Publications by authors named "Michiel van den Brand"

28 Publications

  • Page 1 of 1

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders.

Blood Adv 2021 08;5(16):3188-3198

Laboratory of Onco-Hematology, Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, University de Paris, Paris, France.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.
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http://dx.doi.org/10.1182/bloodadvances.2020004056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8405189PMC
August 2021

Next-Generation Sequencing-Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGS.

J Mol Diagn 2021 Sep 26;23(9):1105-1115. Epub 2021 Jun 26.

Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands.

Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.
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http://dx.doi.org/10.1016/j.jmoldx.2021.06.005DOI Listing
September 2021

Multiple Immunoglobulin κ Gene Rearrangements within a Single Clone Unraveled by Next-Generation Sequencing-Based Clonality Assessment.

J Mol Diagn 2021 Sep 19;23(9):1097-1104. Epub 2021 May 19.

Department of Pathology, Radboud University Medical Centre, Nijmegen, the Netherlands. Electronic address:

Clonality assessment of the Ig heavy- and light-chain genes (IGH and IGK) using GeneScan analysis is an important supplemental assay in diagnostic testing for lymphoma. Occasionally cases with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma are encountered, whereas the occurrence of biclonal lymphomas is rare, and the result of the IGH locus of these cases is in line with monoclonality. Three such ambiguous cases were assessed for clonality using next-generation sequencing. Information on the sequences of the rearrangements, combined with knowledge of the complex organization of the IGK locus, pointed to two explanations that can attribute seemingly biclonal IGK rearrangements to a single clone. In two cases, this explanation involved inversion rearrangements on the IGK locus, whereas in the third case, the cross-reactivity of primers generated an additional clonal product. In conclusion, next-generation sequencing-based clonality assessment allows for the detection of both inversion rearrangements and the cross-reactivity of primers, and can therefore facilitate the interpretation of cases of lymphoma with complex IGK rearrangement patterns.
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http://dx.doi.org/10.1016/j.jmoldx.2021.05.002DOI Listing
September 2021

Molecular Genetics of Relapsed Diffuse Large B-Cell Lymphoma: Insight into Mechanisms of Therapy Resistance.

Cancers (Basel) 2020 Nov 28;12(12). Epub 2020 Nov 28.

Department of Pathology, Radboud University Medical Center, 6525GA Nijmegen, The Netherlands.

The majority of patients with diffuse large B-cell lymphoma (DLBCL) can be treated successfully with a combination of chemotherapy and the monoclonal anti-CD20 antibody rituximab. Nonetheless, approximately one-third of the patients with DLBCL still experience relapse or refractory (R/R) disease after first-line immunochemotherapy. Whole-exome sequencing on large cohorts of primary DLBCL has revealed the mutational landscape of DLBCL, which has provided a framework to define novel prognostic subtypes in DLBCL. Several studies have investigated the genetic alterations specifically associated with R/R DLBCL, thereby uncovering molecular pathways linked to therapy resistance. Here, we summarize the current state of knowledge regarding the genetic alterations that are enriched in R/R DLBCL, and the corresponding pathways affected by these gene mutations. Furthermore, we elaborate on their potential role in mediating therapy resistance, also in connection with findings in other B-cell malignancies, and discuss alternative treatment options. Hence, this review provides a comprehensive overview on the gene lesions and molecular mechanisms underlying R/R DLBCL, which are considered valuable parameters to guide treatment.
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http://dx.doi.org/10.3390/cancers12123553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760867PMC
November 2020

Artificial intelligence to detect MYC translocation in slides of diffuse large B-cell lymphoma.

Virchows Arch 2021 Sep 26;479(3):617-621. Epub 2020 Sep 26.

Department of Pathology, Radboud University Medical Center, Geert Grooteplein 10, P.O. Box 9101, 6500 HB, Nijmegen, The Netherlands.

