Publications by authors named "Philipp Jurmeister"

29 Publications

  • Page 1 of 1

Machine Learning Models Predict the Primary Sites of Head and Neck Squamous Cell Carcinoma Metastases Based on DNA Methylation.

J Pathol 2021 Dec 8. Epub 2021 Dec 8.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Institute of Pathology, Berlin, Germany.

In head and neck squamous cell cancers (HNSCs) that present as metastases with an unknown primary (HNSC-CUPs), the identification of a primary tumor improves therapy options and increases patient survival. However, the currently available diagnostic methods are laborious and do not offer a sufficient detection rate. Predictive machine learning models based on DNA methylation profiles have recently emerged as a promising technique for tumor classification. We applied this technique to HNSC to develop a tool that can improve the diagnostic workup for HNSC-CUPs. On a reference cohort of 405 primary HNSC samples, we developed four classifiers based on different machine learning models (random forest (RF), neural network (NN), elastic net penalized logistic regression (LOGREG), support vector machine (SVM)) that predict the primary site of HNSC tumors from their DNA methylation profile. The classifiers achieved high classification accuracies (RF=83%, NN=88%, LOGREG=SVM=89%) on an independent cohort of 64 HNSC metastases. Further, the NN, LOGREG, and SVM models significantly outperformed p16 status as a marker for an origin in the oropharynx. In conclusion, the DNA methylation profiles of HNSC metastases are characteristic for their primary sites and the classifiers developed in this study, which are made available to the scientific community, can provide valuable information to guide the diagnostic workup of HNSC-CUP. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/path.5845DOI Listing
December 2021

Comparative investigation of cell cycle and immunomodulatory genes in mucosal and cutaneous melanomas: Preliminary data suggest a potential promising clinical role for p16 and the PD-1/PD-L1 axis.

Pathol Res Pract 2021 Nov 22;229:153689. Epub 2021 Nov 22.

Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Berlin, Germany; Institute of Pathology, Ludwig Maximilians University Hospital Munich, Thalkirchner Str. 36, 80337 Munich, Germany. Electronic address:

Mucosal melanomas arise from the mucosal lining of various organs. Their etiology is currently unknown and there are no tissue-based methods to differentiate it from cutaneous melanomas. Furthermore, prognostic and predictive markers (e.g. for immune checkpoint inhibition) are lacking. In this study, we aimed to assess the protein expression levels of cell cycle-associated proteins and immune checkpoint markers in a cohort of mucosal melanomas in comparison to cutaneous melanomas and evaluated the effect of potential regulatory mechanisms. We performed immunohistochemistry, DNA methylation analysis and copy number profiling of 47 mucosal and 28 cutaneous melanoma samples. Protein expression of CD117, Ki67 and p16 was higher in mucosal melanomas, while BCL2, Cyclin D1, PD-1 and PD-L1 were overexpressed in cutaneous melanomas. CDKN2A deletions were the most prevalent numeric chromosomal alterations in both mucosal and cutaneous melanoma and were associated with decreased p16 expression. KIT was frequently amplified in mucosal melanomas, but not associated with CD117 expression. On the other hand, amplification of CCND1 lead to Cyclin D1 overexpression. In mucosal melanoma patients high PD-1 expression and high PD-L1 promoter methylation levels were associated with improved survival. PD-L1 expression correlated with response to immune checkpoint inhibitor therapy in the combined group of melanoma patients. Mucosal and cutaneous melanomas show different expression levels of cell cycle-associated and immunomodulatory proteins that are partially regulated by DNA methylation and copy number alterations. PD-1 expression and PD-L1 promoter methylation levels might be a prognostic marker for mucosal melanomas.
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http://dx.doi.org/10.1016/j.prp.2021.153689DOI Listing
November 2021

Aberrant Expression and Subcellular Localization of ECT2 Drives Colorectal Cancer Progression and Growth.

Cancer Res 2022 Jan 4;82(1):90-104. Epub 2021 Nov 4.

Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.

ECT2 is an activator of RHO GTPases that is essential for cytokinesis. In addition, ECT2 was identified as an oncoprotein when expressed ectopically in NIH/3T3 fibroblasts. However, oncogenic activation of ECT2 resulted from N-terminal truncation, and such truncated ECT2 proteins have not been found in patients with cancer. In this study, we observed elevated expression of full-length ECT2 protein in preneoplastic colon adenomas, driven by increased mRNA abundance and associated with tumor-suppressor loss. Elevated ECT2 levels were detected in the cytoplasm and nucleus of colorectal cancer tissue, suggesting cytoplasmic mislocalization as one mechanism of early oncogenic ECT2 activation. Importantly, elevated nuclear ECT2 correlated with poorly differentiated tumors, and a low cytoplasmic:nuclear ratio of ECT2 protein correlated with poor patient survival, suggesting that nuclear and cytoplasmic ECT2 play distinct roles in colorectal cancer. Depletion of ECT2 reduced anchorage-independent cancer cell growth and invasion independent of its function in cytokinesis, and loss of extended survival in a -null colon cancer mouse model. Expression of ECT2 variants with impaired nuclear localization or guanine nucleotide exchange catalytic activity failed to restore cancer cell growth or invasion, indicating that active, nuclear ECT2 is required to support tumor progression. Nuclear ECT2 promoted ribosomal DNA transcription and ribosome biogenesis in colorectal cancer. These results support a driver role for both cytoplasmic and nuclear ECT2 overexpression in colorectal cancer and emphasize the critical role of precise subcellular localization in dictating ECT2 function in neoplastic cells. SIGNIFICANCE: ECT2 overexpression and mislocalization support its role as a driver in colon cancer that is independent from its function in normal cell cytokinesis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-4218DOI Listing
January 2022

Single-cell RNA sequencing reveals distinct tumor microenvironmental patterns in lung adenocarcinoma.

