Abstract: Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.
Abstract: Adamantinoma of long bones and osteofibrous dysplasia are rare, osteolytic primary bone tumours of uncertain origin containing areas of fibrous and fibro-osseous proliferation. We investigated the nature of the stromal cells in adamantinoma of long bones and osteofibrous dysplasia, and determined cellular and molecular mechanisms of osteolysis in these tumours. Cell culture, molecular (RT-PCR, western blot) and immunohistochemical studies on cases of adamantinoma of long bones and of osteofibrous dysplasia were undertaken to determine the expression of epithelial, osteoblast and osteoclast markers. Ultrastructural and immunophenotypic studies on cultured adamantinoma and osteofibrous dysplasia stromal cells showed that these cells were mainly fibroblast-like with few cells expressing epithelial markers. Osteofibrous dysplasia but not adamantinoma cells expressed alkaline phosphatase. Both osteofibrous dysplasia and adamantinoma cells expressed the ostoclastogenic factors M-CSF and RANKL. Adamantinoma and osteofibrous dysplasia cells also expressed messenger RNA for osteocalcin, osteonectin, osteopontin, osterix and collagen type 1. Adamantinoma and osteofibrous dysplasia cells cultured alone on dentine slices were not capable of lacunar resorption, but in co-cultures with monocytes induced formation of osteoclast-like cells was observered. Cultured osteofibrous dysplasia and adamantinoma stromal cells show similar ultrastructural and immunophenotypic characteristics, and differentially express osteoblast markers. Promotion of osteoclastogenesis by stromal cells may contribute to osteolysis in adamantinoma of long bones and osteofibrous dysplasia.
Abstract: PURPOSE: To review the reliability of MR imaging features for the purpose of distinguishing lipoma and atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL). MATERIALS AND METHODS: A retrospective review of 87 patients with histologically proven lipomatous tumors was performed. All underwent MR imaging, assessing lipomatous content, septation, and nodules. The associations between these features and tumor diagnosis based on morphology and the presence or absence of MDM2 amplification were explored. The age of the patient and the size and location of the lesion were also recorded for statistical analysis. RESULTS: Of the 87 patients, 54 were classified as lipomas and 33 as ALT/WDL. MR identified ALT/WDL with a sensitivity of 90.9 % (CI 74.5-97.6) and a specificity of 37.0 % (CI 24.6-51.3). The positive and negative predictive values were 46.9 % (CI 34.5-59.7) and 86.9 % (CI 65.3-96.6), respectively. The mean age of patients with ALT/WDL was greater (60 years [range 40-83 years]) than those with lipoma (52 years [range 10-79 years]) (p�=�0.025). The mean size of ALT/WDL (18.7 cm [range 5-36 cm]) was significantly greater than lipoma (13.9 cm [range 3-32 cm]) (p�=�0.003). Features that increased the likelihood of ALT/WDL included: patient age over 60 years, maximal lesion dimension over 10 cm, location in lower limb, and presence of non-fatty areas, by a factor of 2.61-6.25 times. CONCLUSIONS: ALT/WDL and lipoma have overlapping MR imaging characteristics. The most reliable imaging discriminators of ALT/WDL were size of lesion and lipomatous content, but due to the overlap in the MRI appearances of lipoma and ALT/WDL, discrimination should be based on molecular pathology rather than imaging.
Abstract: This study assessed whether analysis of MDM2 copy number by fluorescence in situ hybridization (FISH) would help distinguish lipomas from atypical lipomatous tumors, otherwise referred to as well-differentiated liposarcomas, using a commercially available MDM2 FISH kit. 227 lipomatous and 201 non-lipomatous tumors were analyzed to assess its sensitivity and specificity. Of 178 mature lipomatous tumors, 86 were classified histologically as lipoma and 92 as atypical lipomatous tumor. Two of the lipomas harboring MDM2 amplification were reclassified as atypical lipomatous tumors. Overall, 13 atypical lipomatous tumors did not reveal MDM2 or CDK4 amplification, although this was reduced to 12 following analysis of multiple slides. Three of these cases revealed very occasional tumor cells harboring high-level MDM2 amplification, two had a dedifferentiated component, and MDM2 amplification was detected when one tumor recurred. The remaining six cases exhibited reactive/inflammatory features and were reclassified as lipomas. The findings indicate that MDM2 amplification is 93.5% sensitive for diagnosing atypical lipomatous tumor. A total of 2 of the 20 dedifferentiated liposarcomas failed to reveal MDM2 amplification. All atypical lipomatous tumors measured >10 cm, two dedifferentiated liposarcoma presented de novo at <10 cm, and ~50% of lipomas measured >10 cm. Spindle cell lipomas, lipoblastomas, hibernomas and pleomorphic liposarcomas did not reveal MDM2 amplification. Of 201 non-lipomatous tumors, eight revealed MDM2 amplification or multiple faint alphoid 12 signals and were reclassified as dedifferentiated liposarcoma. Multiple faint alphoid 12 signals were observed in nine tumors from seven patients, an observation not previously reported on paraffin sections: these included four atypical lipomatous tumors, and three dedifferentiated liposarcomas, one previously diagnosed as a myxofibrosarcoma, all of which also revealed amplification of CDK4, although two lacked MDM2 amplification. MDM2 FISH test is a useful adjunct to histology for distinguishing lipoma from atypical lipomatous tumor. The limitations of molecular genetic tests must be known before introducing them into a clinical service.
