Publications by authors named "Gunhild Mechtersheimer"

116 Publications

Malignant transformation of uterine leiomyoma to myxoid leiomyosarcoma after morcellation associated with rearrangement and loss of 14q.

Oncotarget 2018 Jun 12;9(45):27595-27604. Epub 2018 Jun 12.

Institute of Medical Genetics, University Rostock Medical Center, Rostock D-18057, Germany.

A 50 year old woman underwent laparoscopic supracervical hysterectomy because of symptomatic fibroids. Histologic examination of samples obtained after morcellation revealed typical uterine leiomyomas in all samples investigated. 28 and 47 months later, respectively, the patient presented with peritoneal spreading of nodules that were surgically removed and histologically classified as leiomyosarcoma. In 3/4 of samples obtained after morcellation copy number/SNP-array hybridization showed complex genomic alterations widely identical to the pattern characterizing the sarcoma. Therefore, we conclude that the leiomyosarcoma had unambiguously developed from one of the leiomyomas as a result of secondary genetic alterations i.e. a rearrangement of and a del(14q). The case is challenging the current risk estimates for spreading of unexpected malignant uterine tumors due to power morcellation and highlights the relevance of certain genetic alterations for rare malignant transformation of uterine benign smooth muscle tumors.
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http://dx.doi.org/10.18632/oncotarget.25137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021249PMC
June 2018

Primary intracranial spindle cell sarcoma with rhabdomyosarcoma-like features share a highly distinct methylation profile and DICER1 mutations.

Acta Neuropathol 2018 08 7;136(2):327-337. Epub 2018 Jun 7.

Department of General Pathology, Institute of Pathology, Heidelberg University Hospital, Heidelberg, Germany.

Patients with DICER1 predisposition syndrome have an increased risk to develop pleuropulmonary blastoma, cystic nephroma, embryonal rhabdomyosarcoma, and several other rare tumor entities. In this study, we identified 22 primary intracranial sarcomas, including 18 in pediatric patients, with a distinct methylation signature detected by array-based DNA-methylation profiling. In addition, two uterine rhabdomyosarcomas sharing identical features were identified. Gene panel sequencing of the 22 intracranial sarcomas revealed the almost unifying feature of DICER1 hotspot mutations (21/22; 95%) and a high frequency of co-occurring TP53 mutations (12/22; 55%). In addition, 17/22 (77%) sarcomas exhibited alterations in the mitogen-activated protein kinase pathway, most frequently affecting the mutational hotspots of KRAS (8/22; 36%) and mutations or deletions of NF1 (7/22; 32%), followed by mutations of FGFR4 (2/22; 9%), NRAS (2/22; 9%), and amplification of EGFR (1/22; 5%). A germline DICER1 mutation was detected in two of five cases with constitutional DNA available. Notably, none of the patients showed evidence of a cancer-related syndrome at the time of diagnosis. In contrast to the genetic findings, the morphological features of these tumors were less distinctive, although rhabdomyoblasts or rhabdomyoblast-like cells could retrospectively be detected in all cases. The identified combination of genetic events indicates a relationship between the intracranial tumors analyzed and DICER1 predisposition syndrome-associated sarcomas such as embryonal rhabdomyosarcoma or the recently described group of anaplastic sarcomas of the kidney. However, the intracranial tumors in our series were initially interpreted to represent various tumor types, but rhabdomyosarcoma was not among the typical differential diagnoses considered. Given the rarity of intracranial sarcomas, this molecularly clearly defined group comprises a considerable fraction thereof. We therefore propose the designation "spindle cell sarcoma with rhabdomyosarcoma-like features, DICER1 mutant" for this intriguing group.
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http://dx.doi.org/10.1007/s00401-018-1871-6DOI Listing
August 2018

Widespread expression of perilipin 5 in normal human tissues and in diseases is restricted to distinct lipid droplet subpopulations.

Cell Tissue Res 2018 Oct 11;374(1):121-136. Epub 2018 May 11.

Department of General Pathology, Institute of Pathology, University Hospital, Heidelberg, Germany.

Diseases associated with the accumulation of lipid droplets are increasing in western countries. Lipid droplet biogenesis, structure and degradation are regulated by proteins of the perilipin family. Perilipin 5 has been shown to regulate basal lipolysis in oxidative tissues. We examine perilipin 5 in normal human tissues and in diseases using protein biochemical and microscopic techniques. Perilipin 5 was constitutively located at small lipid droplets in skeletal myocytes, cardiomyocytes and brown adipocytes. In addition, perilipin 5 was detected in the epithelia of the gastrointestinal and urogenital tract, especially in hepatocytes, the mitochondria-rich parietal cells of the stomach, tubular kidney cells and ductal cells of the salivary gland and pancreas. Granular cytoplasmic expression, without a lipid droplet-bound localization was detected elsewhere. In cardiomyopathies, in skeletal muscle diseases and during hepatocyte steatogenesis, perilipin 5 was upregulated and localized to larger and more numerous lipid droplets. In steatotic human hepatocytes, perilipin 5 was moderately increased and colocalized with perilipins 1 and 2 but not with perilipin 3 at lipid droplets. In liver diseases implicated in alterations of mitochondria, such as mitochondriopathies, alcoholic liver disease, Wilson's disease and acute liver injury, perilipin 5 was frequently localized to small lipid droplets and less in the cytoplasm. In tumorigenesis, perilipin 5 was especially upregulated in lipo-, leio- and rhabdomyosarcoma and hepatocellular and renal cell carcinoma. In summary, our study provides evidence that perilipin 5 is not restricted to certain cell types but localizes to distinct lipid droplet subpopulations reflecting a possible function in oxidative energy supply in normal tissues and in diseases.
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http://dx.doi.org/10.1007/s00441-018-2845-7DOI Listing
October 2018

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information.

Mod Pathol 2018 08 23;31(8):1246-1256. Epub 2018 Mar 23.

Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed "Ewing-like sarcomas" or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.
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http://dx.doi.org/10.1038/s41379-018-0045-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484949PMC
August 2018

Spontaneous Regression of Primary Cutaneous Diffuse Large B-cell Lymphoma, Leg Type.

