Publications by authors named "Thomas Grünewald"

101 Publications

Single-cell transcriptomic analyses provide insights into the developmental origins of neuroblastoma.

Nat Genet 2021 Mar 25. Epub 2021 Mar 25.

Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany.

Neuroblastoma is a pediatric tumor of the developing sympathetic nervous system. However, the cellular origin of neuroblastoma has yet to be defined. Here we studied the single-cell transcriptomes of neuroblastomas and normal human developing adrenal glands at various stages of embryonic and fetal development. We defined normal differentiation trajectories from Schwann cell precursors over intermediate states to neuroblasts or chromaffin cells and showed that neuroblastomas transcriptionally resemble normal fetal adrenal neuroblasts. Importantly, neuroblastomas with varying clinical phenotypes matched different temporal states along normal neuroblast differentiation trajectories, with the degree of differentiation corresponding to clinical prognosis. Our work highlights the roles of oncogenic MYCN and loss of TFAP2B in blocking differentiation and may provide the basis for designing therapeutic interventions to overcome differentiation blocks.
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http://dx.doi.org/10.1038/s41588-021-00806-1DOI Listing
March 2021

Fat induces glucose metabolism in non-transformed liver cells and promotes liver tumorigenesis.

Cancer Res 2021 Mar 9. Epub 2021 Mar 9.

Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology

Hepatic fat accumulation is associated with diabetes and hepatocellular carcinoma (HCC). Here we characterize the metabolic response that high fat availability elicits in livers prior to disease development. After a short term on a high fat diet, otherwise healthy mice showed elevated hepatic glucose uptake and increased glucose contribution to serine and pyruvate carboxylase activity compared to control diet mice. This glucose phenotype occurred independently from transcriptional or proteomic programming, which identifies increased peroxisomal and lipid metabolism pathways. High fat diet-fed mice exhibited increased lactate production when challenged with glucose. Consistently, administration of an oral glucose bolus to healthy individuals revealed a correlation between waist circumference and lactate secretion in a human cohort. In vitro, palmitate exposure stimulated production of reactive oxygen species and subsequent glucose uptake and lactate secretion in hepatocytes and liver cancer cells. Furthermore, high fat diet enhanced the formation of HCC compared to control diet in mice exposed to a hepatic carcinogen. Regardless of the dietary background, all murine tumors showed similar alterations in glucose metabolism to those identified in fat exposed non-transformed mouse livers; however, particular lipid species were elevated in high fat diet tumor and non-tumor-bearing high fat diet liver tissue. These findings suggest that fat can induce glucose-mediated metabolic changes in non-transformed liver cells similar to those found in HCC.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1954DOI Listing
March 2021

Sarcoma classification by DNA methylation profiling.

Authors:
Christian Koelsche Daniel Schrimpf Damian Stichel Martin Sill Felix Sahm David E Reuss Mirjam Blattner Barbara Worst Christoph E Heilig Katja Beck Peter Horak Simon Kreutzfeldt Elke Paff Sebastian Stark Pascal Johann Florian Selt Jonas Ecker Dominik Sturm Kristian W Pajtler Annekathrin Reinhardt Annika K Wefers Philipp Sievers Azadeh Ebrahimi Abigail Suwala Francisco Fernández-Klett Belén Casalini Andrey Korshunov Volker Hovestadt Felix K F Kommoss Mark Kriegsmann Matthias Schick Melanie Bewerunge-Hudler Till Milde Olaf Witt Andreas E Kulozik Marcel Kool Laura Romero-Pérez Thomas G P Grünewald Thomas Kirchner Wolfgang Wick Michael Platten Andreas Unterberg Matthias Uhl Amir Abdollahi Jürgen Debus Burkhard Lehner Christian Thomas Martin Hasselblatt Werner Paulus Christian Hartmann Ori Staszewski Marco Prinz Jürgen Hench Stephan Frank Yvonne M H Versleijen-Jonkers Marije E Weidema Thomas Mentzel Klaus Griewank Enrique de Álava Juan Díaz Martín Miguel A Idoate Gastearena Kenneth Tou-En Chang Sharon Yin Yee Low Adrian Cuevas-Bourdier Michel Mittelbronn Martin Mynarek Stefan Rutkowski Ulrich Schüller Viktor F Mautner Jens Schittenhelm Jonathan Serrano Matija Snuderl Reinhard Büttner Thomas Klingebiel Rolf Buslei Manfred Gessler Pieter Wesseling Winand N M Dinjens Sebastian Brandner Zane Jaunmuktane Iben Lyskjær Peter Schirmacher Albrecht Stenzinger Benedikt Brors Hanno Glimm Christoph Heining Oscar M Tirado Miguel Sáinz-Jaspeado Jaume Mora Javier Alonso Xavier Garcia Del Muro Sebastian Moran Manel Esteller Jamal K Benhamida Marc Ladanyi Eva Wardelmann Cristina Antonescu Adrienne Flanagan Uta Dirksen Peter Hohenberger Daniel Baumhoer Wolfgang Hartmann Christian Vokuhl Uta Flucke Iver Petersen Gunhild Mechtersheimer David Capper David T W Jones Stefan Fröhling Stefan M Pfister Andreas von Deimling