Abstract: Alpha isoform of smooth muscle actin (SMA) expression has been reported in giant cell tumour of bone (GCTB) and other benign and malignant bone tumours, but the pattern of SMA expression and the precise nature of SMA-expressing cells in these lesions is uncertain. We determined by immunohistochemistry the expression of SMA and other muscle and vascular markers in normal bone, GCTB and a wide range of primary benign and malignant bone tumours. Cultured stromal cells of GCTB, chondroblastoma (CB), and aneurysmal bone cyst (ABC) were also analysed for SMA expression. SMA was only noted in blood vessels in normal bone. SMA was expressed by mononuclear stromal cells (MSC) cultured from GCTB, ABC and CB. SMA was strongly and diffusely expressed by MSC in non-ossifying fibroma, fibrous dysplasia, and "brown tumour" of hyperparathyroidism. SMA expression was also noted in GCTB, ABC, CB, chondromyxoid fibroma, malignant fibrous histiocytoma of bone and osteosarcoma. Little or no SMA was noted in Langerhans cell histiocytosis, simple bone cyst, Ewing's sarcoma, osteoblastoma, osteoid osteoma, enchondroma, osteochondroma, chondrosarcoma, myeloma, lymphoma, chordoma and adamantinoma. Our findings show that there is differential SMA expression in primary bone tumours and that identifying the presence or absence of SMA is useful in the differential diagnosis of these lesions. The nature of SMA-expressing cells in bone tumours is uncertain but they are negative for desmin and caldesmon and could represent either myofibroblasts or perivascular cells, such as pericytes.
Abstract: Paget's disease (PD) of the bone is a disorder of bone remodelling that may be polyostotic or monostotic. Although development of a sarcoma in PD is well-recognised, it is less well recognised that pseudosarcomas in bone and soft tissue can also arise in this condition. In this report we document the case of a large giant-cell-rich pseudotumour that developed in the tibia and overlying soft tissues in a case of polyostotic PD. Bone and soft tissues were highly vascular and contained abundant haemorrhage with focal areas of new bone formation and a diffuse infiltrate of osteoclastic giant cells. The lesion has not recurred or produced metastases 3Â years after removal. Clinicians should be aware that a benign giant-cell-rich pseudotumour can develop in PD and that it needs to be distinguished from other giant-cell-rich tumours.
Abstract: Giant cell tumour of bone (GCTB) is a primary bone tumour that contains numerous very large, hyper-nucleated osteoclastic giant cells. Osteoclasts form from CD14+ monocytes and macrophages in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). GCTB contains numerous growth factors, some of which have been reported to influence osteoclastogenesis and resorption. We investigated whether these growth factors are capable of substituting for M-CSF to support osteoclast formation from cultured human monocytes and whether they influence osteoclast cytomorphology and resorption. Vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand (FL), placental growth factor (PlGF) and hepatocyte growth factor (HGF) supported RANKL-induced osteoclastogenesis in the absence of M-CSF, resulting in the formation of numerous TRAP+ multinucleated cells capable of lacunar resorption. Monocytes cultured in the presence of M-CSF, HGF, VEGF-A and RANKL together resulted in the formation of very large, hyper-nucleated (GCTB-like) osteoclasts that were hyper-resorptive. M-CSF and M-CSF substitute growth factors were identified immunohistochemically in GCTB tissue sections and these factors stimulated the resorption of osteoclasts derived from a subset of GCTBs. Our findings indicate that there are growth factors that are capable of substituting for M-CSF to induce human osteoclast formation and that these factors are present in GCTB where they influence osteoclast cytomorphology and have a role in osteoclast formation and resorption activity.
