Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells - evidence for a role in hepatocellular carcinoma growth in vivo.

BACKGROUND: Previous studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model. METHODS: PAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca(2+)]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays. RESULTS: Following verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-ß1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen-coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration. CONCLUSION: PAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC.

Neurofibromin specific antibody differentiates malignant peripheral nerve sheath tumors (MPNST) from other spindle cell neoplasms.

Malignant peripheral nerve sheath tumors (MPNST) derive from the Schwann cell or perineurial cell lineage and occur either sporadically or in association with the tumor syndrome neurofibromatosis type 1 (NF1). MPNST often pose a diagnostic challenge due to their frequent lack of pathognomonic morphological or immunohistochemical features. Mutations in the NF1 tumor suppressor gene are found in all NF1-associated and many sporadic MPNST. The presence of NF1 mutation may have the potential to differentiate MPNST from several morphologically similar neoplasms; however, mutation detection is hampered by the size of the gene and the lack of mutational hot spots. Here we describe a newly developed monoclonal antibody binding to the C-terminus of neurofibromin (clone NFC) which was selected for optimal performance in routinely processed formalin-fixed and paraffin-embedded tissue. NFC immunohistochemistry revealed loss of neurofibromin in 22/25 (88 %) of NF1-associated and 26/61 (43 %) of sporadic MPNST. There was a strong association of neurofibromin loss with deletions affecting the NF1 gene (P < 0.01). In a series of 256 soft tissue tumors of different histotypes NFC staining showed loss of neurofibromin in 2/8 myxofibrosarcomas, 2/12 (16 %) pleomorphic liposarcomas, 1/16 (6 %) leiomyosarcomas, and 4/28 (14 %) unclassified undifferentiated pleomorphic sarcomas. However, loss of neurofibromin was not observed in 22 synovial sarcomas, 27 schwannomas, 23 solitary fibrous tumors, 14 low-grade fibromyxoid sarcomas, 50 dedifferentiated liposarcomas, 27 myxoid liposarcomas, 13 angiosarcomas, 9 extraskeletal myxoid chondrosarcomas, and 7 epitheloid sarcomas. Immunohistochemistry using antibody NFC may substantially facilitate sarcoma research and diagnostics.

Proteinase-activated receptor 2 (PAR(2)) in cholangiocarcinoma (CCA) cells: effects on signaling and cellular level.

In this study, we demonstrate functional expression of the proteinase-activated receptor 2 (PAR(2)), a member of a G-protein receptor subfamily in primary cholangiocarcinoma (PCCA) cell cultures. Treatment of PCCA cells with the serine proteinase trypsin and the PAR(2)-selective activating peptide, furoyl-LIGRLO-NH(2), increased migration across a collagen membrane barrier. This effect was inhibited by a PAR(2)-selective pepducin antagonist peptide (P2pal-18S) and it was also blocked with the Met receptor tyrosine kinase (Met) inhibitors SU 11274 and PHA 665752, the MAPKinase inhibitors PD 98059 and SL 327, and the Stat3 inhibitor Stattic. The involvement of Met, p42/p44 MAPKinases and Stat3 in PAR(2)-mediated PCCA cell signaling was further supported by the findings that trypsin and the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated activating phosphorylation of these signaling molecules in cholangiocarcinoma cells. With our results, we provide a novel signal transduction module in cholangiocarcinoma cell migration involving PAR(2)-driven activation of Met, p42/p44 MAPKinases and Stat3.

New PLAG1-fusion transcripts in the spectrum of pediatric fibrotic, lipofibrotic, and mature lipomatous tumors.