Acta Derm Venereol 2018 Jun;98(6):608-609

Department of Dermatology, Venerology, and Allergology, University Medical Center, Ruprecht-Karls-University Heidelberg, DE-69120 Heidelberg, Germany.

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http://dx.doi.org/10.2340/00015555-2921DOI Listing
June 2018

Intraoperative Electron Radiation Therapy in Retroperitoneal Sarcoma.

Int J Radiat Oncol Biol Phys 2018 02 31;100(2):516-527. Epub 2017 Oct 31.

Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany.

Purpose: To report our experience with surgery, intraoperative radiation therapy (IORT), and external beam radiation therapy (EBRT) in retroperitoneal soft-tissue sarcoma.

Methods And Materials: We conducted a retrospective evaluation of 156 patients (69 primary, 87 recurrent) treated with IORT since 1991. The dominant histology was dedifferentiated liposarcoma (49%); 89% of lesions were high grade. Median tumor size was 11 cm. Surgery resulted in gross complete resection in 92%, and 65% had microscopically positive margins. Median IORT dose was 15 Gy. A total of 114 patients (73%) received additional EBRT (preoperatively n=38, postoperatively n=76, median dose 45 Gy).

Results: Median follow-up was 38 months (49 months in survivors). The 3- and 5-year local control (LC) rates were 57% and 50%, respectively. On univariate analysis, LC was significantly associated with primary versus recurrent status, histology, grade, Union for International Cancer Control (UICC) stage, resection margin, and addition of EBRT. The 5-year LC was 71% in the primary situation and 79% after R0 resection. On multivariate analysis only disease status, grade, resection margin, and addition of EBRT remained statistically significant. The 3- and 5-year overall survival (OS) rates were 66% and 56%. On univariate analysis, OS was significantly associated with primary versus recurrent status, histology, grade, UICC stage, resection margin, and timing of EBRT. The 5-year OS was 63% in the primary situation and 68% after R0 resection. On multivariate analysis only disease status, grade, and resection margin remained independent prognostic factors. Perioperative mortality was 1%, and major complications occurred in 34% (mainly wound complications).

Conclusions: Treatment with surgery, IORT, and EBRT is feasible and resulted in good LC and OS, with acceptable morbidity in this unfavorable patient cohort. Incomplete resection and recurrent status resulted in clearly inferior outcomes. Reasonable efforts should be made during primary treatment to prevent the onset of a local recurrence.
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http://dx.doi.org/10.1016/j.ijrobp.2017.10.034DOI Listing
February 2018

Integrative genomic and transcriptomic analysis of leiomyosarcoma.

Nat Commun 2018 01 10;9(1):144. Epub 2018 Jan 10.

Department of Orthopedics, Heidelberg University Hospital, 69118, Heidelberg, Germany.

Leiomyosarcoma (LMS) is an aggressive mesenchymal malignancy with few therapeutic options. The mechanisms underlying LMS development, including clinically actionable genetic vulnerabilities, are largely unknown. Here we show, using whole-exome and transcriptome sequencing, that LMS tumors are characterized by substantial mutational heterogeneity, near-universal inactivation of TP53 and RB1, widespread DNA copy number alterations including chromothripsis, and frequent whole-genome duplication. Furthermore, we detect alternative telomere lengthening in 78% of cases and identify recurrent alterations in telomere maintenance genes such as ATRX, RBL2, and SP100, providing insight into the genetic basis of this mechanism. Finally, most tumors display hallmarks of "BRCAness", including alterations in homologous recombination DNA repair genes, multiple structural rearrangements, and enrichment of specific mutational signatures, and cultured LMS cells are sensitive towards olaparib and cisplatin. This comprehensive study of LMS genomics has uncovered key biological features that may inform future experimental research and enable the design of novel therapies.
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http://dx.doi.org/10.1038/s41467-017-02602-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762758PMC
January 2018

Histone 3.3 hotspot mutations in conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases.

Clin Sarcoma Res 2017 4;7. Epub 2017 May 4.

Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.

Background: Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown.

Methods: In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes and . H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and G34W/L mutant GCTBs.

Results: Six osteosarcomas (6/106) carried hotspot mutations. No mutations were found in . All patients with mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in mutant osteosarcomas. Unlike a single osteosarcoma with a K27M mutation, the DNA methylation profiles of G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between G34W/R mutant and H3.3 wild-type osteosarcomas were in (p < 0.00005) and (p < 0.0005).

Conclusions: H3.3 mutations in osteosarcomas may occur in at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying G34W/R mutations are associated with epigenetic dysregulation of and .
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http://dx.doi.org/10.1186/s13569-017-0075-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418758PMC
May 2017

Predictive and prognostic factors associated with soft tissue sarcoma response to chemotherapy: a subgroup analysis of the European Organisation for Research and Treatment of Cancer 62012 study.

Acta Oncol 2017 Jul 21;56(7):1013-1020. Epub 2017 Apr 21.

d Royal Marsden Hospital , London , UK.

Background: The European Organization for Research and Treatment of Cancer (EORTC) 62012 study was a Phase III trial of doxorubicin versus doxorubicin-ifosfamide chemotherapy in 455 patients with advanced soft tissue sarcoma (STS). Analysis of the main study showed that combination chemotherapy improved tumor response and progression-free survival, but differences in overall survival (OS) were not statistically significant. We analyzed factors prognostic for tumor response and OS, and assessed histological subgroup and tumor grade as predictive factors to identify patients more likely to benefit from combination chemotherapy.

Methods: Central pathology review was performed by six reference pathologists. Gender, age, performance status, time from first presentation with sarcoma to starting palliative chemotherapy, tumor grade, histological subgroup, primary tumor site involvement, and sites of metastases were assessed as prognostic factors.