Oncogene 2021 12 18;40(50):6748-6758. Epub 2021 Oct 18.

Institute of Pathology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.

Recent developments in immuno-oncology demonstrate that not only cancer cells, but also the tumor microenvironment can guide precision medicine. A comprehensive and in-depth characterization of the tumor microenvironment is challenging since its cell populations are diverse and can be important even if scarce. To identify clinically relevant microenvironmental and cancer features, we applied single-cell RNA sequencing to ten human lung adenocarcinomas and ten normal control tissues. Our analyses revealed heterogeneous carcinoma cell transcriptomes reflecting histological grade and oncogenic pathway activities, and two distinct microenvironmental patterns. The immune-activated CP²E microenvironment was composed of cancer-associated myofibroblasts, proinflammatory monocyte-derived macrophages, plasmacytoid dendritic cells and exhausted CD8+ T cells, and was prognostically unfavorable. In contrast, the inert N³MC microenvironment was characterized by normal-like myofibroblasts, non-inflammatory monocyte-derived macrophages, NK cells, myeloid dendritic cells and conventional T cells, and was associated with a favorable prognosis. Microenvironmental marker genes and signatures identified in single-cell profiles had progonostic value in bulk tumor profiles. In summary, single-cell RNA profiling of lung adenocarcinoma provides additional prognostic information based on the microenvironment, and may help to predict therapy response and to reveal possible target cell populations for future therapeutic approaches.
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http://dx.doi.org/10.1038/s41388-021-02054-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8677623PMC
December 2021

DNA methylation profiling identifies two distinct subgroups in breast cancers with low hormone receptor expression, mainly associated with HER2 amplification status.

Clin Epigenetics 2021 10 3;13(1):184. Epub 2021 Oct 3.

German Cancer Consortium (DKTK), Partner Site Berlin, German Cancer Research Center (DKFZ), 69210, Heidelberg, Germany.

Background: Current clinical guidelines suggest that breast cancers with low hormone receptor expression (LowHR) in 1-10% of tumor cells should be regarded as hormone receptor positive. However, clinical data show that these patients have worse outcome compared to patients with hormone receptor expression above 10%. We performed DNA methylation profiling on 23 LowHR breast cancer specimens, including 13 samples with HER2 amplification and compared our results with a reference breast cancer cohort from The Cancer Genome Atlas to clarify the status for this infrequent but important patient subgroup.

Results: In unsupervised clustering and dimensionality reduction, breast cancers with low hormone receptor expression that lacked HER2 amplification usually clustered with triple negative breast cancer (TNBC) reference samples (8/10; "LowHR TNBC-like"). In contrast, most specimens with low hormone receptor expression and HER2 amplification grouped with hormone receptor positive cancers (11/13; "LowHR HRpos-like"). We observed highly similar DNA methylation patterns of LowHR TNBC-like samples and true TNBCs. Furthermore, the Ki67 proliferation index of LowHR TNBC-like samples and clinical outcome parameters were more similar to TNBCs and differed from LowHR HRpos-like cases.

Conclusions: We here demonstrate that LowHR breast cancer comprises two epigenetically distinct groups. Our data strongly suggest that LowHR TNBC-like samples are molecularly, histologically and clinically closely related to TNBC, while LowHR HRpos-like specimens are closely related to hormone receptor positive tumors.
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http://dx.doi.org/10.1186/s13148-021-01176-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489064PMC
October 2021

Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations.

J Pathol 2022 01 25;256(1):61-70. Epub 2021 Oct 25.

German Cancer Consortium (DKTK), Partner Site Berlin, and German Cancer Research Center (DKFZ), Heidelberg, Germany.

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5808DOI Listing
January 2022

Arthroprosthetic titanium and vanadium exposure - Confounder or cause of multiorgan morbidity.

Joint Bone Spine 2022 Jan 26;89(1):105256. Epub 2021 Jul 26.

Center for Musculoskeletal Surgery, Charité-University Medicine Berlin, Berlin, Germany; Department of Orthopaedics and Orthopaedic Surgery, University Medicine Greifswald, Greifswald, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jbspin.2021.105256DOI Listing
January 2022

DNA methylation-based prediction of response to immune checkpoint inhibition in metastatic melanoma.

J Immunother Cancer 2021 07;9(7)

Neurological Institute (Edinger Institute), University Hospital, Frankfurt am Main, Germany

Background: Therapies based on targeting immune checkpoints have revolutionized the treatment of metastatic melanoma in recent years. Still, biomarkers predicting long-term therapy responses are lacking.

Methods: A novel approach of reference-free deconvolution of large-scale DNA methylation data enabled us to develop a machine learning classifier based on CpG sites, specific for latent methylation components (LMC), that allowed for patient allocation to prognostic clusters. DNA methylation data were processed using reference-free analyses (MeDeCom) and reference-based computational tumor deconvolution (MethylCIBERSORT, LUMP).

Results: We provide evidence that DNA methylation signatures of tumor tissue from cutaneous metastases are predictive for therapy response to immune checkpoint inhibition in patients with stage IV metastatic melanoma.

Conclusions: These results demonstrate that LMC-based segregation of large-scale DNA methylation data is a promising tool for classifier development and treatment response estimation in cancer patients under targeted immunotherapy.
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http://dx.doi.org/10.1136/jitc-2020-002226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8291310PMC
July 2021

IDH2 R172 Mutations Across Poorly Differentiated Sinonasal Tract Malignancies: Forty Molecularly Homogenous and Histologically Variable Cases With Favorable Outcome.

Am J Surg Pathol 2021 09;45(9):1190-1204

Departments of Neuropathology.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.
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http://dx.doi.org/10.1097/PAS.0000000000001697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373679PMC
September 2021

Status quo of ALK testing in lung cancer: results of an EQA scheme based on in-situ hybridization, immunohistochemistry, and RNA/DNA sequencing.