In patients with suspected lymphoma, the tissue biopsy provides lymphoma confirmation, classification, and prognostic factors, including genetic changes. We developed a deep learning algorithm to detect MYC rearrangement in scanned histological slides of diffuse large B-cell lymphoma. The H&E-stained slides of 287 cases from 11 hospitals were used for training and evaluation. The overall sensitivity to detect MYC rearrangement was 0.93 and the specificity 0.52, showing that prediction of MYC translocation based on morphology alone was possible in 93% of MYC-rearranged cases. This would allow a simple and fast prescreening, saving approximately 34% of genetic tests with the current algorithm.
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http://dx.doi.org/10.1007/s00428-020-02931-4DOI Listing
September 2021

Discrepancies in digital hematopathology diagnoses for consultation and expert panel analysis.

Virchows Arch 2021 Mar 25;478(3):535-540. Epub 2020 Aug 25.

Department of Pathology, Radboud University Medical Center, P. O. Box 9101, 6500, HB, Nijmegen, The Netherlands.

Digital pathology with whole-slide imaging (WSI) has a large potential to make the process of expert consultation and expert panel diagnosis more rapid and more efficient. However, comparison with the current methods is necessary for validation of the technique. In this study, we determined if digital assessment of whole-slide images of hematopathology specimens with a focus on the assessment of lymphoma can be used for consultation and panel diagnostics. Ninety-three histological specimens with a suspicion for lymphoma were assessed both with conventional microscopy and digital microscopy with a wash out period between assessments. A consensus diagnosis was based on full concordance between the pathologists or, in case of discordances, was reached at a joint session at a multi-headed microscope. In 81% of the cases, there was a full concordance between digital and light microscopical assessment for all three pathologists. Discordances between conventional microscopy and digital pathology were present in 3% of assessments. In comparison with the consensus diagnosis, discordant diagnoses were made in 5 cases with digital microscopy and in 3 cases with light microscopy. The reported level of confidence and need for additional investigations were similar between assessment by conventional and by digital microscopy. In conclusion, the performance of assessment by digital pathology is in general comparable with that of conventional light microscopy and pathologists feel confident using digital pathology for this subspecialty.
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http://dx.doi.org/10.1007/s00428-020-02907-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973407PMC
March 2021

Fallopian tube abnormalities in uterine serous carcinoma.

Gynecol Oncol 2020 08 12;158(2):339-346. Epub 2020 May 12.

Department of Obstetrics and Gynecology, Radboud University Medical Center, Nijmegen, the Netherlands; Department of Obstetrics and Gynecology, Canisius-Wilhelmina Hospital, Nijmegen, the Netherlands.

Objective: Uterine serous carcinoma (USC) is presumed to arise from endometrial intra-epithelial carcinoma (EIC), whereas tubo-ovarian high-grade serous carcinomas have similar precursor lesions in the Fallopian tube, i.e. serous tubal intra-epithelial carcinoma (STIC). The presence of Fallopian tube abnormalities and their clonal relationship to the concurrent USC was investigated.

Methods: In this multicenter study, all patients treated for USC between 1992 and 2017 were retrospectively identified. Histopathological diagnosis of USC, EIC and STIC was revised by an expert pathologist. Additionally, all Fallopian tube sections were immunohistochemically stained (p53 and Ki-67). Fallopian tube abnormalities were classified as either p53 signature, serous tubal intra-epithelial lesion (STIL) or STIC. The USCs and Fallopian tube abnormalities were analyzed by targeted next-generation sequencing.

Results: In 168 included patients, Fallopian tube abnormalities were found in 27.4% (46/168): p53-signatures in 17.9% (30/168), STILs in 3.0% (5/168) and STICs in 6.5% (11/168). In subgroup analysis, STICs were found in 9.5% (11/115) of patients with at least one section of the fimbriated end embedded. Next-generation sequencing showed identical TP53-mutations in the STIC and corresponding USC.

Conclusions: In conclusion, the presence of Fallopian tube abnormalities was shown in a high percentage of patients with USC, representing either true precursor lesions or metastasized disease.
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http://dx.doi.org/10.1016/j.ygyno.2020.04.707DOI Listing
August 2020

High frequency of inactivating tetraspanin mutations in diffuse large B-cell lymphoma at immune-privileged sites.

Blood 2019 09 31;134(12):946-950. Epub 2019 Jul 31.

Department of Tumor Immunology and.