Results: Three hundred and ten patients were included in this study. Discordance between local and central pathology opinion of tumor histology and tumor grade was observed in 98 (32%) and 122 (39%) cases, respectively. In multivariate analysis, liposarcoma patients had improved tumor response compared to other histological subgroups, whilst patients with metastases other than lung, liver or bone had a poorer response [odds ratio (OR) 0.42, 95% confidence interval (CI) 0.23-0.78; p = 0.006]. Patients with bone metastases had reduced OS [hazard ratio (HR) 1.56, 95% CI 1.16-2.09; p = 0.003]. By central pathology review, patients with undifferentiated pleomorphic sarcoma (UPS) had improved tumor response and OS with doxorubicin-ifosfamide compared to single-agent doxorubicin (OR 9.90, 95% CI 1.93-50.7 and HR 0.44, 95% CI 0.26-0.79, respectively). Grade III tumors had improved response with combination chemotherapy but there was no interaction between chemotherapy and grade on OS.

Conclusions: Prospective central pathology review of tumor histology should be integrated into future STS clinical trials. Doxorubicin-ifosfamide may be most appropriate for young, fit patients with poorly differentiated Grade III tumors including UPS.
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http://dx.doi.org/10.1080/0284186X.2017.1315173DOI Listing
July 2017

PD-L1 (CD274) copy number gain, expression, and immune cell infiltration as candidate predictors for response to immune checkpoint inhibitors in soft-tissue sarcoma.

Oncoimmunology 2017;6(3):e1279777. Epub 2017 Jan 27.

German Cancer Consortium (DKTK), partner sites Heidelberg and Berlin, and German Cancer Research Center (DKFZ), Heidelberg, Germany; Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Soft-tissue sarcomas (STS) are rare malignancies that account for 1% of adult cancers and comprise more than 50 entities. Current therapeutic options for advanced-stage STS are limited. Immune checkpoint inhibitors targeting the PD-1/PD-L1 signaling axis are being explored as new treatment modality in STS; however, the determinants of response to these agents are largely unknown. Using the sarcoma data set of The Cancer Genome Altas (TCGA) and an independent cohort of untreated high-grade STS, we analyzed DNA copy number status and mRNA expression of in a total of 335 STS cases. Copy number gains (CNG) were detected in 54 TCGA cases (21.1%), of which 21 (8.2%) harbored focal CNG and that were most prevalent in myxofibrosarcoma (35%) and undifferentiated pleomorphic sarcoma (34%). In the untreated high-grade STS cohort, we detected CNG in six cases (7.6%). Analysis of co-amplified genes identified a 5.6-Mb core region comprising 27 genes, including . Patients with CNG had higher expression compared with STS without CNG (fold change, 1.8; = 0.02), an effect that was most pronounced in the setting of focal CNG (fold change, 3.0; = 0.0027). STS with CNG showed a significantly higher mutational load compared with tumors with a diploid locus (median number of mutated genes; 58 vs. 40; = 3.6E-06), and CNG were associated with inferior survival (HR = 1.82; = 0.025). In contrast, T-cell infiltrates quantified by mRNA expression of were associated with improved survival (HR = 0.88; = 0.024) and consequently influenced the prognostic power of CNG, with low levels conferring poor survival in cases with CNG (HR = 1.8; = 0.049). These data demonstrate that GNG and elevated expression of occur in a substantial proportion of STS, have prognostic impact that is modulated by T-cell infiltrates, and thus warrant investigation as response predictors for immune checkpoint inhibition.
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http://dx.doi.org/10.1080/2162402X.2017.1279777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384369PMC
January 2017

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

Oncotarget 2016 10;7(41):66344-66359

Center for Medical Genetics Ghent, Ghent University, De Pintelaan, Ghent, Belgium.

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.
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http://dx.doi.org/10.18632/oncotarget.11662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341807PMC
October 2016

Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma.

Clin Cancer Res 2017 Feb 17;23(4):962-973. Epub 2016 Aug 17.

Department of Translational Oncology, National Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Altered FGFR1 signaling has emerged as a therapeutic target in epithelial malignancies. In contrast, the role of FGFR1 in soft-tissue sarcoma (STS) has not been established. Prompted by the detection and subsequent therapeutic inhibition of amplified FGFR1 in a patient with metastatic leiomyosarcoma, we investigated the oncogenic properties of FGFR1 and its potential as a drug target in patients with STS. The frequency of amplification and overexpression, as assessed by FISH, microarray-based comparative genomic hybridization and mRNA expression profiling, SNP array profiling, and RNA sequencing, was determined in three patient cohorts. The sensitivity of STS cell lines with or without FGFR1 alterations to genetic and pharmacologic FGFR1 inhibition and the signaling pathways engaged by FGFR1 were investigated using viability assays, colony formation assays, and biochemical analysis. Increased copy number was detected in 74 of 190 (38.9%; cohort 1), 13 of 79 (16.5%; cohort 2), and 80 of 254 (31.5%; cohort 3) patients. overexpression occurred in 16 of 79 (20.2%, cohort 2) and 39 of 254 (15.4%; cohort 3) patients. Targeting of FGFR1 by RNA interference and small-molecule inhibitors (PD173074, AZD4547, BGJ398) revealed that the requirement for FGFR1 signaling in STS cells is dictated by FGFR1 expression levels, and identified the MAPK-ERK1/2 axis as critical FGFR1 effector pathway. These data identify as a driver gene in multiple STS subtypes and support FGFR1 inhibition, guided by patient selection according to the FGFR1 expression and monitoring of MAPK-ERK1/2 signaling, as a therapeutic option in this challenging group of diseases. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-0860DOI Listing
February 2017

Vorinostat in refractory soft tissue sarcomas - Results of a multi-centre phase II trial of the German Soft Tissue Sarcoma and Bone Tumour Working Group (AIO).

Eur J Cancer 2016 09 28;64:74-82. Epub 2016 Jun 28.

Department of Hematology, Oncology, and Rheumatology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Electronic address:

Introduction: New treatment options for patients with metastatic Soft Tissue Sarcoma are urgently needed. Preclinical studies suggested activity of vorinostat, a histone deacetylase inhibitor.

Methods: A multi-centre, open-label, non-randomised phase II trial to investigate the efficacy and safety of vorinostat in patients with locally advanced or metastatic Soft Tissue Sarcoma failing 1st-line anthracycline-based chemotherapy was initiated. Patients were treated with vorinostat 400 mg po qd for 28 d followed by a treatment-free period of 7 d, representing a treatment cycle of 5 weeks. Restaging was performed every three cycles or at clinical progression.