Nat Commun 2021 01 21;12(1):498. Epub 2021 Jan 21.

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

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications.
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http://dx.doi.org/10.1038/s41467-020-20603-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819999PMC
January 2021

In Vivo Evidence for Serine Biosynthesis-Defined Sensitivity of Lung Metastasis, but Not of Primary Breast Tumors, to mTORC1 Inhibition.

Mol Cell 2021 01 18;81(2):386-397.e7. Epub 2020 Dec 18.

Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology, VIB, Herestraat 49, 3000 Leuven, Belgium; Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Herestraat 49, 3000 Leuven, Belgium. Electronic address:

In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast-cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast-cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast-cancer-derived lung tumors, but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.
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http://dx.doi.org/10.1016/j.molcel.2020.11.027DOI Listing
January 2021

(Immuno)histological Analysis of Ewing Sarcoma.

Methods Mol Biol 2021 ;2226:49-64

Institute of Biomedicine of Sevilla (IBiS), Virgen del Rocio University Hospital/CSIC/University of Sevilla/CIBERONC, Seville, Spain.

The diagnosis of Ewing sarcoma requires the integration of the information generated from numerous techniques, some of them being very sophisticated. However, the first steps of the diagnostic process are crucial to achieve the maximum possible diagnostic performance. In this chapter we will review how to handle the diagnostic specimen from its collection, how to prepare it for diagnosis, how to make a complete pathology report, and provide guidance for the reasonable use of immunohistochemical techniques in this malignancy.
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http://dx.doi.org/10.1007/978-1-0716-1020-6_5DOI Listing
April 2021

Tissue Preservation and FFPE Samples: Optimized Nucleic Acids Isolation in Ewing Sarcoma.

Methods Mol Biol 2021 ;2226:27-38

Division of Translational Pediatric Sarcoma Research, German Cancer Research Center (DKFZ), Hopp Children's Cancer Center (KiTZ), Heidelberg, Germany.

Different methods have been described for the preservation of biopsy or resection samples. In the routine pathology, the cheapest and most commonly used is fixation of samples in formalin and embedding in paraffin (FFPE samples). This method preserves tissue samples for a very long time and is suitable for several specialized techniques such as fluorescence in situ hybridization (FISH) and immunohistochemistry, the latter being the most frequent and often the only additional method used for establishment of final diagnosis. However, in light of the growing need of next-generation sequencing and microarray technologies that are often very helpful to establish and/or confirm diagnoses in the field of pediatric sarcoma (including Ewing sarcoma), preservation of high-quality and quantity of nucleic acids (DNA/RNA) is desirable. Herein, we describe how to ideally preserve samples, as well as how to proceed to isolate nucleic acids for successful subsequent molecular assays with a special focus on Ewing sarcoma samples.
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http://dx.doi.org/10.1007/978-1-0716-1020-6_3DOI Listing
April 2021

Germline Variation and Somatic Alterations in Ewing Sarcoma.

Methods Mol Biol 2021 ;2226:3-14

Division of Translational Pediatric Sarcoma Research, German Cancer Research Center (DKFZ), Hopp Children's Cancer Center (KiTZ), Heidelberg, Germany.

Ewing sarcoma (EwS) is a rare bone or soft tissue tumor that occurs early in life and as such genetic variation is a major contributor to EwS risk. To date, genetic investigations have identified key somatic mutations and germline variants of importance for EwS risk. While substantial progress is being made in uncovering the genetic etiology of EwS, considerable gaps in knowledge remain. Herein, we provide a summary of methodological approaches for future genomic investigations of EwS. We anticipate this recommended analytical framework for germline and somatic investigations, along with genomic data from growing EwS case series, will aid in accelerating new genomic discoveries in EwS and expand knowledge of the genetic architecture of EwS.
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http://dx.doi.org/10.1007/978-1-0716-1020-6_1DOI Listing
April 2021

[Integrative molecular pathology of cancer].