Virchows Arch 2021 Aug 25;479(2):247-255. Epub 2021 Jun 25.

Institute of Pathology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin and Berlin Institute of Health, 10117, Berlin, Germany.

With this external quality assessment (EQA) scheme, we aim to investigate the diagnostic performance of the currently available methods for the detection of ALK alterations in non-small cell lung cancer on a national scale, namely, in situ hybridization (ISH), immunohistochemistry (IHC), and RNA/DNA sequencing (NGS). The EQA scheme cohort consisted of ten specimens, including four ALK positive and six ALK negative samples, which were thoroughly pretested using IHC, ISH, and RNA/DNA NGS. Unstained tumor sections were provided to the 57 participants, and the results were retrieved via an online questionnaire. ISH was used by 29, IHC by 38, and RNA/DNA sequencing by 19 participants. Twenty-eight institutions (97%) passed the ring trial using ISH, 33 (87%) by using IHC, and 18 (95%) by using NGS. The highest sensitivity and interrater agreement (Fleiss ' kappa) was observed for RNA/DNA sequencing (99%, 0.975), followed by ISH (94%, 0.898) and IHC (92%, 0.888). However, the proportion of samples that were not evaluable due to bad tissue quality was also higher for RNA/DNA sequencing (4%) compared with ISH (0.7%) and IHC (0.5%). While all three methods produced reliable results between the different institutions, the highest sensitivity and concordance were observed for RNA/DNA sequencing. These findings encourage the broad implementation of this method in routine diagnostic, although the application might be limited by technical capacity, economical restrictions, and tissue quality of formalin-fixed samples.
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http://dx.doi.org/10.1007/s00428-021-03106-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8364534PMC
August 2021

Artificial intelligence and pathology: From principles to practice and future applications in histomorphology and molecular profiling.

Semin Cancer Biol 2021 Feb 22. Epub 2021 Feb 22.

Institute of Pathology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany; German Cancer Consortium (DKTK), Partner Site Berlin, and German Cancer Research Center (DKFZ), Heidelberg, Germany; Institute of Pathology, Ludwig-Maximilians-Universität München, Thalkirchner Strasse 36, München, 80337, Germany. Electronic address:

The complexity of diagnostic (surgical) pathology has increased substantially over the last decades with respect to histomorphological and molecular profiling. Pathology has steadily expanded its role in tumor diagnostics and beyond from disease entity identification via prognosis estimation to precision therapy prediction. It is therefore not surprising that pathology is among the disciplines in medicine with high expectations in the application of artificial intelligence (AI) or machine learning approaches given their capabilities to analyze complex data in a quantitative and standardized manner to further enhance scope and precision of diagnostics. While an obvious application is the analysis of histological images, recent applications for the analysis of molecular profiling data from different sources and clinical data support the notion that AI will enhance both histopathology and molecular pathology in the future. At the same time, current literature should not be misunderstood in a way that pathologists will likely be replaced by AI applications in the foreseeable future. Although AI will transform pathology in the coming years, recent studies reporting AI algorithms to diagnose cancer or predict certain molecular properties deal with relatively simple diagnostic problems that fall short of the diagnostic complexity pathologists face in clinical routine. Here, we review the pertinent literature of AI methods and their applications to pathology, and put the current achievements and what can be expected in the future in the context of the requirements for research and routine diagnostics.
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http://dx.doi.org/10.1016/j.semcancer.2021.02.011DOI Listing
February 2021

Primary Extracellular Matrix Enables Long-Term Cultivation of Human Tumor Oral Mucosa Models.

Front Bioeng Biotechnol 2020 4;8:579896. Epub 2020 Dec 4.

Institute of Pharmacy (Pharmacology and Toxicology), Freie Universität Berlin, Berlin, Germany.

3D tumor models clearly outperform 2D cell cultures in recapitulating tissue architecture and drug response. However, their potential in understanding treatment efficacy and resistance development should be better exploited if also long-term effects of treatment could be assessed . The main disadvantages of the matrices commonly used for culture are their limited cultivation time and the low comparability with patient-specific matrix properties. Extended cultivation periods are feasible when primary human cells produce the extracellular matrix . Herein, we adapted the hyalograft-3D approach from reconstructed human skin to normal and tumor oral mucosa models and compared the results to bovine collagen-based models. The hyalograft models showed similar morphology and cell proliferation after 7 weeks compared to collagen-based models after 2 weeks of cultivation. Tumor thickness and VEGF expression increased in hyalograft-based tumor models, whereas expression of laminin-332, tenascin C, and hypoxia-inducible factor 1α was lower than in collagen-based models. Taken together, the produced extracellular matrix better confined tumor invasion in the first part of the cultivation period, with continuous tumor proliferation and increasing invasion later on. This proof-of-concept study showed the successful transfer of the hyalograft approach to tumor oral mucosa models and lays the foundation for the assessment of long-term drug treatment effects. Moreover, the use of an animal-derived extracellular matrix is avoided.
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http://dx.doi.org/10.3389/fbioe.2020.579896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746540PMC
December 2020

[Quality assurance in diagnostic in situ hybridization-experience of QuIP].

Pathologe 2020 Nov;41(6):614-620

Qualitätssicherungs-Initiative Pathologie QuIP GmbH, Berlin, Deutschland.