Results: Between 06/10 and 09/13, 40 Soft Tissue Sarcoma patients were treated with vorinostat at seven participating centres. Patients had received 1 (n=8, 20%), 2 (n=10, 25%) or ≥3 (n=22, 55%) previous lines of chemotherapy. Best response after three cycles of treatment was stable disease (n=9, 23%). Median progression-free survival and overall survival were 3.2 and 12.3 months, respectively. Six patients showed long-lasting disease stabilisation for up to ten cycles. Statistical analyses failed to identify baseline predictive markers in this subgroup. Major toxicities (grade ≥III) included haematological toxicity (n=6, 15%) gastrointestinal disorders (n=5, 13%), fatigue (n=4, 10%), musculoskeletal pain (n=4, 10%), and pneumonia (n=2, 5%).

Conclusion: In a heavily pre-treated patient population, objective response to vorinostat was low. However, a small subgroup of patients had long-lasting disease stabilisation. Further studies aiming to identify predictive markers for treatment response as well as exploration of combination regimens are warranted.

Trial Registration: NCT00918489 (ClinicalTrials.gov) EudraCT-number: 2008-008513-19.
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http://dx.doi.org/10.1016/j.ejca.2016.05.018DOI Listing
September 2016

Methylation-based classification of benign and malignant peripheral nerve sheath tumors.

Acta Neuropathol 2016 06 8;131(6):877-87. Epub 2016 Feb 8.

Department of Neuropathology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.

The vast majority of peripheral nerve sheath tumors derive from the Schwann cell lineage and comprise diverse histological entities ranging from benign schwannomas and neurofibromas to high-grade malignant peripheral nerve sheath tumors (MPNST), each with several variants. There is increasing evidence for methylation profiling being able to delineate biologically relevant tumor groups even within the same cellular lineage. Therefore, we used DNA methylation arrays for methylome- and chromosomal profile-based characterization of 171 peripheral nerve sheath tumors. We analyzed 28 conventional high-grade MPNST, three malignant Triton tumors, six low-grade MPNST, four epithelioid MPNST, 33 neurofibromas (15 dermal, 8 intraneural, 10 plexiform), six atypical neurofibromas, 43 schwannomas (including 5 NF2 and 5 schwannomatosis associated cases), 11 cellular schwannomas, 10 melanotic schwannomas, 7 neurofibroma/schwannoma hybrid tumors, 10 nerve sheath myxomas and 10 ganglioneuromas. Schwannomas formed different epigenomic subgroups including a vestibular schwannoma subgroup. Cellular schwannomas were not distinct from conventional schwannomas. Nerve sheath myxomas and neurofibroma/schwannoma hybrid tumors were most similar to schwannomas. Dermal, intraneural and plexiform neurofibromas as well as ganglioneuromas all showed distinct methylation profiles. Atypical neurofibromas and low-grade MPNST were indistinguishable with a common methylation profile and frequent losses of CDKN2A. Epigenomic analysis finds two groups of conventional high-grade MPNST sharing a frequent loss of neurofibromin. The larger of the two groups shows an additional loss of trimethylation of histone H3 at lysine 27 (H3K27me3). The smaller one retains H3K27me3 and is found in spinal locations. Sporadic MPNST with retained neurofibromin expression did not form an epigenetic group and most cases could be reclassified as cellular schwannomas or soft tissue sarcomas. Widespread immunohistochemical loss of H3K27me3 was exclusively seen in MPNST of the main methylation cluster, which defines it as an additional useful marker for the differentiation of cellular schwannoma and MPNST.
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http://dx.doi.org/10.1007/s00401-016-1540-6DOI Listing
June 2016

Intraoperative electron radiation therapy combined with external beam radiation therapy and limb sparing surgery in extremity soft tissue sarcoma: a retrospective single center analysis of 183 cases.

Radiother Oncol 2016 Apr 1;119(1):22-9. Epub 2015 Dec 1.

Department of Radiation Oncology, University of Heidelberg, Germany. Electronic address:

Background And Purpose: To report our experience with limb-sparing surgery, IOERT and EBRT in extremity STS.

Materials And Methods: 183 patients were retrospectively analyzed. 78% presented in primary situation, with 80% located in the lower limb. Stage at presentation was: I: 6%, IIa: 25%, IIb: 21%, III: 42%, IV: 7%. The majority showed high-grade lesions (grade 1: 5%, 2: 31%, 3: 64%). IOERT was applied to the tumor bed (median 15Gy) and preceded (9%) or followed (91%) by EBRT (median 45Gy) in all patients.

Results: Median follow-up was 64months (78months in survivors). Surgery was complete in 68%, while 32% had microscopic residual disease. 5- and 10-year-LC was 86% and 84%, respectively. LC was significantly higher in primary compared to recurrent disease and tended to be higher after complete resection. Estimated 5- and 10-year-DC was 68% and 66%, while corresponding OS was 77% and 66%, respectively. OS was significantly affected by grading and stage. Severe postoperative complications and late toxicities were observed in 19% and 20%, respectively. Limb-preservation rate was 95% with good function in 83%.

Conclusions: Combination of limb-sparing surgery, IOERT and EBRT achieved encouraging LC and OS in this unfavorable patient group with acceptable postoperative complications and low rates of late toxicities resulting in a high limb-preservation rate and good functional outcome.
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http://dx.doi.org/10.1016/j.radonc.2015.11.014DOI Listing
April 2016

Differential nuclear ATRX expression in sarcomas.

Histopathology 2016 Apr 21;68(5):738-45. Epub 2015 Oct 21.

Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Aim: Nuclear α-thalassemia/mental retardation X-linked (ATRX) loss and alternative lengthening of telomeres (ALT) are linked in distinct malignancies. We therefore aimed to determine the nuclear ATRX expression correlated with ALT in a comprehensive series of sarcomas.