Pathologe 2020 Dec;41(Suppl 2):67-69

Abteilung Translationale Pädiatrische Sarkomforschung, Deutsches Krebsforschungszentrum (DKFZ), German Cancer Consortium (DKTK), Im Neuenheimer Feld 280, 69120, Heidelberg, Deutschland.

The field of molecular pathology has revolutionized our understanding of relevant oncogenic alterations in cancer and yielded new diagnostic tools and therapeutic approaches for personalized oncology, especially for malignancies of adulthood. However, many pediatric tumors, such as Ewing sarcoma, are characterized by a remarkable paucity of recurrent driver mutations, which are usually not suitable as drug targets. Despite the relative homogeneity of the somatic mutational profiles, these tumors nevertheless exhibit a relatively strong clinical heterogeneity, indicating additional modulating factors. In this regard, a recent study could demonstrate that the mode of action of the EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukema integration 1) fusion oncoprotein, which is pathognomonic for Ewing sarcoma, is influenced by inherited genetic variants in regulatory DNA elements, which may ultimately affect the course of the disease and also enable new therapeutic options. Thus, these investigations demonstrate in the Ewing sarcoma model that the function of a driver mutation needs to be interpreted in its germline context, which should be taken into account in an integrative approach by the molecular pathology of the future.
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http://dx.doi.org/10.1007/s00292-020-00870-0DOI Listing
December 2020

Expression of the EWSR1-FLI1 fusion oncogene in pancreas cells drives pancreatic atrophy and lipomatosis.

Pancreatology 2020 Dec 10;20(8):1673-1681. Epub 2020 Oct 10.

Department of Visceral, Vascular and Endocrine Surgery, Martin-Luther-University Halle-Wittenberg, University Medical Center Halle, Halle, Germany. Electronic address:

Background: Pancreatic ductal adenocarcinoma (PDAC) harbors mutant KRAS as the most common driver mutation. Studies on mouse models have uncovered the tumorigenic characteristics of the Kras oncogene driving pancreatic carcinogenesis. Similarly, Ewing sarcoma predominantly depends on the occurrence of the EWSR1-FLI1 fusion oncogene. The expression of EWSR1-FLI1 affects pro-tumorigenic pathways and induces cell transformation. In this study, we investigated whether mutant Kras could be exchanged by another potent oncogene, such as EWSR1-FLI1, to initiate pancreatic cancer development.

Methods: We generated two conditional mouse models expressing mutant Kras (KC) or the EWSR1-FLI1 oncogene (E/F) in pancreas cells. Pancreatic tissue was collected from the mice at 4-6 weeks and 11-13 weeks of age as well as from survival cohorts to determine the development of spontaneous acinar-to-ductal metaplasia (ADM) and neoplastic lesions. Immunohistochemistry and immunofluorescence staining were performed to characterize and quantify changes in tissue morphology.

Results: The expression of the EWSR1-FLI1 fusion protein in pancreas cells was confirmed by positive FLI1 immunohistochemistry staining. Notably, the EWSR1-FLI1 expression in pancreas cells resulted in a strong depletion of the acinar cell mass and an extensive lipomatosis. Although the E/F mice exhibited spontaneous ADM formation and a shorter overall survival rate compared to KC mice, no development of neoplastic lesion was observed in aging E/F mice.

Conclusions: The expression of the EWSR1-FLI1 oncogene leads to a strong pancreatic atrophy and lipomatosis. ADM formation indicates that pancreatic acinar cells are susceptible for EWSR1-FLI1-mediated oncogenic transformation to a limited extent. However, the EWSR1-FLI1 oncogene is insufficient to induce pancreatic cancer development.
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http://dx.doi.org/10.1016/j.pan.2020.10.033DOI Listing
December 2020

Sarcoma treatment in the era of molecular medicine.

EMBO Mol Med 2020 11 13;12(11):e11131. Epub 2020 Oct 13.

Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.
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http://dx.doi.org/10.15252/emmm.201911131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645378PMC
November 2020

SOX6: a double-edged sword for Ewing sarcoma.

Mol Cell Oncol 2020 14;7(5):1783081. Epub 2020 Jul 14.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany.

Developmental pathways play an important role in cancer. We have recently demonstrated that the constitutive activation of the developmental transcription factor SOX6 via the fusion oncoproteinne EWSR1-FLI1 (Ewing sarcoma breakpoint region 1 - Friend leukemia virus integration 1) contributes to the aggressive phenotype of Ewing sarcoma but on another hand provides an opportunity for targeted therapy.
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http://dx.doi.org/10.1080/23723556.2020.1783081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469519PMC
July 2020

Leukemia escape in immune desert: intraocular relapse of pediatric pro-B-ALL during systemic control by CD19-CAR T cells.

