteoclasts. Representative images from n = 3 cases. (D) Cathepsin K-positive (green) osteoclasts in the OA synovium do not express detectable ANGPTL4 (red). All scale bars represent 100 µM.</p

ANGPTL4 and HIF-1α are expressed in RA synovial tissue.

<p>(A) The RA synovium is strongly positive for ANGPTL4, as are adjacent blood vessels and surrounding stromal cells; (B) normal synovium shows weak, heterogeneous expression of ANGPTL4. (C) RA synovium is strongly positive for HIF-1α, as are adjacent blood vessels; (D) normal synovium generally does not express HIF-1α. (E) Synovial lining cells and stromal cells adjacent to lymphoid aggregates express ANGPTL4. Inset: ANGPTL4-positive plasma cells. (F) ANGPTL4 (red) is not expressed by B cells (CD20, green). (G) ANGPTL4 (red) is expressed by CD68-positive (green) macrophages adjacent to a lymphoid aggregate. (H) The OA synovium expresses elevated levels of both ANGPTL4 (left panel) and HIF-1α (right panel) compared with the normal synovium. Scale bars (A–F, H) represent 100 µM, scale bar (G) represents 50 µM.</p

Clinical characteristics of RA patients in serum cohort.

<p>DAS  =  disease activity score. CRP  =  C-reactive protein.</p><p>Clinical characteristics of RA patients in serum cohort.</p