Methods And Results: A total of 573 formalin-fixed paraffin-embedded sarcomas comprising 28 entities were investigated for nuclear ATRX expression by immunohistochemistry. Telomere-specific fluorescence in-situ hybridization (FISH) was used to determine the ALT phenotype in 50 sarcomas with complete or heterogeneous ATRX loss. Complete nuclear ATRX loss was detected in 58 of 573 sarcomas, all high-grade, with the highest prevalence in undifferentiated pleomorphic sarcomas (38%) and pleomorphic liposarcomas (38%), followed by dedifferentiated liposarcomas (24%), osteosarcomas (21%), leiomyosarcomas (17%), myxofibrosarcomas (11%) and malignant peripheral nerve sheath tumours (4%). Interestingly, a further 20 sarcomas, all belonging to the aforementioned entities with complete ATRX loss, presented with a heterogeneous ATRX expression pattern. ALT was observed in 41 of 42 sarcomas with complete ATRX loss, but only in two of eight sarcomas with heterogeneous expression.

Conclusion: Nuclear ATRX loss, either complete or heterogeneous, is encountered in a considerable number of high-grade sarcomas with non-specific genetic alterations. A causal relationship with ALT might be indicated at least in cases with a complete nuclear ATRX loss.
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http://dx.doi.org/10.1111/his.12812DOI Listing
April 2016

SRC inhibition represents a potential therapeutic strategy in liposarcoma.

Int J Cancer 2015 Dec 6;137(11):2578-88. Epub 2015 Jul 6.

Department of Pathology, University Hospital Cologne, Cologne, Germany.

Liposarcomas (LS) are the most common malignant mesenchymal tumors, with an overall long-term mortality rate of 60%. LS comprise three major subtypes, i.e., well-differentiated/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma (MLS) and pleomorphic liposarcoma (PLS). Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional significance of SRC in primary human LS and in LS-derived cell lines. Immunohistochemical and Western blot analyses reveal relevant levels of activated p-(Tyr416)-SRC in LS of the different subtypes with particular activation in MLS and PLS. Dysregulation of the SRC modifiers CSK and PTP1B was excluded as major reason for the activation of the kinase. Consistent siRNA-mediated knockdown of SRC or inhibition by the SRC inhibitor Dasatinib led to decreased proliferation of LS cell lines of the different subtypes, with MLS cells reacting particularly sensitive in MTT assays. Flow cytometric analyses revealed that this effect was due to a significant decrease in mitotic activity and an induction of apoptosis. SRC inhibition by Dasatinib resulted in dephosphorylation of SRC itself, its interacting partners FAK and IGF-IR as well as its downstream target AKT. Consistent with a particular role of SRC in cell motility, Dasatinib reduced the migratory and invasive potential of MLS cells in Boyden chamber and Matrigel chamber assays. In summary, we provide evidence that SRC activation plays an important role in LS biology and therefore represents a potential therapeutic target, particularly in MLS and PLS.
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http://dx.doi.org/10.1002/ijc.29645DOI Listing
December 2015

FGFR2 is overexpressed in myxoid liposarcoma and inhibition of FGFR signaling impairs tumor growth in vitro.

Oncotarget 2015 Aug;6(24):20215-30

Institute of Pathology, University Hospital Cologne, Cologne, Germany.

Myxoid liposarcomas account for more than one third of liposarcomas and about 10% of all adult soft tissue sarcomas. The tumors are characterized by specific chromosomal translocations leading to the chimeric oncogenes FUS-DDIT3 or EWS1R-DDIT3. The encoded fusion proteins act as aberrant transcription factors. Therefore, we implemented comparative expression analyses using whole-genome microarrays in tumor and fat tissue samples. We aimed at identifying differentially expressed genes which may serve as diagnostic or prognostic biomarkers or as therapeutic targets. Microarray analyses revealed overexpression of FGFR2 and other members of the FGF/FGFR family. Overexpression of FGFR2 was validated by qPCR, immunohistochemistry and western blot analysis in primary tumor samples. Treatment of the myxoid liposarcoma cell lines MLS 402 and MLS 1765 with the FGFR inhibitors PD173074, TKI258 (dovitinib) and BGJ398 as well as specific siRNAs reduced cell proliferation, induced apoptosis and delayed cell migration. Combination of FGFR inhibitors with trabectedin further increased the effect. Our study demonstrates overexpression of FGFR2 and a functional role of FGFR signaling in myxoid liposarcoma. As FGFR inhibition showed effects on proliferation and cell migration and induced apoptosis in vitro, our data indicate the potential use of FGFR inhibitors as a targeted therapy for these tumors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652999PMC
http://dx.doi.org/10.18632/oncotarget.4046DOI Listing
August 2015

CD8/FOXP3-ratio in osteosarcoma microenvironment separates survivors from non-survivors: a multicenter validated retrospective study.

Oncoimmunology 2015 Mar 2;4(3):e990800. Epub 2015 Apr 2.

Department of Orthopedics and Traumatology; University Hospital Heidelberg , Heidelberg, Germany.

Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8T-cells to FOXP3T-cells (CD8/FOXP3-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004-2012). Highly standardized immunohistochemistry of CD8 and FOXP3, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8/FOXP3-ratios above the median (3.08) compared to patients with lower CD8/FOXP3-ratios ( = 0.000001). No patients with a CD8/FOXP3-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival ( = 0.001) in patients with CD8/FOXP3-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8/FOXP3-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.
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http://dx.doi.org/10.4161/2162402X.2014.990800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404826PMC
March 2015

Melanotic tumors of the nervous system are characterized by distinct mutational, chromosomal and epigenomic profiles.

Brain Pathol 2015 Mar 15;25(2):202-8. Epub 2014 Dec 15.

Department of Neuropathology, Institute of Pathology, University Medical Center, Heidelberg, Germany; German Cancer Consortium (DKTK), Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Melanotic tumors of the nervous system show overlapping histological characteristics but differ substantially in their biological behavior. In order to achieve a better delineation of such tumors, we performed an in-depth molecular characterization. Eighteen melanocytomas, 12 melanomas, and 14 melanotic and 14 conventional schwannomas (control group) were investigated for methylome patterns (450k array), gene mutations associated with melanotic tumors and copy number variants (CNVs). The methylome fingerprints assigned tumors to entity-specific groups. Methylation groups also showed a substantial overlap with histology-based diagnosis suggesting that they represent true biological entities. On the molecular level, melanotic schwannomas were characterized by a complex karyotype with recurrent monosomy of chromosome 22q and variable whole chromosomal gains and recurrent losses commonly involving chromosomes 1, 17p and 21. Melanocytomas carried GNAQ/11 mutations and presented with CNV involving chromosomes 3 and 6. Melanomas were frequently mutated in the TERT promoter, harbored additional oncogene mutations and showed recurrent chromosomal losses involving chromosomes 9, 10 and 6q, as well as gains of 22q. Together, melanotic nervous system tumors have several distinct mutational and chromosomal alterations and can reliably be distinguished by methylome profiling.
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http://dx.doi.org/10.1111/bpa.12228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8029316PMC
March 2015

Acinar cell carcinomas of the pancreas: a molecular analysis in a series of 57 cases.