J Immunother Cancer 2020 09;8(2)

Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich, Bavaria, Germany

Background: Relapsed/refractory B-precursor acute lymphoblastic leukemia (BCP-ALL) remains a major therapeutic challenge in pediatric hematology. Chimeric antigen receptor (CAR) T cells targeting CD19 have shown remarkable initial response rates in BCP-ALL patients, while long-term leukemia control rate is only about 50%. So far, main mechanisms of BCP-ALL relapse after CD19-CAR T-cell therapy have been either insufficient CAR T-cell persistence in vivo or loss of surface CD19.

Case Report: Here, we report an exceptional presentation of BCP-ALL relapse in the eye during the systemic control through CAR T-cell therapy. We report a case of fatal intraocular relapse in a pediatric patient with pro-B-ALL after initial response to CD19-CAR T-cell therapy. One month after CD19-CAR T-cell therapy, remission was documented by bone marrow aspirate analysis with absence of CD19 cells and CD19-CAR T cells could be detected in both peripheral blood and bone marrow. At the same time, however, the patient presented with progressive visual disturbance and CD19 cells were found within the anterior chamber of the eye. Despite local and systemic therapy, ocular relapse led to BCP-ALL dissemination and systemic relapse within weeks. The eye represents a rare site for local manifestation of BCP-ALL, but isolated intraocular relapse is a clinically unreckoned presentation of BCP-ALL in the era of CD19-CAR T cells.

Conclusion: During systemic control of BCP-ALL through CD19-CAR T cells, relapse can emerge in the eye as an immune-privileged organ. Ocular symptoms after CD19-CAR T-cell therapy should guide the clinician to elucidate the etiology in a timely fashion in order to adjust leukemia treatment strategy. Both, local immune escape as well as insufficient CAR T-cell persistence may have contributed to relapse in the reported patient. Mechanisms of relapse in an immune desert under CAR T-cell therapy require future clinical and experimental attention. In particular, ocular symptoms after CAR T-cell therapy should be considered a potentially early sign of leukemia relapse.
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http://dx.doi.org/10.1136/jitc-2020-001052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497522PMC
September 2020

Low-frequency variation near common germline susceptibility loci are associated with risk of Ewing sarcoma.

PLoS One 2020 3;15(9):e0237792. Epub 2020 Sep 3.

Division of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, MD, United States of America.

Background: Ewing sarcoma (EwS) is a rare, aggressive solid tumor of childhood, adolescence and young adulthood associated with pathognomonic EWSR1-ETS fusion oncoproteins altering transcriptional regulation. Genome-wide association studies (GWAS) have identified 6 common germline susceptibility loci but have not investigated low-frequency inherited variants with minor allele frequencies below 5% due to limited genotyped cases of this rare tumor.

Methods: We investigated the contribution of rare and low-frequency variation to EwS susceptibility in the largest EwS genome-wide association study to date (733 EwS cases and 1,346 unaffected controls of European ancestry).

Results: We identified two low-frequency variants, rs112837127 and rs2296730, on chromosome 20 that were associated with EwS risk (OR = 0.186 and 2.038, respectively; P-value < 5×10-8) and located near previously reported common susceptibility loci. After adjusting for the most associated common variant at the locus, only rs112837127 remained a statistically significant independent signal (OR = 0.200, P-value = 5.84×10-8).

Conclusions: These findings suggest rare variation residing on common haplotypes are important contributors to EwS risk.

Impact: Motivate future targeted sequencing studies for a comprehensive evaluation of low-frequency and rare variation around common EwS susceptibility loci.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237792PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470401PMC
October 2020

MHC Class I-Restricted TCR-Transgenic CD4 T Cells Against STEAP1 Mediate Local Tumor Control of Ewing Sarcoma In Vivo.

Cells 2020 06 29;9(7). Epub 2020 Jun 29.

Department of Pediatrics, Children's Cancer Research Center, Kinderklinik München Schwabing, School of Medicine, Technical University of Munich, 80804 Munich, Germany.