The histomorphology of liboblastoma is highly variable and comprises different patterns that are found admixed or in pure form within a tumor. The most important features - mature lipomatous, fibrotic, lipofibrous, and myxoid - overlap with the histomorphology of several other pediatric tumor entities. Regarding the morphologic overlaps, molecular diagnostics with identification of fusion transcripts involving PLAG1 or HMGA2 is essential to identify lipoblastomas. This paper describes the diagnostic procedure in general and two new fusion transcripts of lipoblastoma, MEG3-PLAG1 and COL1A1-PLAG1. In conclusion, the algorithm to diagnose lipoblastomas among this group of pediatric fibrotic, lipofibrous and mature lipomatous tumors essentially includes histomorphology, immunohistochemistry, and molecular diagnostics.

MYC high level gene amplification is a distinctive feature of angiosarcomas after irradiation or chronic lymphedema.

Angiosarcomas (AS) are rare vascular malignancies that arise either de novo as primary tumors or secondary to irradiation or chronic lymphedema. The cytogenetics of angiosarcomas are poorly characterized. We applied array-comparative genomic hybridization as a screening method to identify recurrent alterations in 22 cases. Recurrent genetic alterations were identified only in secondary but not in primary AS. The most frequent recurrent alterations were high level amplifications on chromosome 8q24.21 (50%), followed by 10p12.33 (33%) and 5q35.3 (11%). Fluorescence in situ hybridization analysis in 28 primary and 33 secondary angiosarcomas (31 tumors secondary to irradiation, 2 tumors secondary to chronic lymphedema) confirmed high level amplification of MYC on chromosome 8q24.21 as a recurrent genetic alteration found exclusively in 55% of AS secondary to irradiation or chronic lymphedema, but not in primary AS. Amplification of MYC did not predispose to high grade morphology or increased cell turnover. In conclusion, despite their identical morphology, secondary AS are genetically different from primary AS and are characterized by a high frequency of high level amplifications of MYC. This finding may have implications both for the diagnosis and treatment of these tumors.

Met receptor tyrosine kinase transactivation is involved in proteinase-activated receptor-2-mediated hepatocellular carcinoma cell invasion.

The expression of proteinase-activated receptor (PAR)(2) in human hepatocellular carcinoma (HCC) was established by reverse transcription-polymerase chain reaction, confocal immunofluorescence and electron microscopy in permanent cell lines, primary HCC cell cultures and HCC tumor tissue. Stimulation of HCC cells with trypsin and the PAR(2)-selective activating peptide, 2-furoyl-LIGRLO-NH(2), increased cell invasion across Matrigel. Both effects were blocked by a PAR(2)-selective pepducin antagonist peptide (pal-PAR(2)) and by PAR(2) silencing with specific small interfering RNA (siRNA). PAR(2)-initiated HCC cell invasion was also blocked by inhibiting the hepatocyte growth factor receptor (Met receptor tyrosine kinase) with the receptor-targeted kinase inhibitors, SU 11274 and PHA 665752, or by downregulation of Met with specific siRNA. The involvement of Met in PAR(2)-mediated HCC invasive signaling was further supported by the finding that treatment of HCC cells with trypsin or the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated Met activation-phosphorylation. In addition, Met-dependent stimulation of p42/p44 mitogen-activated protein Kinases was found to be critical for the PAR(2)-Met receptor tyrosine kinase-invasive signaling axis in HCC cells. Our study establishes an important link between the PAR(2) and Met receptor tyrosine kinase signaling in promoting HCC cell invasion.

D2-40 labeling in lymphangiomyoma/lymphangiomyomatosis of the soft tissue: further evidence of lymphangiogenic tumor histogenesis.

We examined ten cases of extrapulmonary lymphangioleiomyoma/lymphangioleiomyomatosis (LAM; all patients female; median age 46.5 years) for immunohistochemical labeling with a monoclonal antibody against podoplanin (D2-40), which is specific for lymphatic endothelial lining. We found positive staining in thin-wall branching vessels reflecting the lymphatic nature of tumor vessels in all cases tested. In contrast, perivascular (HMB-45 positive) myoid cells were not detected by D2-40. The D2-40 labeling confirms the current concept of lymphangiogenic origin of the tumor vessels in LAM. In addition, this study makes a further contribution to the immunohistochemical mapping of this antibody in vascular tumors. Finally, the use of this commercially available antibody provides an additional help in the differential diagnosis of LAM from other soft tissue tumors.