Abstract: Multinucleated cells termed chondroclasts have been observed on the deep surface of resorbed hyaline cartilage but the relationship of these cells to macrophages and osteoclasts and their role in rheumatoid arthritis (RA) and other arthritic conditions is uncertain. Multinucleated cells in RA and other arthritic conditions showing evidence of cartilage resorption were characterised immunohistochemically for expression of macrophage/osteoclast markers. Mature human osteoclasts formed from circulating monocytes and tissue macrophages were cultured for up to 4Â days on slices of human cartilage and glycosaminoglycan (GAG) release was measured. Multinucleated cells resorbing unmineralised cartilage were seen in osteoarthritis, RA, septic arthritis, avascular necrosis and in four cases of giant cell tumour of bone that had extended through the subchondral bone plate. Chondroclasts expressed an osteoclast-like phenotype (TRAP+, cathepsin K+, MMP9+, CD14-, HLA-DR-, CD45+, CD51+ and CD68+). Both macrophages and osteoclasts cultured on cartilage released GAG. These findings indicate that chondroclasts have an osteoclast-like phenotype and that mature human osteoclasts are capable of cartilage matrix resorption. Resorption of unmineralised subchondral cartilage by chondroclasts and macrophages can be a feature of joint destruction in inflammatory and non-inflammatory arthropathies as well as inflammatory and neoplastic subchondral bone lesions.
Abstract: Glomus coccygeum is a glomus body which is found in the pericoccygeal soft tissue. This specialised arteriovenous anastomosis is a non-pathological vestigial structure usually larger than its equivalent in the distal extremities. Its prevalence is uncertain. Glomus coccygeum has been associated with coccygodynia and can cause diagnostic problems to pathologists unfamiliar with this entity.
Abstract: Podoplanin is a type-I transmembrane sialomucin-like protein, which is expressed in a wide range of cell types and is involved in platelet aggregation and tumor metastasis. Here, we investigated the function, regulation, and expression of podoplanin in osteosarcoma. Podoplanin expression was observed in three osteosarcoma cell lines (MG-63, HOS, and U-2 OS) with platelet aggregation-inducing ability, which was blocked by podoplanin small-interfering RNA or a neutralizing antibody. Overexpression of podoplanin in nonmetastatic Dunn osteosarcoma cells promoted cell migration without attenuating cell proliferation. Both podoplanin and TGF-β1 were up-regulated by c-Fos induction in MC3T3-E1 osteoblastic cells, and were highly expressed in c-Fos transgenic mouse osteosarcomas and c-Fos-transformed osteosarcoma cell lines. Immunohistochemistry of human osteosarcoma tissue microarrays (n = 133) showed staining of tumor cells embedded in an excess of irregular neoplastic bone matrix in 100% of tumors undergoing so-called "normalization/maturation." Podoplanin was also expressed in osteosarcoma subtypes, with 65% of osteoblastic, 100% of chondroblastic, and 79% of fibroblastic tumors. CD44 and pERM immunohistochemistry showed coexpression with podoplanin in both mouse and human osteosarcoma. Podoplanin expression was significantly higher in metastatic osteosarcomas (n = 6) than in primary osteosarcomas (n = 10). Our data suggest that podoplanin, which is not expressed in normal osteoblasts but in osteocytes, is aberrantly expressed in transformed osteoblasts and in osteosarcoma, and is under AP-1 transcriptional control. Thus podoplanin is a candidate molecule for therapeutic targeting.
Abstract: S100A4, a 10-12 kDa calcium-binding protein, plays functional roles in tumor progression and metastasis. The present study aimed to investigate the function of S100A4 in osteosarcoma (OS) metastasis, using a loss-of-function approach. Our previous expression profiling analysis revealed that S100a4 was preferentially expressed in the highly metastatic mouse OS cell line, LM8. Introducing a short hairpin ribonucleic acid (shRNA) targeting S100a4 using a newly established vector containing insulators and transposons, we established stable LM8 subclones with almost 100% silencing of endogenous S100a4 protein. These transfectants showed a significant suppression of cell migration in vitro as well as a marked reduction in their ability to colonize the lung and form pulmonary metastases in vivo following intravenous inoculation, whereas there was no significant change in cell proliferation or cell attachment to fibronectin, laminin, and type I collagen. Expression and phosphorylation of ezrin, an emerging OS metastasis-associated factor, and expression of MMPs, remained the same in S100a4-shRNA clones. In 61 human OS, immunohistochemical analysis showed that lesional cells in 85.2% samples expressed S100A4 protein, and the immunoreactivity was primarily cytoplasmic, but it also showed occasional nuclear localization. Chondroblastic and osteoblastic OS subtypes expressed more S100A4 than fibroblastic subtypes. The causative role of S100A4 in OS lung metastasis shown in the murine xenograft model, together with the high proportion of primary human OS expressing S100A4, suggest that S100A4 protein represents an important potential target for future OS therapy.