Virchows Arch 2014 Dec 9;465(6):661-72. Epub 2014 Oct 9.

Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, D-69120, Heidelberg, Germany,

Pancreatic acinar cell carcinomas (PACs) are rare but are distinct aggressive neoplasms that phenotypically differ from pancreatic ductal adenocarcinomas (PDACs) and pancreatic neuroendocrine neoplasms (PNENs). Despite recent work on the genetic changes of PACs, their molecular pathogenesis is still poorly understood. In this study, we focus on a comparative genomic hybridization analysis. Based on frequent chromosomal imbalances, the involvement of DCC and c-MYC in the pathogenesis of PACs is further investigated. Moreover, we examine markers harboring potential therapeutic relevance (K-RAS, BRAF, EGFR, MGMT, HSP90, L1CAM, Her2). PACs revealed a microsatellite stable, chromosomal unstable genotype, defined by recurrent chromosomal losses of 1p, 3p, 4q, 5q, 6q, 8p, 9p, 11q, 13q, 16q, and 18, as well as gains of 1q, 7, 8q, 12, 17q, and 20q. Subsets of PAC displayed reduction/loss of DCC (79 %) and c-MYC-amplification (17 %). Significant EGFR expression occurred in 42 %, HSP90 expression in 98 %, L1CAM expression in 72 %, and loss of MGMT in 26 %. Two cases carried a K-RAS mutation. Mutations of EGFR or BRAF were not detected. All cases were Her2/neu-negative. PACs display characteristic chromosomal imbalances which are distinctly different from those in pancreatic ductal adenocarcinomas and pancreatic neuroendocrine neoplasms. Our findings suggest that DCC and c-MYC alterations may play an important role in the pathogenesis of PACs. Furthermore, EGFR, MGMT, HSP90, and L1CAM may be useful as therapeutic markers and predictors of response to therapy in a subset of PACs.
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http://dx.doi.org/10.1007/s00428-014-1657-8DOI Listing
December 2014

Clinical phase I/II trial to investigate preoperative dose-escalated intensity-modulated radiation therapy (IMRT) and intraoperative radiation therapy (IORT) in patients with retroperitoneal soft tissue sarcoma: interim analysis.

BMC Cancer 2014 Aug 27;14:617. Epub 2014 Aug 27.

Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Background: To report an unplanned interim analysis of a prospective, one-armed, single center phase I/II trial (NCT01566123).

Methods: Between 2007 and 2013, 27 patients (pts) with primary/recurrent retroperitoneal sarcomas (size > 5 cm, M0, at least marginally resectable) were enrolled. The protocol attempted neoadjuvant IMRT using an integrated boost with doses of 45-50 Gy to PTV and 50-56 Gy to GTV in 25 fractions, followed by surgery and IOERT (10-12 Gy). Primary endpoint was 5-year-LC, secondary endpoints included PFS, OS, resectability, and acute/late toxicity. The majority of patients showed high grade lesions (FNCLCC G1:18%, G2:52%, G3:30%), predominantly liposarcomas (70%). Median tumor size was 15 cm (6-31).

Results: Median follow-up was 33 months (5-75). Neoadjuvant IMRT was performed as planned (median dose 50 Gy, 26-55) in all except 2 pts (93%). Gross total resection was feasible in all except one patient. Final margin status was R0 in 6 (22%) and R1 in 20 pts (74%). Contiguous-organ resection was needed in all grossly resected patients. IOERT was performed in 23 pts (85%) with a median dose of 12 Gy (10-20 Gy).We observed 7 local recurrences, transferring into estimated 3- and 5-year-LC rates of 72%. Two were located outside the EBRT area and two were observed after more than 5 years. Locally recurrent situation had a significantly negative impact on local control. Distant failure was found in 8 pts, resulting in 3- and 5-year-DC rates of 63%. Patients with leiomyosarcoma had a significantly increased risk of distant failure. Estimated 3- and 5-year-rates were 40% for PFS and 74% for OS. Severe acute toxicity (grade 3) was present in 4 pts (15%). Severe postoperative complications were found in 9 pts (33%), of whom 2 finally died after multiple re-interventions. Severe late toxicity (grade 3) was scored in 6% of surviving patients after 1 year and none after 2 years.

Conclusion: Combination of neoadjuvant IMRT, surgery and IOERT is feasible with acceptable toxicity and yields good results in terms of LC and OS in patients with high-risk retroperitoneal sarcomas. Long term follow-up seems mandatory given the observation of late recurrences. Accrual of patients will be continued with extended follow-up.

Trial Registration: NCT01566123.
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http://dx.doi.org/10.1186/1471-2407-14-617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156610PMC
August 2014

Identification of novel SHOX target genes in the developing limb using a transgenic mouse model.

PLoS One 2014 2;9(6):e98543. Epub 2014 Jun 2.

Department of Human Molecular Genetics, Heidelberg University Hospital, Heidelberg, Germany.

Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098543PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041798PMC
January 2015

Excellent local control with IOERT and postoperative EBRT in high grade extremity sarcoma: results from a subgroup analysis of a prospective trial.

BMC Cancer 2014 May 20;14:350. Epub 2014 May 20.

Clinical Cooperation Unit Radiation Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Background: To report the results of a subgroup analysis of a prospective phase II trial focussing on radiation therapy and outcome in patients with extremity soft tissue sarcomas (STS).