[Fulminate liver failure in a 39-year-old female patient with leukocytosis, unclear fever, and arthralgic pain]. / Fulminantes Leberversagen bei einer 39-jährigen Patientin mit Leukozytose, unklaren Fieberschüben und Arthralgien.

BACKGROUND: Fulminate liver insufficiency can have many causes and is a challenge for differential diagnosis. CASE REPORT: A 39-year-old woman was admitted because of a nonitching macular-papular exanthema on both thighs with spreading to the trunk. In addition, the patient complained of dysphagia, symmetrical arthralgias, myalgias, fever of 38 degrees C, and night sweats. An outpatient treatment with nonsteroidal antirheumatics, antihistamines and penicillin was started for 3 days before admission. On admission, a neutrophilic leukocytosis (23.6 Gpt/l), an increase in C-reactive protein (185 mg/l), and a ferritin level of 1,740 microg/l were found. Liver enzymes were increased (alanine aminotransferase 1.03 micromol/l.s, aspartate aminotransferase 1.06 micromol/l.s, gamma-glutamyltransferase 2.73 micromol/l.s, and lactate dehydrogenase 12.48 micromol/l.s). Sonographic examination showed a mild hepatosplenomegaly, but otherwise normal findings. X-rays of the lungs, hands, and ankles were normal. An echocardiography was within normal limits. Extensive serologic investigations including assays for hepatitides A, B and C as well as repeated blood cultures were negative. Antibiotic therapy was continued without any improvement. In addition, acetaminophen (4 x 1,000 mg/day) and ibuprofen (3 x 600 mg/day) were given. Liver function worsened and an icterus developed. The patient was transferred to the authors' university hospital. Because of the clinical findings of fever episodes, a typical macular exanthema, lymphadenopathy, hepatosplenomegaly, arthralgias, myalgias, dysphagia, and the presence of neutrophilic leukocytosis, fever, an increase in ferritin, but negative serologic titers and no bacteremia, a working diagnosis of Still's disease was made. The patient was treated with high-dose methylprednisone (250 mg/day for 3 days, then 100 mg/day). Liver biopsy revealed subacute hepatitis with necrosis and accompanying cholangitis. The prednisone therapy induced a fast remission and improvement of liver function, liver transplantation was not necessary. The patient is, 16 months after the incident, without symptoms under prednisone 3 mg/day, and the liver function is normal. CONCLUSION: The etiology of Still's disease is unknown and the disease is characterized by fever episodes, a typical macular-papular exanthema, lymphadenopathy, hepatosplenomegaly, and arthralgias. A mild to moderate increase in liver enzymes is often found as part of this disease. Rarely, a fulminate liver failure has been described, particularly in the presence of co-administration of nonsteroidal antirheumatics or acetaminophen. Still's disease must be considered as part of the differential diagnosis of acute liver failure, because an early diagnosis and consequent therapy with prednisone may prevent the need for liver transplantation.

D2-40 staining in sinonasal-type hemangiopericytoma--further evidence of distinction from conventional hemangiopericytoma and solitary fibrous tumor.

D2-40 is a monoclonal antibody, which reacts with a fixative-resistant epitope of lymphatic endothelium. Sinonasal-type hemangiopericytoma (SHP) and tumors of the (conventional) hemangiopericytoma/solitary fibrous tumor family (HP/SFT) are characterized by prominent vasculature. However, data concerning D2-40 labeling of these tumors are very sparse. In the present study, we investigated D2-40 staining in tissue specimens of 17 patients with SHP (male to female ratio of 2.4:1, median age of 63 years) and compared the immunolabeling with 20 cases of HP/SFT, including three SFT cases from nasal mucosa. D2-40 was detected in vascular channels of all SHP patients examined. By contrast, all cases of HP/SFT did not reveal any vascular channel being positive for D2-40, neither in the nasal cases nor in the remaining patients. This study presented for the first time data on D2-40 labeling in a series of SHP, HP/SFT, and supports the distinction of SHP from HP/SFT.