In this study we report the functional comparison of T cell receptor (TCR)-engineered major histocompatibility complex (MHC) class I-restricted CD4 versus CD8 T cells targeting a peptide from (STEAP1) in the context of HLA-A*02:01. STEAP1 is a tumor-associated antigen, which is overexpressed in many cancers, including Ewing sarcoma (EwS). Based on previous observations, we postulated strong antitumor potential of tumor-redirected CD4 T cells transduced with an HLA class I-restricted TCR against a STEAP1-derived peptide. We compared CD4 T cell populations to their CD8 counterparts in vitro using impedance-based xCELLigence and cytokine/granzyme release assays. We further compared antitumor activity of STEAP-TCR transgenic (tg) CD4 versus CD8 T cells in tumor-bearing xenografted Rag2gc mice. TCR tgCD4 T cells showed increased cytotoxic features over time with similar functional avidity compared to tgCD8 cells after 5-6 weeks of culture. In vivo, local tumor control was equal. Assessing metastatic organotropism of intraveniously (i.v.) injected tumors, only tgCD8 cells were associated with reduced metastases. In this analysis, EwS-redirected tgCD4 T cells contribute to local tumor control, but fail to control metastatic outgrowth in a model of xenografted EwS.
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http://dx.doi.org/10.3390/cells9071581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408051PMC
June 2020

mTOR Signaling and SREBP Activity Increase FADS2 Expression and Can Activate Sapienate Biosynthesis.

Cell Rep 2020 06;31(12):107806

Laboratory of Cellular Metabolism and Metabolic Regulation, VIB-KU Leuven Center for Cancer Biology, VIB, Herestraat 49, 3000 Leuven, Belgium; Laboratory of Cellular Metabolism and Metabolic Regulation, Department of Oncology, KU Leuven and Leuven Cancer Institute (LKI), Herestraat 49, 3000 Leuven, Belgium. Electronic address:

Cancer cells display an increased plasticity in their lipid metabolism, which includes the conversion of palmitate to sapienate via the enzyme fatty acid desaturase 2 (FADS2). We find that FADS2 expression correlates with mammalian target of rapamycin (mTOR) signaling and sterol regulatory element-binding protein 1 (SREBP-1) activity across multiple cancer types and is prognostic in some cancer types. Accordingly, activating mTOR signaling by deleting tuberous sclerosis complex 2 (Tsc2) or overexpression of SREBP-1/2 is sufficient to increase FADS2 mRNA expression and sapienate metabolism in mouse embryonic fibroblasts (MEFs) and U87 glioblastoma cells, respectively. Conversely, inhibiting mTOR signaling decreases FADS2 expression and sapienate biosynthesis in MEFs with Tsc2 deletion, HUH7 hepatocellular carcinoma cells, and orthotopic HUH7 liver xenografts. In conclusion, we show that mTOR signaling and SREBP activity are sufficient to activate sapienate metabolism by increasing FADS2 expression. Consequently, targeting mTOR signaling can reduce sapienate metabolism in vivo.
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http://dx.doi.org/10.1016/j.celrep.2020.107806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326293PMC
June 2020

Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR.

Blood 2020 09;136(12):1407-1418

Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital, Ludwig Maximilians University Munich, Munich, Germany.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.
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http://dx.doi.org/10.1182/blood.2020005185DOI Listing
September 2020

Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

Nat Commun 2020 05 15;11(1):2423. Epub 2020 May 15.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany.

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites as enhancers.Here, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-microsatellite, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments including 3D and PDX models, we discover that constitutively high SOX6 expression promotes elevated levels of oxidative stress that create a therapeutic vulnerability toward the oxidative stress-inducing drug Elesclomol.Collectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.
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http://dx.doi.org/10.1038/s41467-020-16244-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228971PMC
May 2020

West Nile Virus Epidemic in Germany Triggered by Epizootic Emergence, 2019.

Viruses 2020 04 15;12(4). Epub 2020 Apr 15.

Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, 20359 Hamburg, Germany.

One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 (12 birds, two horses), resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures.
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http://dx.doi.org/10.3390/v12040448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232143PMC
April 2020

Pan-Cancer Analysis of Mitochondria Chaperone-Client Co-Expression Reveals Chaperone Functional Partitioning.

Cancers (Basel) 2020 Mar 30;12(4). Epub 2020 Mar 30.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 8410501, Israel.