Methods: Between 2005 and 2010, 50 patients (pts) with high risk STS (size ≥ 5 cm, deep/extracompartimental location, grade II-III (FNCLCC)) were enrolled. The protocol comprised 4 cycles of neoadjuvant chemotherapy with EIA (etoposide, ifosfamide and doxorubicin), definitive surgery with IOERT, postoperative EBRT and 4 adjuvant cycles of EIA. 34 pts, who suffered from extremity tumors and received radiation therapy after limb-sparing surgery, formed the basis of this subgroup analysis.

Results: Median follow-up from inclusion was 48 months in survivors. Margin status was R0 in 30 pts (88%) and R1 in 4 pts (12%). IOERT was performed as planned in 31 pts (91%) with a median dose of 15 Gy, a median electron energy of 6 MeV and a median cone size of 9 cm. All patients received postoperative EBRT with a median dose of 46 Gy after IOERT or 60 Gy without IOERT. Median time from surgery to EBRT and median EBRT duration was 36 days, respectively. One patient developed a local recurrence while 11 patients showed nodal or distant failures. The estimated 5-year rates of local control, distant control and overall survival were 97%, 66% and 79%, respectively. Postoperative wound complications were found in 7 pts (20%), resulting in delayed EBRT (>60 day interval) in 3 pts. Acute radiation toxicity mainly consisted of radiation dermatitis (grade II: 24%, no grade III reactions). 4 pts developed grade I/II radiation recall dermatitis during adjuvant chemotherapy, which resolved during the following cycles. Severe late toxicity was observed in 6 pts (18%). Long-term limb preservation was achieved in 32 pts (94%) with good functional outcome in 81%.

Conclusion: Multimodal therapy including IOERT and postoperative EBRT resulted in excellent local control and good overall survival in patients with high risk STS of the extremities with acceptable acute and late radiation side effects. Limb preservation with good functional outcome was achieved in the majority of patients.

Trial Registration: ClinicalTrials.gov NCT01382030, EudraCT 2004-002501-72, 17.06.2011.
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http://dx.doi.org/10.1186/1471-2407-14-350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032585PMC
May 2014

TERT promoter hotspot mutations are recurrent in myxoid liposarcomas but rare in other soft tissue sarcoma entities.

J Exp Clin Cancer Res 2014 Apr 11;33:33. Epub 2014 Apr 11.

Department of General Pathology, Institute of Pathology, University Hospital, Im Neuenheimer Feld 224, Heidelberg D-69120, Germany.

Background: Recently, recurrent point mutations in the telomerase reverse transcriptase (TERT) promoter region have been found in many human cancers, leading to a new transcription factor binding site, increased induction of TERT and subsequently to telomere maintenance. We determined the prevalence of TERT promoter mutations in soft tissue sarcomas of 341 patients comprising 16 entities and in 16 sarcoma cell lines covering 7 different soft tissue sarcoma types.

Methods: The sarcoma tissue samples were collected from the archives of the Institute of Pathology, University of Heidelberg and were composed of 39 myxoid liposarcomas (MLS), 61 dedifferentiated liposarcomas, 15 pleomorphic liposarcomas, 27 leiomyosarcomas, 25 synovial sarcomas (SS), 35 malignant peripheral nerve sheath tumors (MPNST), 40 undifferentiated pleomorphic sarcomas, 17 myxofibrosarcomas, 9 low grade fibromyxoid sarcomas, 10 cases of dermatofibrosarcoma protuberans, 31 solitary fibrous tumors (SFT), 8 extraskeletal myxoid chondrosarcomas, 9 angiosarcomas, 6 alveolar soft part sarcomas, 5 clear cell sarcomas and 4 epithelioid sarcomas. Sarcoma cell lines were obtained from the raising laboratories. A 193 bp fragment of the TERT promoter region covering the hot-spot mutations C228T and C250T was amplified, and direct sequencing of the PCR products was performed.

Results: TERT promoter mutations were detected in 36/341 sarcomas. They were highly recurrent in MLS (29/39; 74%) and were in the present MLS series not associated with the phenotype (myxoid vs. round cell variant), tumor grade, tumor site and patients' median age or gender. In the remaining cases, TERT promoter mutations were found only in 7/302 sarcoma samples and confined to SFTs (4/31; 13%), MPNSTs (2/35; 6%), and SSs (1/25; 4%). Within the collection of sarcoma cell lines examined, TERT promoter mutations were detected in two MLS and in one of three MPNST cell lines.

Conclusions: TERT promoter mutations are frequent in MLSs including their round cell variants, representing the most prevalent mutation identified in this sarcoma entity to date, and in a minor fraction of SFTs, MPNSTs and SSs. The majority of sarcomas are devoid of TERT promoter hotspot mutations. These data suggest that telomere maintenance through increased expression of telomerase plays an important role in the pathogenesis especially of MLS.
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http://dx.doi.org/10.1186/1756-9966-33-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4022359PMC
April 2014

Nuclear relocation of STAT6 reliably predicts NAB2-STAT6 fusion for the diagnosis of solitary fibrous tumour.

Histopathology 2014 Nov 2;65(5):613-22. Epub 2014 Sep 2.

Department of Neuropathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany; German Cancer Consortium (DKTK), CCU Neuropathology German Cancer Research Centre (DKFZ), Heidelberg, Germany.

Aims: Nuclear relocation of STAT6 has been shown in tumours with NAB2-STAT6 fusion, and has been proposed as an ancillary marker for the diagnosis of solitary fibrous tumours (SFTs). The aim of this study was to verify the utility of STAT6 immunohistology in diagnosing SFT.

Methods And Results: A total of 689 formalin-fixed paraffin-embedded tumours comprising 35 pleural SFTs and 654 other mesenchymal tumours were investigated for STAT6 expression using immunohistochemistry. Nine dedifferentiated liposarcomas (DDLSs) and five SFTs were also examined for the presence of NAB2-STAT6 fusion at the protein level using the proximity ligation assay (PLA), and for copy number variants (CNVs) with the Illumina Infinium Human Methylation450 array. Thirty-four of 35 SFTs showed strong nuclear STAT6 expression. Furthermore, five of 68 DDLSs, two of 130 undifferentiated pleomorphic sarcomas and one of 63 cases of nodular fasciitis showed moderate to strong nuclear STAT6 expression. The PLA indicated the presence of NAB2-STAT6 fusion protein in SFTs, but signal was also detected in some DDLSs. Copy number analysis showed an overall low frequency of chromosomal imbalances in SFTs, but complex karyotypes in DDLSs, including amplification of STAT6 and MDM2 loci.