The Quality Assurance Initiative Pathology (QuIP) gives pathologists the opportunity to check the methodological processes of immunohistological and molecular diagnostics in a result-oriented manner and obtain a certificate reflecting the quality. For in situ hybridization (ISH), 5 round robin tests were organized in 2019, two recurrent (HER2-ISH gastric carcinomas and HER2-ISH breast carcinomas) and three prototypical (ROS1-NSCLC, ALK1-NSCLC, NTRK). The different round robin tests, which were provided by QuIP, are based on the development in diagnostics and the importance of the therapeutic relevance of the molecules which are tested. The results of the round robin tests in 2019 showed a sensitivity of at least 94.4%, a specificity of at least 96.6%, and a success rate of 85-99%. This reflected the high standard of quality of the round robin test and the participating institutes.
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http://dx.doi.org/10.1007/s00292-020-00832-6DOI Listing
November 2020

A multilayered epithelial mucosa model of head neck squamous cell carcinoma for analysis of tumor-microenvironment interactions and drug development.

Biomaterials 2020 11 6;258:120277. Epub 2020 Aug 6.

Freie Universität Berlin, Institute of Pharmacy (Pharmacology & Toxicology), Königin-Luise-Str. 2+4, 14195, Berlin, Germany. Electronic address:

Pharmacotherapy of head and neck squamous cell carcinoma (HNSCC) often fails due to the development of chemoresistance and severe systemic side effects of current regimens limiting dose escalation. Preclinical models comprising all major elements of treatment resistance are urgently needed for the development of new strategies to overcome these limitations. For model establishment, we used tumor cells from patient-derived HNSCC xenografts or cell lines (SCC-25, UM-SCC-22B) and characterized the model phenotype. Docetaxel and cetuximab were selected for comparative analysis of drug-related effects at topical and systemic administration. Cetuximab cell binding was mapped by cluster-based fluorescence lifetime imaging microscopy.The tumor oral mucosa (TOM) models displayed unstructured, hyper-proliferative, and pleomorphic cell layers, reflecting well the original tumor morphology and grading. Dose- and time-dependent effects of docetaxel on tumor size, apoptosis, hypoxia, and interleukin-6 release were observed. Although the spectrum of effects was comparable, significantly lower doses were required to achieve similar docetaxel-induced changes at topical compared to systemic application. Despite displaying anti-proliferative effects in monolayer cultures, cetuximab treatment showed only minor effects in TOM models. This was not due to inefficient cetuximab uptake or target cell binding but likely mediated by microenvironmental components.We developed multi-layered HNSCC models, closely reflecting tumor morphology and displaying complex interactions between the tumor and its microenvironment. Topical application of docetaxel emerged as promising option for HNSCC treatment. Aside from the development of novel strategies for topical drug delivery, our tumor model might help to better understand key regulators of drug-tumor-interactions.
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http://dx.doi.org/10.1016/j.biomaterials.2020.120277DOI Listing
November 2020

Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis.

Lab Invest 2020 10 29;100(10):1288-1299. Epub 2020 Jun 29.

Institute of Pathology, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.

Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.
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http://dx.doi.org/10.1038/s41374-020-0455-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498367PMC
October 2020

Dissecting the spatial heterogeneity of different immune cell subsets in non-small cell lung cancer.

Pathol Res Pract 2020 May 28;216(5):152904. Epub 2020 Feb 28.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Institute of Pathology, Berlin, Germany; Institute of Pathology, Technische Universität Dresden, Dresden, Germany. Electronic address:

To date, most studies investigating the immune tumor microenvironment of non-small cell lung cancers (NSCLC) only consider a small number of immune cell subsets or do not reflect the distribution of these cells between different tumor compartments as they were performed on tissue microarrays (TMA). To address this, we analyzed the immune infiltrate in surgically resected NSCLCs, focusing on potential spatial heterogeneity. We evaluated 45 NSCLCs based on whole-slide sections using immunohistochemistry with eleven different antibodies (CD3, CD4, CD8, CD20, CD68, Gata3, FOXP3, T-bet, kappa, lambda, PD-L1). While most markers were relatively evenly distributed among different tumor compartments as well as within the same tumor compartment, some immune cell subsets showed a considerable variance. Notably, the immune infiltrate at the tumor invasion front was dominated by B cells. Concerning markers for T cell differentiation, FoxP3 (Th2) was predominantly expressed in stromal lymphocytes, while T-bet (Th1) was most commonly expressed in intraepithelial immune cells. Although most immune cell subtypes showed a heterogenous distribution within in the intraepithelial compartment, the results from a simulated TMA and core biopsy were mostly in line with the results from whole slide evaluation. Regarding disease specific survival, there were no clear correlations. Interestingly, patients with intraepithelial T-bet positive lymphocytes had a significantly better outcome (p = 0.039), however, this difference was not preserved in multivariate analysis. In conclusion, our study shows that the immune tumor microenvironment of NSCLCs is complex and partially heterogenous, especially concerning markers for T cell differentiation.
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http://dx.doi.org/10.1016/j.prp.2020.152904DOI Listing
May 2020

A Non-interventional Clinical Trial Assessing Immune Responses After Radiofrequency Ablation of Liver Metastases From Colorectal Cancer.

Front Immunol 2019 19;10:2526. Epub 2019 Nov 19.

Department of Immunology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany.