Abstract: Adamantinoma of long bones (ALB) and osteofibrous dysplasia (OFD) are rare osteolytic bone tumours that principally arise in the tibia. Both ALB and OFD contain epithelial and stromal elements, as well as areas of fibro-osseous proliferation. We assessed expression of podoplanin, a glycoprotein found in osteocytes, in OFD and ALB as well as in fibrous dysplasia and metastatic cancer. Forty-two cases of ALB and OFD, 20 cases of fibrous dysplasia and 20 cases of metastatic carcinoma to bone were stained by immunohistochemistry for expression of podoplanin, epithelial (cytokeratin, epithelial membrane antigen) and vascular (CD34, LYVE-1) markers. Podoplanin was expressed in epithelial cells and tumour glands in ALB as well as in scattered intertrabecular stromal cells in both ALB and OFD. Podoplanin was not expressed by intertrabecular stromal cells in fibrous dysplasia or in metastatic adenocarcinoma. Podoplanin was expressed by osteocytes but not osteoblasts of woven and lamellar bone trabeculae in ALB, OFD, fibrous dysplasia and skeletal metastases. The finding of a common osteocyte marker in OFD/ALB stromal cells is in keeping with a close histogenetic relationship between OFD and ALB; this may reflect the prominence of fibro-osseous proliferation in these tumours. The expression of podoplanin in an osteolytic tumour of the tibia may be useful as a diagnostic discriminant in distinguishing OFD from fibrous dysplasia and ALB from metastatic adenocarcinoma.
Abstract: Pigmented villonodular synovitis (PVNS) is a synovial tumour-like lesion that frequently causes osteolysis. PVNS contains numerous macrophages and osteoclast-like giant cells. In this study, we have analysed the cytochemical and functional characteristics of mononuclear and multinucleated cells in PVNS and determined the cellular and humoral mechanisms underlying giant cell formation and resorption in PVNS. Giant cells and CD14(+) and CD14(-) mononuclear cell populations were isolated from PVNS synovial tissue and cultured alone or in the presence and absence of the osteoclastogenic factors, RANKL and M-CSF. Osteoclast formation and activity was assessed by expression of TRAP and evidence of lacunar resorption. Giant cells in PVNS expressed an osteoclast-phenotype (CD51(+) , TRAP(+) , CD14(-) , HLA-DR(-) ) and were formed only in cultures of mononuclear cells that expressed the macrophage marker CD14. Osteoclast formation required RANKL and occurred in both the presence and absence of exogenous M-CSF. CD14(-) cells in PVNS expressed RANKL. Lacunar resorption by PVNS-derived giant cells was abolished by the addition of the bisphosphonate, zoledronate. Our findings indicate that osteoclasts form by a RANKL-dependent mechanism from CD14(+) mononuclear phagocytes in PVNS. Osteoclast formation occurred even in the absence of exogenous M-CSF, a finding which is in keeping with over-expression of M-CSF playing a pathogenic role in this condition. Anti-osteoclast resorptive treatment may be useful to control osteolysis in PVNS.
Abstract: The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1(tr/tr)) have approximately 50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1(tr/tr) osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1(tr/tr) cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.
Abstract: Fibrous dysplasia is a benign bone disease caused by a mutation in the gene for the stimulatory guanine nucleotide-binding protein Gs alpha, leading to high cyclic adenosine monophosphate levels. Histologically, fibrous dysplasia is characterized by the production of fibrous tissue accompanied by the deposition of ectopic type I collagen and other bone-associated extracellular matrix proteins, as well as by irregular woven intramembranous bone onto which type I collagen-containing Sharpey fibers are often attached. Fibrous dysplasia is also characterized by high expression of c-Fos/c-Jun, known targets for cyclic adenosine monophosphate signaling. In this study, we examined the expression of the bone-related extracellular matrix protein, periostin, and its known receptor, integrin alpha v beta 3 (CD51/61), in normal bones as well as in fibrous dysplasia. Immunohistochemistry and in situ hybridization studies revealed that periostin was expressed in the extracellular matrix during intramembranous but not endochondral ossification, as well as in the fibrous component of fibrous dysplasia; and all cells adjacent to periostin-positive regions expressed CD51/61. Importantly, periostin was abundantly localized to Sharpey fibers. To investigate the contribution of c-Fos, we examined transgenic mice overexpressing c-fos, which develop sclerotic lesions closely resembling those found in fibrous dysplasia. In all lesions, transformed osteoblasts expressed high levels of periostin, whereas normal osteoblasts did not. Our results show that periostin is a novel marker for intramembranous ossification, and is a good candidate as a diagnostic tool and/or a therapeutic target in fibrous dysplasia. Moreover, the Gs alpha-cyclic adenosine monophosphate-c-Fos pathway might represent one mechanism of periostin up-regulation in fibrous dysplasia, resulting in altered collagen fibrillogenesis characteristic of this disease.