Tetraspanin CD37 is predominantly expressed on the cell surface of mature B lymphocytes and is currently being studied as novel therapeutic target for B-cell lymphoma. Recently, we demonstrated that loss of CD37 induces spontaneous B-cell lymphoma in -knockout mice and correlates with inferior survival in patients with diffuse large B-cell lymphoma (DLBCL). Here, mutation analysis was performed in a cohort of 137 primary DLBCL samples, including 44 primary immune-privileged site-associated DLBCL (IP-DLBCL) samples originating in the testis or central nervous system. mutations were exclusively identified in IP-DLBCL cases (10/44, 23%) but absent in non-IP-DLBCL cases. The aberrations included 10 missense mutations, 1 deletion, and 3 splice-site mutations. Modeling and functional analysis of missense mutations revealed loss of function by impaired CD37 protein expression at the plasma membrane of human lymphoma B cells. This study provides novel insight into the molecular pathogenesis of IP-DLBCL and indicates that anti-CD37 therapies will be more beneficial for DLBCL patients without mutations.
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http://dx.doi.org/10.1182/blood.2019001185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789512PMC
September 2019

Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.

Leukemia 2019 09 13;33(9):2227-2240. Epub 2019 Jun 13.

Department of Pathology, Radboud University Medical Center, 6525 GA, Nijmegen, The Netherlands.

One of the hallmarks of B lymphoid malignancies is a B cell clone characterized by a unique footprint of clonal immunoglobulin (IG) gene rearrangements that serves as a diagnostic marker for clonality assessment. The EuroClonality/BIOMED-2 assay is currently the gold standard for analyzing IG heavy chain (IGH) and κ light chain (IGK) gene rearrangements of suspected B cell lymphomas. Here, the EuroClonality-NGS Working Group presents a multicentre technical feasibility study of a novel approach involving next-generation sequencing (NGS) of IGH and IGK loci rearrangements that is highly suitable for detecting IG gene rearrangements in frozen and formalin-fixed paraffin-embedded tissue specimens. By employing gene-specific primers for IGH and IGK amplifying smaller amplicon sizes in combination with deep sequencing technology, this NGS-based IG clonality analysis showed robust performance, even in DNA samples of suboptimal DNA integrity, and a high clinical sensitivity for the detection of clonal rearrangements. Bioinformatics analyses of the high-throughput sequencing data with ARResT/Interrogate, a platform developed within the EuroClonality-NGS Working Group, allowed accurate identification of clonotypes in both polyclonal cell populations and monoclonal lymphoproliferative disorders. This multicentre feasibility study is an important step towards implementation of NGS-based clonality assessment in clinical practice, which will eventually improve lymphoma diagnostics.
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http://dx.doi.org/10.1038/s41375-019-0508-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756030PMC
September 2019

Pathology Image Exchange: The Dutch Digital Pathology Platform for Exchange of Whole-Slide Images for Efficient Teleconsultation, Telerevision, and Virtual Expert Panels.

JCO Clin Cancer Inform 2019 06;3:1-7

Radboud University Medical Center, Nijmegen, the Netherlands.

Among the many uses of digital pathology, remote consultation, remote revision, and virtual slide panels may be the most important ones. This requires basic slide scanner infrastructure in participating laboratories to produce whole-slide images. More importantly, a software platform is needed for exchange of these images and functionality to support the processes around discussing and reporting on these images without breaching patient privacy. This poses high demands on the setup of such a platform, given the inherent complexity of the handling of digital pathology images. In this article, we describe the setup and validation of the Pathology Image Exchange project, which aimed to create a vendor-independent platform for exchange of whole-slide images between Dutch pathology laboratories to facilitate efficient teleconsultation, telerevision, and virtual slide panels. Pathology Image Exchange was released in April 2018 after technical validation, and a first successful validation in real life has been performed for hematopathology cases.
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http://dx.doi.org/10.1200/CCI.18.00146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873950PMC
June 2019

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

J Pathol 2019 04 30;247(4):416-421. Epub 2019 Jan 30.

IMGT®, the international ImMunoGeneTics Information System®, Université de Montpellier, LIGM, Institut de Génétique Humaine IGH, UMR CNRS UM, Montpellier, France.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5209DOI Listing
April 2019

Novel developments in the pathogenesis and diagnosis of extranodal marginal zone lymphoma.