Radiofrequency ablation (RFA) is an established treatment option for malignancies located in the liver. RFA-induced irreversible coagulation necrosis leads to the release of danger signals and cellular content. Hence, RFA may constitute an endogenous tumor vaccination, stimulating innate and adaptive immune responses, including tumor-antigen specific T cells. This may explain a phenomenon termed abscopal effect, namely tumor regression in untreated lesions evidenced after distant thermal ablation or irradiation. In this study, we therefore assessed systemic and local immune responses in individual patients treated with RFA. For this prospective clinical trial, patients with liver metastasis from colorectal carcinoma (mCRC) receiving RFA and undergoing metachronous liver surgery for another lesion were recruited ( = 9) during a 5-year period. Tumor and non-malignant liver tissue samples from six patients were investigated by whole transcriptome sequencing and tandem-mass spectrometry, characterizing naturally presented HLA ligands. Tumor antigen-derived HLA-restricted peptides were selected by different predefined approaches. Further, candidate HLA ligands were manually curated. Peripheral blood mononuclear cells were stimulated with epitope candidate peptides, and functional T cell responses were assessed by intracellular cytokine staining. Immunohistochemical markers were additionally investigated in surgically resected mCRC from patients treated with ( = 9) or without RFA ( = 7). In all six investigated patients, either induced immune responses and/or pre-existing T cell immunity against the selected targets were observed. Multi-cytokine responses were directed against known tumor antigens such as cyclin D1 but also against a (predicted) mutation contained in ERBB3. Immunohistochemistry did not show a relevant influx of immune cells into distant malignant lesions after RFA treatment ( = 9) as compared to the surgery only mCRC group ( = 7). Using an individualized approach for target selection, RFA induced and/or boosted T cell responses specific for individual tumor antigens were more frequently detectable as compared to previously published observations with well-characterized tumor antigens. However, the witnessed modest RFA-induced immunological effects alone may not be sufficient for the rejection of established tumors. Therefore, these findings warrant further clinical investigation including the assessment of RFA combination therapies e.g., with immune stimulatory agents, cancer vaccination, and/or immune checkpoint inhibitors.
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http://dx.doi.org/10.3389/fimmu.2019.02526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877671PMC
November 2020

Next generation sequencing of lung adenocarcinoma subtypes with intestinal differentiation reveals distinct molecular signatures associated with histomorphology and therapeutic options.

Lung Cancer 2019 12 11;138:43-51. Epub 2019 Oct 11.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Institute of Pathology, Berlin, Germany; German Cancer Consortium (DKTK), Partner Site Berlin, German Cancer Research Center (DKFZ), Heidelberg, Germany. Electronic address:

Objectives: We aim to provide a better understanding of the molecular landscape of primary lung adenocarcinomas with intestinal differentiation.

Material And Methods: Five invasive mucinous adenocarcinomas (IMA) and seven pulmonary enteric adenocarcinomas (PEAD) were included in this study. Furthermore, we analyzed six pulmonary colloid adenocarcinomas (CAD), including one primary tumor, one metastasis, and two sample pairs consisting of the primary colloid lung tumor and a matching metastasis and an acinar component, respectively. All samples were characterized using immunohistochemistry (TTF-1, CK7, CK20, CDX2, Ki-67, ALK and PD-L1) and a next generation sequencing panel covering 404 cancer-related genes (FoundationOne® gene panel).

Results And Conclusion: While Ki-67 expression was comparably low in IMA (range: 8-15%) and in primary CAD (range: 5-8%), we observed considerably higher proliferation rates in the non-colloid tumor compartment (16%) and metastases (72%) from CAD, as well as in the PEAD-group (36-71%). The overall tumor mutational burden was lowest in IMA (2.5 mutations per megabase), intermediate in CAD (5.8 mutations per megabase) and highest in PEAD (16.8 mutations per megabase). KRAS mutations were frequent in all three tumor subtypes, but TP53 mutations were mostly limited to PEAD. While chromosomal alterations were rare in IMA, we discovered MYC amplifications in three of four CAD. Comparing primary and metastatic CAD, we observed the acquisition of multiple mutations and chromosomal alterations. PEAD had a variety of chromosomal alterations, including two cases with RICTOR amplification. PD-L1 expression (20%, 50% and 80% of tumor cells) was limited to three PEAD samples, only. In conclusion, we provide a detailed insight into the molecular alterations across and within the different subtypes of pulmonary adenocarcinomas with intestinal differentiation. From a clinical perspective, we provide data on potential treatment strategies for patients with PEAD, including immunotherapy.
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http://dx.doi.org/10.1016/j.lungcan.2019.10.005DOI Listing
December 2019

Machine learning analysis of DNA methylation profiles distinguishes primary lung squamous cell carcinomas from head and neck metastases.

Sci Transl Med 2019 09;11(509)

Institute of Pathology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, 10117 Berlin, Germany.

Head and neck squamous cell carcinoma (HNSC) patients are at risk of suffering from both pulmonary metastases or a second squamous cell carcinoma of the lung (LUSC). Differentiating pulmonary metastases from primary lung cancers is of high clinical importance, but not possible in most cases with current diagnostics. To address this, we performed DNA methylation profiling of primary tumors and trained three different machine learning methods to distinguish metastatic HNSC from primary LUSC. We developed an artificial neural network that correctly classified 96.4% of the cases in a validation cohort of 279 patients with HNSC and LUSC as well as normal lung controls, outperforming support vector machines (95.7%) and random forests (87.8%). Prediction accuracies of more than 99% were achieved for 92.1% (neural network), 90% (support vector machine), and 43% (random forest) of these cases by applying thresholds to the resulting probability scores and excluding samples with low confidence. As independent clinical validation of the approach, we analyzed a series of 51 patients with a history of HNSC and a second lung tumor, demonstrating the correct classifications based on clinicopathological properties. In summary, our approach may facilitate the reliable diagnostic differentiation of pulmonary metastases of HNSC from primary LUSC to guide therapeutic decisions.
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http://dx.doi.org/10.1126/scitranslmed.aaw8513DOI Listing
September 2019

Immunohistochemical analysis of Bcl-2, nuclear S100A4, MITF and Ki67 for risk stratification of early-stage melanoma - A combined IHC score for melanoma risk stratification.

J Dtsch Dermatol Ges 2019 Aug;17(8):800-808

Institute of Pathology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Background And Objectives: Overall survival (OS) in patients with early-stage malignant melanoma differs. To date, there are no established prognostic markers. We aimed to contribute to a better understanding of potential prognostic immunohistochemical markers for risk stratification.