Anthracofibrosis Manifesting as False-Positive Iodine Accumulation in a Patient With Recent History of Thyroid Carcinoma.

A 33-year-old Indonesian woman presented for follow-up after a recent history of papillary thyroid carcinoma treated with total thyroidectomy and radioiodine therapy. A 131I whole-body scintigraphy showed an elongated iodine accumulation in the right hemithorax. On suspicion of pulmonary metastasis, further diagnostics with 124I PET/CT showed thickening of the bronchial wall and retention of secretion in a middle lobe bronchus. Bronchoscopy and histology allowed a diagnosis of stenosing anthracofibrosis with chronic inflammatory changes.

Mammalian target of rapamycin pathway activity in alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a distinct type of soft tissue sarcoma holding a specific ASPL-TFE3 fusion transcript. Curative therapy is based on surgical removal, whereas lately, antiangiogenic targeted therapy regimens have proven effective. In ASPS, analysis of small series additionally display mTOR (mammalian target of rapamycin) pathway activity, thus making mTOR a possible additive target in ASPS, because it is in other tumor entities. Therefore, we systematically evaluated mTOR pathway activity in a large series of ASPS in comparison with soft tissue sarcomas of other differentiation (non-ASPS). Upstream and downstream factors of mTOR signaling and ancillary targets were analyzed in 103 cases (22 ASPS, 81 non-ASPS) by immunohistochemistry mostly using phospho-specific antibodies. TFE3 (transcription factor for immunoglobulin heavy-chain enhancer 3) translocation status was determined by FISH and RT-PCR. All ASPS were positive in TFE3 break-apart FISH and exhibited specific fusion products when RNA was available (type 1: 9x, type 2: 11x), whereas TFE3-immunoreactive non-ASPS did not. In ASPS, TFE3-, cMET-, pAKT T308- (all P < .0001), pp70S6K- (P = .002), and p4EBP1 (P = .087) expression levels were elevated, whereas pAKT S473 was decreased (P < .0001). In addition, ASPS exhibited higher TFE3-, cMET-, pAKT T308-, and pp70S6K- expression levels compared with TFE3-immunopositive non-ASPS sarcomas (all P < .001). We demonstrate elevated mTOR complex 1 (mTORC1) activity in ASPS independent of mTOR complex 2 (mTORC2) activation. mTORC1 activity seems to be related to the existence of ASPL-TFE3 fusion transcripts because TFE3-immunoreactive non-ASPS without ASPL-TFE3 fusion transcripts exhibit significantly lower mTORC1 activation status. Small molecule-based targeting of mTOR might therefore represent a potential mechanism in ASPS alone or in combination with contemporary upstream approaches.

TLE1 is a robust diagnostic biomarker for synovial sarcomas and correlates with t(X;18): analysis of 319 cases.