Metabolic reprogramming is a hallmark of cancer. Such reprogramming entails the up-regulation of the expression of specific mitochondrial proteins, thus increasing the burden on the mitochondrial protein quality control. However, very little is known about the specificity of interactions between mitochondrial chaperones and their clients, or to what extent the mitochondrial chaperone-client co-expression is coordinated. We hypothesized that a physical interaction between a chaperone and its client in mitochondria ought to be manifested in the co-expression pattern of both transcripts. Using The Cancer Genome Atlas (TCGA) gene expression data from 13 tumor entities, we constructed the mitochondrial chaperone-client co-expression network. We determined that the network is comprised of three distinct modules, each populated with unique chaperone-clients co-expression pairs belonging to distinct functional groups. Surprisingly, chaperonins and , which are known to comprise a functional complex, each occupied a different module: co-expressed with tricarboxylic acid cycle cycle enzymes, while co-expressed with proteins involved in oxidative phosphorylation. Importantly, we found that the genes in each module were enriched for discrete transcription factor binding sites, suggesting the mechanism for the coordinated co-expression. We propose that our mitochondrial chaperone-client interactome can facilitate the identification of chaperones supporting specific mitochondrial pathways and bring forth a fundamental principle in metabolic adaptation.
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http://dx.doi.org/10.3390/cancers12040825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226338PMC
March 2020

A comparative view on the expression patterns of PD-L1 and PD-1 in soft tissue sarcomas.

Cancer Immunol Immunother 2020 Jul 28;69(7):1353-1362. Epub 2020 Mar 28.

Institute of Pathology, Faculty of Medicine, Ludwig-Maximilians University Munich, Thalkirchner Str. 36, 80337, Munich, Germany.

Soft tissue sarcomas (STSs) are heterogeneous cancers associated with poor prognosis due to high rates of local recurrence and metastasis. The programmed death receptor ligand 1 (PD-L1) is expressed in several cancers. PD-L1 interacts with its receptor, PD-1, on the surface of tumor-infiltrating lymphocytes (TILs), thereby attenuating anti-cancer immune response. Immune checkpoint inhibitors targeting this interaction have been established as effective anti-cancer drugs. However, studies on the PD-L1 and PD-1 expression status in STS are commonly limited by small sample size, analysis of single STS subtypes, or lack of combinatorial marker assessment. To overcome these limitations, we evaluated the expression patterns of intratumoral PD-L1, the number of TILs, their PD-1 expression, and associations with clinicopathological parameters in a large and comprehensive cohort of 225 samples comprising six STS subtypes. We found that nearly all STS subtypes showed PD-L1 expression on the tumor cells, albeit with a broad range of positivity across subtypes (50% angiosarcomas to 3% synovial sarcomas). Co-expression and correlation analyses uncovered that PD-L1 expression was associated with more PD-1-positive TILs (P < 0.001), higher tumor grading (P = 0.016), and worse patients' 5-year overall survival (P = 0.028). The results were in line with several publications on single STS subtypes, especially when comparing findings for STS with low and high mutational burden. In sum, the substantial portion of PD-L1 positivity, the co-occurrence of PD-1-positive TILs, and the association of PD-L1 with unfavorable clinical outcome provide rationales for immune checkpoint inhibition in patients with PD-L1-positive STS.
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http://dx.doi.org/10.1007/s00262-020-02552-5DOI Listing
July 2020

High Specificity of BCL11B and GLG1 for EWSR1-FLI1 and EWSR1-ERG Positive Ewing Sarcoma.

Cancers (Basel) 2020 Mar 10;12(3). Epub 2020 Mar 10.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, 80337 Munich, Germany.

Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for -positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including -positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by -positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.
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http://dx.doi.org/10.3390/cancers12030644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139395PMC
March 2020

Transcriptional Programs Define Intratumoral Heterogeneity of Ewing Sarcoma at Single-Cell Resolution.

Cell Rep 2020 02;30(6):1767-1779.e6

INSERM U900, 75005 Paris, France; Mines ParisTech, PSL Research University, CBIO-Centre for Computational Biology, 75006 Paris, France; Institut Curie, PSL Research University, 75005 Paris, France. Electronic address:

EWSR1-FLI1, the chimeric oncogene specific for Ewing sarcoma (EwS), induces a cascade of signaling events leading to cell transformation. However, it remains elusive how genetically homogeneous EwS cells can drive the heterogeneity of transcriptional programs. Here, we combine independent component analysis of single-cell RNA sequencing data from diverse cell types and model systems with time-resolved mapping of EWSR1-FLI1 binding sites and of open chromatin regions to characterize dynamic cellular processes associated with EWSR1-FLI1 activity. We thus define an exquisitely specific and direct enhancer-driven EWSR1-FLI1 program. In EwS tumors, cell proliferation and strong oxidative phosphorylation metabolism are associated with a well-defined range of EWSR1-FLI1 activity. In contrast, a subpopulation of cells from below and above the intermediary EWSR1-FLI1 activity is characterized by increased hypoxia. Overall, our study reveals sources of intratumoral heterogeneity within EwS tumors.
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http://dx.doi.org/10.1016/j.celrep.2020.01.049DOI Listing
February 2020

Interaction between somatic mutations and germline variants contributes to clinical heterogeneity in cancer.