J Hematop 2017 Dec 25;10(3-4):91-107. Epub 2017 Sep 25.

Department of Pathology, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 AG Nijmegen, The Netherlands.

Extranodal marginal zone lymphoma (EMZL), mostly represented by mucosa-associated lymphoid tissue (MALT) type, also referred to as MALT lymphoma, is a clinically heterogeneous entity within the group of low-grade B cell lymphomas that arises in a wide range of different extranodal sites, including the stomach, lung, ocular adnexa, and skin. It represents the third most common non-Hodgkin lymphoma in the Western world, and the median age of occurrence is around 60 years. One characteristic aspect in a subset of EMZL detectable in about 25% of the cases is the presence of specific chromosomal translocations involving the genes and , which lead to activation of the NF-κB signaling pathway. Another unique aspect is that several infectious agents, such as in the case of gastric EMZL, and autoimmune disorders, like Sjögren syndrome, have been implicated in the pathogenesis of this cancer. Recent findings as summarized in this review have further improved our understanding of the complex pathobiology of this disease and have been essential to better define novel treatment strategies. In addition, many of these specific features are currently being implemented for the diagnosis of EMZL.
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http://dx.doi.org/10.1007/s12308-017-0302-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712330PMC
December 2017

Pathways towards indolent B-cell lymphoma - Etiology and therapeutic strategies.

Blood Rev 2017 11 5;31(6):426-435. Epub 2017 Aug 5.

Department of Pathology, Radboud university medical center, Geert Grooteplein Zuid 10, 6525GA Nijmegen, The Netherlands. Electronic address:

Although patients with indolent B-cell lymphomas have a relatively good survival rate, conventional chemotherapy is not curative. Disease courses are typically characterized by multiple relapses and progressively shorter response duration with subsequent lines of therapy. There has been an explosion of innovative targeted agents in the past years. This review discusses current knowledge on the etiology of indolent B-cell lymphomas with respect to the role of micro-organisms, auto-immune diseases, and deregulated pathways caused by mutations. In particular, knowledge on the mutational landscape of indolent B-cell lymphomas has strongly increased in recent years and harbors great promise for more accurate decision making in the current wide range of therapeutic options. Despite this promise, only in chronic lymphocytic leukemia the detection of TP53 mutations and/or del17p currently have a direct effect on treatment decisions. Nevertheless, it is expected that in the near future the role of genetic testing will increase for prediction of response to targeted treatment as well as for more accurate prediction of prognosis in indolent B-cell lymphomas.
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http://dx.doi.org/10.1016/j.blre.2017.08.002DOI Listing
November 2017

Assessment of CD37 B-cell antigen and cell of origin significantly improves risk prediction in diffuse large B-cell lymphoma.

Blood 2016 12 19;128(26):3083-3100. Epub 2016 Oct 19.

Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH; and.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37, including TP53 mutation, NF-κB, Myc, phosphorylated STAT3, survivin, p63, and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37 status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37 DLBCL, ICOSLG upregulation in CD37 GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL.
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http://dx.doi.org/10.1182/blood-2016-05-715094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5201094PMC
December 2016

Identification of SLC41A3 as a novel player in magnesium homeostasis.

Sci Rep 2016 06 28;6:28565. Epub 2016 Jun 28.

Department of Physiology Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

Regulation of the body Mg(2+) balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg(2+) excretion. The basolateral Mg(2+) extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg(2+) extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg(2+) homeostasis using the Slc41a3 knockout (Slc41a3(-/-)) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3(-/-) mice suffer from hypomagnesemia. The intestinal Mg(2+) absorption capacity was measured using the stable (25)Mg(2+) isotope in mice fed a low Mg(2+) diet. (25)Mg(2+) uptake was similar in wildtype (Slc41a3(+/+)) and Slc41a3(-/-) mice, although Slc41a3(-/-) animals exhibited increased intestinal mRNA expression of Mg(2+) transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3(-/-) mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg(2+) handling.
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http://dx.doi.org/10.1038/srep28565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923877PMC
June 2016

Recurrent mutations in genes involved in nuclear factor-κB signalling in nodal marginal zone lymphoma-diagnostic and therapeutic implications.

