Kathrin Katenkamp

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.

Low‐grade fibrosarcoma—report on 39 not otherwise specified cases and comparison with defined low‐grade fibrosarcoma types

Aims:  Low‐grade fibrosarcomas are tumours that mainly affect the extremities and trunk of adults of either sex. Among these, low‐grade fibromyxoid sarcoma (FMS), hyalinizing spindle cell tumour with giant collagen rosettes (HST) and sclerosing epithelioid fibrosarcoma (SEF) are well‐established entities. In this study, our aim was to describe a group of low‐grade fibrosarcomatous tumours, which could not be encompassed by these entities. These low‐grade fibrosarcomas, not otherwise specified (FNOS) were provisionally designated as ‘fibrosarcoma, low‐grade fibroblastic type’.

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.

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.

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

BackgroundPrevious 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.MethodsPAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca2+]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.ResultsFollowing 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-β1 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.ConclusionPAR2 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.

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 (PAR2), 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 PAR2-selective activating peptide, furoyl-LIGRLO-NH2, increased migration across a collagen membrane barrier. This effect was inhibited by a PAR2-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 PAR2-mediated PCCA cell signaling was further supported by the findings that trypsin and the PAR2-selective agonist peptide, 2-furoyl-LIGRLO-NH2, 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 PAR2-driven activation of Met, p42/p44 MAPKinases and Stat3.

Extraadrenal Biatrial Cardiac Paraganglioma: Diagnosis, Histological Criteria and Surgical Management

Fig 2. Pthat originate from the neural crest. We report a case of successful surgical treatment of a noncatecholamineproducing cardiac paraganglioma in a 25-year-old white male patient with dyspnea on exertion and intermittent palpitations. Echocardiography revealed solid 4.64.3-cm tumor mass located in close relation to the left and right atria. A magnetic resonance image of the heart demonstrated the solid tumor mass adjacent mainly to the right atrium and in part to the left atrium with an early and strong arterial uptake of the contrast medium (see arrow in Fig 1). A coronary angiogram revealed a feeding blood supply primarily by the right and also left coronary artery systems. The highly vascularized tumor was located mainly in the epicardium with infiltrative growth. The intraoperative situs showed a large, primary epicardially capsulated tumor between the right and left atria compressing the inferior vena cava and the right upper pulmonary vein. The tumor was removed en bloc after clipping and oversewing the feeding vessels of the proximal right coronary artery and the first marginal