Conclusions: The detection of nuclear relocation of STAT6 with immunohistochemistry is a characteristic of SFTs, and may serve as a diagnostic marker that indicates NAB2-STAT6 fusion and helps to discriminate SFTs from histological mimics.
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http://dx.doi.org/10.1111/his.12431DOI Listing
November 2014

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

Acta Neuropathol 2014 Apr 25;127(4):565-72. Epub 2014 Jan 25.

German Cancer Consortium (DKTK), Clinical Cooperation Unit Neuropathology, German Cancer Research Center, DKFZ, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1007/s00401-014-1246-6DOI Listing
April 2014

Integrative DNA methylation and gene expression analysis in high-grade soft tissue sarcomas.

Genome Biol 2013 Dec 17;14(12):r137. Epub 2013 Dec 17.

Background: High-grade soft tissue sarcomas are a heterogeneous, complex group of aggressive malignant tumors showing mesenchymal differentiation. Recently, soft tissue sarcomas have increasingly been classified on the basis of underlying genetic alterations; however, the role of aberrant DNA methylation in these tumors is not well understood and, consequently, the usefulness of methylation-based classification is unclear.

Results: We used the Infinium HumanMethylation27 platform to profile DNA methylation in 80 primary, untreated high-grade soft tissue sarcomas, representing eight relevant subtypes, two non-neoplastic fat samples and 14 representative sarcoma cell lines. The primary samples were partitioned into seven stable clusters. A classification algorithm identified 216 CpG sites, mapping to 246 genes, showing different degrees of DNA methylation between these seven groups. The differences between the clusters were best represented by a set of eight CpG sites located in the genes SPEG, NNAT, FBLN2, PYROXD2, ZNF217, COL14A1, DMRT2 and CDKN2A. By integrating DNA methylation and mRNA expression data, we identified 27 genes showing negative and three genes showing positive correlation. Compared with non-neoplastic fat, NNAT showed DNA hypomethylation and inverse gene expression in myxoid liposarcomas, and DNA hypermethylation and inverse gene expression in dedifferentiated and pleomorphic liposarcomas. Recovery of NNAT in a hypermethylated myxoid liposarcoma cell line decreased cell migration and viability.

Conclusions: Our analysis represents the first comprehensive integration of DNA methylation and transcriptional data in primary high-grade soft tissue sarcomas. We propose novel biomarkers and genes relevant for pathogenesis, including NNAT as a potential tumor suppressor in myxoid liposarcomas.
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http://dx.doi.org/10.1186/gb-2013-14-12-r137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054884PMC
December 2013

Follicle-stimulating hormone receptor expression in soft tissue sarcomas.

Histopathology 2013 Jul 9;63(1):29-35. Epub 2013 May 9.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Aims: In adult humans, the follicle-stimulating hormone receptor (FSHR) is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. Recently, it has been shown that FSHR is expressed selectively on the surface of blood vessels in a wide range of tumours. So far, the expression of FSHR in mesenchymal tumours has not been studied.

Methods And Results: We performed a semiquantitative evaluation of FSHR protein expression in a large cohort of soft tissue sarcomas (STS; n = 335), including 11 subtypes. FSHR-positive vessels were detected in all sarcoma subtypes analysed. Among liposarcomas, significantly more cases of dedifferentiated liposarcomas (28 of 44) showed FSHR expression compared to well-differentiated liposarcomas (WDLS; four of 21; P < 0.001). Vessels in lipomas (n = 9) and non-neoplastic fat were FSHR-negative. FSHR expression was also detected in tumour cells of all sarcoma subtypes examined, with the lowest incidence in WDLS (three of 21; 14.3%) and the highest frequency in undifferentiated high-grade pleomorphic sarcomas (41 of 60; 68.3%).

Conclusions: These data supplement the previously reported results of FSHR expression in endothelial cells of various cancer types and form a solid basis for further studies of FSHR in mesenchymal neoplasms.
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http://dx.doi.org/10.1111/his.12135DOI Listing
July 2013

SRC signaling is crucial in the growth of synovial sarcoma cells.

Cancer Res 2013 Apr 11;73(8):2518-28. Epub 2013 Apr 11.

Department of Pathology, University Hospital Cologne, Cologne, Germany.

Synovial sarcoma is a soft-tissue malignancy characterized by a reciprocal t(X;18) translocation encoding a chimeric transcriptional modifier. Several receptor tyrosine kinases have been found activated in synovial sarcoma; however, no convincing therapeutic concept has emerged from these findings. On the basis of the results of phosphokinase screening arrays, we here investigate the functional and therapeutic relevance of the SRC kinase in synovial sarcoma. Immunohistochemistry of phosphorylated SRC and its regulators CSK and PTP1B (PTPN1) was conducted in 30 synovial sarcomas. Functional aspects of SRC, including dependence of SRC activation on the SS18/SSX fusion proteins, were analyzed in vitro. Eventually, synovial sarcoma xenografts were treated with the SRC inhibitor dasatinib in vivo. Activated phospho (p)-(Tyr416)-SRC was detected in the majority of tumors; dysregulation of CSK or PTP1B was excluded as the reason for the activation of the kinase. Expression of the SS18/SSX fusion proteins in T-REx-293 cells was associated with increased p-(Tyr416)-SRC levels, linked with an induction of the insulin-like growth factor pathway. Treatment of synovial sarcoma cells with dasatinib led to apoptosis and inhibition of cellular proliferation, associated with reduced phosphorylation of FAK (PTK2), STAT3, IGF-IR, and AKT. Concurrent exposure of cells to dasatinib and chemotherapeutic agents resulted in additive effects. Cellular migration and invasion were dependent on signals transmitted by SRC involving regulation of the Rho GTPases Rac and RhoA. Treatment of nude mice with SYO-1 xenografts with dasatinib significantly inhibited tumor growth in vivo. In summary, SRC is of crucial biologic importance and represents a promising therapeutic target in synovial sarcoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3023DOI Listing
April 2013
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