Histopathology 2017 Jan 9;70(2):174-184. Epub 2016 Sep 9.

Department of Pathology, Radboud University Medical Centre, Nijmegen, The Netherlands.

Aims: To investigate the spectrum of mutations in 20 genes involved in B-cell receptor and/or Toll-like receptor signalling resulting in activation of nuclear factor-κB (NF-κB) in 20 nodal marginal zone lymphomas (NMZLs), 20 follicular lymphomas (FLs), and 11 cases of B-cell lymphoma, unclassifiable (BCL-u).

Methods And Results: Nodal marginal zone lymphomas were diagnosed according to strict criteria, including the expression of at least one putative marginal zone marker (MNDA and/or IRTA1). Cases that showed features of NMZL but did not fulfil all criteria were included as BCL-u. All FLs were required to have a BCL2 rearrangement. Mutations were found in: nine NMZLs, with recurrent mutations in TNFAIP3 and CD79B; 12 FLs, with recurrent mutations in TNFRSF14, TNFAIP3, and CARD11; and five cases of BCL-u, with recurrent mutations in TNFRSF14. TNFRSF14 mutations were present in FL and BCL-u, but not in any of the NMZLs. In the BCL-u group, TNFRSF14 mutations clustered with a FL immunophenotype.

Conclusions: These results suggest that TNFRSF14 mutations point towards a diagnosis of FL, and can be used in the sometimes difficult distinction between NMZL and FL, but to apply this in diagnostics would require confirmation in an independent cohort. In addition, the presence or absence of specific mutations in pathways converging on NF-κB could be important for decisions regarding targeted treatment.
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http://dx.doi.org/10.1111/his.13015DOI Listing
January 2017

A subset of low-grade B cell lymphomas with a follicular growth pattern but without a translocation shows features suggestive of nodal marginal zone lymphoma.

J Hematop 2016 Mar 2;9(1):3-8. Epub 2015 Sep 2.

Department of Pathology, Radboud University Medical Center, box 9101, 6500 HB Nijmegen, The Netherlands.

In our consultation practice, it was noted that many cases that were considered to represent follicular lymphoma (FL) without a translocation were ultimately classified as nodal marginal zone lymphoma (NMZL). This study set out to define recurrent morphological features of these cases. Thirty-three low-grade B cell lymphomas without a rearrangement were studied for recurrent morphological features. These features were then applied on 20 randomly selected cases to verify if these criteria are able to distinguish between lymphomas with and without a rearrangement, assigning them to one of five categories ranging from "certain FL" to "certain NMZL." Highly recurrent morphological features were noted in the lymphomas without a rearrangement, which were strongly overlapping with the morphological features of NMZL. All six cases that were assigned to the category of certainly FL or most likely FL indeed harbored a rearrangement, whereas all 12 cases assigned to the category of most likely NMZL or certain NMZL had no break. Of the two cases in the ambiguous category, one had received a final diagnosis of FL and the other of NMZL. This study raises the hypothesis that a subset of low-grade B cell lymphomas with a follicular growth pattern but without a translocation actually represents NMZL. This is at present difficult to prove, because no gold standard is available to differentiate between NMZL and FL without a rearrangement, so further investigations are needed.
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http://dx.doi.org/10.1007/s12308-015-0259-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764621PMC
March 2016

Tetraspanin CD37 protects against the development of B cell lymphoma.

J Clin Invest 2016 Feb 19;126(2):653-66. Epub 2016 Jan 19.