Mol Cell Oncol 2020 6;7(1):1682924. Epub 2019 Nov 6.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany.

Deciphering principles of inter-tumoral heterogeneity is crucial for refinement of precision oncology. We have recently demonstrated that 'oncogenic cooperation' between somatic mutations and regulatory germline variants can serve as a major cause for inter-tumoral heterogeneity, suggesting the requirement of integrating the regulatory genome into 'omics'-based precision oncology.
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http://dx.doi.org/10.1080/23723556.2019.1682924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961672PMC
November 2019

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies.

Cancer Metastasis Rev 2019 12;38(4):625-642

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Thalkirchner Str. 36, 80337, Munich, Germany.

While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the "undruggable" into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments. Graphical abstract Scheme depicting the different targeting strategies of fusion oncogenes in pediatric fusion-driven sarcomas. Fusion oncogenes can be targeted on their DNA level (1), RNA level (2), protein level (3), and by targeting downstream functions and interaction partners (4).
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http://dx.doi.org/10.1007/s10555-019-09839-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994515PMC
December 2019

Hippo pathway effectors YAP1/TAZ induce an EWS-FLI1-opposing gene signature and associate with disease progression in Ewing sarcoma.

J Pathol 2020 04 4;250(4):374-386. Epub 2020 Feb 4.

Department of Pathology, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla, CSIC-Universidad de Sevilla, Seville, Spain.

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of the Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving the gene EWSR1 and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis, but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome. We did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation of the RASSF1 locus (a regulator of the Hippo pathway) was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the pro-tumorigenic isoform RASSF1C, which promotes YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be explained partly by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5379DOI Listing
April 2020

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Mol Oncol 2020 02 21;14(2):248-260. Epub 2019 Dec 21.

Gerhard Domagk Institute of Pathology, University Hospital Münster, Germany.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1-ETS fusion oncogenes, most often EWSR1-FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F-dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate-independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho-dependent cell migration, and impaired caspase-3-mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) enhanced caspase-mediated apoptosis and EwS cell migration, independent from the respective EWSR1-ETS fusion type, mimicking an anoikis-like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.
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http://dx.doi.org/10.1002/1878-0261.12610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998388PMC
February 2020

DNA methylation-based profiling of uterine neoplasms: a novel tool to improve gynecologic cancer diagnostics.

J Cancer Res Clin Oncol 2020 Jan 25;146(1):97-104. Epub 2019 Nov 25.

Department of Pathology, Institute of Pathology, Heidelberg University Hospital, INF 224, 69120, Heidelberg, Germany.

Purpose: Uterine neoplasms comprise a broad spectrum of lesions, some of which may pose a diagnostic challenge even to experienced pathologists. Recently, genome-wide DNA methylation-based classification of central nervous system tumors has been shown to increase diagnostic precision in clinical practice when combined with standard histopathology. In this study, we describe DNA methylation patterns of a diverse set of uterine neoplasms and test the applicability of array-based DNA methylation profiling.

Methods: A multicenter cohort including prototypical epithelial and mesenchymal uterine neoplasms was collected. Tumors were subject to pathology review and array-based DNA methylation profiling (Illumina Infinium HumanMethylation450 or EPIC [850k] BeadChip). Methylation data were analyzed by unsupervised hierarchical clustering and t-SNE analysis.

Results: After sample retrieval and pathology review the study cohort consisted of 49 endometrial carcinomas (EC), 5 carcinosarcomas (MMMT), 8 uterine leiomyomas (ULMO), 7 uterine leiomyosarcomas (ULMS), 15 uterine tumor resembling ovarian sex cord tumors (UTROSCT), 17 low-grade endometrial stromal sarcomas (LGESS) and 9 high-grade endometrial stromal sarcomas (HGESS). Analysis of methylation data identified distinct methylation clusters, which correlated with established diagnostic categories of uterine neoplasms. MMMT clustered together with EC, while ULMO, ULMS and UTROSCT each formed distinct clusters. The LGESS cluster differed from that of HGESS, and within the branch of HGESS, we observed a notable subgrouping of YWHAE- and BCOR-rearranged tumors.