Patients And Methods: 161 surgically resected early-stage malignant melanomas (stage pT1 and pT2) were analyzed for expression of 20 different proteins using immunohistochemistry. The results were correlated with OS. The cohort was randomly split into a discovery and a validation cohort.

Results: High Bcl-2 expression, high nuclear S100A4 expression as well as a Ki67 proliferation index of ≥ 20 % were associated with shorter OS. Strong MITF immunoreactivity was a predictor for favorable prognosis. A combination of these four markers resulted in a multi-marker score with significant prognostic value in multivariate survival analysis (HR: 3.704; 95 % CI 1.484 to 9.246; p = 0.005). Furthermore, the score was able to differentiate a low-risk group with excellent OS rates (five-year survival rate: 100 %), an intermediate-risk group (five-year survival rate: 81.8 %) and a high-risk group (five-year survival rate: 52.6 %). The prognostic value was confirmed within the validation cohort.

Conclusions: Combined immunohistochemical analysis of Bcl-2, nuclear S100A4, Ki67 and MITF could contribute to better risk stratification of early-stage malignant melanoma patients.
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http://dx.doi.org/10.1111/ddg.13917DOI Listing
August 2019

Assessment of the extracellular volume fraction for the grading of clear cell renal cell carcinoma: first results and histopathological findings.

Eur Radiol 2019 Nov 18;29(11):5832-5843. Epub 2019 Mar 18.

Department of Radiology, Charité, Charitéplatz 1, 10117, Berlin, Germany.

Objectives: To assess the potential of T1 mapping-based extracellular volume fraction (ECV) for the identification of higher grade clear cell renal cell carcinoma (cRCC), based on histopathology as the reference standard.

Methods: For this single-center, institutional review board-approved prospective study, 27 patients (17 men, median age 62 ± 12.4 years) with pathologic diagnosis of cRCC (nucleolar International Society of Urological Pathology (ISUP) grading) received abdominal MRI scans at 1.5 T using a modified Look-Locker inversion recovery (MOLLI) sequence between January 2017 and June 2018. Quantitative T1 values were measured at different time points (pre- and postcontrast agent administration) and quantification of the ECV was performed on MRI and histological sections (H&E staining).

Results: Reduction in T1 value after contrast agent administration and MR-derived ECV were reliable predictors for differentiating higher from lower grade cRCC. Postcontrast T1 values (T1 = T1 difference between the native and nephrogenic phase) and MR-derived ECV were significantly higher for higher grade cRCC (ISUP grades 3-4) compared with lower grade cRCC (ISUP grades 1-2) (p < 0.001). A cutoff value of 700 ms could distinguish higher grade from lower grade tumors with 100% (95% CI 0.69-1.00) sensitivity and 82% (95% CI 0.57-0.96) specificity. There was a positive and strong correlation between MR-derived ECV and histological ECV (p < 0.01, r = 0.88). Interobserver agreement for quantitative longitudinal relaxation times in the T1 maps was excellent.

Conclusions: T1 mapping with ECV measurement could represent a novel in vivo biomarker for the classification of cRCC regarding their nucleolar grade, providing incremental diagnostic value as a quantitative MR marker.

Key Points: • Reduction in MRI T1 relaxation times after contrast agent administration and MR-derived extracellular volume fraction are useful parameters for grading of clear cell renal cell carcinoma (cRCC). • T1 differences between the native and the nephrogenic phase are higher for higher grade cRCC compared with lower grade cRCC and MRI-derived extracellular volume fraction (ECV) and histological ECV show a strong correlation. • T1 mapping with ECV measurement may be helpful for the noninvasive assessment of cRCC pathology, being a safe and feasible method, and it has potential to optimize individualized treatment options, e.g., in the decision of active surveillance.
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http://dx.doi.org/10.1007/s00330-019-06087-xDOI Listing
November 2019

DNA methylation profiling reliably distinguishes pulmonary enteric adenocarcinoma from metastatic colorectal cancer.

Mod Pathol 2019 06 5;32(6):855-865. Epub 2019 Feb 5.

Department of Neuropathology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.

Pulmonary enteric adenocarcinoma is a rare non-small cell lung cancer subtype. It is poorly characterized and cannot be distinguished from metastatic colorectal or upper gastrointestinal adenocarcinomas by means of routine pathological methods. As DNA methylation patterns are known to be highly tissue specific, we aimed to develop a methylation-based algorithm to differentiate these entities. To this end, genome-wide methylation profiles of 600 primary pulmonary, colorectal, and upper gastrointestinal adenocarcinomas obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database were used as a reference cohort to train a machine learning algorithm. The resulting classifier correctly classified all samples from a validation cohort consisting of 680 primary pulmonary, colorectal and upper gastrointestinal adenocarcinomas, demonstrating the ability of the algorithm to reliably distinguish these three entities. We then analyzed methylation data of 15 pulmonary enteric adenocarcinomas as well as four pulmonary metastases and four primary colorectal adenocarcinomas with the algorithm. All 15 pulmonary enteric adenocarcinomas were reliably classified as primary pulmonary tumors and all four metastases as well as all four primary colorectal cancer samples were identified as colorectal adenocarcinomas. In a t-distributed stochastic neighbor embedding analysis, the pulmonary enteric adenocarcinoma samples did not form a separate methylation subclass but rather diffusely intermixed with other pulmonary cancers. Additional characterization of the pulmonary enteric adenocarcinoma series using fluorescence in situ hybridization, next-generation sequencing and copy number analysis revealed KRAS mutations in nine of 15 samples (60%) and a high number of structural chromosomal changes. Except for an unusually high rate of chromosome 20 gain (67%), the molecular data was mostly reminiscent of standard pulmonary adenocarcinomas. In conclusion, we provide sound evidence of the pulmonary origin of pulmonary enteric adenocarcinomas and in addition provide a publicly available machine learning-based algorithm to reliably distinguish these tumors from metastatic colorectal cancer.
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http://dx.doi.org/10.1038/s41379-019-0207-yDOI Listing
June 2019

RNA-based analysis of ALK fusions in non-small cell lung cancer cases showing IHC/FISH discordance.