Worldwide, B cell non-Hodgkin lymphoma is the most common hematological malignancy and represents a substantial clinical problem. The molecular events that lead to B cell lymphoma are only partially defined. Here, we have provided evidence that deficiency of tetraspanin superfamily member CD37, which is important for B cell function, induces the development of B cell lymphoma. Mice lacking CD37 developed germinal center-derived B cell lymphoma in lymph nodes and spleens with a higher incidence than Bcl2 transgenic mice. We discovered that CD37 interacts with suppressor of cytokine signaling 3 (SOCS3); therefore, absence of CD37 drives tumor development through constitutive activation of the IL-6 signaling pathway. Moreover, animals deficient for both Cd37 and Il6 were fully protected against lymphoma development, confirming the involvement of the IL-6 pathway in driving tumorigenesis. Loss of CD37 on neoplastic cells in patients with diffuse large B cell lymphoma (DLBCL) directly correlated with activation of the IL-6 signaling pathway and with worse progression-free and overall survival. Together, this study identifies CD37 as a tumor suppressor that directly protects against B cell lymphomagenesis and provides a strong rationale for blocking the IL-6 pathway in patients with CD37- B cell malignancies as a possible therapeutic intervention.
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http://dx.doi.org/10.1172/JCI81041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4731177PMC
February 2016

Partial lack of BCL2 in follicular lymphoma: An unusual immunohistochemical staining pattern explained by ongoing BCL2 mutation.

Pathol Res Pract 2016 Feb 9;212(2):148-50. Epub 2015 Dec 9.

Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.

Follicular lymphomas are characterized by overexpression of BCL2 which, in the large majority of cases, is due to a t(14;18) translocation which juxtaposes the BCL2 locus to the immunoglobulin heavy chain locus (IGH). Here, we report partial absence of BCL2 immunohistochemical staining in a case of FL, due to a mutation in the part of BCL2 that encodes the epitope for the most frequently used antibody against BCL2. This finding shows that mutations in BCL2 occur in an ongoing process in follicular which can give rise to unusual immunohistochemical staining patterns.
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http://dx.doi.org/10.1016/j.prp.2015.12.001DOI Listing
February 2016

Clinical features of patients with nodal marginal zone lymphoma compared to follicular lymphoma: similar presentation, but differences in prognostic factors and rate of transformation.

Leuk Lymphoma 2016 07 23;57(7):1649-56. Epub 2015 Dec 23.

a Department of Pathology , Radboud University Medical Center , Nijmegen , The Netherlands ;

Nodal marginal zone lymphoma (NMZL) is a rare type of B-cell non-Hodgkin lymphoma. This study assessed the clinical features of 56 patients with NMZL in comparison to 46 patients with follicular lymphoma (FL). Patients with NMZL and FL had a largely similar clinical presentation, but patients with FL had a higher disease stage at presentation, more frequent abdominal lymphadenopathy and bone marrow involvement, and showed more common transformation into diffuse large B-cell lymphoma (DLBCL) during the course of disease. Overall survival and event-free survival were similar for patients with NMZL and FL, but factors associated with worse prognosis differed between the two groups. Transformation into DLBCL was associated with a significantly poorer outcome in both groups, but the phenotypes were different: DLBCL arising in FL was mainly of germinal center B-cell phenotype, whereas DLBCL arising in NMZL was mainly of non-germinal center B-cell phenotype.
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http://dx.doi.org/10.3109/10428194.2015.1106535DOI Listing
July 2016

Sequential immunohistochemistry: a promising new tool for the pathology laboratory.

Histopathology 2014 Nov 6;65(5):651-7. Epub 2014 Aug 6.

Department of Pathology, Radboud University Medical Centre, Nijmegen, The Netherlands.

Aims: Current immunohistochemical methods to study the expression of multiple proteins in a single tissue section suffer from several limitations. In this article, we report on sequential immunohistochemistry (S-IHC), a novel, easy method that allows the study of numerous proteins in a single tissue section, while requiring very limited optimization.

Methods And Results: In S-IHC, a tissue section is stained for multiple antibodies, with intermediate scanning of the section and elution of chromogen and antibodies. Overlays are made of the digital images, allowing assessment of multiple proteins in the same tissue section. We used S-IHC to study nine nodular lymphocyte-predominant Hodgkin lymphomas (NLPHLs) and 10 T-cell-rich and histiocyte-rich diffuse large B-cell lymphomas (T/HRBCLs) for expression of cyclin D1, CD20, and CD68. We observed cyclin D1 expression in single tumour cells in 44% of NLPHLs and 60% of T/HRBCLs. Comparison of S-IHC with classic single immunohistochemical staining revealed discrepancies in eight cases (42%), demonstrating the difficulty of differentiating tumour cells from histiocytes on morphological grounds, and stressing the additional value of S-IHC.