Abstract: Acute myocardial infarction (AMI) is a common and lethal heart disease, and the recruitment of fibroblastic cells to the infarct region is essential for the cardiac healing process. Although stiffness of the extracellular matrix in the infarct myocardium is associated with cardiac healing, the molecular mechanism of cardiac healing is not fully understood. We show that periostin, which is a matricellular protein, is important for the cardiac healing process after AMI. The expression of periostin protein was abundant in the infarct border of human and mouse hearts with AMI. We generated periostin(-/-) mice and found no morphologically abnormal cardiomyocyte phenotypes; however, after AMI, cardiac healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness caused by a reduced number of alpha smooth muscle actin-positive cells, impaired collagen fibril formation, and decreased phosphorylation of FAK. These phenotypes were rescued by gene transfer of a spliced form of periostin. Moreover, the inhibition of FAK or alphav-integrin, which blocked the periostin-promoted cell migration, revealed that alphav-integrin, FAK, and Akt are involved in periostin signaling. Our novel findings show the effects of periostin on recruitment of activated fibroblasts through FAK-integrin signaling and on their collagen fibril formation specific to healing after AMI.
Abstract: Periostin is a unique extracellular matrix protein, deposition of which is enhanced by mechanical stress and the tissue repair process. Its significance in normal and neoplastic colon has not been fully clarified yet. Using immunohistochemistry and immunoelectron microscopy with a highly specific monoclonal antibody, periostin deposition was observed in close proximity to pericryptal fibroblasts of colonic crypts. The pericryptal pattern of periostin deposition was decreased in adenoma and adenocarcinoma, preceding the decrease of the number of pericryptal fibroblasts. Periostin immunoreactivity appeared again at the invasive front of the carcinoma and increased along the appearance of cancer-associated fibroblasts. ISH showed periostin signals in cancer-associated fibroblasts but not in cancer cells. Ki-67-positive epithelial cells were significantly decreased in the colonic crypts of periostin-/- mice (approximately 0.6-fold) compared with periostin+/+ mice. In three-dimensional co-culture within type I collagen gel, both colony size and number of human colon cancer cell line HCT116 cells were significantly larger ( approximately 1.5-fold) when cultured with fibroblasts derived from periostin+/+ mice or periostin-transfected NIH3T3 cells than with those from periostin-/- mice or periostin-non-producing NIH3T3 cells, respectively. Periostin is secreted by pericryptal and cancer-associated fibroblasts in the colon, both of which support the growth of epithelial components.
Abstract: After bleeding from trauma or surgery, most of the hematomas undergo spontaneous reabsorption. But, in some rare cases, hematomas persist for long periods as slowly expanding masses for months or years. These hematomas were termed as chronic expanding hematomas. In this report, we describe a case of chronic expanding hematoma with a pseudoaneurysm that underwent surgical biopsy, which led to an increase in its expansion speed.
Abstract: Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis. Aggrus/podoplanin is a platelet aggregation-inducing factor that is up-regulated in a number of human cancers and has been implicated in tumor progression. We studied herein the role of Aggrus in tumor growth, metastasis , and survival in vivo. Aggrus expression in Chinese hamster ovary cells promoted pulmonary metastasis in both an experimental and a spontaneous mouse model. No differences in the size of metastatic foci or in primary tumor growth were found in either set of mice. Aggrus expressing cells , which were covered with platelets, arrested in the lung microvasculature 30 minutes after injection. In addition, lung metastasis resulting from Aggrus expression decreased the survival of the mice. By generating several Aggrus point mutants, we revealed that point mutation at the platelet aggregation-stimulating domain of Aggrus(Thr34 and Thr52) obliterated both platelet aggregation and metastasis. Furthermore, administration of aspirin to mice reduced the number of metastatic foci. These results indicate that Aggrus contributes to the establishment of metastasis by promoting platelet aggregation without affecting subsequent growth. Thus, Aggrus could serve as an ideal therapeutic target for drug development to block metastasis.
Abstract: Renal cell carcinoma (RCC) is one of the most drug-refractory cancers. The aim of this study is to discover a novel therapeutic target molecule for clear cell RCC (CCRCC), which accounts for the majority of RCC.