INTRODUCTION: Genomewide expression profiling has identified a number of genes expressed at higher levels in synovial sarcoma than in other sarcomas. Our objectives in this study were (1) to test whether the differentially expressed gene, Transducin-Like Enhancer of split (TLE1) belonging to the groucho/TLE family, is also distinct on the protein level; (2) to evaluate this biomarker in a series of well-characterised synovial sarcomas on standard, full-sized tissue sections and (3) to correlate the expression of TLE1 with t(X;18) and other established biomarkers. METHODS: Three-hundred and eighty four spindle cell sarcomas from the German consultation and reference centre of soft tissue tumours initially suspected for synovial sarcoma were revisited. Three-hundred and nineteen of these specimens were analysed immunohistochemically using a monoclonal antibody TLE1 and standard, full-sized tissue sections. The nuclear staining was scored semiquantitatively as -, negative; +, weak; ++, moderate and +++, strong positive. Furthermore, 118 specimens among these were further analysed using FISH and/or PCR to detect t(X;18). We correlated the TLE1 expression with the t(X;18) translocation and other established biomarkers (EMA, PanCK, CK7, CD34 and BCL2). RESULTS: TLE1 expression was observed in 96% of the synovial sarcomas (score+, 249/259) and discriminates them from other soft tissue tumours (p<0.001). Multivariate analysis showed that positive TLE1 staining was a statistically independent diagnostic marker. Furthermore molecular analysis showed that t(X;18) was clearly correlated with TLE1 protein expression (p<0.001). CONCLUSIONS: Expression of TLE1 is significantly correlated with t(X;18) and may serve as a new robust diagnostic biomarker in synovial sarcomas and potential therapeutic target.

Soft tissue tumors: new perspectives on classification and diagnosis.

BACKGROUND: In recent years, new tumor entities have been described and previously known tumor types have undergone a reassessment. This article offers an overview of recent developments in the classification and interpretation of soft tissue tumors. METHODS: Selective review of publications from 1990 until 2008 from the literature database of the Consultation and Referral Center for Soft Tissue Tumors in Jena. The current status of the classification and morphological diagnosis of these tumors is described. RESULTS: The description of the biological behavior of soft tissue tumors has become more detailed with the introduction of two intermediate categories ("intermediate, locally aggressive" and "intermediate, rarely metastasizing"). There have also been some changes in terminology. Previously established terms such as "malignant fibrous histiocytoma" or "hemangiopericytoma" will be used much less often in future, because these tumor types have been reinterpreted. The WHO recommends that highly differentiated liposarcoma be renamed "atypical lipomatous tumor." Molecular diagnostic techniques have become firmly established as an ancillary diagnostic method. The importance of molecular tumor characterization for individually tailored therapy is already becoming clear. CONCLUSIONS: Optimal diagnosis is the prerequisite for effective therapy and can be achieved only with state-of-the-art knowledge of the pathology of soft tissue tumors.

mRNA expression and protein distribution of the unspliced tenascin-C isoform in prostatic adenocarcinoma.

The inclusion or omission of the alternatively spliced region in the tenascin-C (Tn-C) mRNA gives rise to the large (Tn-C(L)) or small (Tn-C(S)) variant, respectively. Tn-C(L) is thought to be a typical component of provisional extracellular matrices (ECMs) and is expressed during tumour stroma remodelling in cancer. Tn-C(L) mRNA expression and protein distribution have been studied in 44 prostatic adenocarcinomas using RNA/RNA in situ hybridization supplemented by reverse transcriptase-polymerase chain reaction (RT-PCR), and immunohistochemistry (clone BC-2). While the Tn-C(L) protein was demonstrated within tumour stroma, Tn-C(L) mRNA expression was mainly observed in carcinoma cells, regardless of the histological grade of the tumour. Carcinoma cells containing Tn-C(L) mRNA were particularly localized at the tumour invasion front. Tn-C(L) mRNA was also identified in benign prostatic hyperplasia, where it was present exclusively in the basal cell layer, and in prostatic intraepithelial neoplasia in which there was partial loss of positive basal cells and increasing positivity of luminal cells. Furthermore, newly formed tumour blood vessels and inflammatory and stromal cells take part in the expression of Tn-C(L) and are involved in the formation of a provisional tumour matrix. It is concluded that deposits of Tn-C(L) indicate rebuilding processes in non-neoplastic as well as in neoplastic prostatic tissues. In respect of the Tn-C(L) synthesis in budding prostatic carcinoma cells, the results demonstrate that tumour cells can directly produce the ECM components of carcinoma stroma, creating conditions that facilitate the process of invasion.