Conclusions: For research and diagnostic purposes, S-IHC is a promising technique that assesses the expression of numerous proteins in single tissue sections with complete architectural information, allowing phenotypic characterization of single cells.
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http://dx.doi.org/10.1111/his.12446DOI Listing
November 2014

Recognizing nodal marginal zone lymphoma: recent advances and pitfalls. A systematic review.

Haematologica 2013 Jul;98(7):1003-13

Department of Pathology, Radboud University Medical Center, Nijmegen, The Netherlands.

The diagnosis of nodal marginal zone lymphoma is one of the remaining problem areas in hematopathology. Because no established positive markers exist for this lymphoma, it is frequently a diagnosis of exclusion, making distinction from other low-grade B-cell lymphomas difficult or even impossible. This systematic review summarizes and discusses the current knowledge on nodal marginal zone lymphoma, including clinical features, epidemiology and etiology, histology, and cytogenetic and molecular features. In particular, recent advances in diagnostics and pathogenesis are discussed. New immunohistochemical markers have become available that could be used as positive markers for nodal marginal zone lymphoma. These markers could be used to ensure more homogeneous study groups in future research. Also, recent gene expression studies and studies describing specific gene mutations have provided clues to the pathogenesis of nodal marginal zone lymphoma, suggesting deregulation of the nuclear factor kappa B pathway. Nevertheless, nodal marginal zone lymphoma remains an enigmatic entity, requiring further study to define its pathogenesis to allow an accurate diagnosis and tailored treatment. However, recent data indicate that it is not related to splenic or extranodal lymphoma, and that it is also not related to lymphoplasmacytic lymphoma. Thus, even though the diagnosis is not always easy, it is clearly a separate entity.
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http://dx.doi.org/10.3324/haematol.2012.083386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696602PMC
July 2013

In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac.

Am J Physiol Renal Physiol 2012 Jun 14;302(11):F1395-401. Epub 2012 Mar 14.

Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Urine concentration involves the hormone vasopressin (AVP), which stimulates cAMP production in renal principal cells, resulting in translocation and transcription of aquaporin-2 (AQP2) water channels, greatly increasing the water permeability, leading to a concentrated urine. As cAMP levels decrease shortly after AVP addition, whereas AQP2 levels still increase and are maintained for days, we investigated in the present study the mechanism responsible for the AQP2 increase after long-term 1-desamino-8-d-arginine vasopressin (dDAVP) application using mouse collecting duct (mpkCCD) cells. While 30 min of dDAVP incubation strongly increased cAMP, cAMP was lower with 1 day and was even further reduced with 4 days of dDAVP, although still significantly higher than in control cells. One day of dDAVP incubation increased AQP2 promoter-dependent transcription, which was blocked by the protein kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE-dependent transcription and CREB phosphorylation were not increased. Exchange factor directly activated by cAMP (Epac) 1 and 2 were found to be endogenously expressed in mpkCCD cells. Application of dDAVP increased the expression of Epac1, while Epac2 was reduced. Incubation with a specific Epac activator after dDAVP pretreatment increased both AQP2 abundance and transcription compared with cells left unstimulated the last day. In conclusion, the PKA-CRE pathway is involved in the initial rise in AQP2 levels after dDAVP stimulation but not in the long-term effect of dDAVP. Instead, long-term regulation of AQP2 may involve the activation of Epac.
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http://dx.doi.org/10.1152/ajprenal.00376.2011DOI Listing
June 2012

Angiosarcoma in a patient with immunodeficiency, centromeric region instability, facial anomalies (ICF) syndrome.

Am J Med Genet A 2011 Mar 18;155A(3):622-5. Epub 2011 Feb 18.

Department of Pathology, Radboud University Nijmegen Medical Center, The Netherlands.