Abstract: Immunohistochemical analyses with the monoclonal antibody D2-40 were performed to ascertain the expression of podoplanin (a.k.a. T1-alpha, gp36, or aggrus) in tumors of the central nervous system (CNS) and to determine the diagnostic utility of the antibody. The analyses were performed on 325 tumors of various histologic types. The chief finding was almost constant immunoreactivity in ependymal tumors (37/40, 92.5%), choroid plexus papillomas (8/8, 100%), and meningiomas (100/100, 100%). The reactivity was considered "tissue-specific," as the corresponding normal tissue of each tumor was also found to express podoplanin. In addition, expression, not committed to the lineages, was found in many other tumor types, including astrocytic tumors, medulloblastomas, and hemangioblastomas, with variable frequency and intensity. The way of expression was not fully understood, but the expression in astrocytic tumors seemed to be associated with pronounced fibrous properties or malignant phenotype, as was shown by high-frequent expression in pilocytic astrocytomas (12/12, 100%) and glioblastomas (29/35, 82.9%). The present study has shown that podoplanin is expressed in several types of CNS tumors with variable frequency and intensity. Given the widespread expression of podoplanin, the antibody D2-40 is of little use in diagnostic practice for CNS tumors.
Abstract: Glypican 3 (GPC3) is a cell surface heparan sulphate proteoglycan expressed specifically in the fetal liver and malignant neoplasms of hepatocyte lineage. The aim was to evaluate the significance of GPC3 in alpha-fetoprotein (AFP)-producing gastric carcinoma (GC) and other forms of GC.
Abstract: We studied the possibility of using high-intensity focused ultrasound (HIFU) together with a microbubble agent to treat hepatocellular carcinoma. Development of liver tumors in rats was induced by administration of Dimethylnitrosamin (100ppm). Rats with liver tumors were anesthetized, underwent laparotomy, and were given the microbubble agent Levovist or saline intravenously. After the injection, the liver was exposed to HIFU for 30s (2.18MHz, 600W/cm(2), 40mm in diameter). Immediately after HIFU exposure, ultrasound images of the HIFU area were evaluated. Then the liver was excised and the volume of coagulated tissue was measured. The mean volumes of hyperechoic areas after HIFU were as follows (mm(3), Levovist versus saline: 355.3+/-180.7 versus 47.4+/-35.6, P<0.001, n=13). The volumes of liver tissue coagulated by HIFU were as follows (mm(3), Levovist versus saline: 275.3+/-120.0 versus 60.1+/-23.6, P<0.001, n=13). On microscopic examination of areas exposed to HIFU, implosion cysts were seen, and many cancer cells were found to have been destroyed completely (loss of cell membranes or nuclei). In conclusion, the microbubble agent Levovist can increase the volume of tissue coagulated by HIFU.
Abstract: Mucins are secreted or transmembrane glycoproteins that are expressed mainly in the digestive tract. This family of proteins has been the focus of much gastric cancer research as some transmembrane mucins are implicated in tumorigenesis and make attractive targets for cancer diagnosis and therapeutics. Mucins have also been utilized to classify gastric cancer by differentiating between gastric and intestinal phenotypes. Here we show that transmembrane mucin MUC13 is upregulated in gastric cancer. By quantitative real-time reverse transcription-polymerase chain reaction and immunoblot analysis, overexpression of MUC13 was verified in more than half of the samples examined. In immunohistochemical analysis, MUC13 staining was observed in 74 of 114 cases of gastric cancer (64.9%), predominantly in intestinal type (P < 0.001), and in 9 of 10 cases of intestinal metaplasia, precancerous lesions of intestinal-type gastric cancer, but not observed in normal gastric mucosa. Moreover, MUC13 staining patterns characteristic of histological type were identified: staining was on the apical side of tubular glands in intestinal type and on the cytoplasm in diffuse type. These results suggest that MUC13 is a good differentiation marker for gastrointestinal mucosa and that it may have a causal role that correlates with two distinct gastric tumorigenesis pathways.
Abstract: Fukuyama-type congenital muscular dystrophy (FCMD) is characterized by muscular dystrophy and cortical dysgenesis of the cerebrum and cerebellum. We investigated the extent and nature of tauopathy in the brains of 7 postfetal (14-34 years of age) and 2 fetal (18- and 20-week gestational age) FCMD cases. In all postfetal cases, tauopathy was found in the areas of cortical dysgenesis in the cerebrum, in addition to predictable sites such as the hippocampus. In fetal cases, the neuropil of malformed cerebral cortex was diffusely immunostained with anti-aberrantly phosphorylated tau antibodies. By immunoelectron microscopy, the epitope of the antibodies was associated with microtubule-like bundles within cellular processes protruding through disrupted glia limitans. In Western blot analysis, a unique 50-kDa band of tau was detected in a fetal and a postfetal case. In addition, 3 to 4 tau bands of 60 to 68 kD, similar to tau in Alzheimer disease, were also detected in the latter. After dephosphorylation, the insoluble tau from the fetal and the postfetal cases showed highly similar immunoblotting patterns. This anomalous phosphorylation of tau may be related to the development of the cortical dysgenesis in FCMD and may shed light on the biologic function of tau in the development of the central nervous system.