BMC Cancer 2018 Nov 22;18(1):1158. Epub 2018 Nov 22.

Charité-Universitätsmedizin Berlin corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Pathology, Charitéplatz 1, 10117, Berlin, Germany.

Background: Rearrangements of the anaplastic lymphoma kinase (ALK) belong to the promising targets in the therapy of advanced non-small cell lung cancer (NSCLC) and are predominantly detected by immunohistochemistry (IHC) and/or fluorescence in-situ hybridization (FISH). However, both methods occasionally produce discordant results, especially in so-called borderline (BL) cases, showing ALK FISH-positive signals in 10-20% of the tumor nuclei around the cutoff (15%). This leads to a diagnostic and thus to a therapeutic dilemma.

Methods: We selected 18 unequivocal (12 ALK IHC/FISH-negative; 6 ALK IHC/FISH-positive) and 15 equivocal samples with discordant results between FISH (Abbott, Vysis LSI ALK Dual Color) and IHC (Ventana, D5F3), including cases with FISH-BL results, for further RNA based-analysis. To detect ALK rearrangement at the transcriptional level, RNA was analyzed using a targeted multiplex-PCR panel followed by IonTorrent sequencing and by direct transcript counting using a digital probe-based assay (NanoString). Sensitivity of both methods was defined using RNA obtained from an ALK-positive cell line dilution series.

Results: Cases with unequivocal IHC/FISH results showed concordant data with both RNA-based methods, whereas the three IHC-negative/FISH-positive samples were negative. The four IHC-negative/FISH-BL-negative cases, as well as the five IHC-negative/FISH-BL-positive samples showed negative results by massive parallel sequencing (MPS) and digital probe-based assay. The two IHC-positive/FISH-BL-positive cases were both positive on the RNA-level, whereas a tumor with questionable IHC and FISH-BL-positive status displayed no ALK fusion transcript.

Conclusions: The comparison of methods for the confirmation of ALK rearrangements revealed that the detection of ALK protein by IHC and ALK fusion transcripts on transcriptional level by MPS and the probe-based assay leads to concordant results. Only a small proportion of clearly ALK FISH-positive cases are unable to express the ALK protein and ALK fusion transcript which might explain a non-responding to ALK inhibitors. Therefore, our findings led us to conclude that ALK testing should initially be based on IHC and/or RNA-based methods.
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http://dx.doi.org/10.1186/s12885-018-5070-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251223PMC
November 2018

Prognostic significance of Ki-67 levels and hormone receptor expression in low-grade serous ovarian carcinoma: an investigation of the Tumor Bank Ovarian Cancer Network.

Hum Pathol 2019 03 11;85:299-308. Epub 2018 Nov 11.

Department of Gynecology, Charité University Hospital, 13353 Berlin, Germany; Department of Gynecology and Gynecologic Oncology, Kliniken Essen-Mitte, Evangelische Huyssens-Stiftung, 45136 Essen, Germany.

Low-grade serous ovarian carcinoma (LGSOC) has recently come up as a distinct rare entity of epithelial ovarian cancer. Predictive and prognostic markers are not well studied yet. Because Ki-67 and hormone receptors (HR) have been established as relevant cancer biomarkers in several malignant tumors, we evaluated Ki-67 and HR expression rates by immunohistochemistry in 68 patients with LGSOC. We used a standardized cutoff finder algorithm to analyze prognostic significance for overall survival (OS) and progression-free survival (PFS). Cox regression showed a significant continuous decrease in OS for higher proliferation rates with an HR  of 1.07% (95% confidence interval, 1.01%-3.67%; P = .048) but not in PFS (P = .86). Cutoff finder analysis revealed the best possible cutoff for OS at 6.28% (P = .04) and for PFS at 1.85% proliferative activity (P = .04). Estrogen receptors (ERs) were expressed in most LGSOC patients (n = 61; 89.7%), progesterone receptor (PR) in about half of patients (n = 33; 48.5%). For both ER/PR, a statistically significant cutoff for PFS could be determined, which was at 75% of positive tumor cells for ER (P = .02) and at 15% of positive tumor cells for PR (P = .03). For OS, HR expression showed a tendency toward better OS for HR-positive tumors but did not turn out statistically significant. Our results show that Ki-67 is a valuable prognostic marker in the subgroup of LGSOC. We could also show that most LGSOCs express HRs but that this expression is associated with a better PFS, a finding valuable in times of antihormonal therapy in LGSOC.
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http://dx.doi.org/10.1016/j.humpath.2018.10.020DOI Listing
March 2019

Native T1 Mapping as an In Vivo Biomarker for the Identification of Higher-Grade Renal Cell Carcinoma: Correlation With Histopathological Findings.

Invest Radiol 2019 02;54(2):118-128

Departments of Radiology.

Objectives: The aims of this study were to identify higher-grade clear cell renal cell carcinoma (cRCC) with native T1 mapping and to histologically correlate the results with the collagen volume fraction.

Materials And Methods: For this institutional review board-approved, single-center prospective study, 68 consecutive patients received abdominal magnetic resonance imaging scans at 1.5 T between January 2017 and July 2018, using a Modified Look-Locker Inversion Recovery (MOLLI) sequence. Thirty patients with cRCC (20 men; mean age, 61.9 ± 13.1 years) who underwent partial or radical nephrectomy and histological grading according to the International Society of Urological Pathology (ISUP) classification and a separate healthy cohort of 30 individuals without renal malignancies or complex cysts (16 men; mean age, 59.7 ± 14.6 years) met the eligibility criteria. T1 values were quantitatively measured with region of interest measurements in T1 maps. Quantification of the collagen volume fraction was performed on histological sections (picrosirius red staining).