The Immunodeficiency, Centromeric region instability, and Facial anomalies (ICF) syndrome (OMIM #242860) is a rare autosomal recessive disorder caused by defective DNA methylation. Hematological disease and malignancy (macrophage activation syndrome, myelodysplastic syndrome, and Hodgkin lymphoma) have been reported in three patients. To date, there have been no reports of either epithelial or mesenchymal malignancies. We present a patient with all clinical and laboratory findings of the ICF syndrome who died of a metastatic angiosarcoma of the liver. This is the first report of a non-hematological malignancy in the ICF syndrome. The young age at which our patient developed an angiosarcoma suggests an effect of the defective DNA methylation observed in the ICF syndrome. Therefore, with improvement of recognition and treatment of the ICF syndrome, malignancy could become more common in this condition.
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http://dx.doi.org/10.1002/ajmg.a.33831DOI Listing
March 2011

Hypotonicity-induced reduction of aquaporin-2 transcription in mpkCCD cells is independent of the tonicity responsive element, vasopressin, and cAMP.

J Biol Chem 2011 Apr 15;286(15):13002-10. Epub 2011 Feb 15.

Department of Physiology, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.

The syndrome of inappropriate antidiuretic hormone secretion is characterized by excessive water uptake and hyponatremia. The extent of hyponatremia, however, is less than anticipated, which is ascribed to a defense mechanism, the vasopressin-escape, and is suggested to involve a tonicity-determined down-regulation of the water channel aquaporin-2 (AQP2). The underlying mechanism, however, is poorly understood. To study this, we used the mouse cortical collecting duct (mpkCCD) cell line. MpkCCD cells, transfected with an AQP2-promoter luciferase construct showed a reduced and increased AQP2 abundance and transcription following culture in hypotonic and hypertonic medium, respectively. This depended on tonicity rather than osmolality and occurred independently of the vasopressin analog dDAVP, cAMP levels, or protein kinase A activity. Although prostaglandins and nitric oxide reduced AQP2 abundance, inhibition of their synthesis did not influence tonicity-induced AQP2 transcription. Also, cells in which the cAMP or tonicity-responsive element (CRE/TonE) in the AQP2-promoter were mutated showed a similar response to hypotonicity. Instead, the tonicity-responsive elements were pin-pointed to nucleotides -283 to -252 and -157 to -126 bp. In conclusion, our data indicate that hypotonicity reduces AQP2 abundance and transcription, which occurs independently of vasopressin, cAMP, and the known TonE and CRE in the AQP2-promoter. Increased prostaglandin and nitric oxide, as found in vivo, may contribute to reduced AQP2 in vasopressin-escape, but do not mediate the effect of hypotonicity on AQP2 transcription. Our data suggest that two novel segments (-283 to -252 and -157 to -126 bp) in the AQP2-promoter mediate the hypotonicity-induced AQP2 down-regulation during vasopressin-escape.
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http://dx.doi.org/10.1074/jbc.M110.207878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075646PMC
April 2011

Activated leukocyte cell adhesion molecule expression predicts lymph node metastasis in oral squamous cell carcinoma.

Oral Oncol 2010 May 3;46(5):393-8. Epub 2010 Apr 3.

Department of Pathology, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB Nijmegen, The Netherlands.

Lymphatic metastasis of oral squamous cell carcinoma (SCC) is important for prognosis and clinical decision making concerning the treatment of the neck but may be difficult to detect. Activated leukocyte cell adhesion molecule (ALCAM), has been shown to correlate with prognosis or tumor grade in different tumor types and may be a predictor of lymphatic metastasis. ALCAM expression at the invasive front in fresh frozen tissue samples of oral SCC's (n=41) was studied immunohistochemically, using a polyclonal antibody directed against ALCAM's extracellular domain. Membranous expression of ALCAM at the invasive front was significantly related to lymph node metastasis (p=0.001, sensitivity 69%, specificity 84%) and tumor grade (p=0.035). There was no significant relationship with tumor thickness (p=0.394). Lymph node status (p=0.030), correlated with 5-year overall survival. A significant relation between ALCAM and prognosis could not be established, due to an insufficient sample size. However, ALCAM expression does appears to increase risk of early death. Membranous ALCAM expression at the invasive front serves as a molecular marker for lymphatic metastasis and may facilitate better treatment choices concerning the neck in patients with oral SCC.
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http://dx.doi.org/10.1016/j.oraloncology.2010.03.001DOI Listing
May 2010
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