Abstract: Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT-PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P-cadherin, E-cadherin, N-cadherin or cadherin-11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N-cadherin and cadherin-11 localized to the cell membrane and able to bind to beta-catenin. Overexpression of N-cadherin or cadherin-11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N-cadherin- and cadherin-11-overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.-inoculated site. These observations demonstrate that disruption of N-cadherin- and cadherin-11-mediated cell-cell adhesion is critical in the pulmonary metastasis of osteosarcoma.
Abstract: To evaluate the diagnostic value of a halo on computed tomography (CT) in the diagnosis of invasive pulmonary aspergillosis (IPA), we retrospectively reviewed chest CT scans and autopsy reports for patients who had been admitted to our hospitals for the treatment of hematological malignancy. Pulmonary complications were suspected in all patients and chest CT scans were taken within a month of death. We examined the association between autopsy and CT findings in 48 patients who were diagnosed as IPA (n = 17), candidosis (n = 4), zygomycosis (n = 2), infiltration of hematological malignancy (n = 12), bacterial pneumonia (n = 6), cytomegalovirus pneumonia (n = 2), pulmonary hemorrhage (n = 2), or pulmonary congestion (n = 1). Patients with IPA showed a variety of CT findings, including halo (n = 13), nodules (n = 14), granular shadows (n = 3), masses (n = 6), consolidations (n = 9), wedge-shaped consolidations (n = 1), and cavitation (n = 2). In contrast, 0, 11 and two of the 31 patients without IPA showed halo, nodules and masses, respectively. These signs were more frequently observed in IPA patients than in non-IPA patients. The CT halo, especially, seemed to be specific for IPA in hospitalized neutropenic patients with hematological malignancies who developed antibiotic-resistant fever. For CT findings other than these three signs, there were no significant differences between IPA- and non-IPA patients.
Abstract: To investigate the utility of blood culture of invasive fungal infections in patients with haematological malignancies, an autopsy survey was conducted in 720 patients who were treated between 1980 and 1999. We identified 252 patients with invasive mycosis. These included Candida (n = 94), Aspergillus (n = 91), Zygomycetes (n = 34), Cryptococcus (n = 7), Trichosporon (n = 11), Fusarium (n = 1), and unknown fungi (n = 20). Of the 94 patients with invasive candidiasis, 20 had positive blood cultures. Of the 11 patients with invasive trichosporonosis, seven had positive blood cultures. The sensitivities of blood cultures were 1.1%, 0% and 14% for detecting invasive aspergillosis, zygomycosis and cryptococcosis respectively. Multiple regression analysis showed a significant correlation between results of Candida blood cultures and some variables, including prophylactic use of absorbable antifungals (P = 0.0181) and infection by Candida albicans (P = 0.0086). The sensitivity of blood cultures decreased when patients received antifungal chemoprophylaxis. Unless these agents are inactivated in culture bottles, conventional blood cultures might produce false-negative results.
Abstract: Disseminated aspergillus infection has a poor prognosis, but few reports have been published on extra-pulmonary involvement in aspergillosis. We reviewed 107 autopsy records of patients with invasive aspergillosis. Fifty-five patients had extra-pulmonary aspergillosis. Organs involved included heart, kidney, central nervous system, gastrointestinal tract, spleen, liver, thyroid gland and pancreas. Extra-pulmonary aspergillosis produces different manifestations according to involved organs. Risk factors associated with dissemination included cytotoxic chemotherapy within a month of death (P=0.0087). Lack of response to empiric or preemptive treatment of amphotericin B predicted IA dissemination (P=0.0328). To improve prognosis of IA, it is important to recognize clinical features of extra-pulmonary aspergillosis and to institute the aggressive anti-fungal treatment.
Abstract: We developed a new quantitative system for diagnosis of invasive pulmonary aspergillosis (IPA) using real-time automated polymerase chain reaction (PCR). Intra-assay and interassay precision rates for in vitro examination were 2.53% and 2.20%, respectively, and the linearity of this assay was obtained when there were >20 copies/well. We examined 323 samples taken from 122 patients with hematological malignancies, including 33 patients with IPA and 89 control patients. Blood samples were subjected to PCR antigen detection methods, using enzyme-linked immunosorbent assay (ELISA) and determination of plasma (1-->3)-beta-D-glucan (BDG) concentration. The sensitivities of PCR, ELISA, and BDG measurement for diagnosis of IPA were 79%, 58%, and 67%, respectively; the specificities were 92%, 97%, and 84%. Positive findings on PCR preceded those of computed tomography by -0.3+/-6.6 days, those of BDG measurement by 6.5+/-4.9 days, and those of ELISA by 2.8+/-4.1 days. Real-time PCR was sensitive for IPA diagnosis, and quantitation was accurate.