Conclusion: Herein, we describe distinct DNA methylation signatures in uterine neoplasms and show that array-based DNA methylation analysis holds promise as an ancillary tool to further characterize uterine neoplasms, especially in cases which are diagnostically challenging by conventional techniques.
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http://dx.doi.org/10.1007/s00432-019-03093-wDOI Listing
January 2020

Integrative clinical transcriptome analysis reveals TMPRSS2-ERG dependency of prognostic biomarkers in prostate adenocarcinoma.

Int J Cancer 2020 04 29;146(7):2036-2046. Epub 2019 Nov 29.

Max-Eder Research Group for Pediatric Sarcoma Biology, Institute of Pathology, Faculty of Medicine, LMU Munich, Munich, Germany.

In prostate adenocarcinoma (PCa), distinction between indolent and aggressive disease is challenging. Around 50% of PCa are characterized by TMPRSS2-ERG (T2E)-fusion oncoproteins defining two molecular subtypes (T2E-positive/negative). However, current prognostic tests do not differ between both molecular subtypes, which might affect outcome prediction. To investigate gene-signatures associated with metastasis in T2E-positive and T2E-negative PCa independently, we integrated tumor transcriptomes and clinicopathological data of two cohorts (total n = 783), and analyzed metastasis-associated gene-signatures regarding the T2E-status. Here, we show that the prognostic value of biomarkers in PCa critically depends on the T2E-status. Using gene-set enrichment analyses, we uncovered that metastatic T2E-positive and T2E-negative PCa are characterized by distinct gene-signatures. In addition, by testing genes shared by several functional gene-signatures for their association with event-free survival in a validation cohort (n = 272), we identified five genes (ASPN, BGN, COL1A1, RRM2 and TYMS)-three of which are included in commercially available prognostic tests-whose high expression was significantly associated with worse outcome exclusively in T2E-negative PCa. Among these genes, RRM2 and TYMS were validated by immunohistochemistry in another validation cohort (n = 135), and several of them proved to add prognostic information to current clinicopathological predictors, such as Gleason score, exclusively for T2E-negative patients. No prognostic biomarkers were identified exclusively for T2E-positive tumors. Collectively, our study discovers that the T2E-status, which is per se not a strong prognostic biomarker, crucially determines the prognostic value of other biomarkers. Our data suggest that the molecular subtype needs to be considered when applying prognostic biomarkers for outcome prediction in PCa.
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http://dx.doi.org/10.1002/ijc.32792DOI Listing
April 2020

Clinical Evidence on the Interaction Between MLK4, KRAS and Microsatellite Instability to Determine the Prognosis of Early-Stage Colorectal Carcinoma.

Cell Physiol Biochem 2019 ;53(5):820-831

Department of Medicine 2, University Hospital Grosshadern, University of Munich, Munich, Germany,

Background/aims: MLK4 (KIAA1804) is the second most frequently mutated kinase in microsatellite stable (MSS) colorectal carcinomas (CRC). This molecule is known to regulate different physiological cellular processes, including cell cycle, senescence and apoptosis, and mechanistic evidence has been provided that MLK4 plays a role in carcinogenesis. However, whether this kinase exerts a tumor suppressive role or an oncogenic function is still an object of debate. This study aims to elucidate the role of MLK4 in the pathogenesis of CRC by investigating human tumor specimens.

Methods: This study assessed MLK4 expression levels by immunohistochemistry in surgical tumor samples from 204 early-stage CRC patients and their correlation with various clinical-pathological features and patients' outcomes. In addition, MLK4 mRNA transcription was analysed in an independent cohort of 786 colon cancer samples.

Results: Loss of MLK4 staining was associated with poor overall (OS) and progression free survival (PFS) in CRC patients during a univariate analysis (OS:101 vs 164 months, p=0.0002; PFS:85 vs 125 months, p=0.0001), as well as in multivariate analysis (OS:HR=1.70; p=0.001; PFS:HR=1,61; p=0.001). This was confirmed by analysis of MLK4 mRNA in the second independent cohort. A subgroup analysis according to KRAS mutation status showed that MLK4 staining was associated with better OS and PFS in KRAS mutated cases (HR=2.77; p=0.0001 and HR=2.31; p=0.0003, respectively) and microsatellite stable tumors (HR=1.87; p=0.002 and HR=1.06; p=0.006) but not in KRAS wildtype and microsatellite unstable tumors.

Conclusion: By providing the first report from clinical specimens on the prognostic significance of MLK4, we define an oncogenic loss-of-function of this kinase and suggest a possible role in the interaction with KRAS signaling in determining an aggressive phenotype of CRC. These findings warrant the further investigation of MLK4 in wider cohorts and various clinical settings.
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http://dx.doi.org/10.33594/000000175DOI Listing
November 2019