Results: Native T1 values were significantly lower for lower-grade cRCC (ISUP 1 and 2) compared with higher-grade cRCC (ISUP 3 and 4; P < 0.001). A cutoff value of 1101 milliseconds distinguished higher-grade from lower-grade tumors with a sensitivity of 100% (95% confidence interval [CI], 0.69-1.00), a specificity of 85% (95% CI, 0.62-0.97), and an accuracy of 90% (95% CI, 0.73-0.98). Native T1 values were significantly associated with the histological collagen volume fraction (P < 0.05). Furthermore, T1 times in the renal cortex, medulla, and tumor tissue showed an excellent interobserver agreement.

Conclusions: Native T1 mapping could represent an in vivo biomarker for the differentiation of lower- and higher-grade cRCCs, providing incremental diagnostic value beyond qualitative magnetic resonance imaging features.
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http://dx.doi.org/10.1097/RLI.0000000000000515DOI Listing
February 2019

Two patients with rare mixed adenoneuroendocrine carcinomas of the rectum.

SAGE Open Med Case Rep 2018 14;6:2050313X18758816. Epub 2018 Mar 14.

Department of Surgery, Campus Charité-Mitte and Campus Virchow-Klinikum, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Mixed adenoneuroendocrine carcinomas of the gastrointestinal tract are until today poorly understood and thus very challenging for interdisciplinary therapy. We herewith report the first case series of patients with a primary mixed adenoneuroendocrine carcinoma of the rectum. Both cases were initially diagnosed as adenocarcinoma and only secondarily with mixed adenoneuroendocrine carcinoma and had a poor outcome due to a rapid tumor progression and resistance to chemotherapy. A 65-year-old female presented with local tumor recurrence and hepatopulmonary metastasis 1 year after primary surgery for adenocarcinoma of the rectum and consecutive radiochemotherapy regimen. Fluorouracil (5-FU) was followed by bevacizumab- and capecitabine-based chemotherapy but had to be discontinued due to side effects and progressive disease. Progressive local pain syndrome accompanied by recurrent bleeding episodes led to a local tumor-debulking operation. Afterward, mixed adenoneuroendocrine carcinoma as the underlying diagnosis in the final histopathological examination was detected. The patient died 3 months after the operation in the context of a fulminant tumor progress. A 63-year-old male patient underwent neoadjuvant radiochemotherapy and laparoscopic rectum resection. After 5 months, postoperative oxaliplatin/capecitabine-based adjuvant chemotherapy was switched to carboplatin/etopsid due to a progressive polyneuropathy and biopsy-proven pulmonary metastasis. The patient then had to be switched to local radiation of cerebral metastases and Topotecan due to cerebral bleeding episodes but died 18 months after the initial diagnosis. In conclusion of our case series, mixed adenoneuroendocrine carcinomas of the rectum should be considered as a rare but aggressive tumor entity. An early and detailed histopathological diagnosis is required in order to establish an individual interdisciplinary treatment concept.
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http://dx.doi.org/10.1177/2050313X18758816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5858677PMC
March 2018

ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making.

Lung Cancer 2015 Dec 23;90(3):465-71. Epub 2015 Oct 23.

Institute of Pathology, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address:

Background: Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems.

Material And Methods: Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200).

Results: 94.3% (710/753) of the cases were classified as unequivocally (<10% or ≥20%) ALK-FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated.

Conclusion: While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical considerations.
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http://dx.doi.org/10.1016/j.lungcan.2015.09.022DOI Listing
December 2015

Parallel screening for ALK, MET and ROS1 alterations in non-small cell lung cancer with implications for daily routine testing.

Lung Cancer 2015 Feb 6;87(2):122-9. Epub 2014 Dec 6.

Institute of Pathology, Campus Charité Mitte, Charité Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Electronic address:

Objectives: ALK, MET and ROS1 are prognostic and predictive markers in NSCLC, which need to be implemented in daily routine. To evaluate different detection approaches and scoring systems for optimal stratification of patients eligible for mutation testing in the future, we screened a large and unselected cohort of NSCLCs for all three alterations.

Material And Methods: Using tissue microarrays, 473 surgically resected NSCLCs were tested for ALK and MET expression by IHC and genomic alterations in the ALK, MET and ROS1 gene by FISH. For MET IHC, two different criteria (MetMAb and H-score), for MET FISH, three different scoring systems (UCCC, Cappuzzo, PathVysion) were investigated.

Results: ALK and ROS1 positivity was seen in 2.6% and 1.3% of all ADCs, respectively, but not in pure SCCs. One ROS1 translocated tumor showed additional ROS1 amplification. MET IHC+/FISH+ cases were found in both histological subtypes (8.6% in all NSCLCs; 10.6% in ADCs; 5.0% in SCCs) and were associated with pleural invasion, lymphatic vessel invasion and lymph node metastasis. MET altered ADCs more frequently showed a papillary growth pattern. Whereas ALK testing revealed homogenous results in IHC and FISH, we saw discordant results for MET in about 10% of cases. Both METIHC scoring systems revealed almost identical results. We did not encounter any combined FISH positivity for ALK, MET or ROS1. However, three ALK positive cases harbored MET overexpression.

Conclusion: In daily routine, IHC could support FISH in the identification of ALK altered NSCLCs. Further research is needed to assess the role of discordant MET results by means of IHC and FISH as well as the relevance of tumors with an increased ROS1 gene copy number.
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http://dx.doi.org/10.1016/j.lungcan.2014.11.018DOI Listing
February 2015
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