Abstract: Blood and radiologic tests are frequently used for diagnosis of invasive pulmonary aspergillosis, but it remains unknown which is more useful for its early diagnosis. Aim of the study was to compare usefulness of computed tomographic (CT) scan of chest, latex agglutination (LA) test and determination of plasma (1-->3)-beta-D-glucan (BDG) levels for early diagnosis of invasive pulmonary aspergillosis (IPA).
Abstract: Two isoforms of the human cadherin-11/OB-cadherin gene, the intact and the variant forms, had been isolated from an osteosarcoma cDNA library. The intact form has a typical cadherin structure, whereas the variant form, generated by alternative splicing, encodes a cytoplasmic domain that is completely different from that of the intact form and lacks a homophilic cell-cell adhesion ability. At the protein level, the secreted form generated from the intact cadherin-11 is present. We examined the expression of the intact and the variant forms of cadherin-11 in 23 primary and metastatic osteosarcomas from 22 patients by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, revealing that all 23 tumors in the patients expressed the variant form and three of them expressed it prominently. On the other hand, Western blot analyses of six tumors showed that the secreted form was strongly expressed, and furthermore, expression of N-cadherin was extremely low. Overexpression of the intact cadherin-11 cDNA in osteosarcoma cell lines demonstrated that the secreted form is derived from the intact form of cadherin-11 in osteosarcoma. Immunohistochemically, cadherin-11, N-cadherin, and beta-catenin were expressed at the cell surface of fetal osteoblasts, whereas in osteosarcoma cells, they were expressed only focally or weakly in the cytoplasm. Considering the function of cadherin in carcinomas, it is suggested that the anomalous expression of human cadherin-11 in osteosarcoma and the reduced expression of N-cadherin play a role in metastasis and the irregular morphology in the highly malignant mesenchymal tumor.
Abstract: Cadherin-11, a member of the type II classic cadherin subfamily, differs from type I family molecules such as P-, E-, and N-cadherins. An isoform of the human cadherin-11 gene, termed the variant form, encodes a truncated protein with a different cytoplasmic domain. The resulting protein does not possess any part of the cytoplasmic domain common to other cadherins. In the present study, analysis of the genomic organization of the cadherin-11 gene revealed that an insertion of 179 bp in an intron generates an alternatively spliced form. The mRNA expression of the variant form of cadherin-11 was examined in normal tissues by reverse transcription-polymerase chain reaction and/or Northern blot analyses. The variant form was expressed in the heart, brain, placenta, lung, and bone, but not in the kidney, skeletal muscle, pancreas, and liver. Western blot analyses revealed that the variant form is expressed as an 85 kDa protein, and that an additional secreted form also exists as an 80 kDa protein originated from cleavage of the intact form. Gene transfer of the variant form into L cells demonstrated that it lacked the adhesion properties characteristic of the intact form of cadherin-11 but enhanced the activity of Ca2+-dependent adhesion of the intact form of cadherin-11. The variant was expressed on the surface together with the intact form and stabilized the interaction between the intact form and beta-catenin. These findings suggest that expression of the variant form of human cadherin-11 may regulate the intact cadherin-11-mediated adhesion and alter the morphogenetic processes during mesenchymal cell differentiation including osteoblasts.
Abstract: We applied microsatellite analysis to prenatal diagnosis of Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive severe muscular dystrophy associated with brain malformations. Recent identification of the FCMD gene locus at 9q31-q33 provided the basis for prenatal diagnosis and carrier detection. We recently developed new microsatellite markers which are closer to the FCMD gene and improved the phenotype probability. Nine fetuses in eight unrelated FCMD families, including a twin pregnancy, were analysed using the newly developed markers. Four fetuses showed over 99% probability of being healthy either as normal homozygote (n = 1) or heterozygote carrier (n = 3) and were born without signs of FCMD. The other five fetuses were diagnosed with a probability of FCMD of 99% or greater; all of the latter parents decided to terminate the pregnancies. Brain malformations characteristic of FCMD in one of the aborted fetuses confirmed the diagnosis of FCMD at 19 weeks of gestation.
Abstract: A case of a biphasic synovial sarcoma, arising on the inner surface of the anterior abdominal wall of a 13-year-old girl, is reported. Although the tumor showed rather typical histological and immunohistochemical features for synovial sarcoma, its unusual clinical presentation and anatomical location caused diagnostic difficulty, especially with regard to differentiation from a malignant mesothelioma. Applying reverse transcriptase polymerase chain reaction (RT-PCR) analysis, the SYT-SSX2 chimeric gene transcripts that result from the translocation, t(X;18)(p11.2;q11.2), found in most synovial sarcomas could be demonstrated. Thus, this RT-PCR approach is a reliable method for confirming the diagnosis of synovial sarcomas in unusual locations.
